Editorial: Editorial Current Drug Targets, 2007, Vol. 8, No. 4 491

Editorial
Current Drug Targets, 2007, Vol. 8, No. 4
491
Editorial
A comprehensive understanding of severe sepsis, enough to develop a unified approach to treatment remains the Holy Grail for the critical care medical community. In simple terms sepsis is a bloodstream infection with progressive severity to produce vascular inflammatory perturbations leading to vascular collapse, organ dysfunction, and death. Sepsis consumes the host blood leukocyte response, endothelial responses and related cytokine responses. A formal clinical definition of Severe Sepsis was developed in 1992 by the joint American College of Chest Physicians/Society of Critical Care Medicine. This definition relies on criteria of clinical signs of a systemic inflammatory response (SIRS criteria) and the presence of at least one organ failure. As a disease state, severe sepsis afflicts 750,000 patients in the U.S annually and causes 250,000 deaths. Previous anti-cytokine approaches (e.g. antibody to TNF, anti-IL-1, etc) in clinical trials were too specific and unsuccessful in achieving meaningful severe sepsis survival. Subsequently, agents addressing the inflammation-coagulation axis have been moderately successful in providing a survival advantage for severe sepsis patients. However, practical physiologic approaches of cardiovascular volume support, intensive insulin strategies, and other intensive organ support in the critical care unit have made a difference for some severe sepsis patients. This is a battle that is far from over. In this issue of Current Drug Targets seven reviews address various aspects of sepsis. The first five manuscripts evaluate specific drug targets in sepsis: 1.) apoptosis, 2.) endothelium, 3.) vascular permeability, 4.) cancer and sepsis and a specific target of intermediary metabolism 5.) phosphoenol pyruvate/ethyl pyruvate. Two other reviews take a more clinical approach to cover important subpopulations of sepsis, 6.) bioterrorism agents and 7.) the solid organ transplant population. In sepsis the host innate immune response calls in monocytes whose cytokine responses direct neutrophil and other leukocyte traffic. T-cells provide secondary responses and antiviral help. The stress response including steroids can reduce T-cell number leading to a degree of immune incompetence. Wesche-Soldato et al. review the leukocyte contribution to the host immune response with emphasis on T-cell apoptosis mechanisms and new promising drug targets aimed at this leukocyte subpopulation. Bill Aird reviews the role of the endothelium in sepsis, a complex regulatory organ essential for maintaining vascular-organ homeostasis and interfacing both coagulation and the progressive inflammation encountered in this condition. Natural anticoagulants and anti-inflammatory drug targets highlight a portion of the potential treatment targets within the endothelium. Vascular permeability occurs early in sepsis and intravascular volume loss accounts for concerning lung acute respiratory distress and the hypotension of distributive shock. Endotoxin, Tumor necrosis factor alpha and Thrombin (FIIa) incite this early endothelial permeability. Jacobsen et al. review subcellular mechanisms and potential drug targets aimed at reducing permeability. Cancer and sepsis is a smaller subgroup. With an aging population and evolving therapies for cancer, this population may achieve some relevance. Since some tumors express Tissue Factor, Cancer Procoagulant, and secrete other procoagulant proteins, the thrombotic tendency in sepsis may play to worse outcomes for the cancer patient. The question posed suggests that anticoagulants may be worth considering in this compromised population. Intermediary metabolism has received more notoriety with the finding that intensive insulin therapy can improve severe sepsis survival. Mitch Fink presents Ethyl Pyruvate (EP) as promising fit. From biochemical to cellular, to metabolism considerations through the relevant animal models EP should be near human dosing. Although presented as a single drug, the manuscript provides an excellent example of the process from molecule to mammal. Clinical subgroups presented here touch on bioterrorism, a thorough summary on sepsis-related agents and relevant mechanisms for consideration from Hepburn et al. (US Army infectious disease core, USAMRAIID). Immunosuppressed populations are a persistent concern in sepsis. Solid organ transplant infectious disease concepts and management are presented by Kalil et al. as an overview with some relevance to mechanism and management. Overall, drug-specific, process-specific, and vascular organ specific approaches are addressed with some clinical subgroups of sepsis adding variety to this series. Sepsis is complex from the biochemical and physiological perspective. Searches for new therapies will likely conclude in a few applicable agents with more than one mechanism as concomitant therapy. The battle has started, but it is far from over.
David E. Joyce MD
Current Drug Targets, 2007, 8, 493-500
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The Apoptotic Pathway as a Therapeutic Target in Sepsis
Doreen E. Wesche-Soldato, Ryan Z. Swan, Chun-Shiang Chung and Alfred Ayala*

Div. Surg. Res./Dept. Surgery, RI Hospital/Brown University Sch. Med., Providence, RI 02903
Abstract: Recent research has yielded many interesting and potentially important therapeutic targets in sepsis. Specifically, the effects of antagonistic anti-cytokine therapies (tumor necrosis factor-alpha [TNF- ], interleukin-1 [IL-1]) and anti-endotoxin strategies utilizing antibodies against endotoxin or endotoxin receptor/carrier molecules (anti-CD14 or anti-LPS-binding protein) have been studied. Unfortunately, these approaches often failed clinically, and in many cases, the efficacy of these treatments was dependent on the severity of sepsis. Recently, clinical trials using insulin to lock blood glucose levels and activated protein C treatment have showed that while they provided some survival benefit, their efficacy does not appear to be predicated solely upon anti-inflammatory effects. Here, we will review work done in animal models of polymicrobial sepsis and clinical findings that support the hypothesis that apoptosis in the immune system is a pathologic event in sepsis that can be a therapeutic target. In this respect, experimental studies looking at the septic animal suggest that loss of lymphocytes during sepsis may be due to dysregulated apoptosis and that this appears to be brought on by a variety of mediators effecting intrinsic as well as extrinsic cell death pathways. From a therapeutic perspective this has provided a number of novel targets for clinically successful current, as well as future therapies, such as caspases (caspase inhibition/protease inhibition), proapoptotic protein-expression (via administration and/or over-expression of Bcl-2) and the death receptor family Fas-FasL (via. FasFP [fas fusion protein] and the application of siRNA against a number pro-apoptotic factors). Key Words: Sepsis, mice, human, apoptosis, death-receptor pathway, mitochondrial pathway, organ dysfunction
INTRODUCTION Sepsis has an incidence of approximately 700,000 cases per year in the United States, and carries an overall mortality rate of nearly 30% [1]. Furthermore, as the American population ages, the incidence of sepsis is projected to increase, as the incidence and mortality rate of sepsis rise steadily with age [1]. The treatment of sepsis, however, utilizing clinical as well as pharmaceutical innovations, has proven to be a difficult task. As human sepsis is a complex and evolving disease, defining both the patient population who may benefit from a potential therapy and the timing of delivery of the therapy is critical. This was evident in the intensive care unit setting, where multiple clinical trials aimed at augmenting hemodynamic parameters of the critically ill patient failed to significantly improve survival [2, 3]. The importance of patient selection and timing of therapy was subsequently demonstrated by Rivers et al [4] in an emergency room setting. Patients with severe sepsis or septic shock, defined as having at least two criteria of the systemic inflammatory response syndrome (SIRS) plus a systolic blood pressure no higher than 90mmHg after fluid bolus or a blood lactate greater than four mmol/L, were promptly identified. These patients were then randomized to standard care or an algorithm aimed at balancing oxygen delivery with demand within six hours. Institution of this algorithm in this defined patient population at this early time point, presumably prior to the onset of irreversible tissue ischemia, significantly improved multiple indicators of organ dysfunction as well as 30-day mortality. Development of drug therapies to impact the substantial morbidity and mortality of sepsis has been complicated by these same obstacles, with many anti-inflammatory and anti-coagulant drugs showing promise in the laboratory setting, yet no survival benefit in recent randomized human trials [5, 6]. However, despite this, recombinant human activated protein C [7], low-dose corticosteroids [8], and intensive insulin therapy [9] have been proven to reduce mortality and have subsequently become widely accepted therapies
*Address correspondence to this author at the Division of Surgical Research, Aldrich 227, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903, Tel: (401) 444-5158; FAX: (401) 444-3278; E-mail: AAYALA@LIFESPAN.org 1389-4501/07 $50.00+.00
for treatment of specific populations of septic patients. These clinically successful therapies reduce mortality in part through modulation of the systemic inflammatory response; however, as these drugs (with the exception of corticosteroids) are not classic antiinflammatory agents, their mechanism of salutary benefit remains to be clarified. Thus, the extent of this interaction as well as the effects on the apoptotic pathway within the immune system is the topic of current investigation. Prior to discussing the apoptotic pathway as a potential target for drug therapy, we will briefly review the current research into the mechanism of the above therapies. Van den Berghe et al. [9] demonstrated that maintaining the blood glucose between 80 and 110 mg/dL by intensive insulin therapy correlated with an absolute decrease in mortality from 8 to 4.6%, a relative adjusted reduction of 32%, in a surgical ICU population. The greatest reduction of mortality was within the group of patients with multi-organ system failure and a proven septic focus. Intensive insulin therapy also correlated with reductions in the rate of septicemia, time spent on the mechanical ventilator, need for renal replacement therapy, and incidence of critical illness polyneuropathy. In addition to glycemic control, insulin is essential to serum lipid metabolism; thus, changes in the serum lipid profile may also be involved in the improved outcomes seen in the intensive insulin therapy group. Looking specifically at those patients in the above study who had an ICU stay of greater than seven days, intensive insulin therapy was also found to effect the serum lipid profile. Critical illness was associated with a rise in serum triglyceride levels and a drop in serum high-density lipoprotein (HDL) and low-density lipoprotein (LDL). These changes were significantly reduced by intensive insulin therapy, and this reduction correlated with improved survival [10]. Through modulation of glucose (and potentially free fatty acid) levels, insulin has a myriad of effects upon the immune response. Hyperglycemia has been shown to alter the immune response in multiple ways, and the administration of insulin has consistently been shown to counteract these effects. For example, hyperglycemia induces expression of leukocyte adhesion molecules, such as intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), which is suppressed by insulin treatment. Another example is the hyperglycemia-induced impairment of neu 2007 Bentham Science Publishers Ltd.
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trophil function, including chemotaxis, phagocytosis, and respiratory burst, which is attenuated by insulin. Insulin also appears to suppress the release of pro-inflammatory cytokines, which is exacerbated by hyperglycemia [11]. The effect of insulin-induced reduction in free fatty acid (FFA) levels upon the immune response is less clear. The effects of hyperglycemia and insulin treatment on the immune response during inflammatory insult are a topic of current experimental interest. In a recent human trial utilizing an intravenous bolus of lipopolysaccharide (LPS) during normoglycemia, hyperglycemic clamp at 15mM (with a subsequent rise in endogenous insulin levels), and hyperinsulinemic euglycemic clamp (HEC), the effects upon TNF- , interleukin-6 (IL-6), free fatty acid levels, and leukocyte count were analyzed [12]. This model showed no effect in either treatment group upon the TNF- level; however, the IL-6 level rose significantly higher in both treatment groups over controls at later time points. In both treatment groups, clamping alone significantly lowered the blood lymphocyte count prior to LPS injection, which persisted throughout the trial, although the blood lymphocyte count declined in all groups. Interestingly, FFA levels were suppressed in both treatment groups. Similarly, in a porcine model, the effects LPS and HEC were recently examined. HEC-LPS significantly lowered TNF- and glucagon levels as compared to LPS alone. There was no significant effect on interleukin-10 (IL-10), IL-6, or interleukin-8 (IL-8) levels, but again FFA levels were suppressed in both the HEC alone and HEC-LPS groups [13]. The above findings implicate an anti-inflammatory mechanism of intensive insulin therapy, possibly mediated by the reduction of TNF- or free fatty acid levels. Importantly, insulin by signaling through insulin growth factor receptor also has marked effects on proliferation as opposed to cell death. These seem to be orchestrated by insulin's capacity to activate/phosphorylate the survival factor Akt/PKB [14]. Potential significance of this link has recently been illustrated by Bommhardt et al. [15] who found that overexpression of Akt had a protective effect against lethal polymicrobial septic challenge. Thus far, however, a clear link has not been established between intensive insulin therapy and the apoptotic process. Interestingly, initial studies using the LPS-HEC porcine model have observed that at least with respect to LPS induced apoptosis of T and B lymphocytes, contrary to their hypothesis, HEC alone increased both T and B lymphocyte apoptosis, and the addition of HEC to LPS augmented apoptosis [16]. These studies were limited in that they utilized a relatively short LPS infusion time in an animal model, which does not duplicate the clinical course of the septic patient; however, they do indicate that insulin therapy and normoglycemia affect the inflammatory response and lymphocyte apoptosis. The role of timing of therapy and the extent of this effect upon lymphoid apoptosis in the setting of the septic patient are currently unknown. The known anti-inflammatory effects of corticosteroids lead to multiple trials utilizing high-dose corticosteroids in septic shock, with variable results. In a meta-analysis of the randomized controlled prospective trials that evaluated the effects of high-dose corticosteroids on mortality in the septic patient, no improvement in mortality was identified [6, 17, 18]. Recently, with the elucidation that severe sepsis can inhibit the hypothalamic-pituitary-adrenal axis, a renewed interest in corticosteroids has emerged. In a population of catecholamine-dependant patients who fail to respond to a corticotropin stimulation test, a seven-day course of low-dose hydrocortisone and fludrocortisone improved 28-day mortality [8]. A recent meta-analysis including five randomized controlled trials since 1997 confirmed the benefit of low-dose corticosteroid use in vasopressor-dependent septic shock [18]. The beneficial effects of low-dose steroids in vasopressordependent patients in septic shock is likely mediated by attenuation
of the systemic inflammatory response as well as direct effects on vasomotor tone and an increased responsiveness to vasopressors [19]. The anti-inflammatory effects of corticosteroids have been when established, the full extent of which is beyond the scope of this review. Briefly, the binding of glucocorticoids to glucocorticoid receptor and interactions with glucocorticoid responsive elements within the nucleus can block production of many inflammatory cytokines in multiple cell types. Glucocorticoids also block the production of inflammatory mediators, such as cyclo-oxygenase-2, and decrease leukocyte adhesion to endothelium [19]. In regards to apoptosis, corticosteroids/glucocorticoids have also shown both potentiating and suppressive effects on the process of apoptosis, which appears to be cell/tissue specific in nature. In thymocytes, proliferating lymphocytes, and mast cells, corticosteroids largely produce pro-apoptotic effects[20, 21], however, in neutrophils, epithelial cells and fibroblasts, the effect is typically anti-apoptotic [22, 23]. With respect to sepsis, the experimental data, thus far, indicate that corticosteroid release during sepsis appears to increase apoptosis of thymocytes [24], which is mediated by induction of caspase-9 [25]. However, the effect of low-dose steroids in the experimental setting of either animal sepsis or in vasopressor-dependent septic shock on the onset of apoptosis in immune or non-immune tissue remains to be understood. The concept of modulating the systemic inflammatory response to severe sepsis lead to many anti-inflammatory agents reaching randomized controlled clinical trials. Anti-endotoxin, anti-CD14, anti-LBP, anti-platelet activating factor, anti-TNF, and anti-IL-1 therapies went to phase III trial, and all met with limited success, showing no effect on mortality [5, 6]. Derangements of the coagulation cascade in severe sepsis have also lead to multiple trials of anti-coagulant drugs within the last decade. Large randomized controlled trials of anti-thrombin-III and Tissue Factor Pathway Inhibitor showed no improvement in survival [5, 6]. Recombinant human activated Protein C (rhaPC), on the other hand, was found to reduce 28-day mortality by 6%, a relative reduction of 19%, in patients with a documented or suspected infectious source, signs of the systemic inflammatory response syndrome, and evidence of organ dysfunction [7]. D-dimer as well as IL-6 levels were significantly reduced during rhaPC infusion, evidence of the anti-thrombotic and anti-inflammatory effects. A subgroup analysis of this study showed a decreased effectiveness with lower APACHE II scores at admission, prompting the Food and Drug Administration to approve the drug for use in patients with severe sepsis with high predicted mortality [26]. This finding was recently confirmed in a follow-up study involving patients with severe sepsis and a low risk of death, defined as an APACHE II score less than 25 or single-organ failure. The study was halted at interim analysis due to low likelihood of demonstrating a benefit of treatment and a higher risk of serious bleeding in the rhaPC group [27]. The successful application of rhaPC and the concurrent failure of other anticoagulant trials lead to investigation into the mechanism of action of rhaPC. The coagulation cascade is intimately tied to the mechanisms of the inflammatory response, and aPC exhibits effects on both, thus inhibiting the inflammatory response through both indirect and direct mechanisms. Inhibition of thrombin formation directly and via inactivation of factor Va and VIIIa attenuates thrombin-induced inflammatory cytokine release and endothelial dysfunction [28]. Recombinant human activated Protein C also directly attenuates the inflammatory response of human endothelial cells via binding to protease-activated receptor-1 (PAR-1) in an endothelial protein C receptor (EPCR) dependant manner [29]. Joyce et al. [30] demonstrated that rhaPC alone down-regulated the expression of nuclear factor-kappaB (NF- B) and attenuated NF- B expression following TNF- induction. Furthermore, TNF- induced expression of NFB regulated adhesion molecules, such as ICAM, VCAM, and E-
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selectin, was inhibited by rhaPC, potentially inhibiting leukocyte adherence in vivo. The effect of rhaPC on microcirculation in vivo was recently demonstrated in a rat model utilizing intravital microscopy following LPS versus LPS plus rhaPC injection [31]. At one, two, and three hours post-injection of LPS alone or LPS plus either low or high-dose rhaPC, the mesenteric microcirculation was examined under intravital microscopy. Both treatment groups had a significantly lower number of adherent leukocytes per field than the control group. The low-dose rhaPC group was found to have a significantly lower number of micro-vascular bleeding events and a higher arteriole and venule red blood cell velocity than both the control and high-dose rhaPC group. In addition to the effects upon microcirculation, the treatment groups were found to have an attenuation of the drop in white blood cell and platelet count that was evident in the LPS treated group. In addition, LPS also induced an early rise in TNF and later rise in IL-6, both of which were attenuated by rhaPC treatment. A possible correlation with ensuing organ damage was also demonstrated by rise in alanine transaminase and blood urea nitrogen in the LPS group, which was blocked by rhaPC treatment. Evidence for alteration of leukocyte migration is also shown in a recent human study in which the accumulation of leukocytes, predominantly neutrophils, in the lungs following intra-bronchial LPS instillation was inhibited in patients treated with rhaPC; furthermore, these neutrophils exhibited decreased ex-vivo chemotaxis [32]. Activated protein C has similarly been shown to counter the induction of apoptosis in animal studies as well as in human endothelial and monocyte cell lines in vitro [28, 30, 33, 34]. Recombinant human activated Protein C prevents staurosporine-induced apoptosis in human endothelial cells and up-regulated expression of multiple anti-apoptotic genes, including endothelial nitric oxide synthase (eNOS), Al, Bcl-2 homologue, and inhibitor of apoptosis-1 [30]. Apoptosis in a human monocyte cell line is also inhibited by rhaPC, an effect that is mediated by EPCR [28]. In human brain endothelial cells subjected to hypoxic injury, Cheng et al. [33] found a 60% reduction in apoptosis with the addition of rhaPC, and demonstrated that this cytoprotective effect depends upon EPCR and PAR-1 binding of rhaPC. In addition, hypoxia increased levels of the proapoptotic molecules p53, Bax, and caspase-3 and decreased Bcl-2 levels, an inhibitor of apoptosis. All of these changes were attenuated by the addition of rhaPC. In an in vivo murine model of ischemic stroke, the application of rhaPC decreased the size of infarction and edema formation, and decreased neutrophil infiltration and endothelial ICAM-1 expression [35]. Cheng et al. [33], in a similar model, demonstrated that the rescue effect of rhaPC is mediated by EPCR and PAR-1. A direct neuroprotective effect of aPC was recently shown in vitro and in vivo following N-methyl-D-aspartate (NMDA) and staurosporine induction of apoptosis [36]. In NMDA induced apoptosis, aPC was found to decrease translocation of apoptosis-inducing factor (AIF) into the nucleus, block caspase-3 and p53 induction, and attenuate the increase in Bax and decrease in Bcl-2. Activated protein C also blocked caspase-8 activation, thus blocking staurosporine-induced apoptosis. In both NMDA and staurosporine-induced apoptosis, the effects of aPC were dependant upon PAR-1 and PAR-3. These insights into the mechanisms of the current clinically effective drug therapies for severe sepsis indicate that the apoptotic pathway itself may prove to be a target for future drug therapies. APOPTOTIC PATHWAYS The apoptotic process is one in which cells are actively eliminated via a programmed pathway during morphogenesis, tissue remodeling, and the resolution of the immune response. Inducers of apoptosis include steroids, cytokines such as TNF- , IL-1 and IL-6, FasL, heat shock, oxygen free radicals, nitric oxide and FasL-
expressing cytotoxic T lymphocytes (CTLs) [37]. Apoptotic cell death occurs primarily through three different pathways: the extrinsic death receptor pathway (type I cells), the intrinsic (mitochondrial) pathway (type II cells) and the endoplasmic reticulum or stress-induced pathway (Fig. 1). In type I cells, Fas antigen (CD95), a major death receptor that belongs to the TNF superfamily of membrane receptors, is the first component of the pathway to receive a death signal. Fas is expressed on a variety of cell types, including thymocytes, activated B cells, T cells, monocytes, macrophages, neutrophils as well as on a variety of non-immune cells in the liver, lung and heart [38]. When Fas binds to its ligand, FasL, it trimerizes and creates a death-induced signaling complex (DISC) which recruits an adaptor molecule also containing a death domain, known as Fas-associated death domain (FADD). FADD binds to these activated death domains and to pro-caspase 8 through death effector domains (DEDs) to form the DISC. The death signal is then transduced from the DISC to a downstream caspase cascade when pro-caspase 8 is cleaved and becomes active caspase 8, which can, in turn, cleave and activate downstream effector caspases, such as caspase 3, 6, or 7. Caspase 3 cleaves inhibitors of caspase activated DNase (ICAD) and cleaves DNA in the nucleus [39], which leads to apoptosis. In type II cells, almost no DISC is formed, and the mitochondria is essential for releasing cellular destruction molecules such as cytochrome c which activates downstream caspases such as caspase 3 and caspase 9 (Fig. 1). The initiation of this pathway, however, is not well defined. The pathway can be activated by loss of growth factors such as IL-2, IL-4, or GM-CSF, the addition of cytokines such as IL-1 and IL-6, or exogenous stressors such as steroids, reactive oxygen intermediates, peroxynitrite or nitric oxide (NO) which in turn activate pro- or anti-apoptotic members of the Bcl-2 family. Pro-apoptotic Bcl-2 family members such as t-Bid or Bax are thought to translocate from the cytosol, where they normally exist in a quiescent state, to the mitochondrial membrane where they act to decrease mitochondrial membrane potential ( m). Antiapoptotic members of the Bcl-2 family (Bcl-2, Bcl-xL) block the release of cytochrome c, Smac/Diablo, and apaf-1 from the mitochondria, which, via apoptosome formation can activate caspase 9, which can in turn activate downstream caspase 3. Since apoptosis in type II cells can depend on the balance of the Bcl-2 family members, a dominance of anti-apoptotic family members such as Bcl-2 and Bcl-xL can promote survival of the cell [40]. The endoplasmic reticulum/stress-induced pathway is the least understood of the apoptotic pathways and appears to involve the activation of caspase 12 by Ca2+ and oxidant stress [41] (Fig. 1) [42]. THE ROLE OF APOPTOSIS IN THE PATHOLOGY OF SEPSIS As we have already alluded to in the introduction, advances have been made in the treatment of the septic patient via the application of rhaPC, low-dose steroid therapy and insulin to lock blood glucose levels. These are still somewhat modest effects, however, which are evident on select patient groups. While these agents all appear to commonly affect inflammation, the failure of more directed anti-inflammatory therapeutics as well as important noninflammatory targets of these treatments, suggests that other aspects of the pathology of sepsis may be targets. In this regard, studies in recent years have suggested that dysregulated apoptotic immune cell death may play a role in contributing to the immune dysfunction and multiple organ failure observed during sepsis and that blocking it can improve survival of experimental animals [43-46]. The immune cells most affected by this dysregulated apoptotic cell death appear to be lymphocytes. This loss of lymphocytes is detrimental to the survival of septic mice, as we also know that RAG1-/mice have a markedly decreased ability to survive cecal ligation and puncture (CLP) as compared to their wild type counterparts [47]. Apoptosis of lymphocytes is frequently seen 12+ hours fol-
Fig. (1). Both the extrinsic and intrinsic arms of the death pathway contain possible targets that can be exploited for therapy.
lowing the onset of experimental polymicrobial sepsis in the thymus, spleen, and gut-associated lymphoid tissues (GALT). It has been suggested that dysregulated lymphocyte apoptosis in these experimental animals results in decreased septic survival through the loss of lymphocytes. This, in turn is speculated to lead to immune suppression leaving the mouse unable to fight the lethal effects of sepsis, resulting in multiple organ failure and eventual death. Lymphocyte apoptosis in the thymus appears to occur early after the onset of sepsis (4 hours) and is independent of the effects of endotoxin or death receptors [48], but appear to be the result of glucocorticoids and NO [24]. It is also thought that the early release of complement C5a may contribute to thymocyte apoptosis [49]. In the bone marrow and lamina propria B cells [50], splenic T cells, intestinal intraepithelial lymphocytes (IELs), and mucosal T and B cells of the Peyers patches [51], apoptosis is mainly death receptordriven. Apoptosis in the spleen particularly seems to be important in septic mortality as an increase in splenic lymphocyte apoptosis in experimental animals after CLP results in reduced survival [52]. Lymphocyte restricted overexpression of Bcl-2, however, ameliorates this condition, further supporting the concept of lymphocyte loss as being an important aspect of increased mortality in experimental sepsis [47]. Other immune cell types, which have been reported to exhibit an increased incidence of apoptosis in sepsis, are CD8+ lymphoidderived dendritic cells of the spleen after CD3+CD4+ T cell activation [47], but the significance of this change is not well understood. Certain non-immune cells have also been shown to exhibit apoptotic changes, including mucosal epithelial cells [53] and to a certain extent endothelial cells [54, 55]. As mentioned earlier discussion of APC these latter non-immune cell targets, like the endothelium, may be important targets of the pathological effects of apoptosis.
APOPTOTIC TARGETS AND THERAPEUTIC CONSIDERATIONS Targeting the Intrinsic and Extrinsic Pathways One of the earliest anti-apoptotic approaches in sepsis research was the attempt to inhibit caspase activation. Caspase inhibitors usually contain fluoromethyl ketones (fmk) or chloromethyl ketones (cmk) that are derivatives of peptides that imitate cleavage sites of known caspase substrates. To inhibit the activity, they irreversibly alkylate the cysteine residue on the active site of the caspase [56]. Caspase-specific inhibitors that have been used include z-DEVD-fmk (caspase 3 and 7) and Ac-YVAD-cmk (caspase 1) [57] (Table 1). It has also been shown that broad spectrum caspase inhibitors such as z-VAD-fmk can prevent lymphocyte apoptosis in sepsis, and in turn, improve septic animal survival by 40-45% [58, 59]. However, at high doses, caspase inhibitors can have non-specific effects and cause cytotoxicity. In this respect, a different kind of pan-caspase inhibitor called Q-VD-Oph has been studied, however, that potently inhibits apoptosis but is not toxic at high doses, unlike z-VAD-fmk or Boc-D-fmk. Since it is also equally effective at preventing apoptosis by the three major apoptotic pathways (i.e. caspases 9/3, caspases 8/10, and caspase12) it seems likely that the efficacy of caspase inhibitors may be enhanced by using carboxyterminal o-phenoxy groups to make them more efficient in the clinical setting [60]. Peptidomimetics represent another potentially useful method of targeting the apoptotic pathway. "Peptidomimetics" are mimics that have similar structure and functional properties of the native parenteral peptides. This approach was adopted since the use of biologically active peptides as pharmaceutical compound have more or less failed due to their inability to stay bioavailable, penetrate cell membranes, and maintain metabolic stability. Synthetic mimics, however, can be generated to be more conformationally stable
497
Table 1.
Caspase Inhibitors
Caspase 1 2 3 4 5 6 7 8 9 10 12 13 family family 3,8,9,10,12 Inhibitor Ac-YVAD-cmk [57] z-VDVAD-fmk z-DEVD-fmk z-LEVD-fmk z-WEHD-fmk z-VEID-fmk z-DEVD-fmk z-IETD-fmk z-LEHD-fmk z-AEVD-fmk z-ATAD-fmk z-LEED-fmk z-VAD-fmk [58, 59] Boc-D-fmk Q-VD-Oph [60]
compounds that resist enzyme degradation, can cross cell membranes, and target specific proteins [61]. This new type of approach is being studied at present for its potential pro-apoptotic effect, particularly in cancer. Here they are being used to mimic proteinprotein interactions that will activate the apoptotic pathway in tumors, and in doing so, kill apoptosis-resistant tumors. Mimics have been synthesized that resemble the death domain of the proapoptotic Bcl-family member Bid [62] and also Smac, the proapoptotic protein which acts as an antagonist of the inhibitor-ofapoptosis (IAP), which normally suppresses caspases [63]. Both of these peptidomimetics permitted caspase activation and induced apoptosis in human cancer cells. The methods used to generate these mimics, hydrocarbon stapling [62] and the use of nonnatural amino acid replacements[63], resulted in protease-resistant molecules that were able to cross the cell membrane and bind efficiently to their target proteins. Since the problem with most peptide-based therapies is proteolytic degradation, the conformation of a peptidomimetic is not favorable for this [64]. Peptidomimetics are also water-soluble and are non-immunogenic. This allows them to be administered for longer periods of time [65]. It, therefore, seems possible to speculate that peptidomimetics may be useful in future therapies that need to modulate protein-protein interactions, and in the case of sepsis, activate anti-apoptotic proteins, which may protect those cells from apoptosis normally lost during the course of the disease. This idea may also be an alternative to gene therapy, which, in the past, has not done well in the clinical setting [66]. Enhancement of anti-apoptotic proteins, such as Bcl-2, has been shown to produce almost complete protection against T cell apoptosis in transgenic mice that overexpress Bcl-2. This, in turn, improved their survival after sepsis [47, 59]. In addition, adoptive transfer of T cells from Bcl-2 overexpressing mice into wild type septic mice also improved their survival [58]. While this clearly illustrates the important role of the lymphocyte in sepsis in controlling infection, it also illustrates a clear therapeutic target, which can be used to restore lymphocytes lost during this state. Yet another target that decreases lymphocyte apoptosis is Akt, a regulator of cell proliferation and death. It has been shown in mice that overexpress Akt that lymphocyte apoptosis is decreased and
survival after CLP is improved to 94% [15]. It may be at this level, (i.e. the activation of Akt) that treatments such as glucose control with insulin therapy or low-dose steroids have an effect on apoptosis in septic individuals. In addition, IFN- (a potent macrophage activator) release capacity is improved with Akt overexpression. The presence of IFN- is thought to be therapeutic itself, as restoration of macrophage function has been shown to alleviate sepsis in humans that showed monocyte deactivation and loss of Th1 cytokines [67]. Based on the finding of increased Fas expression in the tissues of septic mice [44, 45, 68, 69], there has been increasing interest in targeting components of the death receptor/extrinsic pathway in an attempt to ameliorate the effects of conditions affected by dysregulated apoptosis, such as sepsis. In this regard, studies from our own lab had initially focused on blocking the pathway at the death receptor itself (Fas). Initially, animals were treated with Fas fusion protein (FasFP; Amgen Inc., Thousand Oaks, CA) to inhibit the receptor ligation. This treatment resulted in a survival benefit [44] and reduced hepatic injury while improving total hepatic, intestinal and cardiac blood flow during sepsis [45]. In these studies it was also observed that when FasFP was given at 12 hours post-CLP but not earlier (0 hour) it was shown to have a positive effect [44]. This observation suggests that pathological events such as the septic aberrations in organ damage and/or blood flow which develop late in the course of sepsis [70], remain amenable to delayed FasFP treatment. In addition, a cell survival tyrosine kinase (MET) has been found to sequester Fas on hepatocytes by inhibiting Fas selfaggregation and Fas ligand binding [71]. However, limitations such as short half-life of these large fusion protein and/or antibody constructs as well as issues of tissue specific bio-availability represent important limitations of this type of intact protein treatment. One solution would be a possible formulation of peptidomimetics, as mentioned previously, that can block Fas. More recently, interfering RNA technology has been utilized to target gene expression of members of the extrinsic death receptor/Fas pathway. The experience with double stranded small interfering RNA (siRNA) (against Fas and caspase 8) has proven somewhat different however. This is most likely related to the biology of siRNA and its function. Both Fas and caspase 8 siRNA have been used in models of fulminant hepatitis [72, 73], and most recently, in sepsis [69]. Fas siRNA given 30 min. after CLP improved survival by 50% while reducing indices of organ damage and apoptosis in both the liver and spleen [69]. The mechanism of this survival benefit is still, for the most part, unknown. Preliminary data from our lab suggest, however, that Fas siRNA can be taken up by CD4+ and CD8+ T cells, as well as B cells. In the case of the spleen, it appears that lymphocyte apoptosis is reduced by silencing Fas, therefore enabling the host to maintain innate and adaptive immune cell crosstalk that would aid it in developing an immune response to ward off the infectious challenge. With respect to the liver, preliminary results suggest that Fas siRNA reduces the recruitment of potentially tissue damaging lymphocytes. It has been suggested in a model of hepatitis C that Fas ligand expressing CD4+ T cells can induce chronic hepatic inflammation [74], which, in the case of sepsis, may be instrumental in initiating multiple organ failure. By the same token, experimental CLP mice lacking CD8+ T cells exhibit improved survival over wild type [75]. It remains to be established what the exact link between increased Fas expression and the recruitment of potentially cytotoxic pro-inflammatory cells in the liver might be that leads to the development of organ damage/failure and eventual death. As intriguing as this approach is, several hurdles need to be overcome beyond cell targeting before siRNA can be applied clinically. Being double stranded RNA, the question can be raised as to whether siRNA is able to induce IFN signaling. Such signaling might induce unwanted pro-inflammatory sequellae in the septic animal undergoing siRNA treatment. While this is still under inves-
Table 2.
Potential Therapeutic Targets in the Apoptotic Pathway
Target Fas death receptor Caspases Bcl-2 family Proteases Akt C5a
Potential therapies FasFP [44], Fas siRNA [69] Inhibitors- broad and specific [58, 59], siRNA [69] Overexpression of anti-apoptotic members (gene therapy) [47, 59] Adrenomedullin/AMBP1 [88], Possible use of peptidomimetics [61] Protease inhibitors [88] Overexpression of (possible gene therapy) [15] anti-C5a neutralizing antibody [49]
tigation, there are studies primarily in mammalian cell lines, which suggest that siRNA can induce interferon under some conditions [76, 77]. On the other hand, we found that the hydrodynamic injection of naked siRNAs (50 g/mouse) in mice was not capable of inducing IFN- or IL-6 [69]. This is in keeping with another recent in vivo study, which has suggested that naked siRNA (as was delivered in our study) is not able to induce IFN through TLR3, unlike poly(I:C) [78]. Probably the biggest hurdle facing the use of siRNA clinically is that of its delivery. While a hydrodynamic-based method (where volume and rate of injection are critical in the uptake of naked constructs) is easily employed in research [79], it clearly cannot be used in human therapy in this way. Because naked siRNAs are degraded within seconds from a low volume injection, there needs to be a carrier or delivery vehicle that will protect the siRNA while not requiring a high volume, rapid injection. Cationic liposomes encoding anti-TNF siRNA have been studied, and appeared to reduce TNF- levels after endotoxemia, and may represent a useful alternative method for encapsulating/delivering siRNA [80, 81]. Other investigators have suggested the use of vectors as another mode of targeted siRNA delivery. However, as vectorized gene delivery these may cause inflammation themselves [76]. With respect to the extent of silencing, we and others have found that hydrodynamic injection of siRNA is able to maintain suppression of its target mRNA, at least in the liver, for up to 10 days following injection, with the signal starting to return at day 14 [69, 72]. With the major target of this hydrodynamic form of injection being the liver, we can only speculate that since the cells in the liver do not divide like cell lines in culture, there is less dilution of functional siRNAs which may allow for the sustained suppression of mRNA signal for a longer period of time [72]. Hypothetically, since this is a prolonged silencing effect and not a permanent one, toxicity would not be of high concern. Other Agents that Affect Apoptosis There have been other agents reported that are not direct inhibitors of programmed cell death per se, but have been discovered to affect the apoptotic pathway indirectly. In this regard, there is strong evidence for complement activation following CLP and in human sepsis [82], and that elevated levels of the anaphylatoxins C3a and C5a are thought to contribute to early thymocyte apoptosis. It was found that blockade of C5a by i.v. injection of rabbit anti-rat C5a antibody in septic rats reduced thymic apoptosis, completely inhibited the activation of caspases 3, 6, and 9 and restored expression of Bcl-xL [49]. Since C5a is not a direct member of the apoptotic pathway, it is thought that since complement activation promotes the release of pro-inflammatory cytokines such as TNF- , IL-1 , IL-6, and IL-8 from leukocytes [83], that it may affect the balance of cytokines that promote apoptosis but without affecting NF- B. Thus, this could be a reasonable explanation as it has been seen that thymocyte apoptosis in sepsis is independent of endotoxin
or TNF receptors such as Fas [24, 84]. Lastly, blocking C5a may also reduce neutrophil chemotaxis that may prevent excessive organ damage in this fashion [85]. Other agents have been tried that may reduce apoptosis after sepsis in vivo, as they are known to inhibit apoptosis in vitro. The protease inhibitor class of anti-retroviral agents were introduced in CLP mice as a pre-treatment and post-treatment and found to increase survival in both cases. This therapy reduced lymphocyte apoptosis, early TNF- levels and late IL-6 and IL-10 levels. Because the treatment had no effect on RAG1-/- mice, this suggests that it is specific for lymphocyte apoptosis [86]. However, like antiC5a, it may also inhibit neutrophil influx into septic animal tissues/organs [87]. Another strategy showing promise includes the use of the vasodilator adrenomedullin and adrenomedullin binding protein (AM/ AMBP1), which have been shown to increase survival of septic mice by 50% by preventing sepsis-induced vascular endothelial cell apoptosis. Treatment of mice with AM/AMBP1 increased protein levels of anti-apoptotic Bcl-2, while limiting tissue injury, improving organ blood flow and preventing the progression of sepsis from the hyperdynamic phase to the hypodynamic phase [55]. Interestingly, while the effect of lymphoid cell apoptosis remains to be established, its effects on endothelial cell apoptosis are in keeping with one of the suggested anti-apoptotic targets of rhaPC [30]. CONCLUSIONS Despite overwhelming research efforts and clinical trials, there has yet to be a therapy offered that significantly modifies the outcome of this disease. Even though there have been promising candidates for therapeutic intervention, sepsis manifests itself as multiple processes, making this task difficult. Here we have considered how recent treatment advances, such as rhaPC administration, lowdose steroids and the application of insulin to control the septic patient's blood glucose levels, may therapeutically speak through action on the apoptotic process. We have also reviewed several experimental studies focusing on the apoptotic arm of sepsis, revealing several targets (Table 2) within the apoptotic pathway, which may be useful in designing stand-alone and/or adjuvant therapies that may have a greater impact on septic mortality. Together these findings will hopefully reveal novel therapeutic targets and approaches that can improve the survival of the critically ill septic patient. ACKNOWLEDGEMENTS This work was supported in part by funds from NIH-RO1s GM53209 and HL73525 (to A.A.), as well as fellowship support from NIH-T32 GM65085 (for R.S.) and GAANN P200A03100 (for D.E.W-S.).
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Received: March 22, 2006
Accepted: May 20, 2006
501
Endothelium as a Therapeutic Target in Sepsis

William C. Aird*
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA
Abstract : The endothelium plays an important role in health and disease. Endothelial dysfunction contributes to sepsis pathophysiology. An important goal is to develop novel therapies that reverse endothelial dysfunction in sepsis. This review will consider the role of the endothelium in sepsis and will highlight its untapped therapeutic potential.
INTRODUCTION Sepsis represents the host response to infection. Severe sepsis, defined as sepsis complicated by organ dysfunction, results in activation of the inflammatory and coagulation cascades. The endothelium plays a key role in mediating the sepsis phenotype. The goals of this review are to discuss how endothelial cells contribute to and are affected by sepsis, and to underscore potential targets for novel therapy. ENDOTHELIAL BIOMEDICINE The endothelium is an expansive, spatially distributed organ system [1]. Endothelial cells participate in a large number of physiological processes including the control of vasomotor tone, the trafficking of cells and nutrients, the maintenance of blood fluidity and the regulation of permeability. However, the endothelium displays remarkable heterogeneity in structure and function. For example, endothelial-mediated regulation of vasomotor tone is a primary function of arteriolar endothelium; post capillary venules mediate transendothelial migration of leukocytes; capillaries from different organs demonstrate distinct barrier properties; and each vascular bed contributes to hemostatic balance through site-specific patterns of anticoagulant and procoagulant gene expression [2]. Phenotypic heterogeneity reflects the capacity of the endothelium to adapt to the varying needs of the underlying tissue. The two most common descriptors used to characterize the endothelium in disease are endothelial cell activation and endothelial cell dysfunction. Endothelial cell activation was originally coined in the 1980s and is now used to describe the phenotypic response of the endothelium to an inflammatory stimulus (though some investigators view proliferating endothelial cells in the context of angiogenesis to be activated). Endothelial activation in not an all-ornothing response. Rather, different agonists result in overlapping yet distinct patterns of signaling and gene expression in endothelial cells; and endothelial cells from different sites of the vasculature respond uniquely to any given extracellular stimulus. Nevertheless, activation typically consists of some combination of proinflammatory and procoagulant properties, and increased permeability. The term endothelial dysfunction, also introduced in the 1980s, is used most commonly to describe abnormalities in endothelial control of vasomotor tone in atherosclerotic coronary arteries. However, the endothelium participates in many homeostatic functions, is spatially distributed, and is involved in virtually every disease state. Thus, it is reasonable to conclude that the term endothelial dysfunction should not be restricted in function to abnormalities
in vasomotor tone, in location to the coronary arteries, or in disease scope to atherosclerosis. Rather, endothelial dysfunction should apply to any phenotype - whether or not it meets the definition of activation that poses a net liability to the host. In so far as the endothelium contributes to morbidity and mortality in severe sepsis, it may be characterized as dysfunctional. The endothelium is under-recognized as an organ [3]. It is hidden from view and is not amendable to traditional bedside diagnostic maneuvers such as inspection, palpation, percussion or auscultation. Unlike other organs such as the kidney and liver, there are no reliable lab markers for dysfunctional endothelium. Various studies have pointed to changes in circulating markers, such as soluble forms of E-selectin, P-selectin, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1 and thrombomodulin in patients with endothelial dysfunction [4, 5]. However, each of these markers has limitations, and none has reached the clinical mainstream. Several important diagnostic assays for endothelial dysfunction are on the horizon. These include the use of proteomics to analyze multiple soluble markers in the same patient, as well as assays for circulating endothelial cells, microparticles, and endothelial precursor cells (reviewed in [6-8]). The endothelium has enormous, though largely untapped therapeutic potential. It is strategically located between the blood and underlying tissue and is rapidly and preferentially exposed to systemically delivered agents. The endothelium is highly adaptable and is therefore modulatable from a therapeutic standpoint. Finally, since the endothelium is functionally linked to the underlying tissue microenvironment, it provides the pharmacotherapist with a direct window into each and every organ of the body. SEPSIS PATHOPHYSIOLOGY When considering sepsis pathophysiology, several important themes emerge. First, it is the host response, rather than the nature of the pathogen, that is the primary determinant of patient outcome. Second, severe sepsis is invariably associated with activation of inflammation and coagulation. This is evidenced by the fact that virtually every patient with severe sepsis has increased circulating levels of interleukin (IL)-6 and D-dimers. Third, monocytes and tissue macrophages are key in initiating the host response to infection. Fourth, the endothelium plays an important role in perpetuating and amplifying the host response. Finally, the combined action of soluble mediators (e.g. components of the inflammatory and clotting cascades) and activated cells (e.g. monocytes and endothelial cells) may result in organ dysfunction. The pathophysiology of sepsis may be simplified according to the scheme shown in Fig. (1). Monocytes, tissue macrophages (and to some extent neutrophils and endothelial cells) bind to pathogens via pattern recognition receptors, most notably members of the Toll-like receptor (TLR) family [9, 10]. Engagement of TLR triggers downstream signaling pathways, which leads to cell typespecific responses that include the release of cytokines, chemokines
2007 Bentham Science Publishers Ltd.
*Address correspondence to this author at the Molecular Medicine, Beth Israel Deaconess Medical Center, RW-663, 330 Brookline Avenue, Boston, MA 02215, USA; Tel.: 617-667-1031; Fax: 617-667-2913; E-mail: waird@bidmc.harvard.edu 1389-4501/07 $50.00+.00
William C. Aird
Fig. (1). The innate immune response. Circulating monocyte or tissue macrophage binds to LPS via TLR4, resulting in activation of inflammatory and coagulation pathways. Components of each pathway (only some of which are listed) participate in autocrine and/or paracrine loops to further activate the monocytes and endothelial cell, respectively. For purposes of illustration, the many receptors for the inflammatory mediators are depicted as a single generic receptor. From the perspective of the endothelium (inset), input may arrive directly by way of LPS or indirectly through monocyte/macrophage-derived paracrine signals. Endothelial output includes a variety of phenotypic changes including alterations in hemostatic balance, leukocyte trafficking, permeability or inflammation (shown is the release of inflammatory mediators; see table for details of additional output). IL, interleukin; TNF , tumor necrosis factor ; IFN-g, interferon g; MCP-1, monocyte chemoattractant protein-1; MIP-1 ; macrophage inflammatory protein-1 ; RANTES, regulated on activation, normal T-cell expressed and secreted; IP-10, interferon-gamma-inducible protein-10; NO, nitric oxide; VEGF, vascular endothelial growth factor; PAF, platelet activating factor; LPS, lipopolysaccharide; TLR4, toll-like receptor 4; PAR, protease activated receptor; PAI-1, plasminogen activator inhibitor. Reproduced with permission from Aird, W.C. (2003) Sci. Med., 9, 108-119.
and other inflammatory mediators. These mediators then function in autocrine or paracrine loops to further amplify the host response. TLR activation also leads to increased expression of tissue factor in monocytes, and possibly some other cell types including endothelial cells. Cytokines and chemokines also interact with their cognate receptors to induce tissue factor expression. Tissue factor activates factor VIIa of the extrinsic pathway, leading to sequential activation of factors X and prothrombin and cleavage of fibrinogen to fibrin. The serine proteases, particularly thrombin, not only participate in the clotting cascade but also bind to specialized receptors, termed
protease-activated receptors (PAR), present on the surface of many cell types including endothelial cells. Thrombin-mediated PAR-1 activation may lead to an accentuation of the inflammatory phenotype [11]. Despite significant advances in our understanding of the soluble mediators and cells involved in mediating sepsis pathophysiology, the precise mechanisms that lead to organ dysfunction and mortality remain unknown. The fact that most therapies directed towards inflammatory mediators have failed to improve survival in sepsis (a possible exception being anti-TNF- antibodies [12]) is consistent
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with the notion that the inflammatory cascade is sufficiently redundant, pleiotropic and interdependent as to preclude single modality therapy. Preclinical studies suggest that selective inhibition of thrombin generation has no effect on mortality [13], suggesting that the clotting cascade in and of itself does not result in death. In contrast, therapies which target both inflammation and coagulation, and/or which attenuate endothelial dysfunction appear to be more promising. Examples of agents with broad spectrum antiinflammatory and anticoagulant action are antithrombin III (ATIII), tissue factor pathway inhibitor (TFPI), and activated protein C. ATIII is a natural anticoagulant produced by the liver which functions with its cofactor, heparan, to inactivate the serine proteases in the clotting cascade, particularly factor Xa and thrombin. In addition, ATIII has been shown to inhibit endothelial cell activation [14, 15]. Despite promising results in phase 1/2 clinical trials with ATIII, a large phase 3 clinical trial failed to demonstrate improved survival in patients with severe sepsis [16]. TFPI is normally expressed by microvascular endothelial cells and plays a critical role in hemostasis by binding to and inactivating components of the extrinsic pathway of the clotting cascade [17]. By blocking serine protease-mediated cell signaling, TFPI may also inhibit inflammation. However, like ATIII, TFPI failed to reduce severe sepsis mortality in a phase 3 clinical trial [18]. Endogenous protein C is activated by thrombin in the presence of endothelial-bound thrombomodulin. Activated protein C cleaves factors Va and VIIIa of the clotting cascade. In addition, activated protein C binds to and activates PAR-1 on endothelial cells through a mechanism that depends on a cell type-restricted co-receptor, endothelial protein C receptor (EPCR) [19]. Activated protein C-mediated signaling has been shown to attenuate endothelial cell activation (discussed below). In a large phase 3 clinical trial, recombinant human activated protein C reduced mortality in high risk patients with severe sepsis [20]. Taken together, the above considerations suggest that the host response to infection is highly complex and that successful therapy will likely require targeting multiple pathways at once. THE ENDOTHELIUM IN SEPSIS Adaptive vs. Non-Adaptive Responses In response to local infection or inflammation, the endothelium undergoes vasodilation, presumably as a mechanism to increase flow (and thus delivery of cells and inflammatory mediators) to the site of injury. In addition, endothelial cells in the vicinity of the stimulus are induced to express cell adhesion molecules that mediate rolling and transmigration of leukocytes. Activated endothelial cells also express increased procoagulants (e.g. tissue factor), and decreased anticoagulants (e.g. thrombomodulin) [21, 22]. The resulting shift in hemostatic balance may serve to wall off the infection and/or to facilitate leukocyte trafficking. It is also possible that once generated, thrombin activates endothelial cells, monocytes, and/or tissue macrophages. Under normal circumstances, the host response prevails in containing and eliminating the pathogen. However, when the host response is excessive and/or sustained, it may spill over into the circulation where it becomes uncoupled from local inhibitory feedback mechanisms. Once in the circulation, the various soluble mediators and activated cells may engage the endothelium at distant sites. Thus, a local adaptive inflammatory response may be converted into a systemic non-adaptive response, and become clinically manifest as (severe) sepsis. Endothelial Heterogeneity The endothelial phenotype in sepsis varies from one site of the vasculature to another. Examples of vascular bed-specific responses are found in animal models. For example, in a rat model of cecal ligation puncture, DNA microarray analyses of various tissues demonstrate remarkable differences in the expression of genes, some of which are specific to the endothelium [23]. As another
example, systemic administration of endotoxin to mice results in organ specific differences in the expression of endothelial cell adhesion molecules [24-26]. Finally, in a baboon model of E. coli bacteremia, tissue factor is increased in endothelial cells of the spleen, but not of other organs [27]. It is likely that other prototypic responses of the activated endothelium also display spatial heterogeneity. Endothelial Cell as an Input-Output Device One way to approach such complexity of the endothelium is to consider each and every one of our 60-trillion endothelial cells as a miniature adaptive input-output device [28]. Input arises from the extracellular environment and includes both biomechanical and biochemical forces. Biomechanical forces consist of shear stress, disturbed or turbulent flow, and cyclical strain. Biochemical mediators include pH, level of oxygenation, chemokines (e.g. monocyte chemoattractant protein (MCP)-1), cytokines (e.g. tumor necrosis factor (TNF)- ), and serine proteases (e.g. thrombin). The endothelium has the capacity to recognize and respond to each of these signals. The response, or output, depends on the scale of investigation. For example a single endothelial cell may undergo a change in calcium flux, shape, protein or mRNA expression. An endothelial cell may migrate, proliferate or undergo apoptosis. Monolayers of endothelial cells express barrier properties and may be assayed for transmigration of leukocytes. Finally, other properties are appreciated only in the context of the blood vessel, the organ or the whole organism, for example endothelial-mediated control of vasomotor tone and redistribution of blood flow. Input is coupled to output by a highly complex non-linear array of signaling pathways. These pathways are the focus of intensive research in vascular biology and include cell surface receptors, signal intermediates and transcription factors. In sepsis there is a net change in signal input, including a reduction in shear stress at the level of the endothelium; hypoxia; and increased circulating levels of chemokines, cytokines, reactive oxygen species, thrombin and complement. This change in input is transduced by endothelial signaling pathways that are commonly implicated in activation states, including the signal intermediate, p38 MAPK, and the transcription factor, NF- B (reviewed in [29, 30]). Output may include some combination of reduced production of nitric oxide, increased expression of cell adhesion molecules, leukocyte adhesion and transmigration, altered hemostatic balance, increased apoptosis, increased permeability, and release of proinflammatory mediators, such as cytokines and chemokines [29,30]. ENDOTHELIUM AS A THERAPEUTIC TARGET There is overwhelming evidence that the endothelium plays an important role in mediating the sepsis phenotype. Thus, the endothelium represents an attractive therapeutic target in this syndrome. Rather than cataloguing every possible endothelial target (for a detailed review of candidate targets, the reader is referred to [29]), this section will develop broad themes related to endothelial-based therapy (Fig. 2). 1. Treatment should be reserved for a dysfunctional endothelium All too often, descriptions of sepsis pathophysiology fail to emphasize that the host response to infection evolved as a protective mechanism and that the clinical phenotype in sepsis represents an exaggeration of an otherwise adaptive response. Thus, from a conceptual standpoint, it is important to distinguish between an adaptive and non-adaptive response and to reserve therapy for those cases in which the endothelium poses a net liability to the host. An important challenge is to determine when the patient (and the endothelium) has crossed the line from a functional to a dysfunctional response. Although patients with severe sepsis who are intubated in the intensive care unit are clearly in a state of dysfunction, there are
William C. Aird
Fig. (2). Endothelium as a therapeutic target in sepsis. Potential targets include blood composition (glucose, pH, oxygen, temperature and flow), soluble inflammatory mediators, endothelial cell receptors for inflammatory mediators, the coagulation pathway (which is critically linked to inflammation) and cellcell-interactions. In addition, intracellular signaling pathways that are involved in transducing the input signals and mediating sepsis phenotype may be targeted (inset). The diagram has been over-simplified for purposes of illustration the various receptors are coupled to multiple downstream intermediates, and the various signal intermediates are all linked to NF- B. Reproduced with permission from Aird, W.C. (2003) Sci. Med., 9, 108-119.
likely to be components of the host response that continue to play an adaptive role. 2. Treatment should be aimed towards recalibrating the endothelial cell The terms endothelial cell activation and activity are not synonymous. Indeed, the normal endothelium is highly active. Thus, when considering therapy aimed towards the endothelium, an important goal is not to shut down the endothelium but rather to nudge it back to its normal state of activity. An important research goal is to delineate and understand the nature of that normal state. 3. Endothelial cell heterogeneity may be leveraged for therapeutic gain There are few features that are common to all endothelial cells. The remarkable heterogeneity of the endothelium poses both chal-
lenges and opportunities. On one hand, the lack of commonality poses tremendous obstacles for investigators who wish to characterize the endothelium and develop pan-endothelial therapies. On the other hand, advances in basic science have enabled investigators to map the complex phenotypes of the endothelium. These studies have yielded so-called vascular addresses or zip code for the endothelium [31]. Such information will be invaluable for developing therapies that are targeted to one or another vascular bed. 4. The input-output device analogy is a valuable framework for approaching therapy If the endothelium plays a role in determining sepsis morbidity (i.e. if sepsis is associated with endothelial dysfunction), then the goal of reversing dysfunction and recalibrating the phenotype will rest on altering some aspect of the input-output relationship (Table 1, and references therein). Examples of targeting the input (and thus
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Table 1.
Treatment INPUT Oxygen pH Shear stress Supplemental oxygen to maintain oxygenation Correction of acidosis Fluid resuscitation and maintenance of blood pressure Body temperature control Tight glucose control Anti-TNF- antibodies; receptor antagonists Anti-IL-antibodies; receptor antagonists PAF receptor antagonists; PAF acetylhydrolases Anti-MCP antibodies; receptor antagonists Anticoagulants ECs are highly sensitive to hypoxia There is increasing evidence that ECs are capable of sensing acidosis independent of hypoxia ECs are highly sensitive to changes in shear stress; early goaldirected therapy may exert its benefit partly through promoting shear stress Fever induces heat shock response Hyperglycemia may have deleterious effects on ECs TNF- is a strong activator of ECs; clinical trials have demonstrated inconsistent results Interleukins activate ECs; clinical trials have failed [41] [42, 43] [36, 44] Comments Ref.
Hyperthermia Hyperglycemia Cytokines TNFIL-1, 6, 8 Chemokines PAF MCP-1 Serine proteases
[45] [35, 46, 47] [12, 48] [49]
PAF is a potent phospholipid agonist that induces EC proliferation and permeability MCP binds to CCR2 on ECs, inducing numerous proinflammatory signals Thrombin binds to PAR on surface of ECs, resulting in proinflammatory phenotype; selective anti-thrombin agents do not improve sepsis survival; rhAPC may exert its benefit partly through inhibition of thrombin-mediated signaling C3a and C5a bind to receptors on ECs Late marker of sepsis; produced by and activates ECs Bradykinin and HMWK activate ECs; clinical trials have failed VEGF binds to EC-specific receptors, and results in a proinflammatory phenotype ECs are highly sensitive to changes in redox state Clinical trials have failed
[50-52] [53, 54] [11, 20]
Complement HMGB1 Bradykinin/HMWK VEGF ROS LPS OUTPUT Leukocyte adhesion and transmigration Barrier dysfunction
Anti-C5a-antibodies; receptor antagonists Anti-HMGB1 antibodies; receptor antagonists Antagonists Anti-VEGF antibodies; receptor antagonists Antioxidants Anti-endotoxin antibodies
[55, 56] [57-59] [60-62] [63] [64, 65]
Anti-adhesion molecule antibodies Sphingosine 1-phosphate; rhAPC; phosphodiesterase 2 inhibition Nitric oxide donors/inhibitors See above under input Caspase inhibitors, rhAPC Chemical inhibitors Double-stranded oligodeoxynucleotides Chemical inhibitors
ICAM-1, VCAM-1, P-selectin, E-selectin are expressed by activated ECs and mediate increased adhesion of leukocytes to endothelium; PECAM-1 plays role in leukocyte transmigration Sphingosine 1-phosphate appears to play a critical role in mediating barrier function; rhAPC may exert its benefit partly through Sphingosine 1-phosphate -dependent reduction in EC permeability Recent evidence suggests that eNOS has a proinflammatory role in sepsis ECs express TNF- , interleukins and many chemokines Sepsis is associated with increased apoptosis of ECs
[66-68]
[37, 69, 70]
Vasomotor tone NOS/NO Inflammatory mediators Apoptosis COUPLING p38 MAPK NF- B GSK3 [79-81] rhAPC may exert its benefit partly through inhibition of NF- B activity [82-84] [85] [71-74]
[75-78]
This table is not exhaustive but rather shows representative examples under each category. Abbreviations: EC, endothelial cell; TNF, tumor necrosis factor; IL, interleukin; PAF, platelet activating factor; MCP, monocyte chemoattractant protein; PAR, protease-activated receptor; rhAPC, recombinant human activated protein C; HMGB1, High mobility group box 1; HMWK, high molecular weight kininogen; VEGF, vascular endothelial growth factor; ROS, reactive oxygen species; LPS, lipopolysaccharide; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; NOS, nitric oxide synthase; NO, nitric oxide
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preventing/attenuating a dysfunctional phenotype) include the administration of antioxidants, inhibition of soluble inflammatory mediators and/or their receptors using antibodies, small molecules or soluble forms of the receptors; anti-complement therapy; anticoagulants; aggressive fluid resuscitation; tight glucose control; and reversal of hypoxia. An alternative approach is to target the endothelial output (or phenotype) itself. These strategies include inhibition of leukocyte adhesion, maintenance of hemostatic balance, optimization of vasomotor regulation, promotion of barrier function, and reduction in apoptosis. Despite significant advances in our understanding of the molecular mechanisms underlying these various endothelial properties in health and disease, there has been little success in translating this knowledge into successful phenotypeselective therapy. Finally, there is increasing interest in targeting the signal transduction pathways that couples input with output. Examples include treatments aimed towards inhibiting p38 MAPK, NF- B, and GSK3 . 5. A common thread in established and promising new sepsis therapies is their capacity to attenuate endothelial dysfunction To date, a total of five phase 3 clinical trials have demonstrated improved survival in critically ill patients or patients with severe sepsis. These include the use of low tidal volume ventilation [32], activated protein C [33], low dose glucocorticoids [34], intensive insulin therapy [35], and early goal-directed therapy [36]. It is possible that each of these regimens exerts its benefit through a protective effect on the endothelium. For example, low tidal volume ventilation may reduce barotrauma to pulmonary endothelium; activated rhAPC has been shown to attenuate the response of endothelial cells to inflammatory mediators, inhibit cell apoptosis and enhance barrier function [37, 38]; low dose steroids may inhibit proinflammatory pathways in endothelial cells including NF- B; intensive insulin therapy reverses any deleterious effect of hyperglycemia on endothelial responses; and early goal directed therapy may result in favorable hemodynamics at the level of the endothelium. There is increasing evidence that statins may play a similar role in attenuating endothelial cell dysfunction [39, 40]. Among the pleiotropic effects of statins are increased nitric oxide bioavailability, reduced activation of NF- B, anti-oxidant effects, and decreased release of inflammatory mediators. Indeed, agents such as recombinant human activated protein C and statins may represent prototypes for a novel class of drugs that fall under the rubric of attenuators of endothelial dysfunction. CONCLUSIONS The endothelium is an under-recognized organ that plays multiple roles in health and disease. The endothelium is an important component of the innate immune response. An excessive or sustained host response may lead to endothelial dysfunction. Endothelial dysfunction in turn contributes to the uncontrolled host response. An important goal in the sepsis field is to develop novel therapies aimed towards reversing endothelial dysfunction. Advances in this field will be contingent upon the development of novel diagnostic assays for measuring endothelial function, and for following its response to therapy. REFERENCES
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Novel Therapies for Microvascular Permeability in Sepsis

J.R. Jacobson* and J.G.N. Garcia
Department of Medicine, University of Chicago, Chicago, Illinois, USA
Abstract: Sepsis is characterized physiologically by an aberrant systemic inflammatory response and microvascular dysfunction. While appropriate antibiotics and supportive care are essential in the management of the septic patient, therapies targeting specific aspects of the pathophysiology could have a significant impact on the morbidity and mortality associated with both sepsis and its sequlea, including acute lung injury (ALI). We have characterized several mediators of endothelial cell (EC) barrier function that may serve as novel therapies for sepsis-induced microvascular dysfunction including simvastatin, adenosine triphosphate (ATP), sphingosine 1-phosphate (S1P), and activated protein C (APC). Notably, APC is already available for the treatment of severe sepsis, however, to date its mechanism of action has been unclear. While distinct in many ways, we have found that these agonists have in common the ability to induce dynamic rearrangement of the EC actin cytoskeleton that corresponds to barrier protection. In addition, we have extended our in vitro findings to relevant animal models of endotoxin-induced acute lung injury and have confirmed beneficial effects of both simvastatin and S1P which are associated with evidence of decreased vascular permeability in this setting. Moreover, our data also indicate that APC effects in sepsis may be largely due to augmentation of EC barrier function affecting decreased microvascular permeability. We speculate that the administration of direct modulators of EC barrier function and microvascular permeability, such as those described here, may ultimately become the standard of care for the septic patient.
Key Words: Acute lung injury, endothelium, simvastatin, adenosine triphosphate, sphingosine 1-phosphate, activated protein C. INTRODUCTION Acute lung injury (ALI) is a well-recognized sequela of sepsisinduced microvascular dysfunction and is associated with significant morbidity and mortality in its own right. Historically, difficulty in arriving at precise numbers regarding the incidence of ALI has arisen in part from the lack of a standard, uniformly recognized definition. In an effort to address this problem, a 1994 AmericanEuropean Consensus Conference established diagnostic criteria for ALI which included a required threshold for hypoxemia (PaO2/FiO2 <300), the presence of bilateral infiltrates on chest xray, and the absence of heart failure [1]. Subsequently, estimates of the incidence of ALI in the United States alone have been as high as 100,000 cases per year and its twenty-eight day mortality has been estimated to be 35-40% [2]. Unfortunately, despite the growing recognition of the enormity of this problem, effective therapies for ALI are lacking. New insights into the pathophysiology of this disease, however, have led to the consideration of agents directed towards novel targets, including specific mediators of microvascular dysfunction, a cardinal feature of sepsis and a critical determinant of the progression to ALI [3]. While the pathophysiology of sepsis has long been attributed to an imbalance in pro- and anti-inflammatory mediators, the full consequences of this imbalance have only recently been recognized [4]. Due to the direct effects of inflammatory mediators, the host immune response, and alterations in vascular permeability, the septic patient is at risk for a number of downstream consequences including ALI. A full characterization of inflammatory mediators in sepsis and their relative contributions to its pathophysiology is an area of active investigation. While several have been identified, the complexity of their collective effects is borne out by the failure to date of the inhibition of individual mediators, including TNF- and IL-1 , to significantly alter clinical outcomes [5-7]. Although a strategy targeting multiple mediators simultaneously has shown some promise [8], a shift of focus to the modulation of other aspects of the septic physiology may prove more fruitful. Accordingly, our lab and others are interested in the mechanisms underlying changes in microvascular permeability particularly as this may lead to new therapeutic strategies for sepsis and ALI. OVERVIEW OF ENDOTHELIAL BARRIER REGULATION The lining of the vasculature is comprised of endothelial cells (EC), multifunctional cellular elements which serve multiple regulatory functions of the microvessel including as a semi-selective barrier to circulating cells and fluid. Vascular permeability is defined by two pathways that determine the movement of fluids and solutes from the vascular space to the interstitum. The transcellular pathway, thought to be a minor contributor to inflammatory vascular permeability [9,10], and the paracellular pathway, characterized by the formation of paracellular gaps in response to various inflammatory mediators and considered the primary determinant of vascular permeability [11]. Regulation of paracellular gap formation can be thought of as a balance of competing intracellular contractile forces and adhesive cell-cell and cell-matrix tethering forces. These forces are regulated through dynamic activation of the actin-based cytoskeleton, the complexities of which have only recently been recognized. The EC cytoskeleton is comprised of three key elements: actin microfilaments, intermediate filaments, and microtubules. While the roles of microtubules and intermediate filaments in EC barrier regulation remain to be fully defined, the critical importance of actin microfilaments is demonstrated by increased EC permeability in response to cytochalasin D [12], an actin disrupter. Conversely, phallacidin, an actin stabilizer, decreases sensitivity to agonist-induced EC barrier disruption [13]. Through multiple focal linkage sites to membrane and intercellular proteins, the actin microfilament system is a critical determinant of EC barrier integrity. At the same time, however, actin is also largely responsible for the generation of tensile intracellular forces via an actomyosin motor, an event which results in EC barrier disruption [14]. Actin rearrangement is driven by the coordinate activities of the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) [15] and Rho Kinase, the effector of the small GTPase Rho [16,17]. Together, MLCK and Rho activity affect myosin light chain (MLC) phosphorylation and actin stress fiber formation. Local intracellular variability in MLC phosphorylation levels and actin polymerization accounts for the phenotypic-specific contracted or relaxed state of the cell. For example, via a pathway independent of MLCK and
*Address correspondence to this author at the Pritzker School of Medicine, University of Chicago, Chicago, Illinois, USA; Tel: 773 702 1051; E-mail: jgarcia@medicine.bsd.uchicago.edu 1389-4501/07 $50.00+.00
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Fig. (1). Cytoskeletal changes of EC barrier-protective agonists. Immunofluorescent imaging of confluent human pulmonary artery EC monolayers treated with simvastatin (5 M, 16 h), ATP (10 M, 30 min), S1P (0.5 M, min), or APC (1 g/ml, 5 min) demonstrate enhanced cortical actin (arrows) and decreased transcellular actin stress fibers relative to control cells. These changes correspond to a protective effect of each of these agonists on thrombin-induced EC barrier disruption as measured by TER.
Rho, another small GTPase, Rac, is involved in cortical actin polymerization and promotes endothelial barrier function [18]. Modulators of the various components of dynamic actin rearrangement may also have an impact on endothelial barrier function and, as a result, affect alterations in vascular permeability. A review of several effectors is provided below including simvastatin, adenosine triphosphate (ATP), the phospholipid sphingosine 1-phosphate (S1P), and activated protein C (APC). While these molecules promote endothelial barrier function via common effects on rearrangement of the actin cytoskeleton, Fig. (1), significant differences in their mechanisms of action may offer important insights into EC barrier regulation. Moreover, each agent holds promise as a potential therapy specific for sepsis-induced microvascular dysfunction. SIMVASTATIN The statins, a class of HMG CoA-reductase inhibitors, are used clinically for their ability to lower serum cholesterol levels via inhibition of the prenylation pathway. While cholesterol synthesis is dependent on farnesylation, the addition of a 15-carbon side chain to proteins, the prenylation pathway may also culminate in geranylgeranylation, the addition of a 20-carbon side chain. The Rho family GTPases undergo this particular modification prior to translocation to the cell membrane which promotes their activation via GTPbinding and enables the regulation of various intracellular signaling events. As the Rho GTPases, including Rho and Rac, are known to be involved in dynamic EC cytoskeletal rearrangement and barrier function, we have examined the relevance of the effects of simvastatin on GTPase-regulated cellular remodeling. Simvastatin promotes EC barrier function through Rho inhibition thus attenuating agonist-induced transcellular stress fiber formation and subsequent cell contraction [19]. Paradoxically, however, we also identified a marked increase in Rac activation (Rac-GTP) by simvastatin which occurs despite the attenuation of Rac translocation to the cell membrane via the inhibition of geranylgeranylation, an important mediator of this event [19]. Moreover, while evidence confirms activation of the PI3 kinase-Akt pathway by statins [20,21], known to induce Rac activation, this pathway also relies on translocation of Rac to the cell periphery and thus seems an unlikely mechanism of Rac activation in this setting. Notably, Rac activation in our experiments was evident only after prolonged periods of simvastatin treatment (5 M, 16 h) and our data implicate specific alterations in EC gene expression by simvastatin, including potential regulators of Rac activation which we suspect may account for this finding although the precise mechanism remains unclear. The cumulative effects of increased Rac activation and Rho inhibition provide the mechanistic basis for the observed augmentation of EC barrier function. Indeed, our in vitro data confirmed EC barrier protection by simvastatin (5 M, 16 h) as thrombin-induced (1 M) barrier disruption was attenuated by 70% relative to control cells and exhibited a more rapid recovery to baseline as measured by transendothelial electrical resistance (TER). These findings led
us to examine the clinical significance of these effects in attenuating the increased vascular permeability characteristic of ALI using an appropriate murine model [22]. Pretreatment of mice with simvastatin prior to the intratracheal administration of lipopolysacharide (LPS) markedly reduced lung inflammation and vascular leak as measured by albumin and cell counts in bronchoalveolar lavage (BAL) fluid and was further evidenced by appreciable effects on inflammation by histology [22]. While statins have pleiotropic properties including anti-inflammatory and immunomodulatory effects, as well as effects on the generation of reactive oxygen species and nitric oxide, our data established significant, direct effects on vascular permeability that suggest a potentially useful role for this class of drugs in the attenuation of the microvascular dysfunction characteristic of sepsis and ALI. Ongoing experiments will characterize the effects of simvastatin treatment in our animal model of ALI after injury has been established as this is a more relevant clinical scenario. Already, mounting evidence from both humans and other animal models have lent support to our findings and underscore the very real potential of this class of drugs in sepsis [23, 24]. These data, however, do not convincingly demonstrate a specific mechanism of the potential protective effects of simvastatin in sepsis and it should be noted that effects on the generation of reactive oxygen species have been suggested in this regard. While simvastatin does inhibit the NADPH oxidase enzyme complex which is responsible for the generation of superoxide anions, we speculate that its vascular protective effects are largely due to direct effects on EC barrier regulation. In separate experiments, employing mice deficient in NADPH oxidase in our ALI model, we plan to explore this issue further. ATP Adenosine triphosphate (ATP) is a nucleotide with direct effects on smooth muscle cells and EC that is supplied extracellularly by a variety of sources including activated platelets and EC themselves. Intracellular EC signaling events are mediated by ATP through activation of specific purinergic receptors [25]. While the activation of P2X receptors promotes the influx of extracellular Ca2+, binding of adenosine nucleotides to P2Y receptors leads to increased inositol 1,4,5-trisphosphate (IP3) via G-protein-mediated phospholipase C activation culminating in the release of Ca2+ from intracellular stores [26], events known to be involved in EC cytoskeletal activation [14]. Given its physiologic importance and its ability to affect relevant EC signaling pathways we sought to define the effects ATP with respect to EC barrier function. ATP (10 M) induces early MLC phosphorylation in association with actin cytoskeletal rearrangement, Fig. (1) [27]. These changes are associated with enhanced endothelial barrier function as measured by TER. In addition, ATP induces both translocation of the actinbinding protein cortactin to the cell periphery and activation of Rac, events associated with cortical actin polymerization and which we found to be necessary for EC barrier enhancement. Moreover, other
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investigators have described the role of the PI3 kinase-Akt pathway in ATP signaling, a mediator of Rac activation [28]. Separately, using silencing RNA we demonstrated the involvement of specific G proteins, G q and G i2, as well as protein kinase A and its substrate VASP in ATP-induced EC barrier enhancement. Moreover, ATP decreases EC MLC phosphorylation at longer time points (30 min), consistent with completion of cytoskeletal rearrangement, in association with activation of myosin-associated phosphatase via G q [30]. Questions regarding the mechanisms involved in cytoskeletal rearrangement by ATP remain. In particular, while ATP induces increased intracellular Ca2+ levels which have been shown to be necessary for Rac activation [29], our data suggests that EC barrier enhancement by ATP occurs despite treatment with BAPTA, a Ca2+ chelator, and is therefore independent of Ca2+ [30]. The effects of ATP on Rac in this setting are unknown and the possibilities exist that Rac activation occurs through a Ca2+-independent mechanism or that Rac activation is associated with but not necessary for EC barrier enhancement. Either of these possibilities would be a novel finding and as such they warrant further investigation. Nonetheless, the known effects of ATP on EC barrier function may have significant clinical applicability and we are actively investigating its ability to attenuate injury in LPS-induced murine ALI. SPHINGOSINE 1-PHOSPHATE We have previously characterized sphingosine 1-phosphate (S1P), a phospholipid released by activated platelets, as having robust EC barrier-enhancing properties [31]. S1P signaling is propagated by ligation of specific S1P receptors (formerly known as endothelial differentiation gene or Edg receptors) on the EC surface [32-34]. To date, five members of this family of receptors have been identified with S1P1 and S1P3 present on EC [35-37]. S1P-induced EC barrier enhancement occurs primarily via S1P1 ligation with subsequent signaling through G protein-dependent pathways. Similar to simvastastin and ATP, S1P affects characteristic cytoskeletal rearrangement, Fig. (1) and is associated with activation of the PI3 kinase-Akt pathway as well as both Rac activation and cortactin translocation [38, 39]. We have reported that S1P signaling occurs via rapid recruitment of several mediators, including S1P1 and p110 PI3 kinase and catalytic subunits, to caveolin-enriched microdomains which is required for dynamic cytoskeletal rearrangement [40]. S1P also drives MLCK translocation to the cell periphery and leads to increased MLC phosphorylation in a cortical distribution, consistent with cytoskeletal activation [39]. TER measurements confirm that these events are associated with dose-dependent increases in EC barrier function as well as an attenuation of thrombin-induced barrier disruption. With respect to downstream signaling, our data implicates the p21-associated Ser/Thr kinase 1 (PAK) as an important target of S1P-induced Rac activation [35]. This signaling pathway is further elucidated by evidence of the S1P-induced translocation to the cortical actin ring of both LIM kinase, a target of PAK and another Ser/Thr kinase, and the actin-binding protein, cofilin, a target of LIM kinase [35, 41]. While the roles of these molecules in S1P effects on EC barrier function is complex, their importance is suggested by the abrogation of S1P-mediated cytoskeletal changes using a PAK dominantnegative construct. Employing our murine model of LPS-induced ALI, we have also confirmed the protective effects of S1P in this setting [42]. S1P (1 M) administered 1 h after intratracheal LPS resulted in marked reductions in BAL protein and cell counts at 6 and 24 h relative to animals treated with LPS alone. S1P-treated mice also demonstrated marked reductions in LPS-induced lung tissue myeloperoxidase (MPO) activity, an index of phagocyte infiltration, and Evans blue dye albumin accumulation, a marker of vascular permeability. These findings were associated with histologic evidence of decreased inflammation in S1P-treated animals. Finally, as sepsis-
induced microvascular dysfunction is a systemic phenomenon, we examined the effects of S1P on acute renal injury in this same animal model. Consistent with our lung findings, S1P affected a significant attenuation of renal tissue MPO activity and Evans blue dye albumin extravasation compared to mice treated with LPS alone. To extend our in vivo findings, we have conducted additional experiments in a canine model of ventilator-associated lung injury. This large animal model is particularly useful for reproducing the regional lung differences characteristic of ALI in humans. Dogs were subjected to high tidal volume mechanical ventilation (17 cc/kg), a known precipitant of ALI in sepsis, prior to the administration of intrabronchial LPS (2 mg/kg). Compared to injured controls, animals concomitantly treated with S1P (85 g/kg, IV over 20 min) were found to have significantly reduced shunts as measured by venous admixture as well as marked reductions in BAL protein accumulation [43]. In addition, a striking difference was evident on computed tomographic (CT) imaging with respect to lung edema formation, Fig. (2). These images demonstrate marked edema formation in gravitationally-dependent lung regions of all LPS-treated animals with the most prominently affected areas in the lung bases and only minimal involvement of the apices. Quantification of S1P effects via measurement of vertical density gradients confirmed attenuation of LPS-induced edema formation throughout all lung regions with the most pronounced effect evident in the mid-lung region. Of significant interest to the critical care community is the availability of a structural analog of S1P, FTY720, which is currently in phase III trials as an immunosuppressive agent. Based on our encouraging results with S1P, we are also interested in the potential therapeutic role of FTY720 in sepsis and ALI. In our murine model of ALI described above, the administration of FTY720 (0.1 mg/kg, intraperitoneal injection) 1h after LPS significantly reduced lung tissue Evans blue dye albumin accumulation compared to injured control animals indicative of a protective effect with respect to LPS-induced microvascular dysfunction [42]. That this agent is already in phase III trials makes it all the more promising. ACTIVATED PROTEIN C The serine protease activated protein C (APC) is an anticoagulant and anti-inflammatory mediator with an established role in the treatment of sepsis. The landmark PROWESS trial established a 28-day survival benefit in patients with severe sepsis who received APC compared to controls with relative and absolute risk reductions of more than 19% and 6%, respectively [44]. While these findings served to identify for the first time an effective treatment for sepsis directed towards the underlying pathophysiology, the precise mechanism of its effects remain unclear. In light of this ambiguity, our interest in mediators of sepsis-induced microvascular dysfunction led us to investigate the potential ability of APC to regulate EC barrier function. Circulating protein C is a proenzyme activated by the thrombomodulin-thrombin complex. Both APC and inactive protein C bind to the endothelial protein C receptor (EPCR) [45]. Ligation of the EPCR receptor promotes the association of protein C with the thrombomodulin-thrombin complex and strongly augments protein C activation [46]. APC binds protein S and subsequently cleaves coagulation cofactors VIIIa and Va which leads to downregulation of thrombin generation. The clinical significance of the protein C pathway is evidenced by the frequent manifestations of hypercoaguability in patients with protein C deficiency. While this pathway is well-described, as are effects of protein C on specific inflammatory mediators, APC administered to humans treated with LPS does not have an appreciable effect on various markers of coagulation or inflammation implicating a separate pathway through which its may effects be mediated [47].
Jacobson and Garcia
Fig. (2). Attenuation of pulmonary edema formation by sphingosine 1-phosphate in a canine model of acute lung injury. CT images from dogs administered intrabronchial LPS (2mg/kg) followed by high tidal volume mechanical ventilation (17 cc/kg, 6 h) are shown. Apical, mid-lung, and basilar images from animals that received LPS alone (A, C, and E, respectively) are matched with those from animals the received S1P (85 g/kg IV) concomitant with LPS (B, D, and F). Reprinted with permission [43].
We examined EC cytoskeletal regulation by APC and reported augmentation of the cortical actin ring similar to that characterized by the effects of other EC barrier-protective stimuli, Fig. (1) [48]. Measurements of TER confirmed the attenuation of thrombininduced EC barrier disruption by APC (1 g/ml) and APC-treatment was associated with a rapid increase in EC MLC phosphorylation, consistent with cytoskeletal activation. As might be predicted, APC also induced a marked increase in Rac activation and the use of a dominant-negative Rac construct significantly attenuated APC barrier protection. To correlate our findings with the known effects of APC we investigated the role of EPCR in EC barrier regulation by APC using RCR-252, an EPCR blocking antibody that inhibits APC binding. Treatment of EC monolayers with RCR-252 prior to the administration of APC affected a significant reduction in barrier
protection as measured by TER and abrogated APC-induced EC MLC phosphorylation. These effects were specific to APC-mediated signaling as both basal EC barrier function and MLC phosphorylation were indistinguishable in RCR-252-treated and control cells. Given the similarities between APC and S1P with respect to EC barrier function and cytoskeletal changes, we sought to identify potential signaling events upstream of these effects that were common to both. In particular, as transactivation of the S1P1 receptor has been described in association with ligation of specific growth factor receptors, including PDGF and VEGF, we postulated that APC binding to EPCR may have a similar effect. Immunoprecipitation experiments confirmed the co-association of EPCR and S1P1 in APC-treated EC as well as threonine phosphorylation of S1P1 by
513
Fig. (3). Agonist-mediated EC barrier regulation. Proposed components and signaling events associated with S1P, APC, and ATP-mediated cytoskeletal activation and EC barrier protection are depicted. S1P signaling occurs via S1P1 ligation with its subsequent threonine phosphorylation and activation by PI3 kinase and AKT. S1P1 activation in turn affects G protein-dependent events including Rac activation which induces both LIM kinase and cofilin translocation via PAK as well as translocation of cortactin which, in association with MLCK, is involved in local MLC phosphorylation and augmentation of cortical actin. Similar effects on cytoskeletal rearrangement are induced by APC upon binding to EPCR via the co-association with and transactivation of S1P1. ATP signaling occurs via G protein-coupled purinergic receptors with subsequent Rac activation, cortactin translocation and cytoskeletal rearrangement. Although activation of the PI3 kinase-AKT pathway has also been described, the role of this pathway in ATP-mediated EC barrier regulation remains to be defined. Of note, while EC barrier effects of simvastatin are also associated with Rac activation, cortactin translocation, and enhanced cortical actin important discrepancies in its effects, including timing of these events, suggest potential functional differences or a role for alternate mediators of EC barrier protection.
APC. These events were inhibited by pre-treatment with the EPCR blocking antibody. In addition, pretreatment with an inhibitor of the PI3 kinase-Akt pathway, LY294002, inhibited threonine phosphorylation of S1P1 by APC, implicating a role for this pathway in APC signaling. Finally, the use of silencing RNA specific for S1P1 markedly attenuated the barrier-protective effects of APC as measured by TER. These data confirm S1P1 transactivation by APC via EPCR and highlight the significant commonality of APC and S1P effects with respect EC signaling and barrier regulation, Fig. (3). CONCLUSION We have identified several agents with the potential to serve as novel and specific therapies for sepsis-induced microvascular dysfunction and ALI. We predict that the shared features of these agents will provide a useful template for the identification of other potential mediators of vascular permeability in the future. Just as important, however, are the points of divergence in the pathways leading to their common effects as well as their differences with respect to modulating EC barrier regulation. For example, while we have found each of the agents discussed to be effective in the attenuation of thrombin-induced EC barrier disruption, it is interesting to note that S1P and ATP induce a dramatic increase in basal TER consistent with augmentation of EC barrier function whereas basal TER is not increased by either simvastatin or APC. In addition, the prolonged time course required for the effects of simvastatin on EC barrier function is wholly unique. The reason for these differences is an important area of ongoing investigation. Accordingly, while the statins, ATP, S1P and its analogue, FTY720, may ultimately prove useful for treating patients with sepsis, further investigation may also lead to more specific therapeutic targets and modalities to affect EC barrier function and treat sepsis. It is for this reason, despite its already proven clinical value, a more complete characterization of the mechanisms of APC effects in sepsis is vital. ABBREVIATIONS ALI = Acute lung injury APC = Activated protein c ATP = Adenosine triphosphate
BAL CT EPCR EC IP3 LPS MLC MLCK MPO PAK S1P TER
= = = = = = = = = = = =
Bronchoalveolar lavage Computed tomography Endothelial protein C receptor Endothelial cells Inositol 1,4,5-trisphosphate Lipopolysaccharide Myosin light chain Myosin light chain kinase Myeloperoxidase p21-associated Ser/Thr kinase 1 Sphingosine 1-phosphate Transendothelial electrical resistance
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Ethyl Pyruvate: A Novel Treatment for Sepsis

Mitchell P. Fink*
1
Departments of Critical Care Medicine, Surgery and Pharmacology, University of Pittsburgh Medical School, 616 Scaife Hall, 3550 Terrace Street, Pittsburgh PA 15261, USA
Abstract: Pyruvic acid is a three-carbon -ketocarboxylic acid that plays a central role in intermediary metabolism, being the final product of glycolysis and the starting substrate for the tricarboxylic acid cycle. Ethyl pyruvate, which is a simple aliphatic ester derived from pyruvic acid, has been shown to improve survival and ameliorate organ system dysfunction in mice with peritonitis induced by cecal ligation and perforation, even when treatment is started as late as 12-24 hours after the onset of sepsis. In studies using lipopolysaccharidestimulated RAW 264.7 murine macrophage-like cells, ethyl pyruvate inhibits activation of the pro-inflammatory transcription factor, NFB, and down-regulates secretion of a number of pro-inflammatory cytokines, such as TNF. In this reductionist in vitro system, ethyl pyruvate also blocks secretion of the late-appearing pro-inflammatory cytokine-like molecule, high mobility group B1 (HMGB1). In murine models of endotoxemia or sepsis, treatment with ethyl pyruvate decreases circulating levels of TNF and HMGB1. While the molecular events responsible for the salutary effects of ethyl pyruvate remain to be elucidated, one mechanism may involve covalent modification of a critical thiol residue in the p65 component of NF- B. Ethyl pyruvate warrants evaluation as a therapeutic agent for the treatment of sepsis in humans.
Key Words: NF-kappaB; lipopolysaccharide; LPS; endotoxin; pyruvic acid; reactive oxygen species; signal transduction; acute renal failure Pyruvate is the trivial name for 2-oxoproprionate (CH3COCOO-). This 3-carbon compound is the product of the terminal step in the glycolytic pathway, which is the reaction catalyzed by the enzyme, pyruvate kinase. Under anaerobic conditions, much of the pyruvate generated by glycolysis is reduced in the lactate dehydrogenase (LDH) reaction to form lactate (i.e., 2-hydroxyproprionate) and the oxidized form of nictonamide adenine dinucleotide (NAD+). Under aerobic conditions, pyruvate is transported from the cytosol into mitochondria where it is oxidized to form acetyl coenzyme A by the enzyzme complex, pyruvate dehydrogenase (PDH). In addition to playing a central role in intermediary metabolism, pyruate probably also functions in cells as an endogenous antioxidant and free radical scavenger [1-5]. The capacity of pyruvate to function as an anti-oxidant was first reported by Holleman, who showed that -keto carboxylates with the general structure, R CO COO -, reduce hydrogen peroxide (H2O2) nonenzymatically in a reaction that yields carbon dioxide and water [6]. In the case of pyruvate, this oxidative decarboxylation reaction can be written as follows: CH3COCOO- + H2O2 CH3COO- + H2O + CO2 . This reaction is both rapid and stoichiometric [7,8]. In addition to H2O2, pyruvate also is capable of scavenging another highly reactive oxygen species (ROS), hydroxyl radical (OH) [9]. The recognition that pyruvate is an effective ROS scavenger has prompted numerous laboratories to try using this compound as therapeutic agent for the treatment of various pathological conditions that are thought to be mediated, at least in part, by redoxdependent phenomena. Perhaps the earliest work in this line of investigation was carried out by Salahudeen et al., who showed that infusing a solution of sodium pyruvate preserves kidney function in rat models of ROS-mediated acute renal failure [10]. Other investigators reported that treatment with pyruvate could ameliorate organ injury or dysfunction in animal models of redox stress, such as transient myocardial [11], intestinal [12], or hepatic [13] ischemia followed by reperfusion. Pyruvate-containing solutions also have been shown to have salutary effects in animal models of galactose[14] or diabetes-induced cataract formation [15], stroke [16] and
*Address correspondence to this author at the Department of Critical Care Medicine, University of Pittsburgh Medical School, 616 Scaife Hall, 3550 Terrace Street, Pittsburgh PA 15261, Tel: 412-647-6965; Fax: 412-6475258; E-mail: finkmp@ccm.upmc.edu 1389-4501/07 $50.00+.00
hemorrhagic shock [17-19] and in vitro models of damage to the lens of the eye caused by exposure to galactose [20], fructose [21] or oxidants [22]. Despite these promising findings, the usefulness of pyruvate as a therapeutic agent may be limited by its poor stability in solution. Aqueous solutions of pyruvate rapidly undergo a spontaneous aldol-like condensation reaction to form 2-hydroxy-2-methyl-4ketoglutarate, a compound that also is known as parapyruvate [2325]. This addition product has been shown to inhibit the enzymatic oxidative decarboxylation of -ketoglutarate to form succinyl coenzyme A [23,26], a key step in the mitochondrial tricarboxylic acid cycle. In order to circumvent this issue, our laboratory formulated a derivative of pyruvic acid, namely ethyl pyruvate, in a calcium- and potassium-containing balanced salt solution, which we called "Ringer's ethyl pyruvate solution" (REPS). In an initial study, Sims et al. found that treatment with this fluid substantially ameliorated much of the structural and functional damage to the intestinal mucosa that normally occurs when rats are subjected to mesenteric ischemia and reperfusion [27]. Interestingly, in this study, treatment with ethyl pyruvate seemed to be more effective than treatment with an equimolar concentration of sodium pyruvate. Similar findings indicating that ethyl pyruvate is more effective than pyruvate were reported by Varma et al., who compared the two compounds in an in vitro study of redox-mediated cellular injury [22]. In a subsequent study from our laboratory, Yang et al. compared the effects of resuscitation with REPS instead of Ringers lactate solution (RLS) on several parameters in a murine model of hemorrhagic shock [28]. The findings from this study provided the first evidence that ethyl pyruvate has anti-inflammatory properties. Treatment with REPS decreased activation of the pro-inflammatory transcription factor, NF- B, in liver and colonic mucosa following reususcitation from hemorrhagic shock and also decreased the expression of several pro-inflammatory genes, including inducible nitric oxide synthase (iNOS), TNF, cyclooxygenase-2, and IL-6 in liver, ileal mucosa and colonic mucosa. ETHYL PYRUVATE IS BENEFICIAL IN ANIMAL MODELS OF SEPSIS Because ethyl pyruvate down-regulated inflammation induced in rodents by hemorrhagic shock and resuscitation [28], Venkata 2007 Bentham Science Publishers Ltd.
Mitchell P. Fink
raman and colleagues compared the effects of resuscitation with REPS or RLS in a rat model of profound arterial hypotension induced by the intravenous injection of a large dose of Escherichia coli lipopolysaccharide (LPS) [29]. When mean arterial pressure (MAP) decreased to 60 mm Hg, the endotoxemic rats were randomized to resuscitation with either REPS or RLS, the volume of fluid being titrated to maintain MAP greater than 60 mm Hg until a total volume of 7 ml/kg was infused. Resuscitation with REPS as compared to RLS prolonged survival time (498 48 versus 362 30 min; p = 0.001). Resuscitation with REPS as compared to RLS also was associated with significantly lower circulating concentrations of nitrite/nitrate (marker of nitric oxide synthesis) and IL-6 and higher plasma levels of IL-10. Thus, delayed treatment with an ethyl pyruvate-containing resuscitation fluid shifted the LPSinduced inflammatory response toward increased production of an anti-inflammatory cytokine (IL-10) and away from production of the pro-inflammatory cytokine (IL-6). Pursuing this line of investigation still further, Ulloa and colleagues investigated the effects of ethyl pyruvate on LPS-induced inflammatory responses in vitro and in vivo [30]. In these studies, incubation of LPS-stimulated RAW 264.7 murine macrophage-like cells with ethyl pyruvate inhibited release of the proinflammatory cytokine, TNF, and decreased steady-state levels of TNF mRNA. In this same system, ethyl pyruvate blocked activation of two molecules, NF- B and p38 mitogen activated protein kinase (MAPK), that have been identified as important elements in two inter-related proinflammatory intracellular signal transduction chains. When mice were pretreated with ethyl pyruvate prior to being challenged with a lethal dose of LPS, survival was significantly improved and circulating concentrations of TNF were decreased. Remarkably, treatment with ethyl pyruvate four hours after the injection of LPS (i.e., well after the monophasic spike in circulating TNF levels) also improved survival mice previously challenged with a lethal dose of bacterial endotoxin. Furthermore, survival was significantly improved when mice were treated with ethyl pyruvate 24 hours after being subjected to cecal ligation and perforation to induce polymicrobial Gram-negative sepsis. Some of the beneficial of effects of ethyl pyruvate in murine endotoxemia and sepsis may be because this drug inhibits secretion of a cytokine-like protein, high mobility group box-1 (HMGB1). First described as a non-histone nuclear protein with high electrophoretic mobility [31], HMGB1 has been identified as a late-acting mediator of LPS-induced [32] or sepsis-induced [33] lethality. Ulloa et al. showed that incubating LPS-stimulated RAW 264.7 cells with ethyl pyruvate blocked the release of HMGB1 in vitro [30]. Furthermore, these investigators documented that treatment of mice with ethyl pyruvate decreased circulating levels of HMGB1 following administration of LPS or induction of sepsis by cecal ligation and perforation [30]. Following the report by Ulloa et al. [30], another laboratory, one directed by Dr. Robert Star at the National Institutes of Health, extended the investigation of ethyl pyruvate as a therapeutic agent for the adjuvant treatment of sepsis [34-35]. In particular, these investigators sought to determine whether treatment with ethyl pyruvate can ameliorate development of sepsis-associated acute renal failure. An important innovation in these studies was the use of aged, rather than young, mice for the experiments. Whereas acute renal dysfunction failed to develop in young mice challenged with LPS or subjected to cecal ligation and perforation, circulating creatinine concentration increased reproducibly after induction of Gram-negative peritonitis in aged mice [34]. This impairment in renal function was prevented if the mice were treated with ethyl pyruvate, even if the therapeutic intervention was delayed for 12 hours after the induction of sepsis [34]. Peritionitis or endotoxemia in rodents may not be ideal animal models for sepsis in humans. Therefore, it is noteworthy that Hauser and colleagues recently described the effects of ethyl pyruvate
in a clinically relevant porcine model of chronic endotoxemia [36]. In this experiment, two groups of pigs were studied. Both were infused continuously with LPS for 24 hours. Beginning at 12 hours after the start of the LPS infusion, one group was treated with ethyl pyruvate (30 mg/kg loading dose over ten minutes, and then 30 mg/kg per hour for 12 hours), whereas the other group received only the vehicle (Ringers solution). Both groups were resuscitated as needed with hydroxyethylstarch to keep mean arterial pressure greater than 60 mm Hg. In this study, delayed treatment with ethyl pyruvate prevented the development of arterial hypotension. Furthermore, ethyl pyruvate ameliorated the development of acute lung injury, as evidenced by reduced intrapulmonary venous admixture and significantly greater PaO2/FiO2 ratios at 18 and 24 hours. MECHANISM(S) FOR THE ANTI-INFLAMMATORY EFFECTS OF ETHYL PYRUVATE The mechanism(s) responsible for the beneficial effects of ethyl pyruvate remain to be established. Since pyruvate is an H2O2 and OH scavenger, we postulated that scavenging of ROS is one important mechanism responsible for the salutary actions of ethyl pyruvate. Indeed, in a rat model of hemorrhagic shock and resuscitation, our laboratory showed that treatment with an ethyl pyruvatecontaining solution blocked the formation of malondialdehyde (MDA), a marker of ROS-mediated lipid peroxidation [37]. Similar findings also have been reported in studies of extrahepatic biliary obstruction in mice [38] and continuous endotoxemia in pigs [36]. Furthermore, ROS have been implicated in the activation or modulation of a number of important intracellular proinflammatory signaling pathways, most notably those dependent upon the transcription factor, NF- B [39,40]. The transcriptionally active form of NF- B is a homo- or heterodimer made up of various proteins belonging to the NF- B family. These proteins include p50, RelA, c-Rel, p52 and RelB. In resting cells, however, these homo- or heterodimeric forms of NF- B exist in the cytoplasm in an inactive form due to binding by a third inhibitory protein, called I B. Upon stimulation of the cell by a proinflammatory trigger (e.g., TNF, IL-1 or LPS), I B is phosphorylated on two key serine residues (Ser32 and Ser36), which targets the molecule for ubiquitination and subsequent proteosomal degradation. Phosphorylation of I B is thought to be mediated by various I B kinases (IKKs). Phosphorylation and degradation of I B permits translocation of the transcriptionally active (dimeric) form of NF- B into the nucleus and subsequent binding of the transcription factor to cis-acting elements in the promoter regions of various NFB-responsive genes. It has been proposed that ROS are important in the upstream events that lead to IKK activation [39,40]. Accordingly, it is conceivable that ethyl pyruvate could inhibit inflammatory signaling by scavenging oxidizing species, and, thereby, inhibiting activation of the NF- B pathway. However, if this were the only important mechanism, then pyruvate should be effective as ethyl pyruvate. However, even in our laboratorys original study, ethyl pyruvate provided more protection against the structural and functional damage to the intestinal mucosa caused by mesenteric ischemia and reperfusion than did an equimolar concentration of pyruvate [27]. Subsequent in vitro and in vivo studies confirmed the notion that ethyl pyruvate is substantially more active as an anti-inflammatory agent than is the parent compound [41,42]. Other experimental findings also argue against the notion that ROS scavenging is the primary mechanism underlying the antiinflammatory properties of ethyl pyruvate. In one study, Sappington and colleagues showed that that pre-treatment of mice with ethyl pyruvate provided durable protection against some of the deleterious effects of LPS (e.g., hepatocellular injury and gut mucosal barrier dysfunction), even when when there was a six-hour delay between the last dose of ethyl pyruvate and the injection of endotoxin [42]. Similarly, in a series of in vitro experiments, pre-treating
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Caco-2 human enterocyte-like cells with ethyl pyruvate ameliorated the increase in epithelial permeability that was induced by incubating the monolayers with a cocktail of pro-inflammatory cytokines called cytomix [41]. Remarkably, this effect was still observed even when the ethyl pyruvate was removed by extensive washing of the cells prior to adding cytomix [41]. Collectively, these observations suggest that persistent exposure to ethyl pyruvate is not necessary in order to observe protection against the deleterious effects of LPS or pro-inflammatory cytokines; rather, transient exposure to the compound appears to be sufficient. These observations call into question the ROS scavenger hypothesis, because, in order to be effective, a pharmacological ROS scavenger must limit the oxidation (or peroxidation) of endogenous molecules by preferentially reacting with superoxide radical (O2-), H2O2, OH, or other related reactive species. This scavenging action requires that the pharmacological agent react rapidly with one or more ROS. The pharmacological agent also must be present in the milieu where ROS are being generated. This last concept is so selfevident that very few prior studies have evaluated the durability of protection provided by various ROS scavengers following washout (in vitro) or after cessation of treatment (in vivo). One study that does provide some information about the duration of protection afforded by an anti-oxidant was carried out by Fan et al. [43]. In this study, acute respiratory distress syndrome (ARDS) was induced in rats by challenging the animals intra-tracheally with a suspension of LPS one hour or 18 hours after resuscitation from hemorrhagic shock. Systemic administration of a large dose (500 mg/kg) of the anti-oxidant, N-acetylcysteine (NAC), at the time of resuscitation from hemorrhagic shock provided significant protection from the development of ARDS, but only when the LPS challenge followed in 60 minutes. If LPS was administered 18 hours after systemic treatment with NAC, then no protection at all was observed. These findings are consistent with the view that the therapeutic effect of NAC is transient and depends on the presence of the compound in cells or the extracellular fluid compartment. But, in contrast to these data, the therapeutic effect of ethyl pyruvate is durable after transient exposure of cells or animals to the compound. This pivotal observation suggests that the mechanism(s) responsible for the antiinflammatory actions of ethyl pyruvate is different from those of a classical anti-oxidant like NAC. This idea is further supported by data from a series of in vitro experiments carried out by Han and colleagues [44]. In these studies, ethyl pyruvate inhibited luciferase expression in lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells transfected with an NF- B-dependent luciferase reporter vector. This finding provides clear evidence that ethyl pyruvate inhibits NF- Bdependent gene transcription. Ethyl pyruvate also decreased NF- B DNA-binding activity in LPS-stimulated RAW 264.7 murine macrophage-like cells and decreased LPS-induced expression of an NF- B-dependent gene, iNOS. Interestingly, in contrast to certain other anti-oxidants, such as vitamin C [45] and curcumin [46], that are known to interfere with NF- B-dependent signaling, ethyl pyruvate had no effect on the degradation of I B or I B in LPSstimulated RAW 264.7 cells. This finding suggests that ethyl pyruvate acts distally to this step in the activation pathway for NF- B. In a cell-free system, binding of p50 homodimers to an NF- B consensus oligonucleotide sequence was unaffected by ethyl pyruvate over a wide range of concentrations, indicating that ethyl pyruvate probably does not modify or interact with the p50 subunit of NFB. In contrast, ethyl pyruvate inhibited spontaneous DNA binding by wild-type p65 homodimers, which were over-expressed in human embryonic kidney 293 cells following transient transfection with a p65 expression vector. However, ethyl pyruvate failed to inhibit the DNA-binding activity of homodimers of an overexpressed mutant form of a p65 with substitution of serine for cys-
teine 38. Collectively, these data suggest that ethyl pyruvate targets a critical thiol moiety (i.e., cysteine 38) in the p65 component of NF- B. This idea is further supported by data from another study, wherein it was shown treating LPS-stimulated RAW 264.7 cells with glutathione ethyl ester, a reducing agent and glutathione precursor, partially inhibits the anti-inflammatory effects of ethyl pyruvate [47]. The idea that ethyl pyruvate exerts therapeutic effects via mechanisms that are not applicable to pyruvate itself is supported by other studies showing that a related pyruvate ester, methyl pyruvate, exhibits pharmacological effects that are distinct from those of the parent carboxylate anion. Specifically, methyl pyruvate stimulates insulin secretion by isolated pancreatic islets [48,49], whereas pyruvate is not insulinogenic [50]. Furthermore, methyl pyruvate but not sodium pyruvate causes closure of ATP-sensitive potassium channels, triggers a sustained rise in intracellular calcium ion concentration, and increases insulin secretion more effectively than glucose [48]. Zawalich and Zawalich speculated that esterification renders pyruvate more membrane-permeable and thereby allows higher levels of the compound to accumulate in mitochondria [49]. This notion is supported by data reported by Malaisse et al., who showed that methyl pyruvate causes less lactate production than sodium pyruvate in pancreatic islets, a finding that is consistent with decreased cytoplasmic metabolism and increased mitochondrial metabolism by the ester [51]. Furthermore, Rocheleau et al. recently reported that the addition of methyl pyruvate (instead of sodium pyruvate) causes a larger and more sustained increase in cellular NAD(P)H content in islet cells. The NAD(P)H response also occurs faster with methyl pyruvate than is observed with either glucose or sodium pyruvate. These data are consistent with the view that the pyruvate ester stimulates mitochondrial production of both NADPH and NADH. Thus, the differential permeability hypothesis seems likely to be right, at least with respect to the effect of pyruvate esters on insulin secretion. Pyruvate, of course, plays a central role in intermediary metabolism. Therefore, ethyl pyruvate might function as a pro-drug leading to pyruvate formation as a result of spontaneous or enzymatically catalyzed hydrolysis of the ester linkage in the extracellular or intracellular milieu. Once formed, pyruvate might function in various shock-like states as metabolic substrate to decrease the cytosolic [NADH]/[NAD+] ratio and maintain the cellular phosphorylation potential, [ATP]/[ADP][Pi]; this mechanism has been proposed to explain some of the beneficial effects of pyruvate administration in a porcine model of hemorrhagic shock [19] and in models of myocardial ischemia and reperfusion [52]. Of course, this mechanism is insufficient to explain the greater activity of ethyl pyruvate as compared to pyruvate in various in vitro or in vivo model systems. SUMMARY In summary, an accumulating body of data supports the view that ethyl pyruvate is an effective anti-inflammatory agent. Treatment with ethyl pyruvate has been shown to have beneficial effects in various rodent models of hemorrhagic shock and ischemia/reperfusion injury. Ethyl pyruvate treatment also has been shown to improve survival or organ system function in murine models of acute endotoxemia and lethal sepsis and an ovine model of resuscitated hyperdynamic endotoxemia. Collectively, these data support the view that ethyl pyruvate warrants further evaluation as a therapeutic agent for a variety of acute conditions, including severe sepsis and septic shock, that are commonly encountered in the practice of critical care medicine. ACKNOWLEDGEMENT This work was supported by grant number NIH R01 GM 68481.
Mitchell P. Fink [28] Yang, R.; Gallo, D.J.; Baust, J.J.; Uchiyama, T.; Watkins, S.K.; Delude, R.L. and Fink, M.P. (2002) Am. J. Physiol. Gastrointest. Liver Physiol., 283, G212-G22. Venkataraman, R.; Kellum, J.A.; Song, M. and Fink, M.P. (2002) Shock, 18, 507-512. Ulloa, L.; Ochani, M.; Yang, H. ; Halperin, D.; Yang, R. Czura, C.J.; Fink, M.P. and Tracey, K.J. (2002) Proc. Natl. Acad. Sci. USA, 99, 12351-12356. Goodwin, G.H.; Sanders, C. and Johns, E.W. (1973) Eur. J. Biochem., 38, 14-19. Wang, H.; Bloom, O.; Zhang, M.; Vishnubhakat, J.M.; Ombrellino, M.; Che, J.; Frazier, A.; Yang, H.; Ivanova, S.; Borovikova, L.; Manogue, K.R.; Faist, E.; Abraham, E.; Andersson, J.; Andersson, U.; Molina, P.E.; Abumrad, N.N.; Sama, A. and Tracey, K.J. (1999) Science, 285, 248-251. Yang, H.; Wang, H.; Li, J.; Tanovic, M.; Harris, H.E.; Susarla, S.; Ulloa, L.; Wang, H.; DiRaimo, R.; Czura, C.J.; Wang, H.; Warren, H.S.; Fink, M.P.; Fenton, M.J.; Andersson, U. and Tracey, K.J. (2004) Proc. Natl. Acad. Sci. USA, 101, 296-301. Miyaji, T.; Hu, X.; Yuen, P.S.T.; Muramatsu, Y.; Iyer, S.; Hewitt, S.M. and Star, R.A. (2003) Kidney Int., 64, 1620-1631. Dear, J.W.; Kobayashi, H.; Jo, S.K.; Holly, M.K.; Hu, X.; Yuen, P.S.; Brechbiel, W.W. and Star, R.A. (2005) Kidney Int., 67, 21592167. Hauser, B.; Kick, J.; Asfar, P.; Ehrmann, U.; Albicini, M.; Vogt, J.; Wachter, U.; Bruckner, U.B.; Fink, M.P.; Radermacher, P. and Bracht, H. (2005) Crit. Care Med., (inpress). Tawadrous, Z.S.; Delude, R.L. and Fink, M.P. (2002) Shock, 17, 473-477. Yang, R.; Uchiyama, T.; Watkins, S.K.; Han, X. and Fink, M.P. (2004) Shock, 22, 369-375. Kabe, Y.; Ando, K.; Hirao, S.; Yoshida, M. and Handa, M. (2005) Antioxid. Redox Signal., 7, 395-403. Haddad, J.J. (2002) Crit. Care, 6, 481-490. Sappington, P.L.; Fink, M.P.; Yang, R.; Delude, R.L. and Fink, M.P. (2003) Shock, 20, 521-528. Sappington, P.L.; Han, X.; Yang, R.; Delude, R.L. and Fink, M.P. (2003) J. Pharmacol. Exp. Ther., 304, 464-476. Fan, J.; Shek, P.N.; Suntres, Z.E.; Li, Y.H.; Oreopoulos, G.D. and Rotstein, O.D. (2000) Surgery, 128, 332-338. Han, Y.; Englert, J.A.; Yang, R.; Delude, R.L. and Fink, M.P. (2005) J. Pharmacol. Exp. Ther., 312, 1097-1115. Bowie, A.G. and O'Neill, L.A. (2000) J. Immunol., 165, 71807188. Brennan, P. and O'Neill, L.A. (1998) Biochem. Pharmacol., 55, 965-973. Song, M.; Kellum, J.A.; Kaldas, H.; Fink, M.P. (2004) J. Pharmacol. Exp. Ther., 308, 307-316. Mertz, R.J.; Worley, J.F.I.; Spencer, B.; Johnson, J.H. and Dukes, I.D. (1996) J. Biol. Chem., 271, 4838-4845. Zawalich, W.S. and Zawalich, K.C. (1997) IJ. Biol. Chem., 272, 3527-3531. Sener, A.; Kawazu, S.; Hutton, J.C.; Boschero, A.C.; Devis, G.; Somers, G.; Herchuelz, A. and Malaisse, W.J. (1978) Biochem. J., 176, 217-232. Malaisse, W.J.; Jijakli, H.; Ulusoy, S.; Cook, L.; Best, L.; Vinambres, C.; Villanueva-Pencarrillo, M.L.; Valverde, I. and Sener, A. (1996) Arch. Biochem. Biophys., 335, 229-244. Mallet, R.T.; Sun, J.; Knott, E.M.; Sharma, A.B. and OlivenciaYurvati, A.H. (2005) Exp. Biol. Med., 230, 435-443.
DISCLAIMER Mitchell P. Fink is a co-founder and shareholder of Critical Therapeutics, Inc., a biotechnology company that is developing ethyl pyruvate, among other products, for the treatment of acute medical conditions. REFERENCES
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Pathogenesis and Sepsis Caused by Organisms Potentially Utilized as Biologic Weapons: Opportunities for Targeted Intervention
Matthew J. Hepburn1,2,*, Bret K. Purcell2 and Jason Paragas2
1 Defence Science and Technology Laboratories, Porton Down, Wiltshire, United Kingdom and 2Division of Medicine, Bacteriology and Virology, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
Abstract: The microorganisms potentially utilized as biologic weapons have a variety of pathogenic mechanisms that lead to overwhelming infection, septic shock and death. Although many of these organisms have unique pathogenic attributes, the development of generic therapies for common pathways would be exceedingly useful as countermeasures. This review will examine the features of pathogenesis leading to sepsis for key biologic threat agents (causative agents of anthrax, plague, tularemia, smallpox and viral hemorrhagic fevers), and highlight current and future therapeutic targets. For some of the biologic threat agents, such as anthrax, substantial research has yielded a number of targeted sites for intervention. For other organisms, further elucidation of the mechanisms of pathogenesis and septic shock is needed to direct therapeutic exploration.
INTRODUCTION The history of warfare is replete with examples of biological agents as weapons [1]. Recently, small- and large-scale terrorism events, most notably the letters containing anthrax spores that were mailed to U.S. government officials and news agencies, highlighted the lethal characteristics of these agents and the associated social disruption and economic impact [2]. In response, levels of funding for biodefense research have increased substantially [3, 4]. In this review, we will discuss the relevant mechanisms of sepsis for some of the key biological agents, and suggest to the reader promising areas for future research. The examination of interventions to interrupt the cascade of events leading to septic shock and fatality is pertinent to biodefense discussions. The challenge for biodefense planners is to develop effective solutions for an enormous variety of potential weapons. The possibility of genetically engineered organisms and unidentified emerging threats further complicates preparations [5]. The ideal countermeasures against these threats would be generic; they would be effective against a wide variety of pathogens, including pathogens that have been genetically manipulated. Some of the common pathogenic mechanisms among organisms potentially used as biologic weapons will be discussed. This review will focus on the biological agents considered to be the most significant threats, designated as the Category A agents by the Centers for Disease Control and Prevention (CDC) [6]. These include Bacillus anthracis, Yersinia pestis and Francisella tularensis among the bacteria, and smallpox and microorganisms causing viral hemorrhagic fevers among the viruses. We will primarily discuss current understanding of the mechanism of pathogenesis that allow evasion of the immune response, replication in humans, and cellular damage as well as the properties that trigger the sepsis cascade. Additionally, we will mention recent and novel therapeutic strategies related to sepsis. This review will not discuss the epidemiology of these agents, nor will it extensively describe current antibiotic choices or vaccine development. Although botulinum neurotoxin is considered a substantial threat as a biological agent [7], its mechanism of action produces profound neurotoxicity, but not bacterial-induced septic shock, and therefore has not been
included. There are numerous other organisms and biological toxins which could be used as weapons, and have substantial relevance to discussions on sepsis. For example, staphylococcal enterotoxin B is a category B agent by CDC classification [6] and functions as a superantigen, provoking robust and non-specific immune activation, potentially leading to septic shock [8]. However, we have opted to provide a detailed discussion of only the five Category A microorganisms. Bacillus anthracis Bacillus anthracis is gram-positive rod-shaped bacillus with a predilection for forming spores, allowing long-term survival without desiccation in environments such as the soil [9]. Anthrax was mass-produced as a biological weapon by both the United States and former Soviet Union [10, 11]. The rapid development of fulminant infection soon after the onset of symptoms, particularly with pulmonary anthrax, is one of the reasons for the high mortality associated with this infection and why this agent was selected in these former weapons programs. Although the most common presentation of anthrax in a clinical setting is a cutaneous eschar, inhalational anthrax is the most lethal [10]. After the anthrax spore reaches the alveoli, alveolar macrophages uptake the spore and travel to the mediastinal lymph node [2]. The spore can remain dormant for a prolonged period of time, as evidenced by infection occurring 58 days after exposure in a non-human primate experimental model [12], and 43 days after exposure in the accidental release of anthrax in the former Soviet Union at Sverdlovsk [13]. Once the anthrax spores germinate within a macrophage [14], multiplication of the organism and release of various toxins ensues, causing the manifestations of clinical infection. Patients infected with B. anthracis by the pulmonary route develop inflammation, hemorrhage and necrosis in the mediastinal lymph nodes, leading to the relatively unique clinical finding of hemorrhagic mediastinitis [15]. Bronchopulmonary pneumonia rarely occurs with inhalational anthrax infection [2]. Although pulmonary infiltrates were frequently observed with the pulmonary anthrax cases in the U.S. in 2001 [16], these findings were attributed to intravascular edema and hyaline membrane formation [17]. Patients with pulmonary anthrax typically develop pleural effusions, which have averaged 1700 ml in one case series [18]. Contrast-enhanced computer tomography images of a victim of the anthrax letters incident revealed diffuse hemorrhagic mediastinal and hilar adenopathy with edema, perihilar infiltrates, bronchial mucosal thickening, pleural and pericardial effusions [19]. During
*Address correspondence to this author at the Defence Science and Technology Laboratories, Porton Down, Wiltshire, United Kingdom; E-mail: Matthew.Hepburn@us.army.mil
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Fig. (1). Legend. Pathogenesis of anthrax toxins (1) Protective antigen (PA), named due to the protective aspects of human antibodies to PA, is released by the bacteria in an inactive form (PA83), binds to a cell-surface receptor, is cleaved by a furin-related protease into its active form (PA63). (2) PA63 then forms a heptamer. (3) This active, oligomerized form binds to either of the exotoxins, lethal factor (LF) or edema factor (EF). The combination of LF and PA is known as lethal toxin (LT), and EF is complexed with PA to form edema toxin (ET). ET and LT enter the cell, leave the endosome after acidification and produce their toxicity. (4) LT has been described as causing human endothelial cell apoptosis [43]. The apoptotic effect of LT is thought to be due to the inhibition of phosphorylation of extracelluar signal regulated kinases (ERKs). (5) LF cleaves mitogen activated protein kinase kinase (MAPKK) molecules at their N-terminus [244, 245], and therefore prevents the downstream activation of related cytokines. (6) Edema factor (EF) is an adenyl cyclase, calmodulin-dependent enzyme, that catalyzes the reaction forming cyclic adenosine monophosphate (cAMP) from adenosine tri-phosphate (ATP) [22]. The accumulation of cAMP results in substantial edema of surrounding tissues [246], and can adversely affect the immune system by accumulation in lymphocytes [51] and neutrophils [247]. These effects impair the respiratory burst activity in neutrophils as well as phagocytosis and production of tumor necrosis factor (TNF) and interleukin-6 (IL-6) by monocytes, furthering the hosts resistance to infection [247, 248]. (7) The intracellular increase in cAMP concentration is dependent on extracellular calcium, and inhibitors of calcium uptake may prevent the edema-toxin induced effects [51]. (8) B. anthracis also secretes proteases, such as the thermolysin-like proteins of the M4 family and the collagenolytic M9B family [63]. These proteases may contribute to the development of sepsis.
the later stages of the disease patients develop sudden fever, dyspnea, diaphoresis, cyanosis, hypotension, shock and death, with complications of meningitis and arteritis in some cases [2]. Disseminated infection tends to occur rapidly, with large numbers of organisms detectable in the blood stream, possibly visible on Grams stain. Features of the septic shock associated with anthrax infection have been studied mostly in animal models. Key features in animals include electrolytes disturbances (hypocalcemia, hyperkalemia), hypoglycemia, acid-base abnormalities (respiratory alkalosis followed by severe acidosis), depression of the respiratory centers, and severe hypoxia [20]. Further research is needed to thoroughly describe and characterize the progression of infection and shock in these animal models. The clinical manifestations of anthrax are thought to be primarily toxin-mediated. Anthrax toxin has two exotoxins and one anti-
phagocytic capsule as well as other factors believed to play a role in pathogenesis. The mechanisms of these toxins are depicted in Figure 1. Protective antigen (PA) is released by the bacteria, cleaved by furin-related protease into its active form, and then binds with either lethal factor (LF) to form lethal toxin (LT) or edema factor (EF) to form edema toxin (ET) (reviewed by Ascenzi et al. [21]). LF is a zinc metalloprotease with various pathological effects, including inhibition of MAPK kinases (see Fig. 1). ET is an adenyl cyclase, calmodulin-dependent enzyme, that catalyzes the reaction forming cyclic adenosine monophosphate (cAMP) from adenosine tri-phosphate (ATP) [22]. Some experiments have suggested the vegetative form of anthrax can multiply in macrophages [23], whereas other studies have suggested enhanced killing of B. anthracis by macrophages when the spores germinate [24]. B. anthracis also has a poly-D-glutamic acid capsule, which helps to prevent phagocytosis and is a well-
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described virulence factor in murine models [25, 26]. The ability of the macrophage to kill B. anthracis may be species-dependent [27], just as the outcome of anthrax infection can vary depending on the species of mice [28]. The effect of lethal toxin (LT) on the macrophage may also depend on the species involved, as well as the state of differentiation of the macrophage. Lethal factor (LF) can produce direct lysis [29] or apoptosis [30] in some murine models, but does not appear to lyse human macrophages and those in other rat strains [27, 31]. In studies of human monocytic cell lines, undifferentiated monocytes were not affected by LT, whereas these cells were susceptible after differentiating into macrophages [32]. Enhancing macrophage function in early infection (phagocytosis, inhibiting spore germination) can be accomplished with appropriate antibodies [33], or with exogenous interferons (gamma and alpha/beta) [34]. B. anthracis may prevent the expression of transcription factors (signal transducer and activators of transcription 1 or STAT1, and ISGF-3) that are necessary for endogenous interferon production [34]. Proteonomics research has recently identified three potential protein targets expressed during early germination (immune inhibitor A, GPR-like spore protease, and alanine racemase) [35]. Augmenting macrophage killing of spores is a potential area for future therapeutic research. LT does not appear to produce an overwhelming response with pro-inflammatory cytokines in mice or rats, and the proposed mechanism of lethality in these animals is shock related to circulatory collapse, but not mediated by cytokines [31, 36]. This bacterial toxin downregulates co-stimulatory molecules and is ineffective in T-cell priming [37, 38]. Both LT and ET directly inhibited T-cell activation in a murine model [39]. LT actually suppresses cytokine production in macrophages by its activity against MAPK kinase signals [38], although this effect may be balanced by the induction of IL-1b, TNF-alpha and IL-6, detected in some murine models [40]. Suppression of a robust cytokine response may be a component of the evasive strategy of B. anthracis. Another aspect is impairment of dendritic cell function, which delays the pathogen recognition components of the adaptive immune response [37, 41]. Specifically, LT impairs pro-inflammatory cytokine release, and also decreases the up-regulation of co-stimulatory molecules (CD40, CD80, CD86) on dendritic cells. ET may facilitate the LT-induced inhibition of cytokine secretion by dendritic cells [42]. By these actions, dendritic cells are not adequately capable of activating nave T-cells. These findings need further elucidation in experimental settings that closely simulate anthrax infection. Other pathological mechanisms of LT include effects on endothelial cells and platelets. LT causes human endothelial cell apoptosis by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK) [43], and the endothelial dysfunction may lead to increased vascular permeability and hypotension associated with septic shock. Prevention of endothelial cell apoptosis may be a future therapeutic strategy. Other researchers have documented increased vascular permeability with human endothelial cells, but their findings of increased permeability did not depend on endothelial cell apoptosis [44]. LT induces hemorrhage in mice, prevents whole blood clotting and inhibits platelet aggregation [45]. The mechanism for these actions on platelet function appear to be mediated by the MAPK kinase pathways. LT has many other systemic effects, such as hemolysis, particularly in the presence of neutrophils [46]. Interestingly, LT may also block the typical endocrinologic response to stress, particularly affecting glucocortocoid and progesterone receptors [47]. Further assessment of glucocortocoid levels in non-human primate anthrax infection models needs to be established before intervention protocols can be pursued. If validated in non-human primate models, glucocortocoids such as hydrocortisone may have therapeutic benefit in the management of anthrax-induced
sepsis. However, in a murine LT-infusion model of infection, dexamethasone actually increased mortality and enhanced the pathologic impact of LT [48], suggesting caution in the use of glucocorticoids for anthrax treatment. The mechanism of pathogenesis of edema toxin (EF+PA) is described in Figure 1. Some potential inhibitors of adenyl cyclase toxins have been identified [49]. One particularly interesting inhibitor in adefovir, an antiviral currently licensed for treating hepatitis B infection, which inhibited the accumulation of cyclic AMP caused by EF in murine macrophages [50]. An additional interesting potential target for intervention is preventing the uptake of intracellular calcium, which is needed for the toxic effects of ET [51]. The development of anti-toxin antibodies is a crucial component in the protective immune response against anthrax. Anti-PA antibodies closely correlate with protection in animal challenge models [52, 53]. Monoclonal antibodies to PA [54, 55] and LF [56] have been developed. Passive PA antibodies are protective in rodent models [55, 57]. Additionally, induction of anti-PA antibodies is thought to be a critical response in the protection induced by anthrax vaccination [58]. Recent investigations in a rat model have suggested that the human monoclonal anti-PA antibody 5H3 can improve survival even if administered 6 hr after lethal toxin infusion is initiated [59]. At this time point, rats are already experiencing heart rate and blood pressure abnormalities consistent with early septic shock. The authors concluded that antibodies to PA may have clinical utility even after the onset of sepsis [59]. However, a better understanding of how LT produces fatal septic shock is needed. Further possible interventions include blockages of the activity of the furin-related protease cleavage [60]. A number of compounds have recently been identified with high throughput screening that may be inhibitors to LF or EF [61, 62], and safety and efficacy data from phase I clinical studies for some of these compounds will hopefully be forthcoming. Although most of the pathogenesis research on anthrax has focused on the toxins, a recent publication has attempted to characterize some of the secreted proteases of B. anthracis [63]. They have suggested some of these proteases belong to the thermolysin-like proteins of the M4 family, while others are members of the collagenolytic M9B family. Further murine experiments suggested that these proteases are lethal in an animal model, and that bacterial protease inhibitors may be protective in murine B. anthracis infection, but only in conjunction with antibiotics [63]. These intriguing findings need to be validated in other experimental scenarios. It is possible that these additional proteases contribute to the septic shock induced by anthrax infection, as recent data suggest that proteases and collagenases (and their corresponding tissue destruction) can result in hemodynamic instability and other clinical findings that resemble bacteria-induced sepsis [64]. These virulence factors also have structural similarities to the virulence factors in other microorganisms, such as Yersinia pestis [65]. Other possible early interventions include quorum sensing inhibitors [66] and monoclonal antibodies to poly gamma-D-glutamic acid capsule [67]. Yersinia pestis Yersinia pestis, the gram-negative bacilli that is the causative agent in plague, was responsible for some of the most devastating epidemics in the history of humanity [68]. Plague continues to cause morbidity and mortality as a naturally-occurring diseases, with a large majority of human cases reported in Africa [69]. There was substantial interest in the former Soviet Union to develop plague as a biologic weapon and this agent was most likely included in their biological weapons arsenal [5]. The rapid lethality of pneumonic plague is a very concerning attribute of this organism [70]. Unlike anthrax, plague can be transmitted person-to-person, although some experts believe simple infection control measures could substantially limit this spread [71]. Although antibiotics such as the aminoglycosides, tetracyclines and flouroquinolones are
Hepburn et al.
Table 1.
Yops Proteins of Yersinia pestis

Yop Yop B, Yop D, LcrV Ysc F Yop N Function and Mechanism Translocation of the other Yop proteins into the host cell [78-81] Protein that facilitates translocation of Yops [76, 82, 83] Regulates and inhibits Yop secretion in the presence of calcium and prior to contact with eukaryotic cells, along with SycN/YscB chaperones, TyeA and LcrG [95-99] Anti-phagocytotic by affecting cytoskeleton through PTPase activity against p130Cas [101], and affecting adhesion-related singal transduction with the Fyn-binding protein Fyb, and SKAP-HOM [102] Inhibits immune function by inhibiting MCP-1 by dephosphorylation enzymes in the phosphotidylinositol 3kinase/Akt pathway [118], which also may affect T and B cell function [118, 119] Anti-phagocytotic activity through inhibition of cytoskeletal function by functioning as a GTP-activating protein, which increase GTP hydrolysis and inhibits RhoA, Rac1, and Cdc42 function [89, 106, 107] Anti-phagocytotic activity as a cysteine protease that cleaves RhoA, Rac and Cdc42 [89, 108, 109] Anti-phagocytotic activity through cytoskeletal disruption by actin binding [89, 110, 111] Inhibits IKK , leading to inhibition of function of NF- and prevention of cytokine activation [112, 115] Inhibits MAPK kinase [113] Causes apoptosis of macrophages [117] Travels to the nucleus on host cell entry, causes depletion of NK cells, possibly by effects on IL-15 [120]
YopH
YopE
YopT YpkA/YopO YopP/YopJ
Yop M
available for treating plague [72], the mortality rate associated with this disease remains high, particularly if antibiotics are initiated after septic shock has ensued. The clinical presentation of plague is classified as bubonic, septicemic or pneumonic, with bubonic plague being the most common naturally occurring form. A plague-related biologic weapon would most likely be distributed by an aerosol route, leading to the pneumonic form. Pneumonic plague tends to have a fulminant course after a short incubation period (1-5 days) [70, 73], with initial symptoms of high fever, dyspnea, malaise, myalgias, leading to hemoptysis and cyanosis. Gastrointestinal symptoms, such as nausea, vomiting and abdominal pain may occur as well [72]. Progressive multiplication of the organism leads to septic shock, multisystem organ failure and disseminated intravascular coagulation that follows a pattern similar to other gram-negative organisms that cause septic shock [70, 74]. Pneumonic plague is considered uniformly fatal if antibiotics are not administered within the first 24 hr of infection [73]. More pathogenesis studies are needed to define the mechanisms of shock in Y. pestis infections. One of the crucial virulence factors in plague infection is the Yersinia outer proteins, or Yops (functions summarized in Table 1). These proteins provide for evasion of the immune system by various mechanisms. In particular, some of these key virulence proteins in this group are the Type III secretion system, which is upregulated when the pathogen is at 37C [75]. The Type III system is not unique to Y. pestis, as similar forms are found in at least 12 other bacteria, including enteric pathogens such as Salmonella enterica, Shigella flexneri and enteropathogenic Escherichia coli [76]. The system is designed to inject virulence proteins into host cells at close contact, causing disruption of the cytoskeleton, downregulation of the release of inflammatory mediators by immune cells, and apoptosis of macrophages [76]. Recent studies have suggested that Y. pestis bacteria specifically target immune cells, such as dendritic cells, macrophages and neutrophils, for injection and thus effectively eliminating the host immune response to infection [77]. Injection is accomplished through organelles known as the Ysc injectisomes, which allow transport of the Yops across the bacterial cell membrane [76]. Adhesins on the bacterial cell surface (YadA and Inv) interact with integrins on the macrophage, allowing for
close contact and eventual injection of the Yops into the macrophage cytosol [76]. Because the injectisomes do not also span the macrophage cell membrane, translocator Yops (Yop B, Yop D and LcrV) [78-81] are needed to open the macrophage cell membrane for other pathogenic proteins. The injectisome and translocator proteins likely form one continuous tunnel that allows the other Yops proteins to travel directly into the macrophage cytoplasm [76]. Ysc F, a surface-expressed protein of the secretion complex, facilitates the translocation of Yop proteins into host cells [82], whether it functions as needle [83] or is needed in the construction of the conduit into the target cell [84]. In the mouse model of plague, vaccination with YscF protects against challenge with Y. pestis [85, 86]. Before injection, there are Yop proteins that require binding with bacterial cytosolic proteins called the specific Yop chaperone (Syc) proteins [87-94]. These proteins represent an additional target for intervention, as Yop secretion is dramatically reduced when they are not present [76]. Additionally, Yop N [95], along with SycN/YscB (chaperone proteins) [96], TyeA [97], LcrG [98], inhibits Yop secretion in the presence of calcium and before host cell contact, providing a regulatory function so that Yops are only secreted when needed to preserve the organism. Bacterial mutants that lack any of these proteins have less efficient transmission of Yops into host cells, most likely due to secretion of Yops before cell contact [99]. The Yop effectors are the proteins that have pathologic effects on the host cell after injection. Their activity can be divided into inhibition of phagocytosis (YopH, YopE, YopT, YopO/YpkA) and the disabling the immune response (YopP/J, YopH) [100]. YopH is a phosphotyrosine phosphatase (PTPase) [100], which dephosphorylates p130Cas, disrupting focal adhesions [101]. It also dephosphorylates the Fyn-binding protein Fyb,[101] and SKAPHOM, inhibiting an adhesion-related signal pathway in the macrophage [102]. The result is the macrophage cannot perform phagocytosis on the Yersinia pathogen. Inhibitors of YopH (targeting PTPase), such as aurintricarboxylic acid (ATA) [103], may have therapeutic potential but non-specific binding is a concern [104]. YopE [105-107], YopT (a cysteine protease) [89, 108, 109] and YpkA/YopO (structurally similar to serine/threonine kinases) [110, 111] target monomeric GTPases such as RhoA, Rac1, and Cdc42 ,
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which are needed for effective cytoskeleton function [105]. YpkA/YopO is activated by binding actin [111]. Other Yop effectors prevent an adequate host immune response, which would normally destroy the bacteria. YopJ affects host cell signal transduction pathways for Y. pestis and Y. pseudotuberculosis, with the homologue YopJ having a similar function for Y. enterocolitica. The Yops inhibits IKK , which prevents the phosphorylation of I [112, 113], a step required to degrade I . I functions to inhibit the activity of NF- , and therefore the YopP/YopJ proteins prevent NF- from being able to perform its essential role as a transcriptional factor in the activation of crucial components of the immune response. For example, cytokine release can be suppressed [112, 114], particularly the release of TNF- by macrophages [115]. YopP/YopJ also inhibits MAPK kinases [113], providing a common pathologic link with LF released by B. anthracis. This effect on MAPK kinase pathways contributes to the observation of blockage of three important pathways related to the immune response: the lipopolysaccharide (LPS) receptor, the interleukin (IL)-1 receptor and the transcription receptor cAMP response element-binding protein (CREB) [116]. YopP/YopJ also induce apoptosis of macrophages [117]. YopH, in addition to its anti-phagocytotic properties, also inhibits release of monocyte chemoattractant protein-1 (MCP-1), which stimulates recruitment of cells of the immune system to the site of infection [118]. YopH may also have effects on B and T lymphocytes, affecting lymphocyte activation proliferation by decreasing IL-2 secretion [118, 119], and antigen-specific B lymphocyte responses [119]. YopM was recently described to cause a depletion of NK cells [120], possibly by effects on IL-15 or the IL-15 receptor . Y. pestis has additional virulence factors, including the V antigen and cell wall LPS. The V antigen (LcrV) is one of the Yop translocator proteins, and can also decrease the release of proinflammatory cytokines by amplifying the production of IL-10. When purified V antigen is given to mice, the LD50 from bacterial LPS is significantly increased [121], suggesting that the antigen prevents the development of fulminant sepsis, perhaps by increasing levels of the anti-inflammatory IL-10 cytokine. These early strategies that evade the immune system must eventually become overwhelmed with the pro-inflammatory effect of the bacterial LPS and other stimulants of the immune system, but how these factors are regulated needs further study. Interestingly, as antibodies to the V antigen inhibit the function of the translocating Yop proteins, the pathogenic effects of both Y. pestis and Pseudomonas aeruginosa (which has a similar injectisome complex) are attenuated in an in vitro model [122]. Vaccines with the F1 (a capsular antigen with anti-phagocytotic properties [123]) and V antigens have been effective in animal models [124] and are currently in human clinical trials. The LPS component of the cell wall of Y. pestis does induce a typical gram-negative bacterial effect, such as stimulating the production of TNF-alpha and IL-6 from mouse macrophages [125]. However, this effect was less than LPS isolated from Escherichia coli strain 0111 in this model. Table 2.
Subcellular proteomic analysis comparing Y. pestis and Y. pseudotuberculosis bacterial-host interactions have demonstrated differential expression of 16 and 13 proteins in interactions with human monocytes, respectively [126]. Only two of these proteins were shared between these two exposures. The proteins identified may be involved in cellular functions ranging from cell signaling to protein synthesis to apoptosis. It is hoped that by utilizing the technologies of proteomic and genomic analyses, additional host and bacterial factors will be identified and therapeutic targets can be developed. Francisella tularensis Although Francisella tularensis is not associated with as high a mortality rate as the organisms that cause plague or anthrax, the high infectivity of the organism compelled the United States and former Soviet Union to develop this agent as a weapon during the Cold War [127]. Antibiotics, such as the aminoglycosides, tetracyclines and flouroquinolones, typically provide successful therapeutic results [128-130]. However, concern for the development of antibiotic-resistant strains has prompted continuing research into the development of alternative countermeasure strategies. A distinct feature of F. tularensis is the low inoculum required for infection. As few as ten organisms can produce an infection when injected subcutaneously into humans, and only 10-50 organisms are required when humans are exposed via aerosol [131, 132]. Mechanisms to evade the immune system are the likely factors that allow for infection at low inoculum. F. tularensis is thought to be almost exclusively intracellular as a pathogen, residing mostly in macrophages, and substantial research has been published studying the macrophage-pathogen interaction with both virulent and avirulent strains of F. tularensis (see Table 2 for mechanisms). A Francisella pathogenicity island protein Ig1C and its regulator Mg1A have been recently discovered and appear to be essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm [133, 134]. F. tularensis initially stimulates cytokine production by macrophages, but after infection of a macrophage is established, the cytokine production ceases [135]. After infection, it appears that F. tularensis inhibits MAPK kinase function [135, 136], similar to B. anthracis and Y. pestis, as previously mentioned. Also similar to B. anthracis and Y. pestis, the NFpathway is inhibited [135]. The clinical disease tularemia is usually manifested by an ulcerative skin lesion, caused by direct inoculation of the organism when the skin barrier is compromised, and regional lymphadenopathy [137]. This ulceroglandular form rarely results in fulminant systemic symptoms, such as shock. The pneumonic form of the disease occurs when the organism is inhaled, and usually presents as pneumonia, without an obvious characteristic pattern, presenting often as a fever and cough [138]. Pneumonic disease can also arise from the ulceroglandular, oculoglandular or oropharnygeal forms of the disease when dissemination of the organism occurs. Additionally, there is a typhoidal form of the disease, with no skin lesions
Survival Mechanisms for F. tularensis within Macrophages

Inhibition of the respiratory burst on macrophage entry, with an acid phosphatase known as AcpA [236, 237]. Prevention of acidification of the phagosome on entry to the macrophage [238]. Escaping the phagosome to reside in the macrophage cytoplasm [238, 239].
Inhibits Toll-like receptor signaling and cytokine secretion, as demonstrated in experiments with murine macrophages and the LVS strain of F. tularensis. [136] Key virulence factor appears to be 23 kDa protein, IglC. The mglAB operon provides regulation of virulence factors for survival in macrophages [240]. MinD protein (functions as a pump to resist oxidative killing) [241].
Hepburn et al.
and minimal adenopathy but systemic symptoms of a severe infection [139]. It is unknown why some patients develop progressive and eventually fulminant symptoms while others do not. Elucidating the pathogenesis of tularemia will hopefully suggest answers to these questions, and allow for improved clinical management strategies. The frequency that F. tularensis causes septic shock is unknown, particularly because most patients have antibiotic intervention before the onset of shock. However, one series describes poor outcomes from tularemia in patients with underlying serious medical conditions, and who experience a delay in antimicrobial therapy [140]. Septicemia does occur, but is rarely reported [141, 142]. The mechanism by which F. tularensis causes sepsis has not been defined. The LPS of F. tularensis does not produce the typical response expected from endotoxin, as it does not produce IL-1 secretion and only weakly causes TNF- production from mononuclear cells [143]. It does not stimulate endothelial cell response in the same pattern as the LPS of other enteric bacteria [144]. The only current countermeasure development strategies for F. tularensis are antibiotics and vaccine development [127]. As an intracellular organism, the cell-mediated response is thought to be more important than the humoral response in the management of this disease [145]. In a human challenge model of pneumonic tularemia, volunteers received a formalin-killed whole-cell vaccine developed by Foshay and colleagues [146]. A strong humoral response was elicited but the vaccine was not protective against cutaneous [132] or respiratory [131] challenge. Therefore, antibodybased countermeasures have not been developed and tested in humans. Further study is needed into the mechanisms by which F. tularensis produces its fulminant manifestations. It is possible that therapies that are effective for septic shock caused by other organisms may be effective for tularemia, but these interventions require further investigation. SMALLPOX In spite of the triumph of the eradication campaign of smallpox throughout the later part of the 20th century, there are concerns that weaponized forms of smallpox still exist, and could still be utilized as a biologic weapon [147, 148]. The former Soviet Union may have developed smallpox as a biologic weapon, and allegedly produced large stockpiles [5, 11, 147]. The devastating clinical infection and the contagious nature of the pathogen make the threat of smallpox particularly ominous. Smallpox was usually spread through airborne transmission, and its use as a biologic weapon would most likely be as an aerosol release. After the aerosolized virus is inhaled, it travels to the regional lymph nodes and rapidly proliferates. Soon after replication in the lymph nodes, viremia and widespread dissemination occurs [149]. In most cases, the duration of viremia is not prolonged, except in the subset of patients who progress to fulminant infection. In particular, a small percentage of patients develop hemorrhages at the site of their smallpox lesions. This hemorrhagic-type smallpox infection is characterized by persistently high levels of viremia, depletion of platelets leading to thrombocytopenia and depletion of megakaryocytes from the bone marrow [150], and a very high mortality rate. The associated hemorrhages are not confined to the skin, but observed diffusely, including gastric and respiratory mucosal surfaces, kidneys, and myocardium as examples [150]. The characteristics that predispose some patients to develop this form of smallpox are not known. Pathology findings in autopsies of patients infected with hemorrhagic smallpox are not consistent, particularly the pathologic effects on the reticuloendothelial system. However, the skin lesions tend to follow a typical presentation [150]. It is also unclear if these manifestations have a similar mechanism to viral hemorrhagic fevers (VHF), and therefore be amenable to therapeutic interventions that may ameliorate the VHF symptoms.
It is unclear if most of the pathogenesis of shock in disseminated smallpox infection is a result of direct virologic invasion and destruction of tissues, or from the immune response to the infection. There are nonhuman primate models for variola and monkeypox virus infections [151-153]. These models are intravenous challenges with high titer inocula or by aerosol exposure. The infectious cycle commences at the primary viremia phase. The affected animals proceed to either develop a VHF-like syndrome or a lethal or non-lethal lesional model. The model outcomes appear to be dose dependent. The pathology is consistent with direct virologic effects in target tissues, but also substantial levels of cytokine expression (cytokine storm) [152]. Low levels of TNF-alpha were measured in this model, along with depressed amounts of transcription of genes associated with TNF-alpha release [154]. These findings suggest that smallpox is able to suppress the TNF-alpha immune response to infection, allowing the virus to replicate during infection. Numerous mouse models for orthopox virus infection have been developed. Mice are susceptible to either vaccinia virus or cowpox virus infections [155]. These viruses cause widespread rapid disease but lack the classical smallpox lesions. A murine model of ectromelia virus, a type of poxvirus, was considered a close approximation of variola infection in humans, and studied extensively (summarized by Buller and Palumbo [156]). In this model, the virus had a predilection for the reticuloendothelial system, with virus and antigen detected on histopathologic examination. This model suggests that direct viral invasion, as opposed to inflammatory response, is responsible for pathological effects of infection. However, other experts conclude that the immune response, rather than direct viral invasion, is the primary contributor to the fatality associated with infection in humans [149]. The molecular pathogenesis of poxviruses relies on the virus ability to utilize host cells to replicate and to evade the host immune system. It appears multiple polypeptides are involved in the initial fusion process, as monoclonal antibodies directed at these polypeptides can block fusion [156]. After entry, uncoating of the virus outer membrane occurs, and this process can be inhibited in cells pre-treated with interferon [156]. Gene transcription and secretion of virulence proteins occur rapidly after entry into the cell, with the poxvirus growth factors and complement-binding proteins being released. Genetic recombination also occurs [156]. The virus begins to replicate, forming concatameric genomes, that separate into individual virions known as intracellular mature virions (IMV). Some of these virions receive an additional coating, being labeled intracellular enveloped virions (IEV) [157]. The IEVs fuse with the host cell membrane, forming cell-associated enveloped virus (CEV), which can either be released directly into the extracellular space, or be transported via actin filaments to nearby cells [157]. Once the CEVs completely detach from the cell or the actin filaments, they are described as extracellular enveloped viruses (EEV). Poxviruses have multiple mechanisms to evade and suppress the host immune system, many of which are currently being studied [158]. There are multiple virus-encoded proteins that substantially impair the host immune response by the use of decoy molecules and interferon antagonists. Much of the data regarding these important molecules still reside in cell culture models. However, studies with the recently published non-human primate models will provide a platform for a systems approach with substantially more relevance to human disease [152, 154]. Observations in cell culture systems suggest that several of these virus-encoded molecules are essential [158], with genetic deletion producing attenuation [159-162]. Most of these molecules function at corrupting host proteins. Therefore, these interactions would be an appealing target for chemotherapeutic interventions [163]. Two well-described proteins for immune evasion for variola are smallpox inhibitor of complement enzymes (SPICE) and chemokine-binding protein type II, which inhibit the function of the
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Table 3.
Potential anti-poxvirus therapy

Compound Mechanism Nucleoside analog, inhibit viral DNA polymerase Nucleoside analog, pro-drug to cidofovir Nucleoside analog, similar to Cidofovir Inhibits IMP dehydrogenase Nucleoside analog Approved use in humans for hepatitis C therapy Comment Approved use in humans for CMV retinitis, can cause severe nephrotoxicity Slightly less potent than cidofovir
Cidofovir (HPMPC) [172-175]
Cyclic cidofovir (cHPMPC) [171, 173] HPMPA [173] Ribavirin [173, 176] 2-amino-7-(1,3 dihydroxy-2propoxymethyl) purine (S2242) [172] 5-methyluracil (FMAU) [172] Tiazofurin [173]
2'-fluoro-Ara nucleosides, nucleoside analog Inhibits IMP dehydrogenase, similar to ribavirin Inhibit SAH hydrolase
Somewhat effective in lethal murine vaccinia model
SAH hydrolase inhibitors: carbocyclic 3deazaadenosine, 3-deazaneplanocin A [173] OMP decsarboxylase and CTP synthase inhibitors [242]
Depletes UTP and CTP pools, inhibiting RNA synthesis
host complement system and CC-chemokine receptors, respectively [164]. SPICE is related to vaccinia virus complement control protein (VCP), which has been suggested as a therapeutic compound for its anti-inflammatory properties [165]. In addition to inhibiting both the classic and alternative complement pathways, VCP also binds to the endothelium, where it may affect antibody binding to infected endothelial cells [166] as well as inhibit interactions between endothelial cells and neutrophils and NK cells [167]. Poxviruses also release multiple intracellular serine protease inhibitors, which can prevent apoptosis [165]. The inhibitory actions of various poxviruses on cytokine response has been reviewed [165]. An example is the T7 protein of the myxoma virus, which has potent anti-IFN activity and inhibitory effects on other chemokines [168]. Control of poxvirus infection requires a broad immunological response, incorporating an innate response, particularly with interferons, NK cells, an effective antibody response and finally cell-mediated mechanisms to successfully resolve the infection [165]. Currently, the only approved therapeutic for poxvirus infection is vaccinia-immune globulin, which targets both the IMV and the EEV forms of the virus [169, 170]. Although substantial experimentation in numerous poxvirus-animal models has been conducted (see [171] for review), none of these models perfectly simulates human smallpox infection, and therefore the actual efficacy of these compounds will not be known unless the smallpox is used as a weapon. Some anti-poxvirus compounds are described in Table 3. Cidofovir has been extensively explored, and appears to be one of the most potent poxvirus inhibitors studied to date [171-175]. Cidofovir is a nucleoside analog, and can effectively inhibit the viral DNA polymerase, as its diphosphate form has a substantially higher affinity for viral DNA polymerase than human DNA polymerase [173]. A cyclic form of cidofovir (cHPMPC) is active against poxviruses but it no longer is being developed by the manufacturer [173]. Another type of therapy with anti-orthopox activity are adenosine analogs that inhibit S-adenosylhomocysteine (SAH) hydrolase. SAH hydrolase is a host cell enzyme, and its inhibition leads to intracellular accumulation of SAH, which is toxic to the virus. Two of these compounds (Table 4) inhibit viral replication in an cell-culture model [173]. Ribavirin and a related compound, tiazofurin, are active against orthopox viruses, including variola [173, 176]. The mechanism of
action of ribavirin that is most likely responsible for its anti-viral properties is inhibition of inosine-5-phosphate (IMP) dehydrogenase, which converts IMP into xanthosine-5-phosphate [177]. These activities prevent guanosine monophosphate (GMP) synthesis, depleting guanosine pools, which eventually prevents viral RNA transcription [173]. Because ribavirin is approved for treating hepatitis C and inhaled ribavirin is licensed as a therapy for respiratory syncytial virus, it presents another possible option to treat smallpox, possibly in combination with other antiviral therapy. Most antiviral compounds directed at poxviruses have focused on inhibiting mechanisms unique to the virus and different than host cells. A more recent approach is to target host cell mechanisms that the virus utilizes to proliferate. Smallpox growth factor (SPGF) interacts with the host cell ErbB-1 [178], an enzyme in the tyrosine kinase superfamily, which causes phosphorylation of key host pathways that promote viral replication. Inhibitors of ErbB-1 kinases (CI-1033) blocked the effect of SPGF, causing a reduction in the release of EEV from infected cells [179], The compound had a survival benefit when administered during a lethal vaccinia murine model [179]. These inhibitors have been developed for cancer therapy [180-182], and demonstrated the utility of inhibition of host-cell signal transduction as potential antiviral therapy [183]. Additionally, antibodies directly at SPGF confer protection in the same murine model [178]. The release of CEVs from the cell requires the participation of the tyrosine kinases of the Abl family [184]. Since Abl family inhibitors are now available for the treatment of chronic myelogenous leukemia, researchers explored the effect of the administration of these kinases on viral replication. The Abl family kinases inhibitor STI-571, a 2-phenyl pyrimidine, effectively blocked the release of EEV from infected cells and decreased viral replication in a murine model [184]. An alternative to developing either peptides or small molecules to block viral or host cell functions would be to modify the disease, particularly once fulminant symptoms such as hypotension or hemorrhage has ensued. It is unknown whether a non-specific intervention, such as activated protein C or tissue factor inhibitors (discussed below under viral hemorrhagic fevers), may alter the morbidity and mortality of the infection. However, substantially more data is needed in the pathology of orthopox viruses in nonhuman primates before these types of interventions can be proposed.
Hepburn et al.
Table 4.
Viruses Causing Viral Hemorrhagic Fevers with Relevance as Biologic Weapons [185]
Arenaviridae (arenavirus) Lassa South American hemorrhagic fever viruses Guanarito (Venezuelan) Junin (Argentine) Machupo (Bolivia) Sabia (Brazil)
Bunyaviridae Crimean-Congo hemorrhagic fevera Hemorrhagic fever with renal syndrome (caused by various viruses)a Rift Valley fever
Flaviviridaeb Kyasanur Forest virus Omsk hemorrhagic fever virus Yellow fever virus
Filoviridae Ebola Marburg
a These viruses can be considered biological threat organisms, but are mentioned as Category C in the CDC classification of organisms, due to their inability to rapidly replicate in culture systems [6, 185, 217]. b Dengue virus is a Flavivirus which causes hemorrhagic fever, but is not listed here as it is not considered a high priority biological threat organism.
VIRAL HEMORRHAGIC FEVER VIRUSES The viral hemorrhagic fever (VHF) viruses represent a diverse collection of organisms that share the common characteristic of severe hemorrhage as a consequence of infection. The former Soviet Union had allegedly weaponized various VHF viruses during the Cold War [185]. The intentional use of one of these viruses would be associated high lethality and panic in the surrounding population [186]. The hemorrhagic fever viruses that are most concerning as biologic weapons are listed in Table 4. We will not discuss dengue virus since it is not included on lists of potential biologic weapons. The hemorrhagic fever viruses have a diversity of potential routes of inoculation, exposure risks in a natural environment, person-to-person transmissibility and associated mortality rates. They share the common characteristic of an initial non-specific clinical presentation (fever, headache, malaise, mylagias, sore throat), causing early diagnosis to be exceedingly challenging [185]. Unique features of the New World arenavirus hemorrhagic fevers (Argentine, Bolivian, Brazilian and Venezuelan) include a predilection to develop central nervous system effects, such as coma and seizures. Yellow fever [187] and Rift Valley fever [188] are most likely to cause hepatotoxicity and jaundice among the VHFs, although Ebola virus and Congo-Crimean hemorrhagic fever [189] have also been associated with substantial liver damage. Many patients will progress to shock, multiorgan system failure and death, usually within 1-2 weeks after the initial symptoms occur [185]. Mortality rates vary between the organisms. The hemorrhagic manifestations of the VHF viruses include mucosal and conjunctival hemorrhage, petechiae, hematuria, hematemesis, and melena [185]. Thrombocytopenia is another common feature for all VHF infections, while platelet dysfunction has been described for some of these viruses whereas it has not been characterized for others (for review, see Chen, JP and Cosgriff, TM [190]). The cause of hemorrhage in these infections is multifactorial and varies between organisms. Generally, disseminated intravascular coagulation (DIC) becomes an increasingly important contributor to hemorrhage as the severity of disease increases, with platelet abnormalities and vasculopathy becoming less important.
DIC may be induced by the infection, or may be a consequence of shock by another mechanism. DIC is an important pathologic manifestation in Rift Valley fever [191], filoviruses [192-194] and CCHF [189, 195], but not Lassa fever [191]. Hepatic dysfunction, particularly by the aforementioned viruses with predilection for hepatotoxicity, contributes to decreased levels of coagulation factors leading to hemorrhagic manifestations [196]. Vasculopathy, particularly the capillary leak syndromes associated with Hemorrhagic Fever with Renal Syndrome (HFRS) [197], is an additional factor in the hemorrhagic signs and symptoms of these infections, as well as resulting in hypovolemic shock. Endothelial damage by these viruses is species-specific. Filoviruses were thought to cause direct damage to the vascular endothelium in non-human primates [198], but more recent evidence utilizing histologic examination and electron microscopy suggests that the hemorrhage associated with Ebola virus is not due to cytolysis caused by direct infection [199]. The pathogenesis of each of the VHF viruses are exquisitely complicated, reflective of the diverse and delicate balance of a host immune response that needs to control infection without resulting in shock and tissue damage from over-expression of pro-inflammatory components [190]. The Arenaviruses appear to have minimal direct cytopathic effect, leading to speculation that their associated pathology [191, 200] is attributable to the immune response to infection. Additionally, HFRS pathogenesis appears to be mediated by an aggressive immune response with immune complex formation, rather than virologic effect [201]. Yellow fever, Rift Valley fever, Lassa and CCHF appear to impair the immune response [190], with impairment of cell-mediated immunity noted in Lassa fever [202], and therefore are more likely to have pathology attributable to direct virologic effects. Dysregulation of cytokine release contributes to the pathology and septic shock in VHF infections, but the importance of cytokines in this process varies between organisms [190]. The underlying mechanisms of shock for many of the viruses have yet to be described. The development of DIC in Ebola infection may be linked to over-expression of the tissue factor (similar to other bacterial sepsis models), and to the disruption of the normal fibrinolytic regulation
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of coagulation. As previously mentioned, it does not appear to result from a direct viral cytolytic effect on endothelial cells [199]. Tissue factor facilitates the initiation of coagulation by binding factor VIIa and factor X [203]. Excessive production of tissue factor has been documented to occur in Ebola-infected rhesus macaque monocytes/macrophages [204]. Additionally, a rapid decline in plasma protein C levels has been documented in these infected nonhuman primates [204]. Due to the role of tissue factor in the development of DIC in Ebola virus infection, researchers have postulated that tissue factor inhibitors may have therapeutic benefit. Treatment of rhesus macaques with a factor VIIa/tissue factor inhibitor (recombinant nematode anticoagulation factor c2 or rNAPc2) led to a survival advantage in an Ebola-infection model [205]. Lower plasma concentrations of interleukin-6 and monocytes chemoattractant protein-1 were documented in the macaques that received rNAPc2 therapy. This suppression of IL-6 and possibly other inflammatory cytokines with the administration of rNPAc2 may have contributed to the beneficial outcomes observed in this study [203]. Further studies elucidating the impact of tissue factor inhibition on filovirus infections are needed. In human trials with sepsis, tifacogin, a tissuefactor inhibitor, did not improve survival [206]. Among antiviral medications, ribavirin (previously described in the smallpox section) has the most evidence of efficacy for the arenaviruses (Lassa fever [207], South American hemorrhagic fever viruses [208, 209]) and bunyaviruses (Congo-Crimean hemorrhagic fever, both in human studies [210, 211] and in vitro [212, 213], hemorrhagic fever with renal syndrome [185, 214, 215], Rift Valley Fever in animal models [216]). Ribavirin, which does not cross the blood-brain barrier, may be less useful for infections that have a propensity to infect the central nervous system especially when initiated late in the course of infection [208, 209], such as the South American VHF viruses. Ribavirin is not effective against the filoviTable 5.
ruses or flaviviruses that cause VHF [208]. The utility of ribavirin after the onset of symptoms of septic shock is unknown. Other antiviral compounds have been studied for viruses such as CCHF, and in vitro data suggest that the Mx family of proteins may have antiviral activity against RNA viruses [217], but further study is needed. Other IMP dehydrogenase inhibitors have been explored for treating arenaviruses, as well as other compounds with in vitro assessment (see Charrel and Lamballerie [218] for review). A variety of antivirals have been tested in a Bunyavirus (Punta Toro virus) murine model, and are reviewed elsewhere [188]. Interferons have been studied in murine models for the Bunyaviridae family, yielding positive results [188]. Interferon alpha2b was tested in a non-human primate Ebola virus model, with small benefit noted [219]. Various interferon combinations may be useful, particularly with VHF infections in which the immune response is impaired. However, interferon compounds may be deleterious in some of these infections, such as Argentine hemorrhagic fever, in which high interferon levels are associated with worse outcomes [220]. There are few other options for the treatment of VHFs, other than supportive care. The use of steroids for treatment is not recommended [185]. Studies on the benefits of passive immunotherapy for treating VHFs have generally yielded mixed results, with studies for Argentine hemorrhagic fever [221, 222], Lassa fever [207, 223], Ebola [219, 224, 225] and Crimean-Congo hemorrhagic fever [195, 226]. None of these studies have demonstrated sufficient benefit to justify recommendations for standard-of-care usage of passive immune serum for treating any of these infections. CONCLUSIONS The opportunities for intervention in patients infected with biological threat agents are numerous, but unfortunately diversified. These organisms display complicated strategies to evade the im-
Potential Areas of Further Investigation in Sepsis Caused Biological Threat Organisms

Role of glucocorticoid inhibition in anthrax-related sepsis [47] Enhancement of macrophage-induced killing with antibodies [33], interferons [34], and other interventions Effectiveness of various toxin inhibitors once sepsis has ensued Bacterial protease inhibitors [63] Targeting proteins expressed during early germination [35] Prevention of endothelial cell apoptosis [43] Early interventions (toxin inhibitors, quorum sensing inhibitors, exogenous interferons [34])
Bacillus anthracis
Yersinia pestis
Efficacy of generic therapies for sepsis (activated protein C) to treat endotoxemia-mediated sepsis Inhibitors of Yop proteins and Type III secretion mechanisms [243] Inhibitors of specific Yop chaperone (Syc) proteins [87, 88] Investigation of additional virulence factors
Francisella tularensis
Mechanisms of shock Role of LPS in pathogenesis Investigation of additional virulence factors
Smallpox
Assessment of viremia (direct virologic effect) versus immune response in smallpox-related sepsis and death Effectiveness of antiviral therapy at delayed time points after infection Evaluation of inhibitors of host cell signal transduction [179, 184] at delayed time points after infection
VHF viruses
Further evaluation of tissue factor inhibitors to treat sepsis for the VHF viruses [205] Elucidation of hemorrhage mechanisms for the various VHF viruses Efficacy of other generic therapies for sepsis (activated protein C) to treat sepsis caused by these pathogens
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mune system, and extensive study of the interaction between the macrophage and bacterial agents (B. anthracis, Y. pestis, and F. tularensis) has elucidated substantial information about how these pathogens are effective. It also suggests targets for specific antibodies and other inhibitors to these virulence factors (antibodies/inhibitors to lethal toxin in anthrax infection, for example), as well as highlights the potential for general immunologic enhancement strategies to promote early killing. The bacterial pathogens each interfere with cellular signaling for releasing pro-inflammatory cytokines, through their interaction with the MAPK kinase and NFpathways. It is unclear if a common therapeutic intervention could prevent the inhibition of these signal transduction pathways. Pediatric patients with defects in the NK- activation will tend to have minimal manifestations of sepsis when infected [227] .However, these patients can also suffer from a variety of acute and chronic infections. Immunologic enhancement strategies can be dangerous as well, with evidence in animal models that certain cytokines actually promote the development of the cascade to septic shock [228]. Once sepsis has developed, generic therapy aimed at the mechanisms of septic shock caused by these pathogens may be effective (tissue factor inhibitors in Ebola infection, for example). Substantially more investigation is needed to further define the development of sepsis in these biological agents, as well as to evaluate new therapeutic strategies (summarized in Table 5). In particular, testing generic therapies for sepsis, such as activated protein C [229-231], naltrexone [232], goal-directed early supportive care [233], and other interventions [234], need to be tested in models of infection with the biological threat organisms discussed in this review. The challenge in the study of these agents is that extensive biocontainment procedures are often required, complicating the investigation of sepsis in a clinical context. Therefore, animal models are needed [235], but further investigation into the applicability and similarities between humans and the animal models is necessary, and is currently lacking. Future research in biodefense presents an excellent opportunity to study the underlying mechanisms of sepsis, its clinical management and possible interventions. For example, the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is developing clinical monitoring platforms using telemetry systems, to provide a detailed description of the hemodynamic impact of septic shock from these agents. Clinical management strategies for optimizing care for primates infected with the various organisms are being pursued, with conclusions analyzed for their possible extrapolation to human infection. These models are excellent platforms to evaluate new therapies. Investigations into understanding the pathogenesis of microorganisms in biodefense research could contribute to the elucidation of the role of the immune response in sepsis. Hopefully, discoveries in biodefense research will not only lead to better countermeasures against these agents, but will also improve the medical communitys ability to treat infectious diseases and sepsis. ACKNOWLEDGEMENTS The opinions or assertions contained herein are those of the authors and are not to be construed as official policy or as reflecting the views of the Department of the Army or the Department of Defense. REFERENCES
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Sepsis and Solid Organ Transplantation

A.C. Kalil ,*, H. Dakroub and A.G. Freifeld
Section of Infectious Diseases , University of Nebraska Medical Center, Omaha, NE, USA
Abstract: Approximately seventy patients undergo solid organ transplantation (SOT) every day in the United States. Sepsis remains the first or second most common cause of death in transplant recipients, depending on the allograft type. The rapid diagnosis and treatment of sepsis is critical to ensure improved survival outcome in this special patient population. However, these patients frequently lack the classic systemic inflammatory response syndrome (SIRS), commonly seen in the immunocompetent patients. In order to minimize delays in the diagnosis of sepsis in SOT recipients, it is paramount to recognize the specific risk factors for infection associated with each allograft type. In addition, the particular surgical techniques involved in each type of transplantation may be closely related to the clinical manifestations of the infection process. This correlation can further advance the diagnosis and treatment of sepsis. In conclusion, precocious diagnosis, rapid initiation of antibiotics, surgical correction when necessary, and reduction of immunosuppression, are the mainstream approach to sepsis in the SOT patient. The recent developments in severe sepsis are discussed in the context of the transplant recipient.
INTRODUCTION Solid-organ transplantation is a constantly growing field of surgery and medicine. The U.S alone performs approximately 25,000 solid-organ transplants annually [1]. Because of the great strides made with respect to surgical techniques, immunosuppression, and infection prophylaxis, most organ recipients are having substantial improvement in both the length and rate of survival. The natural consequence of this success is the substantial increment of immunocompromised patients who are visiting outpatient clinics, being seen at emergency departments, and being admitted to hospitals and intensive care units. In fact, the hospital admission rate from the emergency departments in transplant recipients is significantly elevated by 3-fold higher than that for the general adult emergency department population in one study [2]. Two recent epidemiological studies [3, 4] have shown an disturbing rate of over half a million cases of sepsis per year just in the United States. Severe sepsis has been extensively studied in more than 20 clinical trials [5], but solid organ transplant patients have been systematically excluded from these trials secondary to the concern of increasing the heterogeneity of the sample population and its potential consequences on the study outcomes. In addition, the use of the standard definitions for sepsis and severe sepsis may be compromised by the depressed febrile response and diminished leukocytosis seen in solid-organ recipients [6]. The goal of this narrative review is to refine our understanding of the incidence, diagnosis, general management and prognosis of sepsis in solid-organ transplant recipients. Even though viruses, especially Cytomegalovirus, may cause sepsis and severe sepsis, the focus of this review is on bacterial and fungal sepsis in the adult population. The specific anti-microbial management is out of the scope of this review. Each type of allograft transplant recipient is analyzed and discussed separately. METHODS Medline, Pubmed, and Embase were searched since their inception until July 2005. The keywords used were the following: transplantation, organ-transplantation, solid-organ transplantation, sepsis, severe sepsis, septic shock, bacteria, bacteremia, infection, gram-positive, gram-negative, fungi, fungemia, yeast, mold, kidney, renal, liver, hepatic, lung, respiratory, heart, cardiac, pancreas,
kidney-pancreas, heart-lung. All articles which were pertinent to the subject of this review were individually assessed by the authors. Because the terms sepsis and severe sepsis are interchanged with severe infections in the transplant literature, the authors assessed each paper and abstracted all studies that defined sepsis and/or analyzed the presence of severe bacterial or fungal infections which most likely represented sepsis (e.g. bacteremia, fungemia, pneumonia, abdominal abscesses). LIVER RECIPIENTS Incidence and Risk Factors Thirty to 70% of liver recipient patients experience at least one episode of infection in the year following surgery [7, 8]. Several factors account for this very high incidence: length and magnitude of the operation; high potential for biliary and enteric contamination; poor overall medical condition in the immediate pre-transplant period; prolonged postoperative intensive care unit stay. Underlying diseases such as primary sclerosing cholangitis are associated with higher rate of post-op biliary complications like anastomotic strictures, which are associated with an increase of both cytomegalovirus disease and bacterial cholangitis [9]. Elevated serum bilirubin at the time of transplantation is associated with higher rates of post-op bacterial infections [10]. This may be related to the severity of illness related to the underlying disease. Three studies [7, 10, 11] have shown that the duration of surgery is a significant risk factor for infections in the early posttransplant period. In fact, the longer the operation time the higher the rate of severe infections [7]. The intraoperative blood requirement is also related to the acquisition of severe infections [10-12]. These two risk factors may reflect other variables which likely increase the predisposition to infections such as technical complications, blood loss, hematoma formation, and tissue hypoperfusion. As a consequence, the tissue concentration of prophylactic antibiotics may become sub-optimal. A Roux-en Y choledochojejunostomy for biliary drainage is associated with higher rate of infection than duct-to-duct biliary anastomosis [7, 13, 14]. This is related to the fact that this procedure has a higher rate of biliary complications than duct-to-duct anastomosis. Bacterial and fungal infections are most common in the first 2 months after liver transplantation. The pathogens often originate either from the biliary tree or from the small bowel. More than one half of early bacterial infections occur within the first 2 weeks of surgery [10]. The incidence of bacteremia ranges from 20 to 30% [7, 15] and the organisms involved are usually gram-negative bacilli (E. coli, Kleibsiella spp., Enterobacter spp., Pseudomonas
*Address correspondence to this author at the Assistant Professor of Medicine, University of Nebraska, 985400 Nebraska Medical Center, Omaha, NE 68198-5400, USA; E-mail: akalil@unmc.edu
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spp.), but gram-positives (Staphylococcus spp. and Enterococcus spp.) predominate in some centers [15]. Enterococcal infections, which may account for up to 30% of all bacterial infections after surgery, are more common in liver recipients than in other graft recipients [8]. Multi-resistant bacteria have been increasingly reported in transplant centers [16, 17]. Fecal carriage of Vancomycinresistant Enterococcus faecium was observed in 23% of liver recipients in one institution, and hospital and intensive care stay were significantly longer in these patients compared with patients who had the vancomycin-sensitive strain [18]. Almost all Staphylococcus, two thirds of gram-negative bacilli, and one half of enterococcus infections have been multi-resistant among liver transplant patients. In fact, the rate of MRSA infections has reached 23% [19] in this population, which reflects a significant increase from the early 1990s. Infections with C. difficile occur in 5-10% of patients and are probably related to the prolonged hospitalizations and frequent use of broad-spectrum antibiotics. Invasive fungal infections also affect 10 to 40% of liver recipients in the early period [7, 15, 20]. Candida spp. are the most common organisms and most of these infections occur in the first sixty days after transplantation. The frequent use of broad-spectrum antibiotics, breaches of mucosal integrity due to surgery, use of central venous catheters, and pre-transplant colonization with Candida spp. are all risks for the high rate of fungal infection in liver recipients. The presence of renal failure before surgery, infections with Cytomegalovirus, and re-transplantation have all been associated with higher rate of post-transplant fungal infections [21]. Importantly, the incidence of infections with non-albicans species, mainly C. glabrata, is increasing in most hospitals in the U.S. [22]. Aspergillus spp. is less common, but recipients undergoing retransplantation have a 30-fold higher risk for this infection [23, 24]. Aspergillosis carries a very high mortality in these recipients, ranging from 70100%. Clinical Manifestations and Diagnosis The most common site of infection for liver recipients is the abdomen. Frequent clinical manifestations of abdominal infections include: cholangitis (which may not manifest the classic Charcots triad of fever, abdominal pain, and obstructive liver function tests pattern); intra or extra-hepatic abscess; secondary peritonitis; and surgical wound infection. An underlying biliary stricture is often associated with cholangitis, which may lead to an ascending infection and development of intrahepatic abscess. Cholangitis may also rise from T-tube cholangiography manipulation and endoscopic retrograde cholangiopancreatography [25]. Because of the often absent classic signs of cholangitis, the presentation may be confused with graft rejection. The diagnosis of cholangitis can be further supported by positive blood cultures, graft histopathology and culture. The differential diagnosis of cholangitis in the early posttransplant period includes preservation injury, ischemia, early rejection, and venous outflow obstruction. In one series, the presence of biliary tree complications was significantly associated with infections by VRE, compared to the patients who did not have these complications [26]. Other etiologies for liver abscesses are portal or systemic bacteremia, as well as hepatic artery thrombosis, which may lead to liver hypoperfusion, graft loss, and recurrent hepatic abscesses if the presentation is insidious. Of note, just about half of the patients with hepatic abscess will have bacteremia. In the early post surgical period, abscesses may also be found in other areas, such as the spleen, pericolic area, and pelvis. The occurrence of peritonitis may signal the presence of a bile leak. Also, the removal of a T tube can lead to a bile peritonitis, which can have a self-limited duration, or transform into a chemical peritonitis and predispose the patient to a secondary bacterial peritonitis [27]. A CT scan (with aspiration and culture if possible) should be done when abdominal infection is suspected. If a biliary stent is present, an evaluation for the presence
of a leak or stricture may be done. Abdominal symptoms not directly related to surgical complications may also occur in these patients. Severe abdominal pain and diarrhea accompanied by hypotension are consistent with acute inflammatory colitis, which is mostly caused by CMV and C. difficile in the transplant population. CMV may also present with profuse watery diarrhea and protein loss when small bowel is involved [28]. An endoscopic examination may differentiate between the two by visualization (pseudomembranous with C. diff, versus multiple ulcerations with CMV) or by histopathology. The stool examination for C. diff toxin and the blood testing for CMV DNA may yield the diagnosis for these infections. The second most frequent site of infection is the lung, which has been reported in up to 34% of these patients [7]. Most of the etiology is nosocomial and occur predominantly in the first month after transplantation. Pseudomonas and Staphylococcus are frequently involved [29]. Legionella spp. is less frequent, but associated with institutional outbreaks due to the water supply contamination. Fungal pneumonias are less frequent, but associated with higher mortality than bacterial pneumonias. Aspergillus pneumonia in this population is almost universally lethal [30, 31]. Endemic mycoses, such as histoplasmosis, coccidioidomycosis, and tuberculosis may also be an etiology depending on the geographic area and exposure [31, 32]. Cytomegalovirus pneumonitis may have an indistinguishable presentation from bacterial or fungal pneumonias and be associated with high mortality if treatment is delayed or not initiated. Pneumocystis is rarely seen nowadays because of the standard prophylaxis with tri-sulfa, but this infection may lead to severe respiratory failure and death [31]. General Management and Prognosis Besides the initiation of appropriate intravenous antibiotics, the management of cholangitis involves the correction of the structural abnormality with percutaneous drainage or stents. Hepatic abscesses associated with hepatic artery thrombosis are very difficult to treat, both because of the poor organ perfusion and the common presence of multiple abscesses, which makes the complete drainage unlikely. In these cases, a prolonged antibiotic therapy may allow the patient to undergo retransplantation, which would be the final therapy for both the infection and thrombosis. The outcome of liver patients with MRSA is related to the site of infection. If the MRSA bacteremia is associated with pneumonia, the mortality is 86%, but if it is catheter-related, the mortality falls to 6% [19]. The presence of vancomycin-resistant E. faecium is associated with a crude mortality of 46%, compared to 25% in patients with vancomycinsusceptible E. faecium [18]. HEART RECIPIENTS Incidence and Risk Factors Bacterial infections are the main cause of early death in heart transplantation [33, 34], and ranks second only to primary allograft dysfunction. The most common site of infection in the first three months is the lung. A study investigating risks for pneumonia in heart recipients [35] found that pre-transplant hospitalization, presence of infiltrate on chest x-ray, post-op intubation longer than 1 day, high dose steroids, lack of cyclosporine, cytomegalovirus infection and rejection, among others to be associated with higher rates of pneumonia in a univariate analysis. The multivariate analysis demonstrated that just post-transplantation reintubation and lack of cyclosporine, compared to other drugs, remained significant factors. Most nosocomial pneumonias occur within the first month post-transplant, while the ones from opportunistic organisms occur from 2 to 9 months [35, 36]. Intra-balloon pumps, total artificial hearts and ventricular-assist devices are associated with an increased risk for bacterial and fungal infections because of the high rate of pre-transplant infections with these devices. These immediate pre-transplant infections may
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also elevate the risk for post-operative mediastinitis and aortic suture infection in the post-surgical period [37]. In a large series of heart recipients, S. aureus was the most frequent cause of mediastinitis and mediastinal abscess [38]. High-dose steroids, contaminated hospital water systems, and handling of potting soils have also been associated with infections caused by Legionella spp. [39]. Prolonged respiratory support, impaired renal function, allograft rejection, and high-dose steroids are associated with Nocardia infections [40]. Construction work around the transplant center, contaminated ventilation systems, use of steroids, and neutropenia are risk factors for Aspergillus in these recipients [41]. Three to 14% of these recipients develop invasive Aspergillosis, and 75% of these occur within 90 days of transplantation [42]. Corticosteroids and induction with anti-lymphocyte antibodies are risks for another type of fungal infection, i.e. P. jirovecci, which can simulate severe community-acquired pneumonia and sepsis. Infections such as histoplasmosis, coccidioidomycosis, Chagas disease, Strongyloides and tuberculosis are dependent on the geographic area in which both donor and recipient have lived until transplantation [40]. In determined geographic areas, heart transplant by itself appears to be a risk factor for tuberculosis [43]. Clinical Manifestations and Diagnosis The lungs are the most commonly affected organs in the immediate post-transplant period of heart recipients and the organisms are typically nosocomial. Pseudomonas aeruginosa, Enterobacteriaciae, and Staphylococcus spp. are the most frequently encountered organisms. Urinary infections are also not uncommon in this period due to the frequent use of urinary catheters. Sternal wound infections and mediastinitis are complications which may lead to substantial morbidity and mortality. The incidence of mediastinitis following heart transplantation is close to 3% [33, 38]. Low-grade fever and leukocytosis may present before the appearance of inflammatory signs and drainage from the sternal incision. S. aureus and S. epidermidis predominate as cause of wound infection and mediastinitis, but unusual organisms such as Mycoplasma hominis [44], Nocardia spp.[33], Legionella spp., and Aspergillus spp. may cause similar infections. The majority of bacteremias and fungemias are nosocomial in origin, caused by Pseudomonas aeruginosa, enteric gram-negatives and Staphyloccocus spp., with an overall mortality of 30% [45, 46] . Bacteremia caused by S. epidermidis may be more common in heart recipients than other transplants, accounting for 21% of all bacteremic episodes in one series [47]. S. aureus has also been reported to cause 23% of all bacteremic episodes in heart recipients [47]. Legionellosis may present with fever without significant respiratory symptoms [48]. Close to 70% of all invasive fungal infections are caused by Aspergillus sp, followed by Candida spp. [42]. The mortality rate associated with invasive aspergillosis and disseminated aspergillosis averages 65% and 90%, respectively [49]. General Management and Prognosis Surgical drainage and adequate antibiotic therapy are crucial for the treatment of post-transplant mediastinitis. The debridement is sometimes followed by a muscle-flap placement. There is no consensus with respect to the best drainage method. Also, intensive insulin therapy with a goal of maintaining serum glucose levels between 80 and 100 mg/dl have significantly decrease mortality in cardiothoracic ICU patients in a randomized clinical trial [50]. Wound closure with skin staples has been associated with a significant decrease in post-op wound infections compared with the traditional subcutaneous sutures in a recent randomized trial [51]. Recent refinement in the surgical techniques of heart transplantation has also been associated with a decrease in the post-transplant mortality. The substitution of the bicaval anastomoses for the earlier biatrial cuff technique [52] is an example of that.
LUNGS AND LUNG-HEART RECIPIENTS Incidence and Risk Factors The clinical status of the recipient at the transplantation time has an important correlation with infections complications in this type of transplant. The previous colonization and multiple infections with various species of bacteria and the consequent use of numerous antibiotic courses are not uncommon due to chronic lung diseases. Also, the long-term use of steroids for underlying diseases increases the degree of immunosuppression even before the initiation of anti-rejection drugs in the post-transplant period. These factors are associated with an increase in bacterial and fungal infections in the post-tx period [53, 54]. The presence of morbid obesity, advanced age, renal failure, and malnutrition are associated with higher rates of post-op infections [55-59]. The role of mechanical ventilation at the time of surgery as a risk factor for early bacterial infections remains unclear [53, 60]. The presence of diverticular disease before and after surgery requires attention due to the higher predisposition to bacterial infections. Frequent surveillance colonoscopies and even partial colectomies have been suggested in potential lung recipients 50 years or older [59, 61]. Of note, the native lungs of single-lung transplantation may harbor many different opportunistic infections, such as aspergillosis, pneumocystosis, tuberculosis and endemic mycoses [62, 63]. Similar to other transplants, the length of surgery is an important risk factor for bacterial and fungal infections in the early posttransplant period [53]. Specific issues related to this type of allograft are associated with the predisposition to infections: denervation of allograft, which leads to an impaired mucociliary clearance and diminished cough reflex; absence of lymphatic drainage, which prevents the immune system from reaching the allograft; anastomosis site, which may be related to dehiscence and infection. Lung recipients who are negative for CMV serology who receive an organ from a CMV positive donor are at the highest risk for CMV pneumonia. This CMV serology mismatch may predispose the patient to other bacterial and fungal infections, as well as to allograft rejection. Among the most significant risks for severe posttransplant infections is the presence of bronchiolitis obliterans, which is commonly associated with substantial immunosuppression and impaired mucus clearance. The most common sites of infection in the early post-operative period are the lungs, pleura and thoracic cavity. Notably, the thoracic cavity does not have the mediastinal space as before the surgery because the integrity of the visceral pleura is not restored after transplantation. Thus, with the occurrence of an infection in this space, the entire thoracic cavity is susceptible to the dissemination of this infection and consequently the mortality rate may become very high. Infections by Nocardia and M. tuberculosis are reported, but remain uncommon in this population. Aspergillus is among the most common causes of fungal infection in lung recipients. Colonization [64], single-lung transplantation [57, 62], CMV infection [64, 65], and airway ischemia are all risk factors for this fungus. Aspergillus can cause colonization, tracheobronchitis, sinusitis, and pneumonia. In one series [66], Aspergillus colonization of the bronchial anastomoses was seen in 25% of patients, and airway complications were significantly more frequently seen in these patients (46.7%) compared with the non-colonized ones (8.7%). Approximately 60% of the Aspergillus infections are tracheobronchitis or bronchial anastomotic infections, which occur in the first three months. The remaining 40% presents as invasive pulmonary and/or disseminated aspergillosis, which occur mostly after 5 months [67]. The rate of invasive aspergillosis varies from 4 to 14% with a case fatality rate from 40 to 90% [64, 65, 68, 69]. The timing and risk factors for invasive Candida infections are similar to other SOTs. However, the manifestations may be more severe if mediastinal, aortic or bronchial anastomosis become affected.
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Clinical Manifestations and Diagnosis Lung recipients have the highest rate of post-transplant pneumonias amongst all transplants, and the allograft is more commonly affected than the native lung. Because of frequently seen colonization with gram-negatives before transplant, most patients receive prophylaxis tailored to the patients needs during and in the immediate postoperative period. Pseudomonas spp. remains a common etiology for pneumonias and treatment will depend on patients previous infections pattern and institutional antibiogram. Burkholderia cepacia appears to be associated with worse outcome [70]. The presence of bronchiolits obliterans is associated with more immunosuppression, higher risk of infections, and lower long-term survival [58]. While bacterial pneumonias occur in all posttransplant periods, fungal pneumonias tend to be more frequent in the first one to two months. Aspergillus is the most common organism and has been demonstrated in 10 to 40% of recipients [71]. Colonization with this organism is frequent, but its recovery from healing bronchial anastomoses is associated with invasive infection in about 75% of the cases. Importantly, fever and typical radiologic signs, such as the nodular infiltrates and the halo sign are rare in these patients. Instead, the radiographic signs are rather unspecific [67]. In addition, cytomegalovirus not only causes severe pneumonia, but also predisposes the patient to other bacterial and fungal infections [72]. Mediastinitis occurs in lung recipients at similar or even higher rates than in heart recipients and usually develops within the first four weeks of surgery [58]. Bloodstream infections develop in up to 25% of these recipients. S. aureus and P. aeruginosa are frequent pathogens [73]. EBV-associated lymphoproliferative disease involving the allografted lung may simulate bacterial and fungal infections. This disease is more common in lung than in other graft recipients and is associated with poor prognosis. Intraabdominal infections also affect lung recipients. Bacterial cholecystitis, viral hepatitis, abdominal abscesses, and invasive colitis have all been described [61] in this population. General Management and Prognosis The culture and histopathologic studies of the resected lung is important in order to provide guidance for the treatment of post-op infections. The gram stain and cultures from the donor airways (often colonized due to intubation and ICU stay) may also guide anti-infective therapy after surgery. In addition, the transplant institution hospital and ICU specific bacterial antibiogram is crucial to the approach of these early postoperative pneumonias. In particular, patients with cystic fibrosis have well documented long-term history of colonization and/or infection with pathogens and their specific sensitivities. The presence of extensive pseudomembranes involving the anastomotic sites is suggestive of invasive fungal infection. Aspergillus spp. and Candida spp. can cause invasive anastomotic infection. The combination of debridement, systemic and inhaled anti-fungal have been successfully used against these infections [61, 74]. In the case of single-lung transplant recipient with the suspicion of acute pneumonia, in whom ischemiareperfusion injury has not been ruled out, the ventilator support has to be aimed to decrease injury to the allograft. Minimizing tidal volumes, lowering PEEP, and keeping the transplanted lung side up, all minimize the overdistension of the native lung and its serious consequences, such as worsening of V/Q mismatch and hemodynamic instability [75]. Bacterial infections are the primary cause of mortality in the early post-transplant period [76]. Invasive Aspergillosis remains the infection with the highest case-fatality, ranging from 40-90%. The presence of Aspergilloma in the explanted native lungs has been associated with reduced post-transplant survival [74]. The casefatality of aspergillosis will also depend on the site of infection. While the mortality rate of Aspergillus tracheobronchitis ranges from 20-30%, the one associated with invasive pulmonary disease ranges from 60-90% [67, 68].
PANCREAS AND PANCREAS-KIDNEY RECIPIENTS Incidence and Risk Factors In spite of improvements in the surgical technique, pancreas transplantation still has the highest incidence of infectious complications amongst most solid organ transplantation because of both the procedure itself and the substantial immune suppression necessary to avoid rejection [77]. Also, most patients have diabetes and many of its complications (i.e. peripheral vascular disease, chronic renal insufficiency), which further increase the risk of complications. Older donor age and donor obesity increase the risk of pancreatitis and graft thrombosis, which increase the rate of abdominal infections [78, 79]. Prolonged cold ischemia increase the risk for infections due to its association with thrombosis, pancreatitis and duodenal leaks [79]. Similar to other transplantations, the prolonged operative time is also a risk factor. There are several risk factors directly associated with specifics of the surgical procedure itself: duodenal devascularization during preparation of the graft leading to post-op duodenal leaks, intra-abdominal and wound infections; bladder-drained allograft and/or neuropathic bladder leading to recurrent urinary tract infections; simultaneous pancreas-kidney transplantation (which requires longer operative times due to the implantation of two allografts) leading to a higher rate of infections than the one seen with two sequential surgeries at different times, or pancreas alone [79]. Overall, enteric drainage has been associated with lower rates of infections compared to bladder drainage [80]. Clinical Manifestations and Diagnosis Other than vascular thrombosis, intra-abdominal infections remain the most common cause of pancreatic allograft loss [81]. Although generalized peritonitis may result from the spillage of enteric contents or urine into the peritoneal cavity, the presentation of abdominal infection in this patient population is through the formation of abscesses in or close to the pancreas. Fever, malaise, and abdominal pain accompanied by nausea and vomiting are symptoms and signs found at the initial clinical presentation. These abscesses are mono or polymicrobial, and involve enteric and nonenteric gram-negative organisms, as well as Candida spp. Another complication called peripancreatic sepsis, may present with persistent ileus, abdominal pain, fever, and leukocytosis between 7 and 14 days after surgery. The treatment involves debridement of necrotic peripancreatic fat and antibiotics [82]. Different from early duodenal leaks, which are commonly related to technical causes, late leaks may occur due to ischemia and infection. Leaks from bladder-drained allograft may be diagnosed by a fluoroscopic cystogram and leaks from enteric-drained allograft may be diagnosed by CT scan, even though image findings are not associated with high specificity [83]. Recurrent urinary infections may be another clinical complication of bladder-drained allograft [84]. The presence of diabetic neuropathic bladder, Foley catheter, and urine alkalinization from bicarbonate in the pancreatic secretions all predispose to urinary tract infections. Wound infection has been reported in up to 18% of pancreas recipients, and approximately half of these patients had concomitant intra-abdominal infection [85]. General Management and Prognosis Drainage and appropriate antibiotics are the primary treatments of most intra-abdominal infections. If the abscess is not amenable to drainage by non-invasive methods as interventional radiology, a laparotomy should be performed. Occasionally, the allograft will need to be removed if the patient does not improve despite standard treatment [83]. Another potential cause of intra-abdominal sepsis is the rupture of mycotic pseudoaneurysms of the iliac artery, usually at the site of the anastomosis. It can lead to shock (rupture into the abdominal cavity), massive hematuria (rupture into the bladder), gastrointestinal bleeding (rupture into the small bowel), or loss of allograft (rupture into the pancreas) [86]. Infections due to leaks on bladder drained grafts may be treated medically, but 20% will re-
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quire surgical repair, and the ones due to leak on enteric drained grafts require surgery more often [87]. In fact, recurrent urosepsis may be an indication to convert bladder drained to enteric drained pancreas [88]. A study of 213 recipients [78] found that the presence of infection was associated with a statistically significant decrease in the 1year allograft survival rate from 82% to 60%. Mortality up to 20% has been observed with intra-abdominal infections in these recipients [89]. KIDNEY RECIPIENTS Incidence and Risk Factors In the first month of infection, the vast majority of infections are related to the technical issues and the recipient exposure to the hospital environment. The risks for infections include contamination of the perfusate and/or allograft, use of intravenous or urinary catheters, anastomotic leaks, wound infections, and older age. Urinary tract infections remain the most common site of infection in these recipients, but have decreased substantially after the introduction of prophylaxis with sulfa-trimetoxazole. Long period of hemodialysis and previous UTIs before transplant, female sex, vesicoureteral reflux, diabetes mellitus, indwelling catheterization, polycystic kidney disease with recurrent UTIs without binephrectomy, chronic viral infections, increased aluminum excretion, advanced age, and cadaveric donor are all risk factors for urinary tract infections after renal transplantation [90, 91]. In fact, once an indwelling catheter is in place in a kidney recipient, the incidence of bacteremia goes up to 5-10% per day. [92] Enterococcus spp. are among the most common causes of UTIs in renal recipients [93]. The hospital setting will vary, but includes the presence of multiresistant gram-positive and gram-negative bacteria, airborne (e.g. Aspergillus), and waterborne (e.g. Legionella) as possible etiologies for postoperative infections. Besides a multitude of viruses (mainly CMV) that predominate between two and six months, most of the bacterial and fungal infections are opportunistic and include Pneumocystis, Aspergillus, Nocardia and Listeria. After six months, community-acquired bacterial (e.g. Pneumococcus; Legionella), viral (e.g. respiratory syncytial virus; influenza; parainfluenza) and fungal (Histoplasmosis; Cocidioidomycosis; Cryptococcosis) predominate. Clinical Manifestations and Diagnosis Technical complications such as anastomotic leaks, hematomas and lymphoceles are related to early bacterial infections. These infections may manifest as urinary tract infection, pyelonephritis, bacteremia, sepsis, and wound infections. Of note, the prolonged use of ureteral stents and venous lines may lead to colonization and infections with nosocomial organisms. Because patients are severely immunosuppressed in this early period, clinical manifestations of, for example, urosepsis, may be substantially more subtle than the ones observed in non-transplant patients. At this period, patients are also more susceptible to nosocomial infections with S. aureus and Pseudomonas spp. S. aureus has been found in 10% of all organisms detected at or near the time of death in an autopsy study in renal recipients [94]. Also, in the experience of the authors, organisms usually considered urine colonizers, such as S. epidermidis and Candida spp., may cause urinary tract infection and severe sepsis. In fact, 40% of the bacteremias in these recipients arise from the urinary tract [46]. Perinephrectic abscesses and obstructive fungal balls should be ruled out by an ultra-sonographic examination. In case of recurrent urinary infections, abnormalities such as uretero-vesicle junction strictures, ureteral reflux, and neurogenic bladder should be investigated. Even though the incidence of pneumonia is the lowest compared to other solid organ transplants, pneumonia due to Pneumocystis, Nocardia spp., as well as community-acquired organisms is well reported in these patients, mostly towards the end of the first
year [95]. Legionella pneumonia is usually related to outbreaks, and aspergillosis and zygomycosis of the lung are rare. Even though invasive aspergillosis is less common in these recipients compared to other allograft recipients, the associated mortality is up to 80% [96-98]. Tuberculosis and endemic mycosis are more frequent in these recipients who live in endemic areas. In contrast to the low frequency of pneumonias, kidney recipients have a high rate of abdominal complications and infections compared to other transplants [99-101]. Diverticular disease complications such as perforation and pericolic abscess formation are more common in older recipients and patients transplanted for polycystic disease because of their long-term intake of phosphate containing antacids [102]. Gallstone disease and pancreatitis are also common in these patients due to several factors: secondary hyperparathyroidism; hypertriglyceridemia; and immunosuppressive drugs. Opportunistic infections affecting the CNS occur later and include Listeria monocytogenes, Toxoplasma goondi, and Cryptococcus spp. Fever and headache may be the only initial manifestations. General Management and Prognosis Urosepsis developing early after the transplantation will require thorough investigation for technical and anatomical complications of the kidney transplant. Based on the severity and recurrence of these infections, prolonged treatments up to four weeks may be required for the eradication of infection [103]. Sepsis associated with diverticular disease may present with mild left lower quadrant pain, diarrhea, fever, without peritoneal signs. Antibiotics and surgical resection may be required to eliminate this infection [104]. In a survey of 604 renal recipients, the occurrence of infection was associate with a significant decrease to 88% in the 3-year survival rate, compared to 92% in patients without infection [105]. More recently, a study evaluating 33,479 renal transplant recipients demonstrated that recipients hospitalized for septicemia had a mean survival of 9.0 years (95% CI 7.4-10.6) compared to 15.7 years for all other recipients (95% CI 14.8-16.7) [106]. A study just published from two transplant centers showed that UTIs after renal transplantation were significantly associated with increased mortality (OR 3.5 [95% CI 1.7-7.2]) [91]. OVERALL MANAGEMENT The diagnostic approach may be facilitated by the understanding of the specific timing by which certain microorganisms most commonly affect the SOT patients (Figs. 1 and 2). In addition, many non-infectious transplant-related complications may mimic the clinical presentation of bacterial and fungal sepsis. Tables 1-3 describe the pathologies that should be included in the differential diagnosis of SOT patients with sepsis. The reduction of immunosuppressive therapy at levels in which rejection can still be avoided is of central importance for patients with suspicion or documented sepsis. It should be emphasized that the well-know universal interventions associated with decrease in the development of nosocomial infections in the general population should also be applied to all allograft recipients. They include, but are not limited, to the following: Appropriate use of perioperative antibiotics Early extubation Early removal of venous and arterial lines Early removal of urinary catheters Aggressive pulmonary toilet and physical therapy Adequate wound care Anti-Pneumocystis/Bacterial prophylaxis Anti-Cytomegalovirus/Viral prophylaxis
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Fig. (1). Timing of Bacterial Infections Following SOT.
Fig. (2). Timing of Fungal Infections Following SOT. Anti-Fungal prophylaxis in high-risk patients Infection surveillance Severe Sepsis Mimics in Transplantation
Acute allograft rejection Post-transplantation lymphoproliferative disease Graft thrombosis Graft versus host disease Immediate post-operative period Acute pancreatitis Cytomegalovirus disease Myocardial infarction Pulmonary embolism Stroke OKT-3 induced meningitis Pulmonary edema Pulmonary embolism Hypersensitivity drug reaction Intrathoracic hemorrhage Post-transplantation lymphoproliferative disease Acute respiratory distress syndrome Allograft rejection Pulmonary calcinosis Bronchiolitis obliterans Tacrolimus-induced pneumonitis
Table 1.
found immunosuppression state present in the early port-transplantation period. On the other hand, most of the recent developments in severe sepsis have not been tested in transplant recipients. Therefore, clinical indications and risks have to be carefully evaluated before deciding about the potential application of these therapies in transplant patients. They include: Table 2. Pneumonia Mimics in Transplantation
In the opinion of the authors, these interventions are of substantial importance to prevent sepsis in these patients due to their pro-
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Early goal-directed therapy [107] Tight control of glycemia [50] Low-dose corticosteroids [108] The use of drotrecogin-alfa activated in allograft recipients has been described only in case-reports [109], but transplant patients were excluded from the pivotal phase III trial [110]. Because of the serious bleeding associated with this therapy and the absence of data in transplantation, this drug should be used very cautiously and evaluated on an individual basis (risks/ benefits) for the transplant patient. Of note, this therapy is not indicated for severe sepsis in surgical patients with a singleorgan failure or APACHE II score less than 25 (FDA label).
[13]
[14]
[15]
[16] [17]
Table 3. Intra-abdominal Infection Mimics in Transplantation

Colonic dilatation Pneumatosis intestinalis Acute pancreatitis Anastomotic leaks Acute arterial or venous occlusion Perforation Bleeding Non-bacterial colitis
[18] [19] [20]
[21]
[22] [23] [24]
CONCLUSION Sepsis and severe sepsis remain the first or second most common causes of death in transplant recipients, depending on the allograft type. This patient population is rapidly growing and becoming a large contingent of any medium-large sized hospital and ICU in this country. The knowledge of the particular risk factors and surgical techniques associated with each type of allograft recipient is crucial to aggressively diagnose and manage sepsis in these patients. The improvement in sepsis outcomes is as urgently needed in transplant patients as it is in the general population. REFERENCES
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The Cancer Related Thrombotic Tendency in Sepsis

David E. Joyce*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA
Abstract: The host inflammatory response is activated in both cancer and infection. This includes enhanced production of acute phase reactants, involvement of coagulation and inflammation and the potential for systemic effects. This overview will identify the prothrombotic links between cancer and sepsis and suggest antithrombotic agents as an approach in the specific treatment of sepsis in cancer patients.
Key Words: Cancer, thrombosis, sepsis, severe sepsis, inflammation, anticoagulant, coagulation, adenocarcinoma INTRODUCTION Infection and Sepsis Definitions Beginning with exposure to an infectious agent, the progression evolves from a focal infection to systemic activation of the host immune response. Intrinsic to the response are of blood cellular components, cytokine mediators, and eventual organ vascular dysfunction. For example, a focal lobar pneumonia may progress to systemic bacteremia initiating a larger host-immune response which involves the vasculature and its capillary microcirculation. Loss of cardiac and peripheral vascular smooth muscle tone and progressive capillary leak lead to hypotension and the multi-organ failure syndrome [1]. The systemic signs of severe infection are referred to as the systemic inflammatory response (SIRS). These SIRS criteria include alterations in temperature, pulse, blood pressure, respirations, or leukocyte count [2]. Severe sepsis is formally defined as the presence of SIRS criteria and at least one organ dysfunction. Typically, severe sepsis patients are treated in the intensive care unit where organ support is readily available. Current approaches aimed at organ failure support in the ICU include mechanical ventilation, vasopressors, dialysis, early goal-directed volume support, and intensive glucose control [3-5]. As a disease state, severe sepsis afflicts 750,000 patients in the U.S annually and causes 250,000 deaths [6]. From the 1999 U.S. severe sepsis population, 12,209 cancer patients are projected to be diagnosed with severe sepsis annually [7]. This is a rate of 16.4 cases per 1,000 persons. In addition, severe sepsis rates are expected to increase as the larger segment of the U.S. population ages [8,9]. Infection and Sepsis Cellular Physiology The mechanisms of the sepsis response are complex [10,11]. On the cellular level, initial activation of infection-specific mediators induce monocytes to express Tissue Factor (TF) and secrete cytokines (e.g. IL-8, IL-6, IL-10, TNF- , and IL-1 ) that recruit cells to the site of injury and activate other cellular processes. Early in sepsis, monocyte CD14 receptor is activated by gram negative bacterial lipopolysaccharide (endotoxin, LPS). The gram positive exotoxin, peptidoglycan, and other protein toxins also initiate this early innate immune response. Toll-like receptors, mainly Toll-2 and Toll-4, thrombin receptors, and TNF- receptors are central to activation of the innate immune response. Associated nuclear factors including NF B, SP-1, AP-1, and Egr-1 propagate the proinflammatory cellular responses of the above receptors. Activated neutrophils (activated by IL-8 or complement) roll on vascular endothelium, firmly adhere, and can incite proteolytic events on the endothelium before they diapedese the endothelial barrier and migrate to affected tissues [12]. Migration of neutrophils across endothelial cells to tissues involves cytoskeletal and matrix interactions, for example neutrophil-integrin binding to fibrinogen [13]. The endothelium can become dysfunctional with loss of barrier integrity thus inducing permeability, a mechanism contributing to hypotension [14]. Membrane lipid changes enhance cell surface thrombotic tendency and predispose to endothelial apoptosis [15,16]. During endothelial activation, induced nitric oxide synthase (iNOS) enhances nitric oxide production thus relaxing adjacent vascular smooth muscle. Progressive severe sepsis leads to disseminated intravascular coagulation (DIC) and subendothelial fibrin deposition producing further vascular dysfunction. In early, distributive, septic, shock, permeability and loss of smooth muscle vascular tone predominate. Later, IL-1 and TNF- can directly inhibit cardiac muscle function contributing to prolonged hypotension. The combination of cardiac muscle dysfunction and distributive shock severely disrupts individual organ homeostasis. Thus, progressive organ-specific vascular dysfunction in the setting of reduced blood flow causes hypoxia, high thrombotic tendency and low pH leading to organ failure and eventual death of the organism. Other components of the host response of innate and adaptive immunity act at the cellular level to combat bacteremia. Bacterial organisms undergo phagocytosis by neutrophils and monocytes leading to surface presentation of bacterial components to lymphocytes for priming the humoral immune system. Activated B-cells go on later to produce immunoglobulin (IgM/IgG) of humoral immunity and specific activated T-cells go on to support the cell mediated immune response. As an arm of humoral immunity, Complement lyses bacteria and contributes to hypotension in sepsis (components C3a, C5a, and bradykinin). Later responses in sepsis can prolong inflammation; for example, high mobility group box-1 protein, a nuclear protein, can be released from dying cells after 24 hr, thus reenacting acute inflammatory responses [17,18]. Other later host immune responses may resemble a compensatory antiinflammatory response (CARS) [19]. Vascular beds recovering from shock exhibit reperfusion characteristics including release of the surface endothelial marker thrombomodulin (sTM) [20]. Links Between Sepsis and Coagulation An important component to sepsis pathophysiology is the coagulation system. The Tissue Factor Pathway (extrinsic pathway) of coagulation more recently has been designated as the primary initiator of thrombosis and traditionally has been associated with vascular injury. Inflammatory mediators induce expression of TF on monocytes and potentially on inflamed endothelium [14]. Tissue
*Address correspondence to this author at the Walther Cancer Research Center, 230 Raclin-Carmichael Hall, University of Notre Dame, Notre Dame, IN 46556, USA; Tel.: 574-631-2958; Fax: 574-631-4048; E-mail: djoyce1@nd.edu 1389-4501/07 $50.00+.00
David E. Joyce
Fig. (1). Prothrombotic Pathways of Coagulation. Initiation of TF from the vessel wall leads to generation of thrombin. Thrombin induces the intrinsic pathway and aids in antithrombotic pathway feedback through antithrombin III and thrombomodulin-Protein C pathways. Fibrinolysis is the endogenous process of clot lysis, mediated by plasmin and inhibited by plasmin activation inhibitor-1 (PAI-1). Procoagulant proteins unique to cancer and acute phase inflammatory processes unique to both cancer and infections inflammation may contribute to a heightened thrombotic tendency. Peripheral mechanisms interacting with coagulation include some chemotherapy agents, DIC, and acute phase reactants.
factor binds factor VII (FVII) which when activated cleaves mostly factor X (FX) and some factor IX (FIX). FXa then cleaves FV and generates Thrombin (FIIa), Figure 1. This is the initial path to thrombin generation. Further thrombin-mediated activation of FXI in the intrinsic pathway rapidly generates abundant thrombin [21]. Thrombin is a multifunctional protein that cleaves fibrinogen to fibrin to form clot. Thrombin induces anti-coagulation feedback through antithrombin III, the thrombomodulin-Protein C pathway and directly by inhibition of upstream activation of clotting factors. Receptors for Thrombin (proteinase activated receptors, PAR-1,3,4) reside on platelets, endothelium, and vascular smooth muscle [22]. On endothelium, thrombin activated PAR-1 is proinflammatory and up regulates surface adhesion protein ICAM-1 expression. Contraction of endothelial cells to reduce endothelial barrier protection and production of inflammatory cytokines IL-6 and IL-8 are both PAR1 dependent phenomenon [23]. Thrombin is a major activator of platelets and is mitogenic to vascular smooth muscle. Reducing inflammation occurs by reducing plasma thrombin levels. This may be through direct binding and removal of thrombin, or through thrombin initiated endogenous anticoagulant pathways. Secondary molecules in these pathways may act through unique antiinflammatory mechanisms directed at the vascular-blood interface. The intrinsic pathway of coagulation is also linked to inflammation by the prekallikrein connection to complement and bradykinin, a potent vasodilator. Historically, the late severe sepsis phenomenon of disseminated intravascular coagulation (DIC) manifests as mucosal bleeding, bruising, or as a severe bleeding diatheses. DIC is associated with advanced (metastatic) cancers and specific subtypes of leukemia (e.g. promyelocytic leukemia). Only until recently has microvascular thrombosis in sepsis been viewed as a causal link to inflammation. Thus, the recognition of antithrombotic therapy has sought newer indications, including the treatment of DIC in severe sepsis [24,25]. Subsequently, recombinant antithrombotic proteins recently tested in sepsis, demonstrated anti-thrombotic effects and potential anti-inflammatory effects.
Fibrinolysis is the process of breaking down formed clot. This tightly regulated protelytic cascade begins with cleavage of clotbound fibrin by plasmin. Activators of the formation of plasmin from plasminogen are central to enhancing this reaction. Tissue plasminoegen activator (tPA) and Urokinase plasminogen activator (uPA) accelerate this formation of plasmin. Plasminogen activators are upregulated by some cancers [26]. The next level of regulation is the inhibition of plasminogen activator formation by the enzyme plasminogen activator inhibitor-1 (PAI-1). In inflammation PAI-1 is an acute phase reactant. In both sepsis and cancer, PAI-1 levels are elevated and restrict fibrinolysis. Because of higher plasma concentrations, PAI-1 influence predominates over the profibrinolytic effect of the plasminogen activators in cancer [26]. PAI-1 is produced in some cancer cells, but endothelial release of PAI-1 under the influence of cytokines IL-1 and TNF- is thought to be its primary source [27]. Acute treatment of sepsis through inhibiting PAI-1 might ameliorate or prevent complications of DIC. DIC in cancer, although seen late, may benefit from PAI-1 reduction. Endogenous Anticoagulants and Severe Sepsis Three endogenous anticoagulant molecules targeted for severe sepsis exhibit separate mechanisms of thrombosis inhibition or antiinflammation. These three have all completed Phase III clinical trials in severe sepsis. A fourth anticoagulant-related agent, platelet activating factor (PAF), has not been shown to be successful [28]. The first agent, Tissue Factor Pathway Inhibitor (TFPI), is secreted from endothelial stores and inhibits the TF:FVIIa:FX/(IX) complex to reduce thrombin generation in sepsis patients [29] Recombinant human TFPI has been tested in severe sepsis in a Phase III trial and does not improve survival in severe sepsis patients [30]. Though it is released from the vessel wall by heparin, it appears to have little anti-inflammatory properties except indirectly inhibiting FVII signaling of PARs. rTFPI however is finishing a separate Phase 3 trial in severe sepsis limited to community-acquired pneumonia patients [31]. The second agent, Antithrombin III binds FIXa, FXa, FXIa, and FXIIa, thrombin and FVIIa-TF inhibits the coagulation cascade
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Fig. (2). Inherent Prothrombotic Tendencies Shared in Sepsis and Cancer. Endotoxin and other mediators (TNF- and IL-1 ) can initiate TF expression on monocytes or tumor to promote a prothrombotc state. Tissue Factor, cancer procoagulant (CP) and activated platelets may be extrinsically deposited on the endothelium to propagate fibrin deposition and thrombosis. Prothrombotic proteins (mucin/sialic acid, cysteine proteases, and CP) from tumor act in the common pathway to enhance thrombin generation by activating Factor X. Factor IX and FVIII are elevated as acute phase reactants.
from the intrinsic and common pathways. The addition of heparin accelerates this inhibition of thrombin generation up to 1000-fold [32]. Antithrombin III exhibits anti-inflammatory effects by reducing leukocyte adhesion and thrombin reduction. ATIII reduces proinflammatory effects of PAR-1 signaling in endothelial cells. However, Antithrombin III was overall unsuccessful in a Phase 3 trial of severe sepsis survival. In this trial, The third agent, Antithrombin III without prophylactic heparin appeared more effective, but the overall primary endpoint of survival was not reached. Activated protein C, is produced in plasma when thrombin cleaves zymogen protein C on the endothelial surface protein thrombomodulin [33]. Endothelial surface thrombomodulin can be shed into plasma during reperfusion and it is down regulated by TNF- [34]. Last, Activated protein C is generated on demand in states of excess thrombin generation. Mechanistically, activated protein C cleaves upstream FVa and FVIIIa to reduce thrombin generation and inhibits PAI-1 in a 1:1 binding complex. Activated protein C exerts anti-inflammatory effects on endothelial inflammation through binding endothelial protein C receptor (EPCR) and proteolytically activating PAR-1 [35]. Mechanisms including reduced leukocyte adhesion and migration, suppressed NFkB binding, inhibition of apoptosis, and suppression of endothelial barrier permeability have been ascribed to aPC [35-38]. Monocytes, neutrophils, and eosinophils express aPC receptor (EPCR) allowing aPC to direct anti-inflammatory responses. A recombinant human form of activated protein C has achieved regulatory approval for the treatment of severe sepsis with high risk of death [39], but for less severely affected patients it is not effective [40]. Its direct anti-thrombotic affects (feedback inactivation of FVIIIa and FVa) anti-fibrinolytic effects (inactivation of PAI-1) and antiinflammatory effects (endothelial PAR-1 anti-inflammatory effects) suggest the potential for this drug to address the combined inflammation and thrombotic tendencies of septic cancer patients. The role of these endogenous anticoagulants have not shown any clinical anti-cancer effects to date. The Thrombotic Tendency in Cancer The diagnosis of cancer associated hypercoagulable state first appeared in 1865 when Trousseau described a syndrome of superfi-
cial thrombophlebitis in cancer patients [41]. Indeed, venous thrombosis is not an uncommon presentation in the initial diagnosis of cancer, approximately 2-6% [42]. Hypercoaguability in cancer, when manifested as a venous thrombotic event, often requires longterm or life-long anticoagulation with heparin [43,44]. In the postsurgical patient with cancer, recent evidence suggests the need for prolonged post-operative low molecular heparin prophylaxis [45] Other studies, although unsuccessful, have tested the hypothesis that anticoagulants may improve cancer survival [46]. Hypercoaguable mechanisms of cancer include: 1.) thrombocytosis from cancer related inflammation, 2.) endothelial apoptosis, 3.) TF and cancer procoagulant (CP) expression on tumor, and 4.) mucin producing adenocarcinomas. Other causes of thrombosis in cancer include microvascular processes such as disseminated intravascular coagulation (DIC), vascular endothelial growth factor (VEGF) effects, and arterial events such as nonbacterial thrombotic endocarditis (NTBE) and septic/tumor emboli which may lead to cerebral infarction [47-50]. Hematologic malignancies and their mechanisms of thrombosis deserve mention (e.g. hyperviscosity in IgM myeloma, essential thrombocytosis, qualitative platelet dysfunction of myelodysplastic syndromes, or enhanced fibrinolysis in acute myelogenous leukemia) [47]. However, the molecular predisposition linking cancer and thrombosis in most instances is not well understood. Cytokines TNF- and Interleukin-1B can induce TF expression on tumor, monocytes and through microparticle transfer and deposition of TF on endothelium. Tissue Factor can be elevated two-fold and initiates clotting by binding Factor VIIa leading to sequential activation of factor FX, FV, and thrombin. Acute phase reactants initiated by secreted IL-6 will stimulate hepatocytes to increase levels of PAI-1, fibrinogen, C-reactive protein, and indirectly the platelet count thus contributing to the prothrombotic potential in both cancer and sepsis, Figure 2. Other cancer specific mechanisms such as cysteine protease cleavage of Factor X or mucin secreting tumors that express mucin sialic acid may initiate thrombosis abnormally by cleaving and activating factor X. Another important activator of FX in cancer is Cancer Procoagulant (CP). This 68-kDa cysteine protease activity found in breast, colon, melanoma and other malignancies was initially detected in the 1950s [51]. CP cleaves FX independent of FVIIa to initiate focal
David E. Joyce
Table 1.
Examples of Malignancies with Acquired Coagulation Derangements. Tissue Factor activates factor VIIa initiating the extrinsic pathway. Cysteine proteases, including CP and mucin/siliac acid both activate at the level of factor Xa. Inflammation may also increase plasminogen activator inhibitor-1 (PAI-1). This list does not include all hematologic malignancies. All of the above excluding leukemia and melanoma may present histologically as adenocarcinoma.
Tissue Factor (VIIa) Brain(glioblastoma) Stomach/pancreas Ovary Kidney/prostate Breast Leukemias Cysteine Protease (CP-FX) Lung Breast Colon Melanoma Kidney Leukemia Mucin/Sialic Acid (FX) Lung Pancreas Gastrointestinal Ovary Prostate Renal Cell
tumor fibrin deposition. Plasminogen activator inhibitor (PAI-1) inhibits fibrinolysis. The balance of factors producing thrombin generation, clot formation and fibrinolysis can become dysregulated in advanced cancer (DIC), producing both bleeding and thrombosis [50]. This is usually chronic when found in the cancer patient, and treating the underlying cause of DIC should help improve this condition. In severe sepsis, DIC is a late event. Patients with malignancy may have multiple underlying prothrombotic risk factors (e.g. atherosclerosis, genetic risk, age, or postoperative risk) similar to the noncancer population. The other extreme of cancer as it relates to coagulation at the molecular level is the inherent thrombotic tendency associated with tumor histology. Table one shows the common tumors associated with enhanced thrombotic potential. In general the mucin secreting adenocarcinomas have such thrombotic potential. Table 1 is a summary of specific tumor types and their over expression of coagulation-related protein [26,47]. Table 2 lists coagulation factors and inflammatory processes related to cancer and sepsis. Currently, no model or biomarker is available to predict the likelihood of an individual thrombotic event in the cancer patient. However, the question still deserving exploration is the specific role of thrombotic mechanisms in infection, bacteremia, and severe sepsis outcomes. Table 2. Prothombotic components of coagulation and inflammation also associated with cancer. Coagulation components elevated during inflammation are listed on the left. Inflammatory processes related to thrombosis are listed on the right. IL-6 stimulates liver to produce acute phase reactant proteins, including C-Reactive Protein (CRP)
Inflammation Related To Coagulation Cytokines (TNF, IL-1, IL-6) Enhanced adhesion Apoptosis/Thrombosis Thrombin receptors WBC proteases Acute phase Proteins Hepsin C-reactive protein (CRP)
CLINICAL CONSIDERATIONS Predisposition to Infection in Cancer Depending on tumor type, tumor stage, bulk of disease, or tumor location, the likelihood of an infection occurring or spreading rapidly is of importance. For example post-obstructive pneumonia secondary to lung cancer in the airway or colon cancer obstructing the bowel may lead to acute bacteremia. As expected, abdominal therapeutic resection for cancer carries the postoperative risk of severe sepsis. Immune system malignancies, leukemias or lymphomas, predispose to systemic infection because the malignancy directly alters leukocyte function. Venous vascular access devices can provide a direct nidus for bacteremia and for thrombus. Chemotherapy-induced neutropenia is also a common presentation of severe sepsis. Patients with fever and absolute neutrophil counts below 500/mm3 typically require hospitalization and intravenous antibiotics. Indeed, cancer and its therapeutic implications (surgery, radiation, chemotherapy, and intravenous access devices) indirectly provide the opportunity for bacteremia and development of severe sepsis. Despite targeted chemotherapies designed to reduce infectious risk, prophylactic antibiotics, [52] and granulocytic colony stimulating factor support, bacteremia remains a concern for the cancer patient. Cancer Patients in the ICU In cancer, coagulation favors a prothrombotic tendency and in the ICU cancer patients have a worse mortality, 50-80%, compared to all ICU severe sepsis patients, 40-60% [53]. Little is known about treatment of inflammation as it relates to coagulation in cancer patients. Studies of hospital intensive care unit (ICU) mortality outcomes have attempted to identify risk factors for cancer patient mortality [54,55]. Little information is available on tumor-specific factors that influence the outcome or predict the appropriate treatment of this population. Moreover, few severe sepsis therapies are directed at the host systemic response. Certain systems based on organ failure scoring (SAPS II) or organ failure and comorbidities (e.g. APACHE II) have not achieved the consistent ability to predict sepsis mortality in patients with cancer [56-58]. Consequently, the general prognostic models for ICU patients underestimate the risk of dying for cancer patients admitted to the ICU. More recent models specific to the cancer patient may more reliably identify subgroups of cancer patients with a very high mortality [55,59]. Performance status before the onset of acute infectious symptoms appears to be one important factor. With the recent evaluation of antithrombotic agents for severe sepsis and regulatory approval of activated protein C (aPC) [60], mechanisms of coagulation in cancer-related sepsis may require further evaluation. Previously, antiinflammatory inhibitory agents such as anti-TNF- , anti-IL-1RA, anti-BPI, and others have been unsuccessful in clinical trials [61].
Coagulation Elevated During Inflammation Platelets Fibrinogen PAI-1 Factor IX Factor VIII Mucin Cysteine Pr./CP Tissue Factor (TF)
The Cancer Related Thrombotic Tendency in Sepsis Table 3.
547
Subgroup analysis of cancer patients treated in the rhaPC registration trial. A post-hoc subgroup analysis was preformed on the 303 cancer patients with acute sepsis-related organ dysfunction and who received standard therapy and either rhaPC at 24ug/kg/hr or placebo for 96 hrs. Patients were excluded for increased bleeding risk, including platelet count<30K/mm3, history of bone marrow transplant, poorly controlled neoplasm, or likely death from a non-sepsis cause within 28-days. Mortality and bleeding were evaluated at the 28-day endpoint and mortality at 1-year. Results: Compared to the entire cohort, cancer patients were older, had more chronic health points, and higher APACHE II scores. Cancer patients had a lower survival rate than the overall trial group. Cancer patients experienced a significant treatment effect (28-day relative risk: 0.68) similar to the overall trial population (RR: 0.80). This benefit was similar at one year. Cancer and the overall trial patient groups experienced a similar rate of serious adverse bleeding events. RhaPC is efficacious and safe in patients with severe sepsis and early stage cancer or a history of malignancy. Despite increased baseline morbidity, rhaPC produced an increase in 28day survival in cancer patients that was similar to the overall trial population. rhAPC Enrolled 850 Pts 145 (17.1%) Placebo Enrolled 840 Pts 158 (18.1%) RhAPC 28D 24.70% 28.30% Placebo 28D 30.80% 41.10% RhAPC 1yr 41.10% 51.50% Placebo 1yr 42.80% 56.40%
Mortality Phase 3 Total Patients Cancer Patient
Their focused specificity may translate into a limited efficacy in treating advanced sepsis at presentation to the ICU. Other antibody therapies against inflammatory proteins are being developed [62]. More recently, antithrombotic agents with direct or secondary antiinflammatory effects have been investigated in severe sepsis e.g. TF pathway inhibitor (TFPI), antithrombin III (ATIII), and activated protein C (aPC). Coagulation Cascade Targets and Anti-Inflammatory Agents that Might Address Cancer and Severe Sepsis Although FXa inhibitors have not been tested fully in sepsis, these agents mechanistically inhibit FXa within the cascade to address activators such as cancer procoagulant, mucin, or other cysteine proteases. Similarly, a small molecule inhibitor, active-site inhibited FVIIa (FVIIai) is early in development and may counter excess TF induced FVIIa [63]. TFPI, although less successful in treating severe sepsis, might be appropriate to test in the subpopulation of cancers over-expressing TF. Antithrombin III was not successful in improving severe sepsis survival, but could be considered for testing in subpopulations of cancer patients with severe sepsis given its endothelial anti-inflammatory properties and multiple inhibitory sites in the common and extrinsic pathway. By disrupting focal endothelial adhesion, ATIII exerts its anti-apoptotic and antiangiogenic effects [64]. Direct thrombin inhibitors (e.g. hirudin or argatroban or melagatran) have not been fully evaluated in severe sepsis and like other anticoagulants have a high risk of bleeding [65]. Therapeutic doses of heparin have not been successful in treating sepsis. However, recently low dose heparin infusion was found to prevent central line bloodstream infections in cancer patients [66]. Bleeding and heparin-induced thrombocytopenia are potential risks with this approach. Although not studied prospectively in severe sepsis oncology patients, recombinant human activated protein C has demonstrated some potential benefit in subgroup analyses from the phase 3 registration trial. Activated Protein C in Severe Sepsis Patients with Cancer Multiple mechanisms of action of rhaPC may disrupt the linkage between inflammation and coagulation resulting in improved severe sepsis survival. This may be due to reduced thrombin generation, direct endothelial receptor-mediated anti-inflammatory properties, and enhanced microvascular fibrinolysis during disseminated intravascular coagulation (DIC). In the Phase 3 registration study of rhaPC (Drotrecogin alfa (activated)) in severe sepsis, the cancer patient subgroup was defined as tumors staged as stage I or II. As might be expected, this subgroup was found to be older and at higher risk of death than the overall population, Table 3 [67,68]. Other baseline characteristics of these cancer patients overall were well balanced between treatment arms, placebo Vs rhaPC. Cancer patients treated with rhaPC
received a survival benefit, despite increased baseline morbidity, 31.3% (28-day) relative risk reduction (RRR) in cancer patients and 19.9% (28-day) RRR in the overall population. Over the first year cancer patients maintained a survival benefit following treatment, 213 days median survival for rhaPC-treated cancer patients and 119 days median survival for placebo-treated cancer patients. Side effects were similar in that cancer patients experienced a similar rate of serious bleeding events as the overall population. Neutropenic cancer patients also received significant benefit from rhaPC treatment [69]. The efficacy of this anticoagulant suggests other similar agents might have an advantage in severe sepsis cancer patients. More markers of inflammation and effective anticoagulation require investigation. Moreover, bone marrow transplant patients were excluded from the registration study. A second prospective rhaPC study evaluating bone marrow transplant patients stopped prematurely [7]. Low accrual and an enhanced risk of bleeding led to early discontinuation of this study. Although not formally categorized by histologic subtype of cancer, or by coagulation markers, this early stage cancer subgroup appeared to receive benefit from rhaPC. Thus further prospective evaluation of severe sepsis cancer patients with emphasis on procoagulant and anti-inflammatory mechanisms seems warranted, not only for rhaPC, but for other similar anticoagulants. A realistic approach should include metastatic disease and quality of life evaluation. CONCLUSION Overall, patients with cancer maintain a balance of thrombus formation and fibrinolysis that may become aberrant, secondary to tumor-specific factors or by the influence of inflammatory mediators. The clinical sequelae of this dysregulation are venous (or arterial) thromboses. Superimposing severe sepsis with its acute infectious inflammation on the thrombotic tendency of cancer makes the predictability of severe sepsis-related mortality also difficult. Developing scoring systems specific to the septic cancer patient is essential to prediction of outcome. Treatment strategies for cancer patients with severe sepsis might consider anticoagulants, both for prophylaxis of venous thromboembolic disease and for direct treatment of severe sepsis mechanisms. Further studies in animal models and ICU cancer patients with sepsis are needed to address the growing needs of future severe sepsis patients at any stage of the disease. REFERENCES
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The Effect of Statins on Postprandial Lipemia

Genovefa D. Kolovou1,*, Katherine K. Anagnostopoulou1, Klelia D. Salpea1, Stella S. Daskalopoulou2 and Dimitri P. Mikhailidis3
1
Cardiology Department, Onassis Cardiac Surgery Center, Athens, Greece; 2Department of Medicine, Division of Clinical Epidemiology, Royal Victoria Hospital, McGill University, Montreal, Canada and 3Department of Clinical Biochemistry (Vascular Disease Prevention Clinics) and Department of Surgery, Royal Free Hospital, Royal Free and University College Medical School, University of London, London, UK
Abstract: Several studies showed that postprandial plasma triglyceride (TG) concentrations are higher in patients with coronary heart disease. TG-rich lipoprotein remnants accumulated in the postprandial state are involved in atherogenesis and in events leading to thrombosis. Lipid lowering drugs, such as 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) are of significant benefit in the primary and secondary prevention of atherosclerosis. Statins can decrease total cholesterol and low density lipoprotein cholesterol as well as TG concentrations and improve postprandial lipoprotein metabolism. Since abnormal postprandial lipemia is associated with pathological conditions, its treatment is relevant. This review considers the effect of statins on postprandial lipemia.
Key Words: Postprandial lipemia, statins, triglycerides, coronary heart disease. INTRODUCTION Due to the number of meals during the day and the long duration of postprandial lipemia, a substantial part of our life is spent in the postprandial state. Therefore, postprandial lipid metabolism has been investigated (e.g. by conducting fat load tests) [1-5]. The changes in lipoproteins observed postprandially include a marked increase in triglyceride (TG)-rich lipoprotein concentrations and changes in low density lipoproteins (LDLs) and high density lipoproteins (HDLs) [5]. Epidemiological data support the concept that elevated fasting plasma TG concentrations are an independent risk factor for coronary heart disease (CHD) [5-7]. Less evidence exists supporting the concept that postprandial lipemia is an independent risk factor for CHD [5,8]. Nevertheless, postprandial TG levels can be highly discriminatory between CHD patients and control subjects [9]. The magnitude of the postprandial response appears to play a role in the etiology and the progression of CHD, since the TG-rich remnant lipoproteins accumulated in the postprandial state are involved in atherogenesis and in events leading to thrombosis [9-11]. Furthermore, an enhanced TG rise postprandially has also been reported in patients with diabetes mellitus [12-14], hypertension [15], obesity, metabolic syndrome [16], and other conditions [17,18]. It has been proposed that the degree of postprandial lipemia is determined by the degree of insulin resistance as an underlying abnormality [13]. At present, normal postprandial TG ranges have not been established. Plasma TGs derive from two major sources; intestinally-derived chylomicrons (CM) and hepatically-derived very low density lipoproteins (VLDL) [19,20]. Almost all the fat ingested during meals is absorbed and incorporated into CMs [21,22]. The CMs pass from the lymphatic to the blood circulation where the TGs are selectively removed firstly, in muscle and adipose tissue by lipoprotein lipase activity with apolipoprotein C-II as cofactor, and secondly, in the liver by endocytosis [23]. VLDL formation (the second source of TGs) depends firstly, on the availability of hepatic cholesterol substrates and secondly, on the fatty acid supplies to the liver as well as hepatic TG pools [24]. The catabolism of VLDLs follows the same pathway as the CM remnants [25,26]. Postprandial hypertriglyceridemia is probably a consequence of competition between CM and VLDL remnants for lipoprotein lipase and the hepatic receptors [27-29]. Physiologically, CM and VLDL remnants are removed rapidly from the circulation by a receptor-mediated process [30]. Evidently, there is a threshold dietary level that does not overwhelm this clearance capacity. It was suggested that in obese humans, fasting plasma lipids can be normal but postprandial lipid metabolism is abnormal with an accumulation of TG-rich remnant lipoproteins [31]. CM remnant catabolism was significantly decreased in men with visceral obesity when compared with lean individuals; this could be attributed to the competition for clearance by LDL receptors between CM remnants and the increased hepatic production of VLDL [31]. Furthermore, Halkes et al. showed that both lean and overweight women had a more favorable fasting lipoprotein profile than lean and overweight men, respectively [32]. Similarly, the diurnal TG profile was lower in lean women than lean men. However, diurnal TG profiles in overweight women and overweight men were similar, suggesting that overweight resulted in a 'male diurnal TG profile' in females probably due to abdominal fat accumulation [32]. Postprandial lipemia is considered in greater detail in another review [5]. In the early postprandial period (first 3 h) smaller sized CMs are secreted and later, in the postprandial period de novo-formed larger CMs appear. The smaller sized CMs are considered to be atherogenic [30]. VLDLs secreted by the liver are not considered to be atherogenic in normal subjects [29]. However, in the hypertriglyceridemic state the secreted VLDL particles are large and result in the formation of small dense LDL particles that have proatherogenic properties (e.g. increased arterial wall retention and susceptibility to oxidation of LDL) [33-36]. Additionally, it has been proposed that elevated plasma TG concentrations promote the cholesteryl ester exchange reactions mediated by cholesteryl ester transfer protein (CETP) [37]. In this case, the HDL particles are TG-enriched via CETP mediated exchange with TG-rich lipoproteins. Such HDL-TG enriched particles are cleared more rapidly from the circulation leading to low serum HDL cholesterol levels [38-40]. HDL has several beneficial pleiotropic effects (antioxidant, anti-inflammatory and others), besides reverse cholesterol transport [41].
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Endogenous cholesterol enters the intestinal lumen in association with bile salts and is immediately available for absorption, while there is a time delay for the absorption of dietary cholesterol since it needs to be solubilised in bile salt micelles [42,43]. As a consequence the amount of dietary cholesterol does not substantially influence CM lipid concentration [44]. As a result of cholesterol homeostasis, in the case of reduced cholesterol absorption, caused by genetic, environmental and lifestyle factors, an increase in cholesterol synthesis occurs. This in turn enhances the secretion of VLDL and the increased amount of LDL particles forms saturated LDL receptors [45]. Therefore, the availability of LDL receptors for CM- and VLDL-remnants, formed postprandially, is limited leading to postprandial hypertriglyceridemia. Furthermore, mixed meals containing slowly digestible carbohydrates that induce low glycemic and insulinemic responses reduced the postprandial accumulation of both hepatically- and intestinally-derived TG-rich lipoproteins (plasma TG concentrations, apolipoprotein B-100 and apolipoprotein B-48) in obese subjects with insulin resistance [46]. In another study, the effect of two hypoenergetic diets [a very low-carbohydrate (<10% energy as carbohydrate) and a low-fat (<30% energy as fat)] on fasting blood lipids and postprandial lipemia was compared in overweight men [47]. Postprandial TG values generally peaked 4 h after the meal and gradually returned to baseline after 7 to 8 h [47]. A very low-carbohydrate diet was more effective at improving characteristics of the metabolic syndrome as shown by a decrease in fasting serum TG, the TG/HDL-C ratio, serum glucose (-44, -42, and -6%, respectively), postprandial lipemia, an increase in LDL particle size, and also greater weight loss (P < 0.05). However, serum LDL cholesterol was reduced (P < 0.05) only by the low-fat diet (-18%). A similar study in overweight women showed that both the very low-carbohydrate and the low-fat diet significantly decreased postprandial lipemia and resulted in similar non-significant changes in the total cholesterol/HDL-C ratio, fasting TG, oxidized LDL, and LDL subclass distribution [48]. Postprandial TG values generally peaked about 3 h after the meal in overweight women and gradually returned to baseline after 7 to 8 h [48]. Furthermore, it was suggested that the determinants of fasting and postprandial lipids are different. The independent atherogenic influence of postprandial lipids may relate more to smoking and diet than to obesity and insulin resistance. Smokers had substantially increased retinyl palmitate and apolipoprotein B-48 responses, while persons who consume more calories or omega-3 fatty acids had reduced CM responses [49]. Reznik et al. showed that type 2 diabetes mellitus patients who were normotriglyceridemic in the fasting state had an amplified response of both TG and retinyl palmitate concentrations after oral fat load when compared with normotriglyceridemic obese controls [50]. Furthermore, postprandial lipoprotein profiles were distinguished in type 2 diabetic patients according to apolipoprotein E phenotype: postprandial retinyl palmitate response was twofold to threefold higher in E2/3 and E3/4 patients than in the common E3/3 phenotype, while lower fasting and postprandial HDL cholesterol and HDL3 cholesterol levels were observed in E3/4 versus E3/3 patients [50]. On the other hand, the apolipoprotein E4 allele has been shown in some studies to be associated with increased response to dietary intervention, while apolipoprotein E2 carriers appear to be more responsive to statin therapy [51]. In obese humans fasting plasma lipids can be normal but postprandial lipid metabolism is abnormal with an accumulation of TGrich remnant lipoproteins [31]. CM remnant catabolism was significantly decreased in men with visceral obesity when compared with lean individuals. This could be attributed to the competition for clearance by LDL receptors between CM remnants and the increased hepatic production of VLDL [31].
Since abnormal postprandial lipemia is associated with pathological conditions, its treatment is relevant. The effect of 3hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) on postprandial lipemia is discussed in this review. STATINS Lipid lowering drugs, such as statins (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin), are of significant benefit in the primary and secondary prevention of atherosclerosis and they can reduce vascular morbidity and mortality [52-55]. Statins are the principal agents for managing hyperlipidemia because of their ability to reduce total and LDL cholesterol levels by up to 60% and decrease clinical events [56-58]. In contrast to fibrates, statins exert less influence on postprandial lipoprotein metabolism (including large and small CM remnants) [14,59-66]. However, in an earlier study cerivastatin (which has been withdrawn) was compared with gemfibrozil in terms of their effect on oral fat loadinduced changes in endothelial function and postprandial lipids [67]. Cerivastatin, but not gemfibrozil, significantly reduced the increase in remnant-like particle-cholesterol after an oral fat-load and also reversed the endothelial dysfunction induced by fat-load [67]. Furthermore, in a later study statins were compared with gemfibrozil in patients with type 2 diabetes and combined dyslipidemia. Statins, in addition to their superior LDL cholesterol-lowering effect, were as effective as gemfibrozil in reducing postprandial accumulation of TG-rich atherogenic remnant lipoprotein particles (34% vs -43%, respectively) [68]. Statins can also improve postprandial lipoprotein metabolism by upregulating LDL receptors and by decreasing hepatic VLDL synthesis [63,64]. Both mechanisms lead to less competition for the clearance mechanisms shared by CMs and VLDL [25]. A major advantage of statin administration is the pleiotropic effects that they exert directly on atherosclerotic plaques [54,56,69]. Some reports link both phenotype and genotype to differential VLDL and LDL lowering ability of statins. Thus, the effect of statins on TGs depends on the baseline TG level. Individuals with low plasma TG levels have almost no reduction on treatment, whereas in those who were initially hypertriglyceridemic the percentage reduction in plasma TG levels was almost equal to the percentage decrease in LDL cholesterol [70,71]. In another report the TGlowering effect of statins was greater in patients with initial TG levels above 170 mg/dl (1.9 mmol/l), while the effect is even greater when TG levels were over 250 mg/dl (3.0 mmol/l) [72]. The metabolic syndrome, a clustering of several abnormalities including fasting hypertriglyceridemia, is associated with abnormal postprandial lipemia [16,73,74]. In hypercholesterolemic patients with the metabolic syndrome, simvastatin and atorvastatin had comparable beneficial effects on apolipoprotein B-containing atherogenic lipids and lipoproteins, and metabolic syndrome status was effectively modified by both drugs. However, although atorvastatin produced slightly greater decreases in TG than simvastatin (31.5% and 29.6%, respectively), simvastatin produced greater increases in HDL-C than atorvastatin (10.7% vs 3.7%, respectively) [75]. The effect of statins on circulating fasting and postprandial TG levels also depends on the dose of statin used (i.e. its LDL cholesterol-lowering capacity). This is discussed in the section below. The Effect of Statins on Postprandial TG Levels Statins are highly effective in reducing plasma LDL [56,76]; however, some statins (atorvastatin, fluvastatin, rosuvastatin and simvastatin) that can cause marked reductions in plasma TG concentrations [77,78]. Interestingly, the TG-lowering effect of statins is related to their LDL cholesterol-lowering potency; the greater the effect exerted on LDL cholesterol, the more will the compound decrease plasma TG [79,80]. The effect of statins on postprandial TG concentration appears to correlate with the degree of reduction
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in fasting plasma TG [79-82]. In miniature pigs, atorvastatin induced a greater reduction in plasma TG levels over the postprandial phase than at baseline [83]. Parhofer et al. [84] reported that postprandial lipoprotein metabolism is enhanced in normolipidemic subjects under statin therapy as a result of elevated catabolism of CM remnants and/or decreased conversion of CMs to CM remnants; in contrast, the formation and secretion of CMs was not affected. Lipid-lowering drugs may influence CM metabolism as a direct consequence of reduction in plasma VLDL levels. Karpe et al. [85] demonstrated a major contribution of liver-derived, TG-rich lipoproteins to postprandial triglyceridemia. Guerin et al. [86] found that atorvastatin did not influence the intestinal production rate of CMs but may induce an acceleration of the catabolism of CMs and their remnants. However, Funatsu et al. [87] in an animal model (sucrose-fed rats) found that atorvastatin decreased not only the increased rate of TG secretion in the VLDL fraction, but also that in the CM-rich fraction. CM production may also be regulated by intestinal cholesterol synthesis [88] and atorvastatin decreased apolipoprotein B-48 secretion from transformed human intestinal cholesterol enterocyte (CaCo2 cells). Since statins [89,90] tend to distribute preferentially into the small intestine in addition to accumulating in the liver, the possibility that they improve the increase in intestinal CM-TG secretion could not be ruled out [91]. Statin treatment can significantly reduce the postprandial CM apolipoprotein B-48 area under the curve (AUC) (P < 0.01) and apolipoprotein B-100 in the CM fraction (P < 0.05) [92]. The effect of statin therapy on postprandial TG concentration can be explained by several mechanisms. Firstly, statins can limit hepatic VLDL production [83,94.] Secondly, they can enhance the clearance and fractional catabolic rate of circulating TG-rich lipoproteins [83,95]. Thirdly, they can reduce apolipoprotein C-III levels [96], and, fourthly, statins can decrease cholesteryl ester transfer [97]. The inhibition of HMG-CoA-reductase leads to an up-regulation of the LDL receptor, resulting in an increased catabolism of LDL particles and, thus, lower LDL cholesterol concentrations [98]. This provides an opportunity for lipoproteins other than LDL, including postprandial TG-rich lipoproteins, to be internalized into hepatocytes via the LDL receptor pathway [22,25,99,100]. An enhanced uptake of VLDL remnants by LDL receptors was observed in subjects with mixed hyperlipidemia treated with lovastatin [101,102]. Animal studies showed that a deficiency in the LDL receptor is associated with a delayed CM remnant removal [103105]. Cabezas et al. [106] demonstrated a small but significant postprandial increase in retinyl palmitate concentration in the CM remnant fraction in heterozygous familial hypercholesterolemia (hFH) patients compared with normolipidemic control subjects. Our group also found exaggerate postprandial lipemia in hFH men [107] and women [108]. However, Kowal et al. [109] reported that only 5% of the binding capacity of the LDL receptor was needed to remove CM particles, suggesting only a minor role for LDL receptors in the removal process of lipoprotein remnants. It is likely, that statins have profound effects on postprandial lipoprotein metabolism, particularly in hypertriglyceridemic patients [72,76]. TG reduction may be related to the fact that VLDL and LDL compete for the same removal mechanisms [22,25]; thus, a reduction in the number of LDLs may also increase the removal of VLDLs. Furthermore, the capacity to eliminate CMs is also increased since VLDLs and CMs compete for the same lipolytic pathway [25]. Earlier studies of the effects of lipid-lowering drugs on postprandial lipid metabolism revealed that a reduction in postprandial lipemia is closely related to the drug-induced decrease in fasting TG concentrations [60,84,92,110-112]. Fibrates, which have a more pronounced effect on fasting TG levels than statins, significantly reduce postprandial hypertriglyceridemia [65,111,112]. Overall,
statins reduce fasting TG levels to a lesser degree than fibrates and therefore have less effect on postprandial lipid levels [60,65,92, 110]. Statins also reduce apolipoprotein C-III levels, a protein that inhibits lipoprotein lipase (LPL)-mediated hydrolysis [96,113]. Plasma apolipoprotein C-III levels were independently and significantly correlated with the secretion rate of VLDL-apolipoprotein B (P = 0.001) and with the percent conversion of VLDL-apolipoprotein B to LDL-apolipoprotein B (P = 0.004) in men with visceral obesity [114]. This reduction is thought to occur via the peroxisome proliferator-activated receptor effect of statin on the AI/CIII/AIV gene cluster, leading to decreased apolipoprotein C-III mRNA and protein concentrations [96]. This increased LPL activity results in a more effective hydrolysis of fasting and postprandial TG- rich lipoproteins. CETP-mediated cholesteryl ester transfer to TG-rich lipoprotein particles (CMs and VLDL) is enhanced during postprandial lipemia in obese or dyslipidemic subjects as a result of an elevation in their mass and particle number. Statin treatment significantly reduces cholesteryl ester transfer from HDL to VLDL particles during the postprandial phase [97]. The effect of statin administration on postprandial lipid metabolism in different subjects and various studies is shown in Table 1 [59,60,62,63,76,84,86,91,110,115-131]. Furthermore, the effect of statins on apolipoprotein B-containing lipoproteins, VLDLs, intermediate-density lipoproteins (IDLs), and LDLs, is mediated in part by the preferential reduction of CETP-mediated cholesteryl ester transfer from HDLs to apolipoprotein B- containing lipoproteins and principally to large VLDL particles [97]. Microsomal TG transfer protein (MTP) is responsible for the assembly of TG-rich lipoproteins and is increased in diabetic animal models [132]. Phillips et al. proposed that MTP plays a role in regulating the cholesterol content of the CM particle [132]. They found that MTP mRNA expression is increased in diabetic when compared with non-diabetic subjects. It is interesting that statin therapy lowered MTP mRNA in both diabetic non-diabetic subjects [132]. Atorvastatin Schaefer et al. evaluated the effects of atorvastatin, fluvastatin, lovastatin, pravastatin and simvastatin on fasting and postprandial lipoproteins in CHD patients [76]. At all doses tested in the fasting and postprandial states, atorvastatin (20, 40 and 80 mg/day) was significantly (P < 0.05) more effective than all statins, except for simvastatin, in lowering TG and remnant lipoprotein cholesterol. At 40 mg/day in the fasting state, atorvastatin was significantly (P < 0.01) more effective than all statins, except for lovastatin and simvastatin, in lowering cholesterol levels in small LDL, and was significantly (P < 0.05) more effective than all statins, except for simvastatin, in increasing cholesterol in large HDL and in lowering LDL particle numbers. Atorvastatin was the most effective statin tested in lowering cholesterol in LDL, non-HDL, and remnant lipoprotein in the fasting and fed states, and getting patients with CHD to established goals. Fluvastatin, pravastatin, lovastatin, and simvastatin had about 33%, 50%, 60%, and 85% of the efficacy of atorvastatin, respectively, at the same dose in the same patients [76]. The same group also reported significant fasting TG reductions of 22%, 26% and 30% (at the 20, 40 and 80 mg daily doses of atorvastatin), and a postprandial TG normalization with a dose of 40 mg atorvastatin/day [118]. More recently this group also showed that atorvastatin (40 mg/dl) was more effective than fluvastatin, lovastatin, pravastatin or simvastatin in decreasing not only LDL cholesterol but also fasting and postprandial high-sensitivity Creactive protein (by 32%, P < 0.01) and lipoprotein-associated phospholipase A2 (by 26%, P < 0.0001) in CHD patients with LDL cholesterol levels >130 mg/dl (3.4 mmol/l) [133]. Furthermore, in
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Table 1.
Effect of Statin Administration on Postprandial Lipid Metabolism in Different Subjects and Various Studies
Meal Content Atorvastatin Subjects Result
1st Author, [study], year
Ceriello, [115], 2005 Schaefer, [76], 2004 Cabezas, [116], 2004 Parhofer, [117], 2003 Boquist, [91], 2002 Schaefer, [118], 2002 Guerin, [86], 2002 Vansant, [119], 2001 Nordoy, [120], 2001 Parhofer, [84], 2000
F:75g, C:5g, P:6g /1m body surface F: 57% by energy F:50g/m2 body surface F:87%, C:7%, P:6% by energy F:60%, C:27%, P:13% by energy F:57% by energy F:18%, C:70%, P:12% by energy F:53%, C:23.5%, P:23.5% by energy F:78g F:87%, C:7%, P:6% by energy Fluvastatin
N=20 (male, female) with type II DM N=97 (male, female) with CHD N=16 with FCH N=10 (male, female) with HTG N=16 (male) with FCH and MI N=88 (male, female) with CHD N=11 (male) HC N=15 (female) homozygotes for 3 N=42 N=10 (male) healthy
(-)PL (short-term) PL hepatic TG, (-) intestinal TG iAUC PL RLP PL, in CE transfer PL (-)PL AUC TG, AUC RE
Schaefer, [76], 2004
F:57% by energy Lovastatin
N=28 (male, female) with CHD
PL
Schaefer, [76], 2004 Simo, [121], 1993 Cianflone, [63], 1990 Weintraub, [110], 1989
F:57% by energy F:69%, C:26%, P:5% by energy F:69%, C:26%, P:5% by energy F:65%, C:20%, P:15% by energy
N=24 (male, female) with CHD N=11 (male) HPAL N=12 (male, female) with HC N=5 (male, female) with HC N=5 (male, female) with HC and HTG Pravastatin
PL (-) PL Mild LpL activity CM remnant clearance (-) PL PL
Schaefer, [76], 2004 Contacos, [122], 1998 Bhatnagar, [60], 1995 O'Keefe, [62], 1995
F:57% by energy F:90%, C:4.8%, P:5.2% by energy F:51g, C:84 g, P:40g F:90g/m2 body surface
N=22 (male, female) with CHD N=19 (male, female) with HC N=16 (male, female) with HC and type II DM N=18 (male, female) with low HDL and HTG Simvastatin
PL CETP activity CETP activity, attenuated TG (-) PL
Van Wijk, [123], 2005
F:40%, C:2.8%, P:57.2% by energy
N=18 (male, female) with CHD
CETP activity, AUC TG AUC RE PL PL
Schaefer, [76], 2004 Ceriello, [124], 2002 Halkes, [125], 2001 Sheu, [126], 2001 Nordoy, [127], 2000 Twickler, [128], 2000
F:57% by energy F:75g, C:5g, P:6g /1m body surface F:50g/m body surface F:33%, C:52%, P:15% by energy F:78g F:40%, C:2.8%, P: 57.2% by energy
2 2
N=25 (male, female) with CHD N=30 (male, female) with type II DM N=20 (male, female) with CHD N=24 (male, female) with type II DM N=17 (male, female) with combined hyperlipidemia N=7 (male, female) with hFH,
PL AUC, iAUC RLP

(Table 1) contd.
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1st Author, [study], year
Meal Content Simvastatin
Subjects
Result
Noutsou, [129], 1999 Vigna, [130], 1999 Cabezas, [131], 1994 Cabezas, [59], 1993
F:30-35%, C:50-55%, P:10-15% by energy F:35g/m2 body surface F:50g/m body surface F:50g/m body surface
2 2
N=8 (male, female) with type I DM N=30 (male) with combined hyperlipidemia N=4 (male) FCH N=7 (male) FCH
AUC TG Minor modification of PL PL (-) AUC RE
AUC=area under the curve, AUC RE= area under the curve of chylomicron remnant concentration, AUC TG=area under the curve of triglyceride concentration, C=carbohydrates, CE=cholesteryl ester, CETP=cholesteryl ester transfer protein, CHD=coronary heart disease, DM=diabetes mellitus, F=fat, FCH=familial combined hyperlipidemia, HC=subjects with hypercholesterolemia, HDL=high density lipoprotein, hFH=heterozygotes familial hypercholesterolemia, HPAL=hypoalphalipoproteinemia, HTG=hypertriglyceridemia, iAUC=incremental AUC, LpL=lipoprotein lipase, MI=myocardial infarction, N=number of subjects, P=proteins, PL=postprandial lipemia, RLP=remnant like particle, TG=triglycerides.
hypercholesterolemic patients atorvastatin 10 mg/day showed an early benefit by decreased remnant-like particles cholesterol (P < 0.01) after 2 weeks of treatment [134]. In obese humans fasting plasma lipids can be normal but postprandial lipid metabolism is often abnormal with an accumulation of TG-rich remnant lipoproteins [31]. In viscerally obese men CM remnant catabolism was markedly decreased when compared with lean individuals. The decreased clearance of CM remnants in viscerally obese subjects may be explained by competition between CM remnants and the increased hepatic production of VLDL for clearance by LDL receptors. Increased food intake in rodent models of obesity was shown to be associated with a delay in the catabolism of remnant lipoprotein particles [31]. In a study involving normolipidemic patients with CHD, atorvastatin 80 mg/day significantly decreased apolipoprotein B-48 (P = 0.019), remnant-like particle-cholesterol (RLP-C) (P = 0.032) and total postprandial apolipoprotein B-48 AUC (P = 0.013) when compared with placebo [135]. Atorvastatin also significantly increased LDL-receptor binding activity (P < 0.001), and this correlated with changes in fasting apolipoprotein B-48 (r = 0.80, P = 0.01) [135]. Parhofer et al. [84] reported that postprandial lipoprotein metabolism was improved in normolipidemic subjects receiving 10 mg of atorvastatin daily probably by increasing the direct removal of CMs from plasma. Atorvastatin significantly (P < 0.05) decreased the incremental AUC for CM remnants (small TG-rich lipoproteins) but not for CM (large TG-rich lipoproteins) [84]. In contrast, in hypertriglyceridemic patients atorvastatin significantly (P < 0.05) decreased the incremental AUC for TG, cholesterol and retinylpalmitate in large TG-rich lipoproteins, but not small TG-rich lipoproteins particles [117]. In this study a significant decrease on fasting TG levels (-43%) was also noted in hypertriglyceridemic patients [117]. Boquist et al. found that atorvastatin (40 mg daily) caused almost a 50% reduction in postprandial plasma concentrations of all TG-rich particles in combined hyperlipidemic patients with premature CHD and also significantly reduced the fasting plasma concentrations of VLDL cholesterol, LDL cholesterol and VLDL-TG (median change: 29%, 44% and 27%, respectively) and increased HDL cholesterol by 19% [91]. The postprandial plasma concentrations of large [Svedberg flotation rate (Sf) 60-400] and small (Sf 20-60) VLDLs as well as CM remnants were almost half compared with baseline values, and postprandial triglyceridemia was reduced by 23% during active treatment [91]. Similarly, in familial combined hyperlipidemia as well as in the impaired fasting glucose state, atorvastatin (40 mg/day) caused a decreased postprandial response of TG (approximately -19%) [136,137]. Similar results were found in obese women homozygous for apolipoprotein E-3 and in subjects with type II B hyperlipidemia [86,119]. Additionally, Cabezas et al.
found that the clearance of VLDL improved by 24% without major changes in the other fractions postprandially [116]. Chan and colleagues detected an increase in the catabolic rate of TG-rich remnants in obese patients with dyslipidemia treated with atorvastatin (40 mg/day) [138]. This was partially attributed to an enhancement of hepatic clearance of CM remnants, as evaluated by the stable isotope breath test [138]. Furthermore, kinetic studies in obese men with the metabolic syndrome showed that atorvastatin can significantly increase the clearance rate of VLDL-apolipoprotein B by 58%, which resulted in a 27% decrease in circulating VLDL levels, while the production rate of the lipoprotein was unaffected by the treatment [139]. It was also shown that in centrally obese individuals atorvastatin can significantly increase the catabolism of all apolipoprotein B-100-containing lipoproteins, VLDL, IDL and LDL without decreasing VLDL-apolipoprotein B secretion [140]. Animal studies produced similar results. A decrease (-24%) in TGs of TG-rich lipoprotein AUC and an increase in TG-rich lipoprotein clearance were observed in miniature pigs [83]. The latter may relate to a decreased competition for removal by hepatic VLDL. Funatsu et al. produced a rat model that replicates postprandial hypertriglyceridemia in humans (prolonged increase in TG concentration accompanied by both an increase in TG secretion and decrease in TG clearance). They showed that atorvastatin (30 mg/kg) reduced both fasting and postprandial TG levels and suppressed rates of TG secretion in CM-rich and VLDL fractions after oral fat loading [87]. It seems that atorvastatin improves postprandial lipoprotein metabolism by reducing TG secretion from the liver or/and intestine and by increasing TG-rich lipoprotein clearance through elimination of saturation [87]. Fluvastatin Fluvastatin was the first wholly synthetic statin to be marketed [141,142]. It is structurally different from the fungal metabolites (lovastatin, pravastatin, and simvastatin) [143,144]. Its effects are similar in most patient groups with 20 to 80 mg daily reducing LDL cholesterol by 22% to 36% and increasing HDL cholesterol by 3.3% to 5.6% [145,146]. In addition to lowering LDL cholesterol levels, 20 to 80 mg dosages of fluvastatin significantly reduced TG levels by 4% to 18%, respectively [145,147]. Furtheremore, fluvastatin exerts non-lipid lowering-associated pleiotropic effects in both clinical and experimental studies [142]. Combination therapy (fluvastatin + fenofibrate) was associated with a more improved lipid profile than fluvastatin monotherapy, as the latter does not always manage to substantially decrease TG concentrations [148,149]. Derosa et al. reported a significant change in TGs (-32%) by the co-administration of fluvastatin 80 mg
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and fenofibrate 200 mg daily in hyperlipidemic patients [148]. Spieker et al. observed an even greater TG change (-47%; P < 0.0001) in patients with persistent hypertriglyceridemia taking fluvastatin 20 mg and bezafibrate 400 mg than fluvastatin alone [146]. Papadakis et al. evaluated the use of combination therapy (ciprofibrate 100 mg or bezafibrate 400 mg plus fluvastatin 40 mg) in 23 patients with established cardiovascular disease [150]; both treatments achieved a significant (p 0.01) decrease in TG (53% and 46%, respectively) and LDL cholesterol (36% and 26%, respectively) levels. Although HDL cholesterol levels were increased (19% for both treatment groups), this rise only achieved significance (P = 0.01) in the ciprofibrate group [150]. However, the effect of fluvastatin on postprandial lipemia has not yet been clarified. Lovastatin Lovastatin reduces LDL cholesterol concentrations by 25-40% in hypercholesterolemic patients [151-153] and decreases plasma TG and VLDL levels in hypertriglyceridemic patients [102,154]. Weintraub et al. found that lovastatin exerts a better TG-lowering effect in patients with mildly elevated TG levels (160-240 mg/dl; 1.8 - 2.7 mmol/l) rather than at lower levels (<160 mg/dl; 1.8 mmol/) [110]. Additionally, they found that lovastatin significantly reduces VLDL and postprandial lipoprotein levels [110]. On the other hand, Simo et al. failed to show any improvement in fasting TG levels after lovastatin (40 mg/day) administration whereas for postprandial TG levels, a non-significance trend towards reduction was observed [121]. Taking into account that the fasting TG concentration is the primary determinant of the magnitude of postprandial lipemia [155], drugs affecting fasting TG levels may effectively diminish postprandial hypertriglyceridemia. Although lovastatin monotherapy may benefit hypertriglyceridemic patients and, thus, postprandial hypertriglyceridemia, an additional benefit can be obtained by combination therapy. Niacin extended-release (ER) plus lovastatin at a dose of 1,000/20 mg or 2,000/40 mg reduced TG levels by 26% and 43%, respectively [156,157]. Niacin ER/lovastatin was comparable to atorvastatin 10 mg and more effective than simvastatin 20 mg in reducing LDL cholesterol and TG levels [158]. Pitavastatin Aoki et al. using a guinea pig model of postprandial lipemia found that the administration of pitavastatin at 3 mg/kg decreased the 0-12 h AUC of TG levels above the initial levels, by 77% [159]. This effect was also shown with 30 mg/kg of atorvastatin but was weaker with the same dose of simvastatin [159]. Pravastatin Contacos et al. suggested that pravastatin may reduce the atherogenicity of lipoproteins in hypercholesterolemia by reducing CETP activity [122]. Furthermore, cholesteryl ester transfer is strongly influenced by postprandial lipemia, which may have a cumulative effect on LDL size [63]. Only the pravastatin/niacin regimen significantly diminished postprandial lipemia (-32% change in the remnant particle TG concentration and decreased VLDL remnant levels). Thus, in this group of patients with clustered risk factors, the combination of pravastatin and niacin resulted in significant improvements in HDL cholesterol and TG levels, total cholesterol to HDL cholesterol ratio, small dense LDL levels and postprandial lipemia. Pravastatin alone or in combination with magnesium resulted in less significant changes that were largely limited to LDL cholesterol reduction [62]. Furthermore, in the West of Scotland Coronary Prevention Study the presence of the metabolic syndrome predicted CHD events (HR = 1.30, 95% CI, 1.00 to 1.67, P = 0.045) in a multivariate model incorporating conventional risk factors; men with 4 or 5 features of the syndrome had a 3.7fold increase in risk for CHD and a 24.5-fold increase for diabetes compared with men with none of the features (both P < 0.0001)
[160]. However, pravastatin reduced the risk for CHD in those with the metabolic syndrome to a similar degree as compared with those without the syndrome (HR = 0.73 and 0.69, respectively) [160]. Furthermore, pravastatin may improve insulin resistance in patients with the metabolic syndrome [161]. Rosuvastatin Rosuvastatin, a relatively new statin, may possess a number of advantageous pharmacological properties, including enhanced HMG-CoA reductase binding characteristics, relative hydrophilicity and selective uptake into hepatic cells [162]. Cytochrome P450 metabolism of rosuvastatin appears to be minimal and is principally mediated by the 2C9 enzyme, with little involvement of 3A4; this finding is consistent with the absence of clinically significant pharmacokinetic drug-to-drug interactions between rosuvastatin and other drugs known to inhibit cytochrome P450 enzymes [162]. This statin can reduce LDL cholesterol levels (by up to 63%) [162,163]. There are no studies investigating the effects of rosuvastatin on postprandial TG response, though it can lower fasting TG levels. Hunninghake and coworkers reported significant TG-mean reductions at all doses (5, 10, 20, 40 or 80 mg) compared to placebo (18% to -40% vs +2.9%) in patients with fasting TG levels 300 to <800 mg/dl ( 3.4 to <9.0 mmol/l) [164]. Its advantageous effects have been extended to diseases involving aberrant TG metabolism such as the metabolic syndrome (TG reduction from 22% to 34%) [165] and combined hyperlipidemia [166]. Both syndromes are often characterized by postprandial hypertriglyceridemia. Furthermore, treatment with rosuvastatin 10 mg when compared with atorvastatin 10 mg, atorvastatin 20 mg, simvastatin 20 mg and pravastatin 40 mg produced similar or greater reductions in TG levels and increases in HDL-C levels in subjects with or without the metabolic syndrome [167]. Additionally, rosuvastatin seems to significantly reduce CETP levels (which are increased in metabolic syndrome and obesity, conditions associated with abnormal postprandial lipemia) by 33% and 37% in normo- and hyper-triglyceridemic subjects respectively, as well as its activity (-59%) in hypertriglyceridemics [70]. Studies in experimental animals showed that rosuvastatin treatment in apolipoprotein E*3 Leiden transgenic mice, an established model for hyperlipidemia and atherosclerosis, on a high fat/cholesterol diet, results in decreased hepatic VLDL-TG and VLDL-apolipoprotein B production without any alteration in VLDL lipid composition rather than a reduction in the number of VLDL particles secreted. In the same study, the highest dose of rosuvastatin reduced plasma TG levels by 42% [168]. Simvastatin Twickler et al. investigated the effects of high-dose simvastatin on postprandial lipoproteins in patients with hFH [128]. They showed that a dose of 80 mg/day significantly reduced fasting and postprandial levels of lipoprotein remnants [128]. Similarly, treatment with a high dose of simvastatin resulted in significantly decreased postprandial lipid-lipoprotein concentrations in familial combined hyperlipidemia [60]. The beneficial effect of simvastatin (20 mg/day) in lowering fasting and postpradial TGs was also reported in subjects with type 2 diabetes and combined hyperlipidemia [126]. After treatment, a decreased AUC of TG was also observed in subjects with type 1 diabetes and elevated LDL cholesterol levels [129]. Furthermore, in normolipidemic patients with premature CHD, simvastatin (80 mg/day) produced significant reductions of fasting TGs and of their AUC by 26% and 30%, respectively, while the CM remnant clearance was improved (AUC) by 36% (P < 0.01) [123]. The same group also compared the daytime (non-fasting) capillary (cTG) profile in normotriglyceridemic patients with premature CHD versus control subjects [169]. Patients with CHD had a 44% higher total AUC cTG than matched controls (P < 0.01). After lo-
557
gistic regression analysis, cTG-AUC was the strongest predictor of the presence of CHD (P < 0.001). It is interesting that although the target for LDL cholesterol was reached (78 mg/dl; 2.0 mmol/l) by simvastatin 20 mg/day, higher doses of simvastatin were needed to normalize daytime triglyceridemia. Simvastatin 40 mg/day decreased the AUC cTG by 28% (P < 0.05 versus baseline), reaching comparable values as in controls, without further improvement with simvastatin 80 mg/day (26% reduction versus baseline; P < 0.05) [169]. After treatment with simvastatin (80 mg), fasting and postprandial complement component 3 concentrations decreased significantly [125]. The magnitude of these changes was correlated with changes in postprandial TG metabolism, underlining the relationship between postprandial triglyceridemia and complement component 3 (C3). Whether treatment with lower doses of statins results in similar changes in postprandial TG and C3 metabolism in similar patients remains to be investigated [125]. Furthermore, it was suggested that when glucose is added to an oral fat load, the postprandial free fatty acid response is reduced, and the fat-specific increase in C3 is prevented; however, ingestion of fat without glucose, and, thus, lack of insulin response may lead to C3-mediated peripheral free fatty acid trapping [170]. Finally, the combination of simvastatin (20 mg/day) plus omega-3 fatty acids apart for improving the degree of postprandial lipemia seems to benefit the hemostatic profile by reducing the tissue factor inhibitor fraction in the fasting state and inhibiting the activation of factor VII that occurs during postprandial lipemia [127]. Postprandial Lipemia and Other Drugs with Lipid-Lowering Effects Orlistat administration resulted in a lower postprandial TG AUC, shorter postprandial lipemia, lower concentration of large TG-rich particles and decrease of VLDL particle size [171]. Thus, the 8 h postprandial mean TG AUC was significantly (P = 0.02) lower with orlistat plus a moderate-fat meal and orlistat plus a highfat meal (0.79 and 1.33 mmol/lh) than with placebo plus a high-fat meal (4.33 mmol/lh; P = 0.02). Mean change in large VLDL subclass concentration during the 4-8 h and mean VLDL size after 8 h was significantly (P < 0.001) lower with orlistat plus a moderate- or high-fat meal than with placebo plus a high-fat meal. Small HDL particle concentration decreased significantly (P < 0.001) with high-fat meal versus moderate-fat meal or high-fat meal [171]. The FenOrli study compared the effect of orlistat and fenofibrate alone or in combination on lipid parameters in overweight or obese patients with the metabolic syndrome [172]. Significant reductions in TGs were observed in all treatment groups when compared with baseline values. The combination of orlistat and micronised fenofibrate was more effective in reducing TGs (-37%) compared with that in the orlistat group (-14%) (P < 0.05) but there was no difference in the effect between the combination treatment and the fenofibrate alone group [172]. The role of fibrates in reducing postprandial lipemia has been mentioned above. Other lipid-lowering drugs, such as omega-3 fatty acids, orlistat, and nicotinic acid, have been shown to have alone, or in combination with statins, a favorable effect on postprandial lipemia [62,171,173,174]. Pooled results have shown that ezetimibe, a selective cholesterol absorption inhibitor, can reduce fasting TG levels by approximately 7% [175,176]. However, the effect of ezetimibe alone or in combination with statins in postprandial lipemia needs to be evaluated [176,177]. Morgan et al. suggested that cholestyramine can significantly lower circulating postprandial TG levels [178]. Rimonabant, a selective cannabinoid1 receptor blocker, was shown to reduce TGs by 13.0 3.5% (repeated-measures method) [179]. Sibutramine was also shown to significantly reduce TG levels [180,181]. Nevertheless, the effects
of the above-mentioned drugs on postprandial lipaemia need to be assessed. CONCLUSIONS The long duration of the postprandial lipemia and the number of meals during the day leads to significant changes in lipoproteins postprandially. It is well-known that the remnants of TG-rich lipoproteins accumulated in the postprandial state are involved in atherogenesis and, therefore, the postprandial TG concentration may be an independent predictor of CHD. Improvement in fasting hypertriglyceridemia is the main feature associated with improvement in postprandial lipemia. The main effect of statins is the decrease of serum levels of LDL cholesterol upregulating LDL receptors. Nevertheless, all statins can lower TG levels although this effect is related to factors such as drug, dose and baseline TG levels. For example, the more effective the statin is at decreasing LDL cholesterol, the more effective it is at decreasing TG levels in patients with hypertriglyceridemia. However, trials designed for elucidating the effect of lipid-lowering compounds on postprandial lipemia in humans are still sparse. In some trials statins failed to induce a significant reduction of postprandial lipemia, whereas in others they resulted in reduced postprandial hypertriglyceridemia (Table 1). Nevertheless, definitive conclusions cannot be drawn, since the studies conducted in this field are few and include small and heterogeneous populations. Although statin monotherapy can benefit hypertriglyceridemic patients, an additional benefit may be obtained by combination therapy that provides greater reductions in TGs. However, combining a statin with either niacin or a fibrate may increase the risk for side effects and, therefore, requires careful follow-up and evaluation of the pros and cons for each patient. Co-administration of statin and fibrate therapy should be considered if monotherapy or adding other drugs (e.g. cholesterol absorption inhibitors, omega-3 fatty acids or possibly nicotinic acid) did not achieve lipid targets or is impractical. Combination treatment should be hospital-based and reserved for high-risk patients with a mixed hyperlipidaemia [149]. The role of other drugs with lipid-lowering effects alone or in combination with statins on postprandial lipemia needs to be further investigated. More clinical trials are needed to determine the optimal treatment in patients with postprandial hypertriglyceridemia. REFERENCES
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Received: May 10, 2006
Accepted: September 30, 2006
561
Lipid Management and Peripheral Arterial Disease

S.S. Daskalopoulou1,2, M.E. Daskalopoulos2, D.P. Mikhailidis3,* and C.D. Liapis2
1
Department of Medicine, Division of Clinical Epidemiology, McGill University, Montreal, Quebec, Canada; 2Department of Vascular Surgery, Athens University Medical School, Athens, Greece and 3Department of Clinical Biochemistry and Department of Surgery (Vascular Disease Prevention Clinics), Royal Free Hospital, Royal Free and University College School of Medicine (University of London), London, UK
Abstract: Peripheral arterial disease (PAD) is a common disorder usually associated with silent or symptomatic arterial disease elsewhere in the circulation and a cluster of cardiovascular risk factors (e.g. smoking, dyslipidemia, hypertension, and insulin resistance/diabetes mellitus). The medical management of PAD should focus on both the relief of symptoms and prevention of secondary cardiovascular complications. This approach must include smoking cessation, optimal cholesterol levels, blood pressure and glycemic control as well as prescribing antiplatelet therapy. This review focuses on the evidence supporting the use of lipid-lowering drugs in PAD. Several trials indicate that getting low density lipoprotein-cholesterol levels to target (<2.6 mmol/l; 100 mg/dl), or even lower, is associated with improvement of symptoms and a reduction in vascular events in patients with PAD.
Key Words: Peripheral arterial disease, risk factors, dyslipidemia, lipid lowering, statins, prevention. INTRODUCTION Peripheral arterial disease (PAD) is a common disorder affecting up to 20% of adults older than 55 years and is associated with silent or symptomatic arterial disease elsewhere in the vascular bed [1-5]. The majority of all patients with PAD are asymptomatic [1,6]. However, both symptomatic and asymptomatic patients with PAD carry a higher risk for vascular events [1-4,7-16]. This risk is so high that PAD is considered as a coronary heart disease (CHD) equivalent [7-9]. Therefore, patients with PAD need to have their modifiable vascular risk factors aggressively controlled. The management of PAD must include smoking cessation, control of hypertension and diabetes mellitus (DM), lipid-lowering treatment, antiplatelet agents, exercise/physical activity and other lifestyle measures [3,6,14,17-26]. Emerging risk factors, such as high levels of homocysteine, C-reactive protein (CRP), fibrinogen, interleukin (IL)-1, IL-6, creatinine, cystatin C, have been associated with PAD [5,6,17,27-37]. Furthermore, in addition to preventing vascular events, there is a need to establish if aggressive treatment can improve symptoms in PAD patients (e.g. claudication distance) [7,8,10-13,17,38]. Dyslipidemia ranks among the modifiable vascular risk factors that can be effectively managed [7-13]. Several trials clearly show that intervention with lipid-lowering drugs in high-risk patients results in a significant reduction in vascular events, including myocardial infarction (MI) and stroke [7-12,39]. Primary prevention trials (involving healthy subjects) also showed that the development of clinically evident CHD (and related events) was significantly reduced as a result of lipid-lowering treatment [7-12,39,40]. According to international guidelines, [7-9] patients with PAD should reach the low density lipoprotein-cholesterol (LDL-C) target [European LDL-C target <2.5 mol/l (96 mg/dl) and National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III LDL-C target <2.6 mmol/l (100 mg/dl)]. This is particularly important since these patients [3-15,17,23, 33,41-43]:
*Address correspondence to this author at the Department of Clinical Biochemistry (Vascular Disease Prevention Clinics), Royal Free Hospital, Royal Free and University College School of Medicine, (University of London), Pond Street, London NW3 2QG, UK; Tel: 0044 (0) 20 7830 2258; Fax: 0044 (0) 20 7830 2235; E-mail: MIKHAILIDIS@aol.com and mikhailidis@hotmail.com
have extensive (clinical or subclinical) vascular disease (e.g. CHD and/or carotid and/or renal artery involvement) and comorbidities, have a high risk of MI or stroke, often have a clustering of risk factors (e.g. smoking, hypertension, dyslipidemia, and/or DM). Furthermore, it is possible that progression to PAD may be prevented or slowed down by treating adverse lipid profiles. This review considers the evidence showing the effect of lipidlowering drugs on three aspects relating to PAD: Prevention of PAD, Improvement of symptoms associated with PAD, Reduction of the risk of vascular events (e.g. MI, stoke or revascularization) associated with PAD. PREVENTION OF PAD (TABLE 1) A post-hoc analysis of the Scandinavian Simvastatin Survival Study (4S) [44] found that new or worsening intermittent claudication was significantly (p = 0.008) reduced (by 38%) in those taking simvastatin when compared with the group assigned to placebo. All participants (n = 4,444) had CHD and were followed-up for a median of 5.4 years [44]. In the Program on the Surgical Control of Hyperlipidemias (POSCH) [45] the effect of cholesterol lowering induced by partial ileal bypass in patients (n = 838) with a previous MI and hyperlipidemia was assessed. The incidence of new cases of claudication was significantly (p = 0.009) lower in the intervention group when compared with the control (non-intervention) group four years after the formal closure of the trial [relative risk (RR): 0.66, 95% confidence interval (CI): 0.20-0.90] [45]. IMPROVEMENT OF SYMPTOMS ASSOCIATED WITH PAD (TABLE 2) As mentioned above, new or worsening intermittent claudication was significantly (p = 0.008) reduced (by 38%) in the active treatment group of the 4S trial [44]. In another study [46], patients (n = 354) with intermittent claudication were assigned to placebo or active treatment with atorvastatin 10 or 80 mg/day. Maximal walking time after 12 months of treatment did not change significantly. However, there was a significant (p = 0.025) improvement in pain-free walking time in the
1389-4501/07 $50.00+.00
Daskalopoulou et al.
Table 1.
Study
Trial-Based Evidence for Prevention of PAD

Treatment group Simvastatin 20 to 40 mg Partial ileal bypass Control group Placebo No intervention Number of patients 4,444 838 Treatment period/ follow-up Median: 5.4 years 4 years after formal closure of the trial Inclusion criteria CHD MI and hyperlipidemia Primary outcome p
4S44 (1998) POSCH45 (1996)
New or worsening PAD: reduced by 38% in the simvastatin group New PAD: reduced by 34% (RR: 0.66, 95%CI: 0.20-0.90) in the intervention group
0.0008 0.009
PAD: peripheral arterial disease, CHD: coronary heart disease, MI: myocardial infarction, RR: relative risk, CI: confidence interval
Table 2.
Trial-Based Evidence of Improved Symptoms Associated with PAD

Treatment group(s) Simvastatin 20 or 40 mg Atorvastatin 10 or 80 mg Control group Placebo Placebo Number of Patients 4,444 354 Treatment Period/ Follow-Up Median: 5.4 years 12 months Inclusion Criteria CHD PAD
Study 4S44 (1998) Mohler46 (2003)
Primary Outcome
New or worsening PAD: reduced by 38% in the simvastatin group Walking time: improvement in the atorvastatin 80 mg group vs placebo Walking distance: improvement in ambulatory activity with atorvastatin 10 and 80 mg Physical activity questionnaire
0.0008 0.025 0.011 NS 0.045 0.006 <0.001
McDermott47 (2003)
Statin use
Non-use
392
Cross-sectional analysis
PAD (ABPI <0.90) and absence of PAD (ABPI 0.9 and 1.50)
Walk performance (6 min): improved Walking velocity: faster Summary performance score: higher All changes in favor of statin use
Mondillo48 (2003)
Simvastatin 40 mg
Placebo
86
6 months
Pain-free walking distance: increased by 90 m (95% CI: 64-116) PAD and hyperlipidemia Total walking distance: increased by 126 m (95% CI: 101-151) ABPI at rest: increased by 0.09 (95% CI: 0.06-0.12) ABPI after exercise: increased by 0.19 (95% CI: 0.14- 0.24) Symptoms: improved All changes in favor of simvastatin
<0.005 <0.001 <0.01 <0.005 <0.05
Aronow49 (2003)
Simvastatin
Placebo
60
12 months
PAD and Simvastatin increased treadmill exercise time by 54 sec from baseline hyperlipidemia (24% increase) after 6-month treatment and by 95 sec (42% increase) after 1-year treatment PAD and non-PAD PAD patients using statins had less annual decline in usual-pace walking velocity (0.002 versus -0.024 m/s/year), rapid-pace walking velocity (-0.006 versus -0.042 m/s/year), 6-min walk performance (-34.5 versus -57.9 feet/year,), and the summary performance score (-0.152 versus -0.376) compared with non-users Among participants without PAD, there were no significant associations between statin use and functional decline
<0.0001 < 0.0001 0.013 0.006 0.088 0.067 NS 0.028
Giri50 (2006)
Statin use
Non-use
552
3 years
Shige51 (2001) Shinohara52 (2005) Leibovitz53 (2001)
Simvastatin 20 or 40 mg/day Atorvastatin 10 mg Titrated dose of atorvastatin to achieve their LDL-C target (<2.6 mmol/l; 100 mg/dl) Statin use
Placebo
20
Cross-over, 4 weeks each 6 months 20 weeks
PAD and hypercholesteremia PAD and DM type 2 PAD and hypercholesterolemia
Simvastatin improved arterial compliance by significantly reducing peripheral pulse wave velocity Significant reduction in femoral-ankle pulse wave velocity The small artery elasticity index was significantly improved
22 17
0.019 < 0.01
Henke54 (2004)
Non-use
293 (338 infrainguinal bypass procedures) 1,568
17 months
PAD and infrainguinal bypass
Statin therapy was associated with improved graft patency (OR 3.7, 95% CI 2.1-6.4) and limb salvage (decreased amputation rate) (OR 0.34, 95% CI 6.15-0.77) Bezafibrate reduced the severity of intermittent claudication for up to three years
<0.001 0.01 for 3 years: 0.02
LEADER55 (2002)
Bezafibrate 400 mg
Placebo
Median: 4.6 years
PAD
CHD: coronary heart disease, PAD: peripheral arterial disease, ABPI: ankle-brachial pressure index, CI: confidence interval, DM: diabetes mellitus, NS: non-significant, OR: odds ratio, CI: confidence interval.
Lipid Management and PAD
563
80 mg group compared with placebo (by 63% versus 38%, respectively) [46]. A physical activity questionnaire demonstrated improvement (p = 0.011) in ambulatory ability for the 10 and 80 mg groups, whereas two quality of life questionnaires did not show significant changes [46]. In a study [47] that included 392 men and women with an ankle brachial pressure index (ABPI) <0.90 and 249 with ABPI 0.90-1.50 the effect of statin use versus non-use was assessed. Adjusting for age, sex, ABPI, co-morbidities, cholesterol, and other confounders, those taking statins had a better walk performance (6 min) (p = 0.045), faster walking velocity (p = 0.006) and a higher summary performance score (p < 0.001) than participants not taking statins [47]. Positive associations were slightly attenuated after additional adjustment for CRP level but remained statistically significant for walking velocity and the summary performance score [47]. These findings suggest that both the cholesterol- and non-cholesterollowering actions of statins may favorably influence functioning in persons with and without PAD [47]. The effect of short-term therapy with simvastatin on walking performance was assessed in hypercholesterolemic (>5.7 mmol/l; 220 mg/dl) patients (n = 86) with PAD [48]. The patients were assigned to either simvastatin 40 mg (n = 43) or placebo (n = 43) [48]. At six months, the mean pain-free walking distance increased by 90 m (95% CI: 64-116; p < 0.005) more in the simvastatin than in the placebo group. Similar results were seen for the mean total walking distance (increased by 126 m; 95% CI: 101-151; p < 0.001) [48]. The ABPI at rest increased by 0.09 (95% CI: 0.06-0.12; p < 0.01) and after exercise by 0.19 (95% CI: 0.14-0.24; p < 0.005) in those taking simvastatin [48]. There was also a greater improvement in claudication symptoms among patients treated with simvastatin; the effects on walking performance, ABPI and questionnaire scores were also significant at three months [48]. In another study [49], simvastatin significantly increased the treadmill exercise time by 54 sec from baseline (24% increase, p <0.0001) after six-month treatment, and by 95 sec (42% increase, p < 0.0001) after one-year treatment; in contrast, no difference was noted in the placebo group. In a recent study, Giri et al. [50] evaluated the effect of statin use (versus non-use) on the annual decline in lower-extremity functioning in patients with and without PAD (n = 552) over three-year follow-up. PAD patients using statins had less annual decline in usual-pace walking velocity (0.002 versus -0.024 m/s/year, p = 0.013), rapid-pace walking velocity (-0.006 versus -0.042 m/s/year, p = 0.006), 6 min walk performance (-34.5 versus -57.9 feet/year, p = 0.088), and the summary performance score (-0.152 versus 0.376, p = 0.067) compared with non-users. In contrast, among subjects without PAD, there were no significant associations between statin use and functional decline [50]. In a smaller double-blind, cross-over randomized control trial (n = 20) PAD patients with hypercholesteremia were assigned to either simvastatin (20 or 40 mg/day) or placebo, each for 4 weeks [51]. Simvastatin improved arterial compliance by significantly (p = 0.028) reducing peripheral pulse wave velocity [51]. Similarly, atorvastatin 10 mg/day significantly (p = 0.019) decreased the femoral-ankle pulse wave velocity in PAD patients with type 2 DM (n = 22) treated for six months, resulting in improved stiffness of leg arteries and of walking performance [52]. Furthermore, the small artery elasticity index was significantly (p < 0.01) improved in patients (n = 17) with PAD and hypercholesterolemia (LDL-C >4.4 mmol/l; 170 mg/dl) treated with a titrated dose of atorvastatin for 20 weeks to achieve their LDL-C target (<2.6 mmol/l; 100 mg/dl) [53]. Henke et al. [54] reported that statin therapy was also associated with improved graft patency [odds ratio (OR) 3.7, 95% CI 2.16.4, p < 0.001] and limb salvage (decreased amputation rate) (OR
0.34, 95% CI 6.15-0.77, p = 0.01) in patients (n = 293) who underwent infrainguinal bypass. In the Lower Extremity Arterial Disease Event Reduction (LEADER) trial [55] bezafibrate 400 mg/day (n = 783 men) or placebo (n = 785 men) were compared (median follow-up 4.6 years). Bezafibrate reduced the severity of intermittent claudication for up to three years (for three years, p = 0.02) [55]. It is important that the presence of co-morbidities, inflammation, and lack of exercise significantly contribute to the functional decline in PAD patients [22,56,57]. REDUCTION OF THE RISK OF VASCULAR EVENTS ASSOCIATED WITH PAD (TABLE 3) The Heart Protection Study (HPS) [58] confirmed that statin treatment reduces the risk of death and adverse cardiovascular events in patients with coronary and non-coronary atherosclerosis, including patients with PAD but without diagnosed CHD (n = 2,701). There was a significant reduction (approximately 25%) in the first major vascular event (major coronary event, stroke or revascularization) among patients with PAD, with or without prior CHD (both p < 0.0001) [58]. In HPS there was a significant 16% (standard error 6.0, 95% CI 5.0-26.0) proportional reduction in the rate of non-coronary revascularization (4.4% versus 5.2%, p = 0.006) [58]. Half of that difference involved a definite reduction in carotid endarterectomy or angioplasty (0.4% versus 0.8%, p = 0.0003) [58]. Furthermore, a recent prospective observational cohort study of 2,420 patients with PAD followed-up for a median of 8 years showed that statins were associated with a reduction in long-term mortality [hazard ratio (HR) 0.46, 95% CI 0.36-0.58]; -blockers, aspirin and angiotensin converting enzyme inhibitors were also protective [59]. In the Mohler et al. study [46], mentioned above, there was a significant (p = 0.003) reduction in vascular events after 12 months treatment with atorvastatin (number needed to treat for one year to prevent one event 15). Only three events occurred in the 240 patients (1.3%) assigned to atorvastatin (10 and 80 mg/day), whereas nine events in the 114 patients (7.9%) assigned to placebo [46]. In a comparison of 318 PAD patients treated with statins versus 342 patients not on lipid-lowering drugs there were significant changes in the event rates after a mean follow-up of 39 months [60]. Sudden coronary death, fatal MI and new coronary events were all significantly reduced (p = 0.0005, p = 0.007, and p < 0.0001, respectively) [60]. Statins were also shown to decrease the risk of vascular events in PAD diabetic patients with and without prior MI [61]. Schillinger et al. [62] showed that statins were also associated with an improved survival of patients with severe PAD with elevated high-sensitivity CRP (hs-CRP) levels (>4.2 mg/l). Patients treated with statins had a lower level of inflammation (hs-CRP: p < 0.001, serum amyloid A: p = 0.001, fibrinogen: p = 0.007, albumin: p < 0.001, neutrophils: p = 0.049) than patients not treated with statins. It is interesting that patients with low inflammatory activity (hs-CRP 4.2 mg/l) had no significant benefit from statin therapy (p = 0.74 for survival; p = 0.83 for event-free survival), whereas in patients with high hs-CRP (>4.2 mg/l) statin therapy was associated with a significantly reduced risk for mortality (adjusted HR 0.58, p = 0.046) and the composite of MI and death (adjusted HR 0.46, p = 0.016) [62]. This observation supports the suggestion that statins may exert their beneficial effect through anti-inflammatory properties [62]. Furthermore, Ward et al. [63] in a retrospective analysis of consecutive infrainguinal vascular bypass surgeries (n = 446) showed that preoperative statin use was associated with fewer 30-day perioperative cardiac and vascular complications (OR 0.36, 95% CI
Table 3.
Trial Based Evidence of Decreased Vascular Events Associated with PAD

Treatment Period/ Follow-Up 5.0 years
Study
Treatment group(s)
Control group
Number of patients
Inclusion Criteria
Primary outcome
HPS58 (2002)
Simvastatin 40 mg
Placebo
2,701
PAD with or without CHD
First major event (major coronary, stroke, revascularization): In patients with CHD: reduction by 25% In pts without CHD: reduction by 25% Reduction in non-coronary revascularization by 16% All changes in favor of simvastatin Vascular events [worsening symptoms of claudication, development of rest ischemia, peripheral revascularization, other (carotid endarterectomy)]: reduction 1.3% in the atorvastatin groups (3 events/240 patients) vs 7.9% in the placebo group (9 events/114 patients)
<0.0001 <0.0001 0.006
Mohler46 (2003)
Atorvastatin 10 or 80 mg
Placebo
354
12 months
PAD
0.003
Aronow60 (2002)
Statin
No Lipidlowering
660
39 months
PAD and New coronary events hyperlipidemia (Sudden coronary death Fatal myocardial infarction Non-fatal myocardial infarction New coronary events) 48% incidence in the statin group 73% incidence in the control group PAD Patients on statins had better survival rates (adjusted HR 0.52) and event-free survival rates (adjusted HR 0.48) than patients not treated with statins
0.0005 0.007 0.079 <0.0001
Schillinger62 (2004)
Statin use
Non-use
515
21 months
0.022 0.004
Ward63 (2005)
Statin use
Non-use
446
Retrospective Infrainguinal Preoperative statin use was associated with fewer 30-day perioperative analysis vascular bypass cardiac and vascular complications (OR 0.36, 95% CI 0.14-0.93), surgeries a shorter length-of-hospital stay (OR 1.49, 95% CI 1.14-1.95), and improved long-term survival (OR 0.52, 95% CI 0.32-0.84) Median: 4.6 years PAD men CHD and stroke: bezafibrate did not significantly reduce the incidence of CHD and stroke combined but reduced non-fatal CHD (RR: 0.60; 95% CI: 0.36-0.99). In men <65 years, bezafibrate reduced all coronary events by 62% (RR: 0.38; 95% CI: 0.20-0.72)
0.035 0.003 < 0.004
LEADER55 (2001)
Bezafibrate 400 mg
Placebo
1,568
0.05
PAD: peripheral arterial disease, CHD: coronary heart disease, HR: hazard ratio, RR: relative risk.
0.14-0.93, p = 0.035), a shorter length-of-hospital stay (OR 1.49, 95% CI 1.14-1.95, p = 0.003), and improved long-term survival (OR 0.52, 95% CI 0.32-0.84, p < 0.004) after adjustment for significant baseline patient characteristics [63]. In the LEADER trial [55], bezafibrate had no effect on the incidence of CHD and of stroke combined. However, the incidence of non-fatal coronary events was reduced (RR 0.60, 95% CI 0.36-0.99, p = 0.05), particularly in those aged <65 years at entry in whom all coronary events may also be reduced (RR 0.38, 95% CI 0.20-0.72) [55]. In this trial, plasma fibrinogen levels fell by 13% (p < 0.0001) in the bezafibrate group [55]. It is well established that elevated plasma levels of this coagulation factor contribute to whole blood viscosity and to an increased risk of vascular events in patients with PAD [64]. FEMORAL ARTERY ANGIOGRAPHIC AND IMAGING STUDIES (TABLE 4) Several angiographic studies showed some regression in atherosclerosis following lipid-lowering treatment with a wide variety of drugs. These findings are in agreement with the clinically relevant benefits following the use of lipid-lowering drugs, as outlined above. Duffield et al. [65] demonstrated in an angiographic randomized control study involving patients (n = 24) with femoral atherosclerosis that after 19 months follow-up the mean increase in plaque area (mm2/segment/year) in the treatment group (dietary advice and cholestyramine, nicotinic acid or clofibrate) was only one third of that in the usual-care group (0.58 versus 1.72). Additionally, in the treated group significantly fewer segments showed detectable progression of the plaque, and twice as many arterial segments showed improvement (decrease in edge irregularity) (both, p < 0.05) [65].
In the Cholesterol-Lowering Atherosclerosis Study (CLAS-I) [66], colestipol and niacin treatment plus diet was compared with placebo plus diet in men (n = 162) with previous coronary bypass surgery. Two-year results showed a decreased progression and increased regression of femoral atherosclerosis (p = 0.02-0.04) [66]. In the POSCH study no appreciable differences were noted in the progression or regression of arteriographic PAD between the intervention and the control group [45]. However, this trial only showed a significant decrease in new PAD cases (see section dealing with prevention of PAD, above) [45]. A decrease in the progression of the femoral intima-media thickness (IMT) was also reported in the Kuopio Atherosclerosis Prevention Study (KAPS) trial (n = 447 men), where less than 10% of the subjects had prior MI and the remainder of the study population consisted of a primary prevention group [67]. All participants were hyperlipidemic and they were randomly assigned to either pravastatin 40 mg or placebo for 3 years [67]. The beneficial effect was more prominent for carotid artery IMT, especially in smokers [67]. The femoral artery changes were not significant [67]. A reduction in common femoral artery (CFA) IMT on ultrasound was reported in hyperlipidemic patients with PAD (n = 25) who were treated with atorvastatin 20 mg [68]. This difference achieved significance (p = 0.0003) after 8 weeks of treatment, but a trend was visible at 4 weeks [68]. In an earlier study [69], femoral atheroma was angiographically evaluated after one year of treatment with diet and nicotinic acid plus fenofibrate in 45 asymptomatic, hyperlipidemic, middle-aged men in a non-randomized controlled study. Progression was found in 24% and 40% in the treatment and control groups, respectively, whereas regression occurred in 29% and 0%, respectively [69].
565
Table 4.
Femoral Artery Angiographic and Imaging Studies

Treatment Period/ Follow-up 19 months
Study
Treatment group
Control group
Number of patients
Inclusion criteria
Primary outcome
Duffield65 (1983) CLAS-I66 (1991) POSCH45 (1996) KAPS67 (1995) Youssef68 (2002) Olsson69 (1990)
Dietary advice + cholestyramine, nicotinic acid, or clifibrate Colestipol-niacin-diet Partial Ileal bypass operation Pravastatin 40 mg
Usual-care
24
PAD and dyslipidemia CABG MI and hyperlipidemia
Femoral atherosclerosis: decreased progression and increased regression in the treatment group Femoral atherosclerosis: decreased progression and increased regression in the treatment group Progression or regression on PAD (arteriography)
0.05
Placebo-diet No intervention Placebo
162 838
2 years 4 years after formal closure of trial 3 years
<0.04 NS
447
Men mainly primary prevention (<10% prior MI) and hyperlipidemia PAD and hyperlipidemia Asymptomatic PAD and hyperlipidemia
Femoral IMT (ultrasound): effect on atherosclerotic progression CFA IMT (ultrasound): reduced Progression was found in 24% and 40% in the treatment and control groups, respectively, whereas regression occurred in 29% and 0%, respectively
NS
Atorvastatin 20 mg Diet + nicotinic acid + fenofibrate
_ No lipidlowering
25 45
8 weeks 12 months
0.0003
PAD: peripheral arterial disease, CABG: coronary artery bypass graft, MI: myocardial infarction, CFA: common femoral artery, IMT: intima-media thickness.
IS LOWER BETTER FOR CHOLESTEROL? Although statins can improve both clinical outcomes and symptoms in PAD it is possible that further benefit can be obtained if LDL-C levels are lowered beyond the current targets for high-risk patients (2.5-2.6 mmol/l; 96-100 mg/dl) [7-9]. Recent trials showed that lowering LDL-C levels to values below those indicated in the guidelines [7-8] results in a greater benefit, either in a surrogate marker or in CHD-related events. This new evidence led to a revision of the guidelines [9,70] that set the new optional target from <2.6 mmo/l (100 mg/dl) to <1.8 mmol/l (70 mg/dl) for very highrisk patients and <3.4 mmol/l (130 mg/dl) to <2.6 mmol/l (100 mg/dl) for moderately high-risk patients. These new targets are challenging for many patients in clinical practice. In the Reversal of Atherosclerosis with Aggressive Lipid Lowering (REVERSAL) study [71] the effect of pravastatin 40 mg versus atorvastatin 80 mg in patients (n = 502) with CHD was assessed. The objective was to compare the effect of these drugs on coronary artery atheroma burden and progression as measured by intravascular ultrasound (IVUS) after treatment for 18 months [71]. The reduction in LDL-C was, as expected, significantly (p = 0.001) greater with atorvastatin (from 3.9 to 2.0 mmo/l; 151 to 77 mg/dl) than with pravastatin (from 3.9 to 2.9 mmol/l; 151 to 112 mg/dl) [71]. There was significant disease progression in the pravastatin group (+2.7%; p = 0.001) and a non-significant (NS) regression in those on atorvastatin (0.4%; p = NS); this difference in disease progression was significant (p = 0.02) [71]. Furthermore, there was disease progression in the patients taking pravastatin, even if they reached the LDL-C goal (<2.6 mmol/l; 100 mg/dl) [7] for high-risk patients. In this group the mean ( standard deviation) LDL-C was 2.3 0.25 mmol/l (87.5 9.8 mg/dl) [71]. In the Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction 22 (PROVE ITTIMI 22) study [72] the same statins, pravastatin 40 mg and atorvastatin 80 mg, were compared in patients (n = 4,162) who were admitted with acute coronary syndrome (ACS) within the previous 10 days [72]. The mean study duration was 24 months. The fall in LDL-C was significantly (p = 0.001) greater with atorvastatin (from 2.7 to 1.6 mmol/l; 104 to 62 mg/dl) than with pravastatin (from 2.7 to 2.5 mmol/l; 104 to 96 mg/dl) [72]. The primary composite endpoint (death, MI, unstable angina, revascu-
larization or stroke) occurred in 26.3% and 22.4% of patients in the pravastatin and atorvastatin groups, respectively (hazard ratio: 16%, p = 0.005) [72]. The results started to diverge by 30 days [72]. The Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) trial [73] (atorvastatin 80 mg versus placebo, follow-up 16 weeks, n = 3,086) showed benefits (reduced recurrent fatal or non-fatal ischemic events) within 16 weeks in patients with ACS. In the HPS (Heart) [58] (n = 20,536 high-risk patients followed up for 5.0 years; simvastatin 40 mg versus placebo) those patients with a LDL-C <2.6 mmol/l (100 mg/dl) before receiving simvastatin benefited to the same extent as those who had higher baseline LDL-C values. In the Anglo-Scandinavian Cardiac Outcomes Trial Lipid Lowering Arm (ASCOT-LLA) [74] primary prevention trial (n = 10,305 patients followed up for 3.3 years; atorvastatin 10 mg versus placebo), the mean LDL-C achieved in the treatment arm was 2.3 mmol/l (89 mg/dl), a value below the LDL-C target set in the international guidelines at that time [7,8]. This was associated with a significant benefit in clinically relevant endpoints [74]. In the GREek Atorvastatin and Coronary-heart-disease Evaluation (GREACE) trial (n = 1,600 patients with CHD, mean followup 3.0 years) [75], forced titration with atorvastatin (10-80 mg) to the NCEP ATP III guidelines (2.6 mmol/l; 100 mg/dl) [7] was associated with significant benefits in clinically relevant endpoints when compared with usual care [75]. The Treating to New Targets (TNT) trial [76] compared the effect of atorvastatin 10 mg with atorvastatin 80 mg/day on the occurrence of a first major cardiovascular event (death from CHD, nonfatal non-procedure-related MI, resuscitation after cardiac arrest, or fatal or non-fatal stroke) in patients (n = 10,001) with stable CHD and LDL-C <3.4 mmol/l (130 mg/dl) (median of 4.9 years). The mean LDL-C levels were 2.0 mmol/l (77 mg/dl) in the atorvastatin 80 mg group, and 2.6 mmol/l (101 mg/dl) in the atorvastatin 10 mg group. A primary event occurred in 8.7% receiving atorvastatin 80 mg, as compared with 10.9% on atorvastatin 10 mg, representing an absolute reduction in the rate of major cardiovascular events of 2.2% and a 22% relative reduction in risk (HR 0.78, 95% CI 0.690.89, p < 0.001) [76]. However, a greater incidence of elevated liver function tests occurred in the atorvastatin 80 mg group.
The Incremental Decrease in End Points Through Aggressive Lipid Lowering (IDEAL) trial [77] showed that in patients (n = 8,888, median follow-up 4.8 years) with previous MI, intensive lowering of LDL-C with atorvastatin 80 mg did not result in a significant reduction in the primary outcome of major coronary events when compared with simvastatin 20 mg. However, intensive treatment reduced the risk of non-fatal acute MI (HR 0.83, 95% CI 0.710.98, p = 0.02) and other composite secondary endpoints (major cardiovascular events: HR 0.87, 95% CI 0.77-0.98, p = 0.02, and occurrence of any coronary event: HR 0.84, 95% CI 0.76-0.91, p < 0.001) [77]. However, patients in the atorvastatin group had higher rates of drug discontinuation due to non-serious side-effects; transaminase elevation resulted in 1.0% versus 0.1% withdrawals (p < 0.001) [77]. In contrast with the clinical benefit of aggressive treatment noted in the studies mentioned above, a relatively small fall (17%) in LDL-C in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) [78,79] (primary prevention study, pravastatin 40 mg versus placebo; n = 10,355, mean follow-up 4.8 years) did not produce any significant differences in the primary endpoint compared with the placebo group. There is also imaging evidence supporting the concept that lower is better as far as LDL-C is concerned. In the Arterial Biology for the Investigation of the Treatment Effects of Reducing Cholesterol (ARBITER) study [80], atorvastatin 80 mg was compared with pravastatin 40 mg in 161 patients (46% with known cardiovascular disease) treated for 12 months. The beneficial effect of atorvastatin was significant with respect to the LDL-C reduction (p < 0.001) and the decrease in carotid IMT (p = 0.03) [80]. The same group showed that the regression of carotid atherosclerosis was directly related to the absolute LDL-C level on statin therapy [81]. The greatest regression was obtained with an LDL-C <1.8 mmol/l (70 mg/dl) [81]. In the Atorvastatin versus Simvastatin on Atherosclerosis Progression (ASAP) study [82] the effect of atorvastatin 80 mg versus simvastatin 40 mg in the carotid IMT was evaluated in 325 patients with familial hypercholesterolemia. After 2 years treatment, the carotid IMT decreased (p = 0.0017) in the atorvastatin group but increased (p = 0.0005) in the simvastatin group [82]. The change in IMT differed significantly between the two groups (p = 0.0001), again indicating that aggressive treatment is more effective [82]. Therefore, we can conclude that less is less regarding LDL-C lowering [79]. Since there is a trend for the LDL-C target values to fall, combination treatment will become necessary to achieve these new goals and also avoid side-effects from the maximum dose of a statin. In this context, ezetimibe, a selective cholesterol transport inhibitor, could prove to be useful [83]. For example, in the Ezetimibe Add-on to Statin for Effectiveness (EASE) study (n = 3,030, follow-up 6 weeks) the addition of ezetimibe (10 mg/day) to a stable dose of a statin resulted in a further fall in LDL-C of 26%, while the reduction in the control group (statin + placebo) was only 3% (p < 0.001) [83]. Other combinations should be administered with caution, for example a statin with either niacin or a fibrate may increase the risk for side-effects and, therefore, requires careful follow-up and risk-benefit evaluation for each patient [84]. ADDITIONAL POTENTIAL ACTIONS OF LIPID-LOWERING DRUGS THAT MAY BENEFIT PAD PATIENTS Statins exert pleiotropic effects, which may be independent of their LDL-C lowering action [85-87]. These effects may appear even before a change in lipid level occurs [87]. A significant reduction of the carotid and femoral IMT was also reported early after statin treatment [85-87]. There is evidence that patients with PAD have endothelial cell injury [88,89]. Inflammation has been independently associated
with PAD [32]. Elevated plasma levels of von Willebrand factor and adhesion molecules [soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cellular adhesion molecule-1 (sVCAM-1)] and endothelial dysfunction have also been documented in PAD [88,89]. Statins have antioxidant properties and anti-inflammatory effects. They can increase nitric oxide (NO) production and improve endothelial function (e.g. increased flow-mediated dilatation) [8587]. Statins inhibit the secretion of several matrix metalloproteinases (MMPs) from both smooth muscle cells (SMCs) and macrophages and most of the statins dose-dependently decrease the migration and proliferation of SMC and macrophages; they can also reduce cholesterol accumulation in macrophages [90,91]. These effects could contribute to plaque-stabilization [90,91]. The anti-inflammatory effects of statins include reduction in the circulating levels of CRP, inflammatory and proinflammatory cytokines (e.g. IL-6, IL-8), adhesion molecules (e.g. ICAM-1, VCAM1) and other acute phase proteins [85-87]. Another atheroprotective effect of statins might be exerted through a decrease in the levels of platelet-derived T-lymphocyte-stimulating soluble CD40 ligand [92]. There is growing evidence that hemostatic factors are involved in the development and progression of atherosclerosis and its complications [93-95]. Lipid-lowering drugs exert beneficial effects on the arterial wall, endothelial function, blood rheology and thrombogenesis [93-95]. Coagulation parameters are altered in PAD [64,96]. This may play a role in the susceptibility to thrombotic complications. Statins can reduce tissue factor expression and platelet activity, whereas fibrinolysis can be enhanced [85-87]. Statins can also decrease plasma fibrinogen levels [62]. Fibrates can also significantly decrease plasma fibrinogen levels and inhibit tissue factor expression and activity in human monocytes and macrophages [97]. In the LEADER trial plasma fibrinogen fell by 13% in the bezafibrate group [55]. Furthermore, in the ADMIT trial, niacin treatment of PAD patients resulted not only in a favourable alteration of their lipid profile, but also in a significant decrease in both fibrinogen (p < 0.001) and prothrombin fragment1+2 (p = 0.04), an indicator of thrombin generation [96,98]. In this study, von Willebrand factor increased after antioxidant vitamin treatment (p = 0.04), while low-dose warfarin resulted in a significant decrease in factor VIIc (p < 0.001) and plasma prothrombin fragment1+2 (p = 0.001) in PAD patients [96]. Statins can improve microalbuminuria, renal function, hypertension and arterial wall stiffness [37,85-87]. PAD is not only associated with higher circulating creatinine levels and chronic kidney disease [35,41,99] but also predicts an increase in creatinine levels over time [100,101]. Hypertensive patients with PAD tend to have more pronounced hyperuricemia (p = 0.01) than hypertensive patients without PAD [102]. Hyperuricemia is also associated with worse functional status of the peripheral circulation [102]. Elevated urate levels may also predict the risk of vascular events [102]. Some statins and fenofibrate can decrease serum uric acid levels [103-105]. In the GREACE trial [106,107], the effect of atorvastatin on serum creatinine and uric acid was rapid and it was reversed equally rapidly if patients happened to discontinue atorvastatin. The fall in creatinine was greater in both those with higher baseline serum levels of this indicator of the glomerular filtration rate and at the higher doses of atorvastatin [106,107]. Atorvastatin 20 mg/day improved renal function in PAD patients with hyperlidemia; a significant reduction in serum creatinine, an increase in calculated clearance creatinine and a significant decrease in serum cystatin C levels were found after treatment for 8 weeks [37]. Simvastatin also significantly reduced serum creatinine and uric acid levels (p < 0.0001) in patients with PAD treated for 3-4 months [108]. The
567
difference in the creatinine levels was more pronounced in the tertile of patients with the highest baseline creatinine levels [108]. CRP is a predictor of the development of PAD [31,109]. Ridker et al. [31] showed that CRP was the strongest non-lipid independent predictor (RR for the highest versus lowest quartile 2.8, 95% CI 1.3-5.9) of development of symptomatic PAD. CRP provided additive prognostic information over standard lipid measures significantly improving risk prediction models based on lipid screening alone (p < 0.001). PAD patients with increased CRP levels had a higher risk for cardiovascular events [33]. Furthermore, inflammation may play a role in functional decline not only in patients with PAD but also in subjects without PAD [56]. Statins can reduce circulating CRP levels [110-113]. Thus, the beneficial effect of statins may include, beyond their lipid-lowering effect, the inhibition of the inflammatory processes of atherosclerosis [59,114]. In fact, it has been shown that a reduction in the inflammatory component through the statin use can improve the clinical outcome in patients with CHD, regardless of the reduction in cholesterol levels [71,72]. In the REVERSAL trial [71,115], CRP fell by 36.4% with atorvastatin but only by 5.2% with pravastatin (p < 0.001). Furthermore, in a subanalysis of the REVERSAL trial, patients with reductions in both LDL-C and CRP that were greater than the median had significantly slower rates of progression than patients with reductions in both biomarkers that were less than the median (p = 0.001) [116]. In the PROVE-IT trial [72] the reduction in CRP was also significantly greater in the atorvastatin when compared with the pravastatin group. In a subanalysis of the PROVE-IT trial [117], patients with low post-treatment CRP levels had better clinical outcomes than those with higher CRP levels, regardless of the resultant level of LDL-C. In a post-hoc analysis, patients who had LDL-C and CRP levels of less than the median values [1.8 mmol/l (70 mg/dl) and 1.0 mg/l, respectively] after statin therapy (81.8% of patients in this subgroup had been assigned to receive atorvastatin) had the lowest rate of recurrent events (1.9/100 person-years) when compared with those with levels above the median (4.5/100 personyears, p < 0.001) [117]. Other lipid-lowering drugs (e.g. ciprofibrate [118], fenofibrate [119,120], and ezetimibe when given in combination with a statin [121-123]) can also decrease circulating CRP levels. It has been also proposed that LDL-apheresis might have anti-inflammatory effects and improve PAD [124]. However, the potential relevance of lowering CRP levels to a greater or lesser extent remains unclear. DM is highly associated with PAD and its progression [2,3,15, 20,21,125]. Colhoun et al. in the Collaborative Atorvastatin Diabetes Study (CARDS) evaluated the effect of atorvastatin 10 mg/day in patients (n = 2,838) with type 2 DM without a history of cardiovascular disease, with LDL-C 4.2 mmol/l (160 mg/dl) and fasting triglyceride levels 6.8 mmol/l (616 mg/dl), and at least one of the following: retinopathy, albuminuria, current smoking, or hypertension [40]. In a post-hoc analysis, the effect of atorvastatin was rapidly apparent (from six months), and at one year it was similar to the 37% relative risk reduction of any major vascular event observed at the end of the trial (median follow-up 3.9 years) [40,126]. Furthermore, patients with the metabolic syndrome have a clustering of many risk factors, such as hypertension, insulin resistance/type 2 DM, dyslipidemia and obesity [127,128]. Therefore, these patients are at increased risk for developing PAD [129-131]. The prevalence of the metabolic syndrome in PAD patients in a cross-sectional survey was 58% [129]. In the same survey it was reported that in patients with manifest vascular disease the presence of the metabolic syndrome is associated with advanced vascular damage [130]. Future studies may throw more light in the role of conventional and emerging risk factors in the development of atherosclerosis and
their associations as well as the role of lipid-lowering drugs in their management [132]. ARE ALL STATINS THE SAME? Statins are of proven efficacy in secondary prevention in highrisk groups such as PAD patients (see above). However, we do not know if all statins are equally effective. This question can only be answered by head to head comparison trials that incorporate clinical endpoints. Potential differences between statins can conveniently be considered in the context of: LDL-C lowering potency, and, pleiotropic actions So far, head to head comparisons clearly show that some statins are considerably more potent, in terms of LDL-C lowering, than others [71,72,115]. However, potency must not be considered in the decision-making process at the expense of safety and event-based evidence. What is less clear is if statins differ in properties that may not be exclusively due to differences in their LDL-C lowering capacity, the so-called pleiotropic actions [71,72,115,133]. CONCLUDING COMMENTS As the aging population increases as are life expectancy and the incidence of DM and the metabolic syndrome, the incidence/ prevalence of PAD is expected to also increase. However, the disease remains underdiagnosed and undertreated despite their high risk for vascular events [12,17,24,134-142]. Since most patients are asymptomatic and carry potentially significant morbidity and mortality risks, screening for PAD should become routine practice at primary care level. Given the overwhelming evidence on the known benefits of cardioprotective medications, their underuse remains puzzling in a population at high risk. Other treatment options (e.g. certain nutrients and rimonabant) might also prove useful in the future for PAD patient [143-145]. A recent meta-analysis of more than 90,000 subjects at high risk for vascular events showed that statins can reduce the 5-year incidence of major vascular events by 21% (RR 0.79, 95% CI 0.770.81, p < 0.001) per 1 mmol/l (38 mg/dl) reduction in LDL-C level, irrespective of the initial lipid profile and other baseline characteristics [39]. There is compelling trial-based evidence showing that lipidlowering treatment is beneficial in patients with PAD in terms of both primary and secondary prevention. Moreover, there is growing evidence suggesting that lipid-lowering treatment can improve PAD-related symptoms and functioning. Furthermore, preoperative use of statins in PAD patients may be associated with a reduction in perioperative mortality and morbidity (this topic has reviewed elsewhere) [146]. There is compelling evidence suggesting that the time has come for those looking after PAD patients to use aggressive preventive treatment. REFERENCES
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Received: June 20, 2006
Accepted: November 25, 2006

Editorial: Editorial Current Drug Targets, 2007, Vol. 8, No. 4 491

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Editorial

Current Drug Targets, 2007, Vol. 8, No. 4

Current Drug Targets, 2007, 8, 493-500

The Apoptotic Pathway as a Therapeutic Target in Sepsis

Doreen E. Wesche-Soldato, Ryan Z. Swan, Chun-Shiang Chung and Alfred Ayala*

494 Current Drug Targets, 2007, Vol. 8, No. 4

Apoptosis as a Target in Sepsis

Current Drug Targets, 2007, Vol. 8, No. 4

496 Current Drug Targets, 2007, Vol. 8, No. 4

Apoptosis as a Target in Sepsis

Current Drug Targets, 2007, Vol. 8, No. 4

498 Current Drug Targets, 2007, Vol. 8, No. 4

Potential Therapeutic Targets in the Apoptotic Pathway

Apoptosis as a Target in Sepsis

Current Drug Targets, 2007, Vol. 8, No. 4 [32]

[33] [34] [35]

[2] [3] [4]

[5] [6] [7]

[10] [11] [12] [13]

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[55] [56] [57] [58]

[59] [60] [61] [62] [63] [64]

[28] [29] [30] [31]

Received: March 22, 2006

Accepted: May 20, 2006

Current Drug Targets, 2007, 8, 501-507

Endothelium as a Therapeutic Target in Sepsis

502 Current Drug Targets, 2007, Vol. 8, No. 4

Endothelium as a Therapeutic Target in Sepsis

Current Drug Targets, 2007, Vol. 8, No. 4

504 Current Drug Targets, 2007, Vol. 8, No. 4

Endothelium as a Therapeutic Target in Sepsis

Current Drug Targets, 2007, Vol. 8, No. 4

Hyperthermia Hyperglycemia Cytokines TNFIL-1, 6, 8 Chemokines PAF MCP-1 Serine proteases

[45] [35, 46, 47] [12, 48] [49]

[50-52] [53, 54] [11, 20]

[55, 56] [57-59] [60-62] [63] [64, 65]

[37, 69, 70]

506 Current Drug Targets, 2007, Vol. 8, No. 4

[13] [14] [15]

Current Drug Targets, 2007, Vol. 8, No. 4

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[62] [63] [64] [65] [66] [67]

[43] [44] [45]

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[54] [55] [56]

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Received: March 22, 2006

Accepted: May 20, 2006

Current Drug Targets, 2007, 8, 509-514

Novel Therapies for Microvascular Permeability in Sepsis

510 Current Drug Targets, 2007, Vol. 8, No. 4

Jacobson and Garcia

Novel Therapies for Microvascular Permeability in Sepsis

Current Drug Targets, 2007, Vol. 8, No. 4