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The Emergence of New Therapeutic Targets in Pulmonary Arterial Hypertension

From Now to the Near Future
Simon Malenfant, Guillaume Margaillan, Jrmy Edwin Loehr, Sbastien Bonnet, Steeve Provencher Expert Rev Resp Med. 2013;7(1):43-55.

Abstract and Introduction

Abstract

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease that pathologically increases pulmonary vascular resistance. Ultimately, this leads to right ventricular failure and premature death. Current therapeutic strategies are mainly designed to induce relaxation of the pulmonary arteries, but are not directly aimed to improve vascular remodeling that characterize PAH. Although these treatments modestly improve patient symptoms, pulmonary hemodynamics and survival, none of them are curative and approximately 15% of patients die within 1 year of medical follow-up despite treatment. Within the last 5 years, tremendous advances in our understanding of the PAH pathophysiology have arisen. These advances have a high potential for the development of better patient care by providing novel therapeutic targets. The goal of this report is to review the current PAH treatments, as well as novel therapies that will pave the future in this devastating disease.
Introduction

Pulmonary arterial hypertension (PAH) is characterized by an intense pulmonary vascular remodeling leading to the progressive increase in pulmonary vascular resistance (PVR), right heart failure and eventually death. PAH may be idiopathic or associated with various conditions. It predominantly affects young adults,[1] and its prevalence is estimated at 50 persons per million.[2,3] Over the last two decades, the authors have seen major advances in our understanding of PAH pathology. As a result, the first PAH-specific therapies targeting the endothelial dysfunction were developed and commercialized, leading to improvement in patients' pulmonary hemodynamics, exercise capacity and survival.[4] Nevertheless, long-term prognosis in PAH remains poor and most patients display persistent and significant dyspnea, exercise intolerance and poor health-related quality of life despite current therapies. Thus, treatment algorithms will probably change dramatically in upcoming years. Newer paradigms in the PAH pathophysiology, including pro-proliferative and antiapoptotic phenotype of pulmonary artery smooth muscle cells (PASMCs) and the confirmation of genetic, inflammatory and metabolic abnormalities will pave the future for the development of novel therapeutic targets. The current treatment algorithm for PAH is reviewed, as well as new therapeutic targets that have a high potential to impact on functional status, quality of life and survival of patients with PAH in the near future.

Actual Therapeutic Targets in PAH

In addition to conventional and supportive therapies, current treatments target the endothelin (ET), prostacyclin and nitric oxide (NO) pathways.[5,6] A schematic overview of the current treatment algorithm is also presented in Figure 1. However, PAH is a heterogeneous disease and clinicians' evaluation and management varies considerably. The PAH Quality Enhancement Research Initiative revealed significant gaps between the clinical practice and the current guideline recommendation.[7] For example, only 6% of the patients had performed all recommended tests, and 10% of patients were diagnosed without the right heart catheterization considered as the gold standard for the diagnosis of PAH. Similarly, a significant proportion of patients with more severe disease did not receive prostacyclin analogs, rising concerns that there are substantial gaps in the management of PAH patients with more advanced disease. For this reason, early referral to pulmonary hypertension (PH) expert centers is strongly encouraged.[6]

Figure 1.

The actual treatment algorithm of pulmonary arterial hypertension (group 1 patients of the current pulmonary hypertension classification only). Early referral to an expert center is recommended. Patient selection for cardiopulmonary rehabilitation remains controversial. Supportive therapies should be adapted according to patients' condition. Acute vasodilator testing should be performed in all patients with idiopathic PAH or PAH associated with anorexigen exposure. Only patients with documented vasoreactivity should be treated with CCBs. Patients with moderate disease are generally treated with oral drugs initially, whereas iv. epoprostenol is preferred for more severe disease unless contraindicated. Note that classes of recommendations are not shown in this treatment algorithm.In selected patients.CCB: Calcium channel blocker; FC: Functional class; iv.: Intravenous; PAH: Pulmonary arterial hypertension; PH: Pulmonary hypertension; sc.: subcutaneous.Adapted with permission of Oxford University Press (UK) from [6] European Society of Cardiology.
Conventional & Supportive Therapies

Diuretics are an important component of the treatment to control fluid retention.[5,6] Based on observational studies suggesting an improved survival in idiopathic PAH,[8] oral anticoagulants are administrated in idiopathic PAH to prevent in situ thrombosis and venous thromboembolism. As hypoxemia is a potent pulmonary vasoconstrictor, hypoxemic PAH patients can also receive supplemental long-term oxygen therapy. Acute administration of digoxin has also been shown to enhance right ventricular function.[9] The role of a cardiopulmonary rehabilitation program still remains controversial in PAH. While exercise training has been found to improve quality of life and functional status in stable PAH patients,[10,11] some patients can experience presyncope and syncope.[11] This fear of worsening severe and unstable patients with rehabilitation is reinforced by preclinical data showing that rodents with stable PAH benefit from endurance training, whereas it worsens pulmonary vascular remodeling and survival in rats with severe PAH.[12] At this point, the benefit from exercise training remains to be determined for PAH patients. During initial right heart catheterization, patients undergo vasoreactivity testing to identify a small minority of patients (5% of idiopathic, heritable and anorexigen-induced PAH) exhibiting a significant improvement in pulmonary hemodynamics with vasodilators. These patients are candidates for long-term therapy with calcium channel blockers.[13,14] Finally, lung transplantation, originally the only salvage treatment, remains indicated when patients condition are worsening despite current therapies.
Current & Novel ET-receptor Antagonists

ET-1 has a dominant role in the physiopathology of PAH, acting as a potent vasoconstrictor and promoting the PASMCs proliferation. ET-1 is overexpressed in plasma and lung tissue of PAH patients.[15] Current oral ETreceptor antagonists (ERAs) include bosentan and ambrisentan. Both ERAs have been shown to improve pulmonary hemodynamics, exercise capacity and functional class, and to increase the time to clinical worsening.[1619] The most common adverse event observed with ERAs is reversible elevation of the liver aminotransferase concentration, nasopharyngitis and fluid retention.[18] Only a limited proportion of current ERAs can cross the lipophilic cell membranes and access the ET receptors within the tissues due to their highly ionized state. More recently, macitentan has been developed as a new dual ERA made with an increased proportion of the nonionized form of the molecule, which better crosses the lipophilic membrane and increases tissue penetration.[20] It has a low propensity for drugdrug interaction and urine and feces are the main route of elimination.[21,22] A recent large scale multicenter, double-blind, randomized, placebo-controlled Phase III trial involving 742 patients from 180 participating centers worldwide documented that macitentan at both the 3- and 10-mg dose was associated with a significant decrease in morbidity and mortality for patients.[23]
Prostacyclin, Prostacyclin Analogs & Non-prostanoid Inositol Phosphate (Prostacyclin) Receptor Agonist

Prostaglandin I2 (prostacyclin) induces vasodilatation by stimulating the production of cAMP in the vascular endothelium.[24] Prostaglandins also inhibit proliferation of PASMCs and aggregation of platelets.[5,25] In PAH, abnormally reduced prostacyclin levels and its metabolites are key features of endothelial dysfunction. Supplemental prostacyclin with intravenous (iv.) epoprostenol was the first therapy that significantly improved patient outcomes, resulting in symptomatic and hemodynamic improvements and survival benefit for patients with idiopathic PAH.[26] Iv. epoprostenol is still considered by most PAH experts to be the most potent drug currently available. However, its administration is complex and bears many inconveniences for the patients, including very short half-life (36 min) requiring continuous administration by a central venous catheter and a pump, as well as potential life-threatening complications like pump failure and catheter-related infections. Moreover, the product is thermolabile and needs icepacks to be well preserved. Other prostacyclin analogs like subcutaneous,[27] inhaled[28] and iv. treprostinil[29] and inhaled iloprost[30] have been developed to overcome the

limitations of the iv. epoprostenol and have been documented to be effective. Conversely, oral beraprost and oral treprostinil have failed to show any clinical benefit over current prostacyclin analogs.[31,32] Recently, thermostable epoprostenol, which allows a longer conservation period without the use of icepacks, has been launched in many countries.[33] Nevertheless, all prostacyclin analogs are associated with a large range of side effects like headache, jaw pain, flushing, nausea, diarrhea and skin rash, eventually causing skin discoloration and musculoskeletal pain.[34] Most recently, selexipag, an orally available non-prostanoid IP (prostacyclin) receptor agonist with a relatively long half-life of 7.9 h that permits twice-daily dosing, was associated with a significant reduction the PVR by 30.3% and a significant increase in the mean cardiac index, in a multicenter, randomized, double-blind, placebo-controlled Phase II proof-of-concept study.[35]
Phosphodiesterase Type 5 Inhibitors & Stimulator of Soluble Guanylate Cyclase

In healthy individuals, NO binds to soluble guanylate cyclase (sGC) and mediates the synthesis of cyclic guanosine monophosphate (cGMP), activating cGMP-dependent protein kinase. This regulates cytosolic calcium ion concentration leading to vasodilation and reduction in smooth muscle cell proliferation. cGMP is metabolized through phosphodiesterase type 5. There are currently two phosphodiesterase type 5 inhibitors available for the treatment of PAH, sildenafil and tadalafil. Sildenafil was shown to improve exercise capacity, quality of life and hemodynamics.[36] Tadalafil was shown to improve all the previously mentioned elements, in addition to improving the time to clinical worsening.[37] These drugs were generally well tolerated although mildto-moderate side effects were observed such as headaches. More recently, a new class of drugs directly stimulating sGC has been developed. Riociguat directly stimulates sGC activity independently of NO.[38] It increases the sensitivity of sGC to endogenous bioavailable NO and mimics the effects of NO when it is absent or insufficiently produced by endothelial cells.[39] In rodent models, riociguat has shown beneficial effects on pulmonary hemodynamics, right heart hypertrophy and pulmonary vascular remodeling.[40,41] A nonrandomized, uncontrolled, single-group assignment, open-label, multicenter Phase II trial was conducted to investigate individual dose titration of oral riociguat for patients with PAH or chronic thromboembolic PH. After 12 weeks, the treatment significantly improved pulmonary hemodynamics and exercise capacity.[42] Phase III clinical trials are currently underway to evaluate the efficacy and safety of different oral doses of riociguat in patients with symptomatic PAH (PATENT-1 study) and chronic thromboembolic PH (CHEST-1 study).
Combination of Approved Therapies

In case of inadequate clinical response to the initial therapy, combination of treatments targeting the ET, prostacyclin and NO pathways should be considered. Two recent meta-analyses have mined the effect of combination therapy on exercise tolerance and prognosis of PAH. Zhu and colleagues have reported an increase in exercise capacity and a reduced risk of clinical worsening for the combination therapy compared with the monotherapy in seven randomized controlled trials (RCTs),[43] while Fox and colleagues reported a modest improvement in exercise capacity for combination therapy compared with monotherapy out of six RCTs.[44] As a result, combination therapy is now widely used in clinical practice. Presently, no combination therapy has proven to be more effective than the other. Moreover, it remains unknown whether combination therapy should be used upfront at the time of diagnosis (initial combination therapy) or a second therapy should be added (add-on therapy) in case of clinical deterioration or when the therapeutic goals are not met (goaloriented therapy).

New Therapeutic Targets in PAH

Current therapies significantly improved exercise capacity, quality of life, pulmonary hemodynamics and shortterm survival.[6] Observational studies also suggest enhanced long-term survival. However, none of the current treatments are actually curative and long-term prognosis remains poor.[45] Hence, there is a clear necessity to develop new therapies. While the endothelial dysfunction is a key feature of PAH, there is growing evidence that the pulmonary vasculopathy is related to inflammation and to a pro-proliferative/antiapoptotic phenotype of PASMCs. Interestingly, cancer hallmarks have been found in PAH, therefore opening the way to new therapeutic strategies developed in oncology.[46] A schematic overview of the main novel pathways and potential therapeutic targets are shown in Figure 2.

Figure 2.

The pathological molecular pathways and potential new therapeutic targets in pulmonary arterial hypertension. DCA: Dichloroacetate; DHEA: Dehydroepiandrosterone; EPC: Endothelial progenitor cell; ET-1: Endothelin-1; Kv1.5: Voltage-gated K+ channel 1.5; NFAT: Nuclear factor of the activated T cells; PAH: Pulmonary arterial hypertension; PASMC: Pulmonary arterial smooth muscle cell; PDH: Pyruvate dehydrogenase; PDK: Pyruvate dehydrogenase kinase; ROCK: Rho-kinases; ROS: Reactive oxygen species; m: Membrane potential.
Vasoactive Intestinal Peptide & HMG-CoA reductase Inhibitors (Statins)

In PAH patients, pulmonary vasoactive intestinal peptide (VIP) serum level are decreased compared with healthy patients, while the expression of VIP-mediating receptors is increased in PASMCs of patients.[47,48] This potentiality as a therapy was investigated with eight patients in a prospective controlled intraindividual trial and has shown significant decrease of mean pulmonary arterial pressure, PVR, dyspnea score and increased cardiac output (CO), mixed venous oxygen saturation and 6-min walk test (6-MWT) distance for a single 200g dose of inhaled VIP.[49] An open-label study had shown a significant pulmonary vasodilatation, stroke volume and mixed venous oxygen saturation improvement for a single 100-g dose of inhaled VIP for only a short period of time.[50] However, the possibility that the hemodynamic and gas exchange improvement observed in a

small group of patients could come from spontaneous fluctuation cannot be ruled out. Also, no dose response curve were made, therefore, limiting the interpretation of the hemodynamic efficacy and the safety of the drug. The HMG-CoA reductase inhibitors (statins) are known to have antiproliferative and anti-inflammatory effects. Recently, some evidence has shown that statins can reverse PH, restore endothelial function similar to NO synthase expression and exert antiproliferative effects on PASMCs in monocrotaline (MCT)-induced PH rat models.[51] Statins also improve prognosis in PH animal models.[52] In human studies, statins were associated with a reduction of right ventricle mass[53] without any effects on the 6-MWT distance.[54] To date, statin use remains unclear in PAH and further studies are needed to assess its possible positive effects in PAH.
Endothelial Dysfunction Therapy

As endothelial dysfunction is a well-known hallmark of PAH, it became an area of interest as a therapeutic target in recent years. Endothelial progenitor cells (EPCs) are a cell population that can circulate in blood, proliferate and differentiate into mature endothelial cells. These cells express some cell surface markers or transcription factors that characterize mature endothelium. A pilot RCT documented the feasibility, safety and potential benefit of intravenous infusion of a heterogeneous autologous population (CD34+, VECAD+) of EPCs as they documented a significant increase of 48.2 m in the 6-MWT distance and 0.38 l/min in CO and significant reduction in mean pulmonary arterial pressure of 4.5 mmHg and in PVR of 185.4 dyne/s/cm5.[55] A new Phase I clinical trial is currently under way to confirm tolerability, safety and efficacy of autologous EPCs programmed to overexpress the endothelial NO synthase.[56] Further studies are, thus, needed to confirm the real potential of this therapy in PAH.
Tyrosine Kinase Inhibitors

Tyrosine kinases (TKs) are enzymes that trigger the phosphorylation of tyrosine residues by transferring the terminal phosphate of ATP to a protein. Therefore, they activate or inhibit a variety of cellular function. Growth factors, which include bFGF, PDGF and EGF, act through the transmembranes receptor TKs to activate major signaling transduction pathways. In addition to human cancers and cardiopulmonary diseases, they also have been implicated in the development and the progression of pulmonary vascular remodeling.[57] PDGF expression is found in many cell types, including endothelial cells and smooth muscle cells. It exerts its action via the activation of two PDGF receptors (PDGFR- and PDGFR-), promoting cellular proliferation, migration, survival and transformation. The expression of mRNA PDGF and its receptors is upregulated in PAH small distal pulmonary arteries.[58] PDGFR is thought to play an important role in the pathophysiology of PAH by initiating and maintaining the underlying pulmonary vascular remodeling.[58,59] The inhibition of the PDGFR signalization through the TK pathway becomes a potential target to inhibit cellular abnormal proliferation, survival and migration. Thus, several drugs have recently been investigated targeting the inhibition of PDGFR. Imatinib mesylate, an orally administrated antineoplastic drug, is a selective antagonist of the BCR-ABL TK, PDGFR and c-kit. It is approved for the treatment of various cancers like chronic myeloid leukemia, acute lymphoblastic leukemia and gastrointestinal stromal tumors.[6062] It binds the BCR-ABL fusion protein, therefore, blocking the active site of the phosphate transferring TK.[63] The potential efficiency of imatinib mesylate in PAH might come from his PDGFR inhibitory effect.[59] Recent clinical trials provide data for its efficacy in the treatment of PAH. A 24-week, randomized, double-blind, multicenter, placebo-controlled Phase II study evaluated the safety, tolerability and efficacy of imatinib mesylate in PAH patients with inadequate response to established therapy. While patients did not significantly improve their 6-MWT distance, they relevantly improved their pulmonary hemodynamic measurements.[64,65] Subgroup analysis suggested that patients with more severe hemodynamic impairment were more likely to benefit from imatinib mesylate therapy. More recently, preliminary results of a multinational, multicenter, double-blind, parallel-group Phase III trial study evaluating the efficacy and tolerability of imatinib mesylate in PAH patients with severe hemodynamic impairment indicate beneficial effects on exercise capacity and functional class. [65,66] In addition to common side effects, such as headache observed with imatinib,[67] concerns were raised about the possibility of an increased incidence of subdural hematoma.[66,68] The place of imatinib mesylate in a future treatment algorithm thus remains to be determined. Sorafenib, an oral multikinase inhibitor targeting the regulator of endothelial apoptosis Raf-1 and the TKs of VEGFR and PDGFR, affects tumor signaling and decreases tumor angiogenesis.[69] It is used mainly in advanced renal cell carcinoma[70] and hepatocellular carcinoma.[71] In the experimental MCT-induced PH rat model, sorafenib impeded pulmonary vascular remodeling and improved cardiac and pulmonary function.[57] An investigator-initiated, 16-week, open-label, Phase Ib study demonstrated that PAH patients with poor exercise

capacity despite continuous infusion of a prostacyclin analog had a significant increase in their 6-MWT distance.[72] However, in PAH patients with baseline 6-MWT >450 m, CO slightly decreased on sorafenib, suggesting a possible deleterious effect of sorafenib on the cardiac function. Nilotinib is a new generation of oral TK inhibitor used for the treatment of gastrointestinal stromal tumors and for newly diagnosed chronic myeloid leukemia or for patients presenting resistance or intolerance to imatinib mesylate. Nilotinib acts through competitive inhibition at the ATP-binding site of BCR-ABL, leading to the inhibition of the tyrosine phosphorylation of proteins involved in the intracellular signal transduction that BCRABL mediates.[73] Nilotinib has 2050-times the inhibitory effects of imatinib mesylate. It is also active in most imatinib-resistant cell lines with mutant ABL kinase[7476] and it has a relatively favorable safety profile.[73] In PAH, a 24-week, randomized, double-blind, multicenter, placebo-controlled Phase II study is currently underway to establish the safety, tolerability and pharmacokinetics of nilotinib. [201] Nonetheless, not all members of the TK inhibitor family give a positive response to PAH treatment. As for imatinib mesylate, sorafenib and nilotinib, disatinib is a PDGFR inhibitor but with a broader spectrum and with an additional inhibition of the sarcoma viral oncogene homolog (Src) kinase family. Reports have most recently presented cases of newly diagnosed severe PAH associated with disatinib therapy.[59,77] Therefore, the TK inhibitor family seems to have heterogeneous effects on PAH. The disatinib metabolic pathway might enlighten us as it inhibits PDGF proliferation and migration of vascular smooth muscle cells by the inhibition of both PDGFR and also the structurally related Src family kinase.[78] However, the Src family kinase are required for the degradation of activated PDGFR[79] and, therefore, the inhibition of Src by dasatinib may lead to an increase in PDGF expression which finally favors the development of PAH. Even if the development of PAH with dasatinib therapy is a rare event, the increased use of the drug for chronic myeloid leukemia treatments will inevitably lead to more PAH cases in the years to come. Physicians must be aware of this possible major complication for their patients.
RhoA/Rho-kinase Inhibitors

The Rho-kinases (ROCK) are effectors of the G-protein RhoA and are implicated in signaling pathways that regulate cellular differentiation, proliferation, apoptosis, motility and migration. [48] They are also implicated in endothelial cell dysfunction, which leads to an abnormal vasoconstriction and vascular remodeling.[80] These effects originate from the activated RhoA/ROCK signaling pathway that inhibits myosine light chain phosphatase activity.[81,82] Furthermore, this signaling pathway may be activated by serotonin[83] and enhances ET-1-mediated PASMCs vasoconstriction. The RhoA/ROCK activity is increased in PASMCs of idiopathic PAH patients.[84] Fasudil, a selective RhoA/ROCK inhibitor, is already used as a vasodilator agent for cerebral vasospasm and is under investigation as a potential therapy for memory improvement in Alzheimer disease.[85] Besides its vasodilator effect, it also suppresses cellular proliferation and enhances apoptosis in MCT-induced PH rat models.[86] A preliminary study involving fasudil has been shown to have beneficial effects on pulmonary vasculopathy.[87] Another recent study showed that both inhaled fasudil and inhaled NO led to a reduced PVR ratio in a small PAH patient cohort.[88] Those studies indicated that the RhoA/ROCK signaling pathway might be a potential new therapeutic target in PAH. However, a systemic administration of fasudil has a potent nonspecific vasodilating action.[85] Larger clinical trials are therefore needed to evaluate the safety and efficacy of fasudil in PAH.
Dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) is a precursor of a steroid hormone clinically used to inhibit the PI3K/Akt [89] and the transcription factor STAT3[90] pathways. PI3K/Akt pathway phosphorylates and inactivates glycogen synthase kinase-3 (GSK-3). GSK-3 decreases the mitochondrial membrane potential, promotes apoptosis and inhibits the activation of the nuclear factor of the activated T (NFAT) cells transcription factor. By contrast, the STAT3 activation relies on the Src pathway,[91] promoting the activation of NFAT through the upregulation of an oncogene provirus integration site for Moloney murine leukemia virus (PIM-1).[92] Finally, these pathways target genes of NFAT, which regulate the expression of many genes involved in cellular proliferation and apoptosis-resistance like Bcl2 and survivin proteins.[93] Because of its actions on the Akt/GSK-3/STAT3/NFAT pathway, DHEA could prevent and reverse the remodeling processes and represents a promising therapeutic option in PAH.[94] Indeed, DHEA prevents injuryinduced vascular remodeling in vitro.[94] Moreover, DHEA decreases the effects of the Src/STAT3 pathway and the PIM-1/NFAT/survivin axis expression, whereas it promotes Bcl2 expression resulting in decreased cellular proliferation and enhanced apoptosis in PAH-PASMCs.[95] Finally, DHEA inhibits the RhoA/ROCK axis signaling.[96] In a pilot study evaluating the efficacy of DHEA 200 mg daily versus placebo over 1 year in adults

with PH associated to chronic obstructive pulmonary disease, patients receiving DHEA experienced significant ameliorations in exercise capacity, pulmonary hemodynamics and carbon monoxide diffusing capacity of the lung without worsening in gas exchange.[97] The patients also exhibited an excellent clinical tolerance for DHEA.
Anti-inflammatory Therapies

Numerous inflammatory and autoimmune conditions like scleroderma and systemic lupus erythematosus are associated with the development of PAH. The activation of NFAT in human and in experimental models has been identified to regulate inflammatory PASMCs remodeling.[98] Pulmonary vessel infiltration by inflammatory cells like dendritic cells, T and B lymphocytes and macrophages and increased plasmatic concentrations of IL1, IL-6 and TNF- have also been reported in patients with PAH and experimental animal models, suggesting inflammation is implicated in the pathophysiology of PAH.[99] The overexpression of TNF- in mouse models has been associated with severe PAH and emphysema.[100] Whereas, the use of a TNF- antagonist, the recombinant TNF- receptor II:IgG Fc fusion protein, successfully attenuates the process of MCT -induced PH rat models through anti-inflammatory activity.[101] Other observations in relevant experimental PH models support the implication of inflammation in PAH pathogenesis. For instance, plasmatic IL-6 is increased in hypoxic and MCT-induced PH rat models.[102] Chemokines are also believed to play an important role since pulmonary endothelial cells overexpress fractalkine, which is also detected in high concentrations in the systemic circulation of severe PAH patients.[103] The CXC-chemokine ligand 10, which inhibits angiogenesis, attracts T lymphocyte cells and promotes abnormal small vessel formation and lymphocyte infiltration, has been measured at a higher concentration in PAH patients' serum.[104] The CC-chemokine ligand 2, also found in higher concentration in PAH patients, nourishes the proinflammatory imbalance by increasing monocytes migration.[105] Furthermore, CD44, a cell adhesion molecule, is overexpressed in pulmonary artery endothelial cells within plexiform lesions and surrounded by T-cell infiltration in PAH patients.[106] Finally, dendritic cells are antigen-presenting cells and inflammatory response initiation cells that might be involved in PAH pathogenesis. Perros and colleagues identified an increased number of dendritic cells in the pulmonary arterial lesions in human lung tissue samples and MCT-induced PH rat models supporting its role in the pathological remodeling of pulmonary arterial vessels.[107] These observations open the possibility to develop new therapeutic strategies targeting the inflammatory component of PAH. Previous observational studies documented that a subset of patients with PAH related to systemic lupus erythematosus and mixed connective tissue disease could benefit from an immunosuppressive treatment combining cyclophosphamide and glucocorticoids.[108,109] Treatment of MCT-induced PH rats with a human IL-1 receptor antagonist also inhibited the development of PAH and right heart hypertrophy.[110] However, the same treatment has been conducted in hypoxic-induced PH rats without significant results.[110] Furthermore, with the use of dexamethasone in MCT-induced PAH in rat models, Price and colleagues have established an improvement in hemodynamics and survival, a reduced expression of IL-6 in the lungs of the rats, a reduction in the adventitial infiltration of IL-6-expressing inflammatory cells and inhibition of PASMC proliferation associated with the reduction of IL-6 expression.[111] However, there are no placebo-controlled studies evaluating the efficacy and safety of immunosuppressive therapy and its potential long-term complications.[112]
miRNA

miRNA are small noncoding RNAs (1723 nucleotides in length). Previously, miRNA were described in cancer to be important regulators of the cellular process and their aberrant expression is associated with tumor progression through their regulatory effect on the mRNA translation and stability. Caruso and colleagues first described a down regulation of miR-21 expression in PH animal models.[113] Shortly after, our laboratory described for the first time miRNA involvement in human PAH.[91] In humans, the expression of 377 different miRNAs was measured. Seven miRNAs were aberrantly expressed in PAH-PASMCs. Among them, only the miR-204 level was down regulated. Src homology 2 domain containing phosphatase 2 (Shp2), a cytoplasmic tyrosine phosphatase implicated in Src activation, is one of miR-204's targets.[114,115] Interaction between miR204 and Shp2 mRNA regulates Shp2 activation and consequently Src activation. In PAH, STAT3 downregulates miR-204, thus, enhances Src activation through Shp2 overexpression. It has also been demonstrated that STAT3 regulates the expression of the cluster miR-17/92.[92] Also, these miRNAs are implicated in the downregulation of the BMPR2 gene expression in pulmonary artery endothelial cells. Moreover, hypoxic stress has been recently shown to enhance miR-34a and miR-210 expression.[116] These miRNAs are involved in the cell cycle and consequently could participate in PAH pathogenesis.[117,118] Recent data also suggest that miRNA are directly involved in the pathophysiology of right heart failure.[119] All these recent advances linking miRNA and PAH strongly suggest that miRNA could be a very interesting therapeutic

target in PAH. This idea is reinforced by our study in which intratracheal nebulization of miR-204 mimics restored basal expression and reversed right ventricle hypertrophy and pulmonary artery remodeling in an MCT-induced PAH rodent model.[91] However, amelioration of the administration techniques of oligonucleotides is needed before miRNA therapy may be applied in humans.
Metabolism

The Warburg effect refers to the use glycolysis as a main energy source, allowing the shutdown of mitochondrial function and is involved in increased proliferation and apoptosis resistance of PAHPASMCs.[120,121] Bonnet and colleagues demonstrated that a disrupted mitochondrial function induced a hypoxiclike condition despite normoxic conditions resulting in hypoxia-inducible factor-1 (HIF-1) activation, which by its transcriptional activity sustained the pro-proliferative and antiapoptotic phenotype seen in PAH-PASMCs.[122] In this perspective, the first strategy was to target the hypoxia sensor HIF-1 using EZN-2968, and a RNA antagonist of HIF-1 mRNA with a selective and durable effect. While EZN-2968 is an attractive therapeutic target for malignant tumors,[123] it may also be of interest in PAH. Malonyl-CoA decarboxilase (MCD) is another therapeutic target in PAH. MCD is an enzyme implicated in acetyl-CoA cytoplasmic production and influences the cellular metabolism. Consequently, MCD participates in the activation of the metabolic pathway to oxidize glucose and promote the Warburg effect. In PAH, this protein is upregulated. Sutendra and colleagues demonstrated that a MCD inhibitor (CBM-301106) reversed PAH in hypoxia and MCT-induced PH murine models.[124] However, the development of new MCD inhibitors with improved innocuity is essential as CBM-301106 is associated with a wide range of serious adverse events. The pyruvate dehydrogenase kinase is another potential therapeutic target in PAH. McMurtry et al. demonstrated in vitro and in MCT-induced PAH models that the inhibition of pyruvate dehydrogenase kinase by dichloroacetate (DCA) allowed restoration of normal apoptosis and PASMCs proliferation activity and reversed the pulmonary arterial vascular remodeling.[125] DCA is now in Phase I clinical trial and results are expected by the end of 2013.[202]
Gene Therapy

Recently, gene therapy was used to restore the abnormal K+ channels expression of PAH-PASMCs.[126] Using the same strategy, adenoviruses were used to insert a survivin-dominant negative copy of this gene in the PH rat genome.[127] This approach resulted in decreased PASMCs proliferation. However, effectiveness of this therapy depends on its distribution mode. The use of adenovirus and lentivirus transporter promoted an immunologic response, leading to a toxic inflammatory side effect. Gene therapy has also been confronted to many problems in cancer therapy such as the development of secondary cancers. In addition, nonviral vectors using plasmids are generally less efficient and could also induce inflammatory response.

Expert Commentary
The current therapies for PAH remain unsatisfactory. While they predictably produce symptomatic improvement, they have not been shown to significantly impact long-term survival. The current therapeutic strategies mainly targeted the pulmonary vascular tone imbalance that characterize PAH. However, scientific studies have now shown that in the majority of patients, cellular proliferation, inflammation, mitochondrial abnormalities and thrombosis are the dominant underlying pathobiologic processes. Consequently, newer treatments that are under consideration are being selected to target these processes. However, future clinical trials for PAH will be difficult as this disease combines the challenges inherent to treating an orphan disease with a disease where the knowledge of the molecular biology is complex and far in advance of current medical therapies. These challenges will include the unknown duration of trials necessary to demonstrate efficacy of antiproliferative drugs, the possible need for multiple therapies and the unknown potential toxicities on right ventricular function. New clinical trial designs and the validation of clinically relevant biomarkers are necessary to evaluate multiple therapies with enrollment of small numbers of patients. A multidisciplinary approach is thus needed to bring forward the actual therapies and the new emerging therapies.

Five-year View
Over the next 5 years, combinations of therapies targeting the ET, prostacyclin and NO pathways will probably become standard treatment for most PAH patients. Drugs with improved pharmacodynamics and convenience will also be launched, including macitentan, thermostable epoprostenol and nonparenteral prostanoids, and potentially direct agonists of non-prostanoid IP (prostacyclin) receptor and sGC. We also anticipate that the

treatment algorithm will evolve to progressively include a variety of proapoptosis and antiproliferative treatments as they may target the microvascular root of PAH with more efficiency. Among the emerging therapies, TK inhibitors, DHEA and DCA are currently investigated in clinical trials. We also speculate that therapeutic targets will go beyond the pulmonary circulation. As functional status is a major component of impaired quality of life in PAH, specific interventions aimed to improve exercise capacity are essential. One potential target is skeletal muscle abnormalities that contribute to exercise tolerance in PAH. [128] Similarly, researchers pay more attention to the mechanisms leading to right ventricular failure.[119] Therefore, proapoptosis, antiproliferative and anti-inflammatory treatments will probably be added to vasodilatory compounds, potentially with interventions specifically targeting the right ventricle and peripheral skeletal muscles.

Sidebar
Key Issues

Scientific studies have shown that in the majority of patients, vascular tone imbalance, cellular proliferation, inflammation, mitochondrial abnormalities and thrombosis are the dominant underlying pathobiologic processes of pulmonary arterial hypertension (PAH). In addition to conventional and supportive therapies, the current therapeutic strategies mainly targeted the pulmonary vascular tone imbalance of PAH. Current therapies for PAH remain unsatisfactory. They predictably produce symptomatic improvement, but they have not been shown to significantly impact long-term survival. The treatment algorithm will evolve to progressively include a variety of proapoptosis and antiproliferative treatments as they may target with more efficiency the microvascular root of PAH. New clinical trial designs and validation of clinically relevant biomarkers are necessary to evaluate multiple therapies with enrollment of small numbers of patients. Drugs with improved pharmacodynamics and convenience will be launched, including macitentan, thermostable epoprostenol and nonparenteral prostanoids, and potentially direct agonists of nonprostanoid IP (prostacyclin) receptors and soluble guanylate cyclase. As pulmonary remodeling in PAH shares common features of the cancer physiopathology, some anticancer agents may be of interest for the treatment of PAH. These include tyrosine kinase inhibitors, Rho-kinase inhibitors, dehydroepiandrosterone, anti-inflammatory therapy and drugs targeting the mitochondrial abnormalities. miRNA, metabolic and gene modulation may also have therapeutic potential. Therapeutic targets will go beyond the pulmonary circulation. As functional status is a major component of impaired quality of life in PAH, specific interventions aimed to improve exercise capacity are essential.

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Papers of special note have been highlighted as: * of interest ** of considerable interest

Acknowledgements Figure 1 was produced with the help of medical illustration department of the CRIUCPQ. Expert Rev Resp Med. 2013;7(1):43-55. 2013 Expert Reviews Ltd.