ADVANCES IN VASCULAR BIOLOGY
Does endothelial dysfunction contribute to the
clinical status of patients with peripheral arterial
disease?
Joseph A Vita MD, Naomi M Hamburg MD
JA Vita, NM Hamburg. Does endothelial dysfunction contribute to
the clinical status of patients with peripheral arterial disease? Can J
Cardiol 2010;26(Suppl A):45A-50A.
Peripheral arterial disease leads to lower extremity ischemia and limb loss,
and is linked to cardiovascular events. The anatomical extent of lower
extremity atherosclerosis fails to fully explain ischemic symptoms or pre-
dict the development of critical limb ischemia. Endothelial dysfunction is
known to contribute to the pathogenesis and clinical expression of coro-
nary artery disease, but the importance of endothelial dysfunction in
peripheral arterial disease remains incompletely understood. Endothelial
dysfunction could contribute to lower extremity ischemia by impairing
blood flow responses to ischemia, collateral formation and arterial remod-
elling, and by promoting vasospasm, thrombosis, plaque rupture and lesion
progression. There is a need for additional studies examining the contribu-
tion of endothelial dysfunction to the pathogenesis of peripheral arterial
disease, and the potential role of endothelial dysfunction as a surrogate
marker with utility in the management of patients.
Key Words: Critical limb ischemia; Endothelium; Inflammation; Nitric oxide;
Peripheral arterial disease
PERIPHERAL ARTERIAL DISEASE
Atherosclerotic peripheral arterial disease (PAD) is a major public
health problem that affects approximately eight million Americans
and up to 20% of individuals older than 70 years of age (1-4). PAD
produces considerable morbidity and health care expense because of
intermittent claudication, critical limb ischemia and amputation. In
addition, patients with PAD have a four- to sixfold increased risk for
cardiovascular events (2), making detection and management of PAD
an important public health priority.
PAD is associated with classical as well as more recently recognized
cardiovascular risk factors (2,5,6). Accordingly, risk reduction therapy
is the primary approach to management, particularly smoking cessa-
tion and lipid-lowering therapy. In randomized trials, statin treatment
has been shown to improve claudication and prevent cardiovascular
events (3,7,8). Current guidelines also recommend aggressive treat-
ment of other risk factors such as diabetes mellitus and hypertension to
slow progression of PAD and to reduce coronary risk (3,9). It is impor-
tant to point out, however, that risk factors for PAD are not identical
to the risk factors for coronary artery disease (CAD) and stroke, sug-
gesting that there may be differences in pathophysiology (10,11).
Other treatments for PAD have gained acceptance. In particular, anti-
platelet therapy reduces the risk for cardiovascular disease events and graft
occlusion (3,12-14). For patients with intermittent claudication, treat-
ment with the phosphodiesterase-3 inhibitor cilostazol (15) improves
walking distance, possibly due to vasodilator and antiplatelet effects.
Exercise programs particularly reduce symptoms and improve quality of
Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, USA
Correspondence: Dr Joseph A Vita, Section of Cardiology, Boston Medical Center, 88 East Newton Street, Boston, Massachusetts 02118, USA.
Telephone 617-638-8742, fax 617-638-8765, e-mail jvita@bu.edu
Received for publication July 14, 2009. Accepted September 25, 2009
Can J Cardiol Vol 26 Suppl A March 2010
La dysfonction endothliale contribue-t-elle
ltat clinique des patients atteints dune
maladie artrielle priphrique ?
Les maladies artrielles priphriques entranent une ischmie des membres
infrieurs et une perte des membres et sont lies la coronaropathie.
Ltendue anatomique de lathrosclrose des membres infrieurs nexplique
pas tout fait les symptmes ischmiques et ne permet pas de prdire
lapparition dune ischmie critique des membres. On sait que la dysfonction
endothliale contribue la pathogense et lexpression clinique des
coronaropathies, mais on comprend mal limportance de la dysfonction
endothliale en cas de maladie artrielle priphrique. La dysfonction
endothliale pourrait contribuer une ischmie des membres infrieurs en
nuisant la rponse du dbit sanguin lischmie, la formation collatrale
et au remodelage artriel et en favorisant le spasme vasculaire, la
thrombose, la rupture de la plaque et la progression des lsions. Dautres
tudes sont ncessaires pour examiner lapport de la dysfonction endothliale
sur la pathogense des maladies artrielles priphriques ainsi que le rle
potentiel de la dysfonction endothliale comme marqueur de substitution
utile pour la prise en charge des patients.
life (1,16,17). Relevant to the present review, improved endothelial con-
trol of vascular tone, arterial remodelling, collateral formation and inflam-
mation are believed to be important mechanisms accounting for the
benefits of exercise, statins and other risk reduction therapies (1,17-20).
Revascularization is indicated for uncontrolled symptoms and criti-
cal limb ischemia (3,21-23). Percutaneous revascularization is useful,
particularly in the iliac arteries, although it is interesting that exercise
training may be equally effective (24). Long-term results in more
distal vessels have been disappointing despite the availability of drug-
eluting stents (16,23). Surgical revascularization with bypass grafting
decreases symptoms and prevents limb loss in selected patients; how-
ever, surgical procedures carry a risk of procedural cardiac events and
have a significant failure rate over time (21,22,25,26). Overall, only
45% of patients with critical limb ischemia are alive with two limbs
at one-year follow-up (3,16). Thus, there is a need to develop new
approaches for management. In this regard, it is important to consider
the mechanisms leading to intermittent claudication and critical limb
ischemia.
Pathogenesis of ischemia and limb complications in PAD
Leg ischemia in PAD has traditionally been attributed to obstruction
of blood flow by fixed atherosclerotic lesions. Classical claudication,
with exercise-induced leg pain that is relieved by rest, could largely be
explained by such a demand ischemia model. Critical limb ischemia
usually occurs in patients with multilevel atherosclerosis and could
reflect gradual progression of atherosclerotic lesions.
2010 Pulsus Group Inc. All rights reserved 45A
Vita and Hamburg
Figure 1) Potential links between endothelial dysfunction and the clinical
expression of peripheral arterial disease (PAD). COX-2 Cyclooxygenase-2;
CRP C-reactive protein; eNOS Endothelial nitric oxide synthase; FMD
Flow-mediated dilation; ICAM-1 Intercellular adhesion molecule-1; IL-1
Interleukin-1-beta; MCP-1 Monocyte chemoattractant protein-1; NFB
Nuclear factor kappa B; ox-LDL Oxidized low-density lipoprotein; RH
Reactive hyperemia; ROS Reactive oxygen species; TNF- Tumor necrosis
factor-alpha
Multiple lines of evidence, however, suggest that the pathogenesis
of limb ischemia in PAD is more complex. First, severe disease may be
present in asymptomatic patients (3,27,28). Second, intermittent
claudication is a poor predictor of revascularization and limb amputa-
tion (29-31). For example, only 15% to 20% of patients with intermit-
tent claudication develop critical limb ischemia and only 10% require
amputation (32). Third, the anatomical severity of obstruction does
not predict clinical status. Thus, ankle-brachial index correlates
poorly with walking distance (33) and is a poor predictor of progressive
limb ischemia, revascularization or cardiovascular events (30,34-36).
Furthermore, the observed benefits of vasodilators, antiplatelet agents
and exercise in PAD fit poorly with a demand ischemia model. In addi-
tion, interventions such as statin therapy and exercise act too quickly
for meaningful regression to have occurred (1,8).
These findings raise the possibility that disturbances of vascular
function may contribute to clinical status in PAD. Indeed, a number of
investigators have proposed that endothelial dysfunction, plaque rup-
ture, thrombosis and impaired collateral formation may all be impor-
tant (3,37-39). While the role of such mechanisms in the coronary
circulation for both stable angina and acute coronary syndromes has
been well established (20,40-42), their specific role in PAD remains
uncertain.
Endothelial dysfunction and CAD
As has been reviewed by the authors and others, the vascular endothe-
lium is a primary regulator of vascular homeostasis (20,42-44). Nitric
oxide and other endothelium-derived vasodilators regulate blood flow
and maintain appropriate arterial tone (45-47). Nitric oxide also
46A
blunts platelet aggregation, proliferation of vascular smooth muscle
and leukocyte adhesion, and is required for appropriate arterial remod-
elling and collateral development (48-50). In pathological states, loss
of nitric oxide and increased production of vasoconstrictors, such as
endothelin, limits blood flow and contributes to ischemia (51-53).
Under pathological conditions, the endothelium may also be
activated to express prothrombotic and proinflammatory factors,
including tissue factor, adhesion molecules, chemotactic factors and
cytokines (54-58) (Figure 1). In general, cardiovascular risk factors are
associated with an alteration of endothelial phenotype that promotes
atherosclerosis (42). Importantly, these observations have been trans-
lated to humans, and it is well known that patients with risk factors
and CAD have impaired endothelium-dependent dilation. However,
the contribution of an activated proinflammatory phenotype to
human disease is less well studied.
Endothelial dysfunction is firmly linked to the pathogenesis of
CAD and acute coronary events. On average, endothelium-dependent
vasodilation is lower in coronary disease patients than in controls
(20,42,59). It is important to note, however, that endothelial func-
tion varies widely even among patients with disease. Those with more
severely impaired endothelial function are more likely to have myo-
cardial ischemia on ambulatory electrocardiogram monitoring (60)
and have increased risk for myocardial infarction, unstable angina and
coronary death (61-67). Interventions proven to reduce cardiovascu-
lar risk also reverse endothelial dysfunction (20), and failure of the
endothelium to respond to therapy identifies patients at higher risk
(68,69). Thus, there is strong evidence linking endothelial dysfunc-
tion to CAD. However, it remains unknown whether endothelial
dysfunction contributes to clinical ischemia in the lower extremities
of patients with PAD.
Endothelial dysfunction and PAD
Endothelial dysfunction could contribute to clinical status in PAD in
a number of ways. Loss of nitric oxide in skeletal muscle microvessels
blunts the hyperemic responses to exercise and ischemia, which would
be expected to limit oxygen delivery under conditions of increased
demand (45-47). As in epicardial coronary arteries, loss of nitric oxide
in the lower extremities could worsen the vasoconstrictor effects of
catecholamines and impair flow-mediated dilation, which might
worsen stenosis severity and increase resistance to blood flow during
exercise (19,70-75). Importantly, such abnormalities can be reversed
over weeks to months with risk reduction therapy even if lesion regres-
sion does not occur (17,19,20,73,76). This time course corresponds to
improvement of claudication symptoms and reduction in cardiovascu-
lar events during intervention studies.
As described by Glagov et al (77), outward remodelling helps
maintain a patent arterial lumen as atherosclerotic plaques increase in
size (77). Experimental studies have shown that compensatory remod-
elling depends on the activity of nitric oxide synthase (49,78,79), and
the authors recently demonstrated that the remodelling response is
blunted in patients with local endothelial dysfunction (80). Chronic
and recurrent limb ischemia stimulates compensatory collateral forma-
tion (81-83) and this response depends on the bioavailabity of nitric
oxide (84-89). Thus, endothelial dysfunction may worsen clinical sta-
tus in PAD because of impaired collateral formation and remodelling.
Endothelial activation with expression of proinflammatory and
prothrombotic factors may also contribute to ischemia by promoting
plaque rupture (39,41). Studies completed by the authors and others
have shown that endothelial dysfunction predicts acute coronary
events in PAD patients, suggesting a relation with plaque rupture in
the coronary circulation (65-67,90). Plaque rupture in the lower
extremities would produce an acute reduction in blood flow, and this
mechanism has been proposed to contribute to the development of
critical limb ischemia (39); the observed benefits of antiplatelet ther-
apy supports this concept (3,12-14). Furthermore, thrombus reorgani-
zation following subacute rupture has been proposed as a mechanism
for lesion progression (39,91-93).
Can J Cardiol Vol 26 Suppl A March 2010

Consistent with these potential mechanisms, several small cross-
sectional studies have demonstrated loss of nitric oxide bioavailability
in patients with PAD. For example, patients with PAD have blunted
vasodilator responses to acetylcholine (94,95) and impaired endothelium-
dependent flow-mediated dilation of the brachial artery (96-98). Flow-
mediated dilation also has been reported to correlate inversely with
ankle-brachial index (98-100). Urine nitrate and cyclic GMP levels
are reduced in patients with PAD, suggesting decreased total body
nitric oxide production (101). Polymorphisms of nitric oxide synthase
genes correlate with ankle-brachial index (102). Finally, PAD is associ-
ated with increased endothelin and plasminogen activator inhibitor-1
(103-105). These studies support a link between endothelial dysfunc-
tion and PAD, but do not address the question of whether endothelial
dysfunction contributes to clinical status among PAD patients.
Inflammation and endothelial dysfunction in PAD
Atherosclerosis is a chronic inflammatory disease (40,57,58) and, not
surprisingly, studies suggest systemic inflammation in PAD. For exam-
ple, monocyte count (106), C-reactive protein and soluble intercellu-
lar adhesion molecule-1 predict future development of PAD
(6,106,107). Polymorphisms in genes related to inflammation are also
associated with PAD (108). Patients with critical limb ischemia have
immune activation (109,110) and increased blood and tissue levels of
inflammatory markers (111,112), although it is difficult to determine
whether inflammation is a cause or consequence of ischemia in this
setting. Indeed, inflammatory markers decrease and endothelial func-
tion improves following revascularization or limb amputation
(112,113). Interestingly, C-reactive protein correlates with functional
status of PAD patients, as measured by 6 min walk distance and 4 m
walking speed (114,115). Furthermore, an increase in C-reactive pro-
tein over time is associated with a decrease in 6 min walk distance
(116).
The vascular endothelium is affected by and contributes to the
inflammatory process (55). Endothelial cells can be activated by vari-
ous relevant factors, including interleukin-1-beta (IL-1), tumor
necrosis factor-alpha (TNF-), oxidized LDL (ox-LDL), and C-reactive
protein to promote an atherogenic phenotype (55,117-119). The acti-
vated endothelium, in turn, expresses adhesion molecules, chemotac-
tic factors, cyclooxygenase-2 and other factors that accelerate the
inflammatory process. An important consequence of endothelial acti-
vation is loss of the biological activity of nitric oxide. In this regard,
TNF-, C-reactive protein, and ox-LDL have been shown to decrease
expression of endothelial nitric oxide synthase (55,117,119,120). In
addition, the proinflammatory and pro-oxidant milieu associated with
endothelial activation may lead to oxidative consumption of nitric
oxide, modification of nitric oxide enzymatic targets and uncoupling of
the enzyme to increase oxidative stress (121,122). Investigators have
argued that a broad alteration of endothelial function, including loss of
nitric oxide under proinflammatory conditions, may be a critical mecha-
nism that links systemic inflammation to atherosclerosis (119).
A number of studies suggest that endothelial activation impairs
nitric oxide bioavailability in humans. For example, vaccination, infu-
sion of endotoxin or TNF-, and in situ exposure of human veins to
cytokines have all been shown to acutely impair endothelium-depen-
dent vasodilation (123-126). Conversely, anti-inflammatory therapy
improves endothelium-dependent vasodilation in models of acute
inflammation (125,127) and in patients with rheumatoid arthritis
(128-130).
The transcription factor nuclear factor kappa B (NFB) controls
expression of a panel of proinflammatory genes and plays a critical role
in the endothelial response to immune activation and cytokine stimu-
lation (118,131). Activation of NFB involves phosphorylation and
subsequent degradation of the inhibitory subunit IB- by IB kinase,
which allows translocation of the regulatory subunits p50 and p65 to
the nucleus (132). In humans, increased expression of p65 and
decreased abundance of IB- has been observed in endothelial cells
isolated from older and obese individuals, and these findings relate to
Can J Cardiol Vol 26 Suppl A March 2010
Endothelial dysfunction and PAD
impaired endothelium-dependent vasodilation (133-135). The anti-
inflammatory drug salsalate has been shown to inhibit IB kinase, and
thus limit activation of NFB (132,136). Treatment of obese patients
with salsalate has been shown to reduce expression of p65, increase
detectable IB- and improve endothelium-dependent vasodilation
(137), but the effects of anti-inflammatory therapy in PAD are
unknown.
CONCLUSIONS AND IMPLICATIONS
PAD is an important public health problem because of the morbidity
associated with lower extremity ischemia and limb loss, and because
PAD is linked with increased risk for CAD. The anatomical extent
of atherosclerosis and a demand model of ischemia fail to fully
explain ischemic symptoms in PAD or predict the development of
critical limb ischemia. Endothelial dysfunction, particularly loss of
endothelium-dependent nitric oxide and activation of the endothe-
lium to a proinflammatory state, has convincingly been shown to
play a role to the clinical expression of CAD. The pathogenesis and
clinical manifestations of PAD and CAD are not identical, and the
contribution of endothelial dysfunction to clinical status and lower
extremity ischemia in PAD is incompletely understood. There is a
need for studies examining the relative importance of the anatomical
extent of atherosclerosis and endothelial dysfunction as determi-
nants of clinical status in PAD and there is a need for investigation
of whether endothelial dysfunction can serve as a surrogate for clini-
cal status in intervention trials. We suggest that such studies would
improve our understanding of the pathogenesis of clinical ischemia
in human PAD and may provide new information about novel
approaches to management.
ACKNOWLEDGEMENTS: Dr Vita is supported by grants from the US
National Institutes of Health (HL083269, HL083801, HL081587, and
HL75795) and the Boston University Medical Center Clinical and
Translational Science Institute (1UL1RR025771). Dr Hamburg is sup-
ported by the Boston University Leadership Program in Vascular Medicine
(K12 HL083781). The authors have no conflicts of interest to declare.
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