The Search for Disease-Modifying Therapies in Pulmonary Hypertension

Apelin

The endogenous peptide apelin, which has vasodilatory effects on the pulmonary vasculature, is related at least in part to the regulation of endothelial NO and NO synthase (NOS) levels. ²⁵ Apelin is also a key downstream target of BMPR2 signaling, mediated through the actions of β-catenin and peroxisome proliferator–activated receptor γ (PPARγ). ²⁶ Previous studies have revealed that administration of apelin prevents PH in preclinical rodent models. ^26,27 Furthermore, clinical trials have shown that short-term infusion of apelin decreases PVR and increases cardiac output and stoke volume in patients with PAH with short-term hemodynamic effects. ^28,29 Although there was no alteration in mPAP and systemic vascular resistance, the long-term effects of apelin in PAH remain undefined.

Serotonin receptor antagonist and tryptophan hydroxylases inhibitor

Serotonin (5-HT) is a signaling molecule that plays a major role in the pathogenesis of PAH. The 5-HT is released from endothelium and further stimulates smooth muscle cell proliferation and vessel constriction directly through 5-HT receptor or 5-HT transporter (SERT). ^30,31 Patients with idiopathic PAH carry high levels of circulating 5-HT. ³² In preclinical models of disease, increased SERT expression in pulmonary artery smooth muscle cells (PASMCs) drove the development of PH. ³³ Of the 14 distinct 5-HT receptors, the 5-HT_2A, 5-HT_2B, and 5-HT_1B receptors are especially related to PAH, ^34,35 and numerous inhibitors and antagonists of 5-HT receptors have been studied in this disease. Treatment with 5-HT_1B receptor antagonist LY393558, alone or in combination with fluoxetine (selective 5-HT reuptake inhibitor) and SB224289 (5-HT_1B receptor antagonist), reduced 5-HT-induced pulmonary vasoconstriction in rats exposed to hypoxia for 2 weeks. ³¹ Additionally, C-22, an antagonist of the 5-HT_2B receptor, can lower pulmonary pressures as well as repress RV hypertrophy and pulmonary vascular remodeling in a monocrotaline-induced PAH model. ³⁶ Terguride is an oral antagonist of 5-HT_2A and 5-HT_2B receptors. In a monocrotaline rat model, it attenuates PAH development through blockading the signaling action of serotonin to the 5-HT_2A and 5-HT_2B receptors on the vasculature. ³⁷ A selective 5-HT_2B receptor antagonist, PRX-08066, has been tested in a phase II clinical trial (NCT00345774), displaying efficacy in inhibiting a rise in PAP in humans exposed to hypoxia.

Inhibition of 5-HT synthesis may be another potential therapeutic strategy for the treatment of PAH. ³⁸ Two tryptophan hydroxylases (TPH1 and TPH2) are critical for 5-HT synthesis. Genetic TPH1 deficiency has been shown to protect mice from experimentally induced PAH. ^{39 -41} Moreover, a recent study demonstrated that TPH1 inhibition by KAR5585 and KAR5418 ameliorates PAH in both moncrotaline (MCT)-induced and SU5416-hypoxia rat models. ⁴² In aggregate, inhibition of either 5-HT synthesis or 5-HT receptors would be a promising therapeutic strategy against PAH; however, due to the pleiotropic effects of 5-HT in other tissues and organ systems, ameliorating untoward side effects and enhancing specificity of action may be key requirements for the future utility of such drugs in this disease.

Vasoactive intestinal polypeptide

Vasoactive intestinal polypeptide (VIP) is a neuropeptide (28 amino acids) hormone that inhibits smooth muscle cell proliferation and platelet aggregation, scavenges free radical oxygen, and induces pulmonary vasodilation via VPAC1, VPAC2, and PAC1 receptor activation. ⁴³ Activation of these receptors stimulates both adenylyl- and guanylyl cyclase-signaling pathways. Genetic deficiency of VIP in mice causes spontaneous development of PH ⁴⁴ due to increased expression of genes associated with pro-inflammation and pulmonary vascular remodeling, as well as decreased expression of genes involved in antiproliferation. ⁴⁵

The expression and receptor-binding affinity of VPAC2 is elevated in PASMCs from patients with PAH. However, VIP levels in the lung and serum are low in PAH. ⁴⁶ As such, supplementation of VIP via nebulization (200 μg daily) was found to improve pulmonary hemodynamics in patients with PAH, resulting in lower PVR and improvement of 6MWD. ⁴⁷ In a clinical study, 20 patients with PAH who inhaled a single dose of 100-μg VIP analog (aviptadil) showed significantly reduced PAP with minimal side effects. In 6 patients, a decrease in PVR of >20% was also reported. ⁴⁸ Furthermore, a combination treatment with VIP and the ET receptor antagonist bosentan has been evaluated in preclinical models of PH, with robust reversal of pulmonary vascular remodeling and improvement of hemodynamics, leading to mortality prevention for at least 45 days. Thus, adding VIP to current vasodilator therapy may be effective, ⁴⁹ but this concept awaits verification in larger clinical trials.

Endothelial progenitor cells

Endothelial progenitor cells (EPCs) originate from hemangioblasts in bone marrow or mesodermal stem cells and are enriched in peripheral blood. ^50,51 Circulating in plasma, they gravitate to sites of ischemia or endothelial injury, then differentiate into mature ECs in situ, contributing to revascularization and vascular homeostasis. ⁵⁰ Because loss of or damage to the pulmonary vascular endothelium is central to development of PH, cell-based therapy using EPCs (EC-based therapy) has been considered a potential therapeutic option for PH. ^{52 -54}

Administration of EPCs has been shown to improve PH. Treatment with umbilical cord blood-derived or autologous EPC has been found to reverse and prevent PH in preclinical rodent models, with improvement of hemodynamics and enriched medial thickening of the small pulmonary arteries. ^{53 -55} The EC-based therapy also improved hemodynamic and histologic PH in rodent models, particularly by EPCs transfected with genes such as endothelial NOS (eNOS) that inhibits smooth muscle cell contraction and proliferation. ^54,56 A combination treatment of EPCs with sildenafil has resulted in synergistic effects on PH manifestation, as compared to EPCs therapy alone. ⁵⁷ Moreover, EC-based therapy may have preventative activity in PH via promoting vascular endothelial repair ⁵⁸ through paracrine stimulation. ^59,60

Two pilot studies have been reported of patients with idiopathic PAH including adults and children were administrated a single IV infusion of autologous mononuclear cells that provide support for the therapeutic potential of EC-based therapy in human patients. ^61,62 The PHACeT trial assessing the safety of administrating autologous, cultured, eNOS-transduced mononuclear cells in patients with PAH demonstrated that such delivery was tolerated hemodynamically by these patients and there was evidence of short-term hemodynamic improvement associated with longer term benefits in quality of life and function. ⁶³ However, the complete actions of EC-based therapy for PAH remain uncertain, specifically whether the complex behavior of progenitor cells in lung tissue is beneficial or harmful, on balance. For example, in hypoxia, inflammatory and progenitor cells have been found to contribute, rather than ameliorate, to pulmonary vascular remodeling ⁶⁴ ; but it is unknown whether this directly applies to PAH.

Rho kinase inhibitors

RhoA (Ras homolog), a small monomeric G-protein that comprises several Cdc42, Rac, and Rho subfamilies, and their downstream effectors (eg, ROCK) interact with each other and further stimulate other intracellular signaling pathways to regulate cell adhesion, migration, proliferation, contraction, apoptosis, and hypertrophy. ^77,78 RhoA/ROCK signaling has been implicated in PH pathogenesis through vascular remodeling and vasoconstriction. ^{79 -81} The first ROCK inhibitor, fasudil, was approved for the treatment of subarachnoid hemorrhage–induced brain vessel vasospasms. ^82,83 Chronic fasudil treatment has also been found to improve pathologic parameters of PH and pulmonary artery remodeling in both hypoxia- and monocrotaline-induced rodent models of PH. ^{84 -86} In patients with PH, low IV administration of fasudil caused modest acute decreases in PVR. ^87,88 Fasudil may also show efficacy in a rat model of end-stage PH due to left ventricular heart disease. ⁸⁹ Despite these promising results, the roles and mechanisms of RhoA/ROCK signaling in the development and progression of PH remain unknown. The combination of fasudil with certain pulmonary vasodilators may show synergistic effects in animal models, but no clinical trial has evaluated the long-term efficacy of fasudil in PAH. ⁹⁰

Receptor tyrosine kinase inhibitors

Multiple growth factors have been associated with the abnormal proliferation and migration of pulmonary artery vascular smooth muscle cells (PAVSMCs) in PH such as epidermal growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). ^{91 -94} As a potent mitogen, PDGF forms homodimers or heterodimers and exerts its effects via 2 receptor tyrosine kinases (RTKs): PDGFR-α and PDGFR-β. ⁹⁵ As shown in cancer therapeutics, blocking these RTKs may have substantial effects in mitigating cellular proliferation. Because of the growing appreciation of molecular similarities of cancer and vascular cells seen in PH, ⁹⁶ the possibility of repurposing RTK inhibitors has been pursued.

Imatinib is a tyrosine kinase inhibitor (TKI) that was first used to treat chronic myelogenous leukemia. ⁹⁷ In experimental PH, imatinib was found to inhibit proliferation of PASMCs and reverse neointima formation in rodents through inhibition of PDGFR phosphorylation and downstream signaling. ⁹¹ Moreover, the effects of imatinib in patients with PAH were studied in a randomized, double-blind, placebo-controlled phase II study. ⁹⁸ Fifty-nine patients (WHO functional classes II-IV) who followed over 3 months of PAH-specific therapy (PGI2 analogs, ERAs or PDE5 inhibitors) were administered an oral imatinib (400 mg daily) or placebo. Upon 24 weeks of treatment, the study failed to meet its primary efficacy end point of improvement in 6MWD. However, some secondary end points such as PVR were improved. A post hoc analysis, which stratified patients according to baseline median PVR, showed improvements in hemodynamics and 6MWD in patients treated with imatinib who had PVR baseline more than 1000 dyn·s/cm⁵. In a phase III trial of imatinib in PAH, 202 patients with elevated PVR (≥800 dyn·s/cm⁵) receiving imatinib showed significant improvement in 6MWD, along with decreased mean PAP and PVR. ⁹⁹ However, this trial also called into question the safety of using imatinib in patients with PAH, particularly an elevated risk of subdural hematoma, for those receiving imatinib and anticoagulation. Approval for use of imatinib in patients with PAH was not granted, but nonetheless these results may suggest a benefit for this type of therapy if a more specific PAH population with a more favorable risk–benefit profile could be identified. Alternatively, other TKIs may be of interest, such as nilotinib, a second-generation TKI with 30-fold more potency than imatinib which was found to robustly reverse pulmonary vascular remodeling in experimental PH. ¹⁰⁰ Related approaches such as FGF2 small interfering RNAs and a pharmacologic FGFR1 inhibitor (SU5402) have been shown to reverse experimental PH. ¹⁰¹

Mammalian target of rapamycin inhibitors

Beyond RTKs, mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an important role in cell proliferation, survival, and metabolism in response to various environmental factors. The mTOR signaling pathway has been implicated in pulmonary vascular remodeling, ¹⁰² and thus mTOR signaling may present an attractive potential therapeutic target for PAH associated with severe pulmonary vascular remodeling.

Rapamycin and its analogs are currently the only FDA-approved mTOR inhibitors to predominantly suppress the mammalian target of rapamycin complex 1 (mTORC1), a kinase complex that regulates survival, growth, and metabolism in response to available cellular nutrients and energy. Rapamycin is in clinical use as an immunosuppressor to prevent transplant rejection and as an antiproliferative agent applied to coronary stents to reduced local restenosis. ¹⁰³ Rapamycin has been shown to inhibit proliferation in 3 types of PA vascular cells (EC, SMC, and fibroblast) by suppression of mTORC1. Moreover, rapamycin treatment over a 24-hour period triggers apoptosis in primary human ECs, human umbilical vein ECs, and aortic ECs, by inhibition of mTORC2. ^104,105 All together, these studies indicate that rapamycin may be applicable as a proapoptotic drug for human pulmonary vascular cell types in PAH as well. In preclinical studies, rapamycin treatment reduced the proliferation of PASMCs from monocrotaline-exposed PH model of rats ¹⁰⁶ and PAVSMCs from patients with idiopathic PAH. ¹⁰⁷ In hypoxia-induced PH mice, rapamycin attenuated thickening and proliferation of the pulmonary vasculature as well as RV hypertrophy to prevent vascular remodeling. ¹⁰⁸ In a small clinical study, a second mTOR inhibitor, everolimus, improved PVR and 6MWD in 8 of the 10 patients with PAH or CTEPH. ¹⁰⁹ An albumin-bound mTOR inhibitor (ABI-009) is also now being evaluated in patients with severe PAH (NCT02587325).

Nuclear factor-κB inhibition

Nuclear factor kappa light chain enhancer of activated B cells (NF-κB) is a ubiquitous transcription factor that is regulated by various stimuli, including growth factors, inflammatory mediators (cytokines), chemotherapeutic drugs, and many others. It is a crucial regulator of fundamental cell functions such as survival, proliferation, and mobility. ^120,121 Several studies have identified NF-κB as crucial for PAH pathogenesis via activation of inflammatory and proliferative stimuli in PASMCs and mediating cytokine-induced release of the vasoconstrictor ET-1. ¹²² In addition, activation of NF-κB is induced in monocrotaline PH rats, and pharmacologic NF-κB blockade has been found to ameliorate PH. ^123,124 Furthermore, by study of transgenic mice with cardiac-specific overexpression of a dominant-negative IκBα gene (an inhibitory binding partner of NF-κB), inhibition of NF-κB transcriptional activity was sufficient to prevent RV hypertrophy induced in monocrotaline PH rats. ¹²⁵ Lastly, NF-κB has been reported to be highly activated in pulmonary lymphocytes, macrophages, ECs, and PASMCs from patients with end-stage idiopathic PAH, ¹²⁶ suggesting that NF-κB may be a potential therapeutic target for PAH.

Bardoxolone methyl (CDDO-Me) is an orally available semisynthetic triterpenoid that inhibits NF-κB activation but also acts as a nuclear factor erythroid 2-related factor 2 (Nrf2) inducer. CDDO-Me exhibits anti-inflammatory effects by activation of Nrf2/Keap1 pathway and downregulation of NF-κB activity. ¹²⁷ In preclinical studies, it was found to increase endothelial NO bioavailability, improve metabolic dysfunction, and inhibit smooth muscle cell proliferation. ^{127 -129} Currently, a phase II clinical trial is examining its efficacy in patients with PH (NCT02036970). ¹³⁰ Another NF-κB relevant drug, dimethyl fumarate (DMF), ¹³¹ has been recently examined as a therapeutic agent for PAH using patient-derived cells and mice models. The DMF works through inhibition of NF-κB but also signal transducer and activator of transcription 3 (STAT3) and c-jun (an activator protein-1 transcriptional factor subunit) signaling, leading to degradation of the pro-fibrogenic mediators: specificity protein 1 (Sp1), transcriptional coactivator with PDZ-binding motif (TAZ), and β-catenin. ¹³² In rodent models of PAH, DMF treatment was effective in reducing inflammation, oxidative damage, fibrosis, and hemodynamic reversal. ¹³² Advancement of DMF to clinical trial in PAH has not yet been explored, but given its favorable safety profile and FDA approval as Tecfidera for the treatment of multiple sclerosis, ¹³³ repurposing for PAH may be reasonable in the near future.

Nuclear factor of activated T cells inhibition

Nuclear factor of activated T cells (NFAT), a calcium (Ca²⁺)-dependent transcription factor, is active in diseased pulmonary vasculature. ^110,134 The NFAT is highly activated in PASMCs and circulating leukocytes in both human and animal instances of PAH. ¹³⁴ Another study indicated that dysregulation of miR-204 ultimately activates NFATc2 and NFAT signaling, leading to hyperproliferative PASMCs and ultimate development of PH in rodents. ¹³⁵ The NFAT inhibition, by cyclosporine (an NFATc2 inhibitor) or via the selective peptide VIVIT that interferes with the interaction between NFAT and calcineurin, was shown to reverse monocrotaline-induced rat model of PH. ¹³⁴ While the selectivity of VIVIT is attractive for further clinical testing, challenges with delivery and stability have substantially limited the use of VIVIT in humans. ¹³⁶

Leukotriene B₄ inhibitor

Leukotriene B₄ (LTB₄) is a pro-inflammatory lipid mediator produced from arachidonic acid by consecutive activities of 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene A₄ hydrolase. ¹³⁷ Several studies have suggested that LTB₄ contributes substantially to PAH initiation and progression.

Use of an LTB₄ receptor antagonist (ONO4057) reduced RV hypertrophy in monocrotaline-exposed rats and prevented these animals from developing PH. ¹³⁸ More recently, bestatin (ubenimex), a nonspecific inhibitor of leukotriene A₄ hydrolase, was shown to reverse established PH in both SU5416-exposed athymic rats and monocrotaline-exposed rats. ¹³⁹ The efficacy of bestatin in patients with PAH was recently studied in a 24-week phase II clinical trial (NCT02664558, LIBERTY trial) which included 61 patients with PAH across 45 clinical sites in North America. Although the trial results await publication, preliminary reports from the trial sponsor (Eiger Biopharmaceuticals, Palo Alto, CA, USA) have indicated that treatment failed to improve PVR in comparison to placebo and was unable to improve exercise capacity of patients, as measured by 6MWD. It is possible that the nonspecificity of this particular drug may have played a role in these negative trial results. Nonetheless, in the wake of these results, it remains to be seen if targeting LTB₄ or other leukotriene metabolites still has a viable path forward in clinical trial for improving the inflammatory component of this deadly disease.

CCR5 antagonist

HIV infection is a key risk factor for developing PAH, ¹⁴⁰ but the pathogenic mechanisms remain enigmatic. CCR5 is a G-protein-coupled receptor that acts as a coreceptor critical for HIV entry into cells; thus, it is a common therapeutic target for HIV infection. ^141,142 Recently, CCR5 has been implicated as a potential therapeutic target for PAH. The CCR5 is activated upon stimulation by CCL3, CCL4, and CCL5 (CCR5 ligands) and is highly expressed in the principal cell types implicated in PH pathogenesis, for example, macrophages, T cells, ECs, and smooth muscle cells. ^{113,114,143,144} Increased CCL5 expression has been observed within the pulmonary vascular wall in patients with idiopathic PH. ¹⁴⁵ It has been also shown that elevated CCR5 level is found in lung tissues isolated from patients with PAH, in mice with hypoxia-exposed PH, and in HIV-infected macaques. ¹⁴⁶ Furthermore, CCR5-knockout mice manifested less severe phenotypes of PH compared to wild-type mice upon chronic hypoxic exposure. ¹⁴⁷ Maraviroc, a pharmacologic CCR5 antagonist, prevented the development of PH in hypoxic mice ¹⁴⁶ and partially reversed PH in a mouse model of disease driven by metabolic syndrome. ¹⁴⁷ As of yet, no clinical trial targeting CCR5/CCL5 in HIV-PAH has been reported.

Pyruvate dehydrogenase kinase inhibitor

Pyruvate dehydrogenase (PDH) is a key enzyme that converses pyruvate into acetyl-coenzyme A (acetyl-CoA) as a substrate for the Krebs cycle, and this process is suppressed in PAH. To do so, pyruvate dehydrogenase kinase (PDK), an enzymatic PDH inhibitor, is able to be activated through the tyrosine kinase axis ¹⁵¹ and hypoxia-inducible transcription factor, such as HIF-1α. ¹⁵² PDH is also repressed when there is decreased mitochondrial Ca²⁺ signaling, ¹⁵³ a striking feature of PAH.

Dichloroacetate (DCA) is a small molecule PDK inhibitor under study in investigator-led clinical trials for various cancers and PAH. ¹⁵⁴ In PAH, inhibition of PDK via DCA attenuated and reversed pulmonary vascular remodeling in monocrotaline and hypoxic rats with PH. ^155,156 In these studies, DCA induced apoptosis and inhibited proliferation through depolarizing mitochondria, which resulted in the release of H₂O₂ and cytochrome c, leading to the subsequent restoration of Kv channel expression and function. ^155,156 In addition to the effects on mitochondria function, DCA was found to inhibit NFAT, suppress HIF-1α, as well as upregulate copper/zinc superoxide dismutase (Cu/Zn SOD; also called SOD1) activity in experimental PH models. ^157,158 A recent phase I clinical study included 20 patients with PAH treated with chronic oral DCA. Findings were complicated, demonstrating that DCA administration led to an improvement in PAP, PVR, and functional capacity in some, but not all, of the patients. ¹⁵⁹ Intriguingly, resistance to DCA resistance correlated with the presence of carrying genetic variants of sirtuin-3 (SIRT3) and uncoupling protein 2 (UCP2), both of which predict decreased protein activity and have been related to the development of PAH. ¹⁶⁰ Thus, these findings give impetus to further study of DCA as well as potentially using genetic screening as a guide for defining appropriate responders and trial participants.

Fatty acid oxidation inhibitor

Fatty acid and glucose metabolism are inversely associated with RV homeostasis and dysfunction. In the failing RV with PAH, inhibition of glucose oxidation is accompanied by increased fatty acid oxidation, a process known as the Randle cycle. ¹⁶¹ As such, inhibition of fatty acid oxidation (and thus the Randle cycle) has been offered as a putative therapeutic possibility to improve RV function and to promote glucose oxidation. A key regulatory enzyme for fatty acid oxidation in the RV is malonyl-CoA decarboxylase (MCD). The MCD-deficient mice have displayed increased glucose oxidation and a decreased tendency to develop hypoxic PH, ¹⁶² thus offering preclinical support for this hypothesis.

Trimetazidine and ranolazine are both small molecules that partially inhibit fatty acid oxidation via restoring PDH activity and glucose oxidation, thereby accelerating ATP levels. ¹⁶³ They have been shown to reduce elevated RV glycogen levels in the hypertrophic rat RV, to increase exercise capacity and cardiac output, and to attenuate exertional lactic acidemia in pulmonary artery banding. ¹⁶³ In addition, trimetazidine has been found to normalize intracellular calcium levels in PASMCs from patients with PAH and to improve PAPs, RV thickness, and pulmonary vascular remodeling in animal models of PAH. ¹⁶² Ranolazine is a structurally related drug, originally marketed as an antianginal drug for coronary artery disease, ¹⁶⁴ but now being studied as a therapeutic that can reverse the Randle cycle specifically in the diseased RV. ¹⁶³ A numbers of clinical trial studies on the use of ranolazine in PH have recently completed. A safety study of ranolazine in patients with PAH appeared safe without worsening hemodynamics (NCT01757808). ¹⁶⁵ An open-label ranolazine study in patients with PAH with angina or an anginal equivalent showed improvement in exercise capacity, ¹⁶⁶ RV function and RV structure (reduced size), but did not alter hemodynamic parameters (NCT01174173). ¹⁶⁶ A phase IV study investigating the efficacy of ranolazine in 10 patients with PH and diastolic dysfunction (WHO group 2 PH) is ongoing but has not yet been published (NCT02133352). Two other clinical trials are in progress, studying the effects of ranolazine on RV ejection fraction and change in metabolic and microRNA (miRNA) profiles in non-WHO patients with group 2 PH with RV dysfunction (NCT01839110 and NCT02829034).

Metformin

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that positively regulates NO production via eNOS, whereupon it suppresses vascular smooth muscle cell proliferation and remodeling. ¹⁶⁷ The AMPK has been reported to be downregulated in the pulmonary arteries from patients with PAH and hypoxia-induced PH mice. ¹⁶⁸ Furthermore, endothelial-specific AMPK knockout mice are predisposed to develop PH upon hypoxic exposure, ¹⁶⁸ suggesting that AMPK could be potential novel therapeutic targets of PH.

The antidiabetic agent metformin is an AMPK activator. Thus, it has been hypothesized as a new therapeutic approach for PH, given the importance of AMPK in PH and the association of metabolic syndrome and diabetes in WHO group 2 PH (namely, PH associated with heart failure with preserved ejection fraction, HFpEF). ¹⁶⁹ Metformin has been shown to protect against the development of PH in hypoxia- and monocrotaline-exposed models of PH in rats ¹⁷⁰ as well as in a preclinical rodent model of PH-HFpEF. ¹⁶⁹ A phase II clinical trial evaluating the efficacy of metformin on pulmonary vascular function in patients with PAH is currently underway (NCT01884051).

Peroxisome proliferator–activated receptor γ agonist

The PPARγ is a nuclear receptor that predominantly regulates lipid and glucose metabolism in adipocytes and is crucial for cardiovascular homeostasis. Evidence indicates that PPARγ serves as a protective modulator in PAH, ¹⁷¹ particularly in diseased PASMCs ^172,173 and PAECs. ^174,175 The level of PPARγ expression was observed to be low in lungs from SU5416-hypoxia- and chronic hypoxia-exposed rats, as well as in lungs from patients with severe PAH and with chronic obstructive pulmonary disease. ^176,177 Mice carrying endothelial-deficient PPARγ were found to suffer more severe PH compared to wild-type mice under hypoxia exposure. ¹⁷⁵ Similarly, mice with PPARγ gene deletion in arterial smooth muscle cells developed PH. ¹⁷⁸ In part, this may be driven by PPARγ alterations of TGF/BMP signaling with downstream effects on cell proliferation and glucose metabolism. ¹⁷³ The miRNA 130/301 family was also found to target PPARγ in multiple vascular cell types, leading to an array of consequences on proliferation, vasomotor tone, and vascular matrix stiffening in PH. ^179,180 In aggregate, these studies provide a mechanistic foundation for the essential role of PPARγ deficiency for PAH disease development, thus prompting speculation about the potential for PPARγ agonists in this disease.

The PPARγ activators (ligands) such as rosiglitazone and pioglitazone (thiazolidinedione, TZD) have indeed shown benefits in animal models of PAH. ^172,181 Rosiglitazone attenuated hypoxia-induced models of PH in mice and rats. ^177,182,183 Treatment with rosiglitazone reversed PAH by inhibition of TGFβ1-Stat3-FoxO1 cascade pathways in TGFβ1-overexpressing mice. ¹⁷³ Despite these promising attributes, the clinical potential of rosiglitazone was stymied by cardiovascular safety concerns in larger phase III trials in diabetic patients. ¹⁸⁴ On the other hand, pioglitazone, another TZD-class insulin sensitizer, has been studied for therapeutic revival in a number of cardiovascular diseases, given its beneficial safety profile versus rosiglitazone ^{185 -187} and even beneficial effects on diastolic left ventricular function. ¹⁸⁸ In preclinical studies, treatment with pioglitazone reversed PAH and prevented RV failure in the SU5416-hypoxia rats through regulating distinct messenger RNAs (mRNAs) and miRNAs, renovating mitochondrial function (Food and Agriculture Organization induction), and preventing lipid accumulation in myocyte. ¹⁸¹ Taken together, next-generation PPARγ-activating drugs may, in fact, be potent agents for the treatment of PAH, RV failure, and abnormality in altered lipid/glucose metabolism, which are characteristic features of cardiopulmonary remodeling.

Beyond TZD drugs, an electrophilic nitroalkene-containing fatty acid nitro-oleic acid (NO₂-OA) is a novel and endogenous PPARγ ligand that directly adducts the Cys285 residue in the PPARγ ligand-binding domain, thus leading to potent agonist for PPARγ. ¹⁸⁹ The NO₂-OA has displayed beneficial metabolic effects by induction of PPARγ transcriptional activity ¹⁸⁹ and attenuated hypoxia-induced PH in mice by inhibiting the proliferation of smooth muscle cells to reduce muscularization of small pulmonary vessels. ¹⁹⁰ At present, NO₂-OA has cleared preclinical toxicology and testing in FDA-approved human safety trials of both oral and IV formulations (IV IND 122583; oral IND 124524), and a phase II human trial with this molecule is underway in PAH.

Antioxidants

A number of recent human studies have implicated increased oxidative stress (ie, the rapid depletion of small molecule antioxidants, lipid peroxidation, and DNA oxidation) in patients with PH. ^{191 -194} It is reasonable to suggest that redox imbalance may contribute to the pathological conditions associated with idiopathic PAH. Several small molecules with antioxidant properties have been reported to have therapeutic outcomes in animal models of PH. In preclinical studies, several antioxidants have been demonstrated to inhibit PH and/or RV dysfunction. Examples include N-acetylcysteine, probucol, tempol, allicin, pyrrolidine dithiocarbamate, superoxide dismutase, allopurinol, sulfur dioxide, and resveratrol. ¹⁹⁴ More recently, new specific oxidase inhibitors are available, such as febuxostat (a putative inhibitor of xanthine oxidase) and statins (inhibitors of HMG-CoA reductase), which have broad effects on modulation of inflammatory vascular oxidases, but have not been tested rigorously in clinical human PAH.

Targeting sex hormones

A gender predisposition has long been appreciated in PAH. The initial National Institutes of Health registry identified a ratio of 1.7:1 female:male patients with PAH, ¹⁹⁵ and a recent French registry for multinational treatment trials reported a ratio of 4:1. ¹⁹⁶ However, men with PAH tend to suffer from more severe disease, and male sex has been identified as a risk factor for mortality in 2 individual PAH registries. ^197,198 A recent study has reported that sex hormone levels are associated with the risk of PAH in men. Specifically, an increase in estradiol (E2) level and a decrease in dehydroepiandrosterone (DHEA) level were linked to more severe hemodynamic burden. ¹⁹⁹ In particular, elevated circulating E2 level is linked to a high risk of PAH in men.

Although the biogenesis and metabolism of E2 and DHEA are complex, findings have indicated the importance of aromatase, one of the several biogenesis enzymes for E2 production, in the development of PH. Namely, the occurrence of a gain-of-function single-nucleotide polymorphism in the aromatase promoter region is not only associated with a higher level of circulating E2 in patients with PH but also with an increased risk of developing PAH with cirrhosis. ²⁰⁰ Administration of the aromatase inhibitor anastrozole has been found to reduce PAPs, pulmonary vascular remodeling, and indices of RV hypertrophy in hypoxia-exposed mice and Su5416-hypoxia PH models in rats. ²⁰¹ A phase II clinical trial evaluating the safety and efficacy of anastrozole in patients with PAH has been completed, and it demonstrated daily treatment with 1 mg anastrozole for 3 months significantly lowered E2 levels in plasma by 40% and increased 6MWD capacity. ²⁰²

The DHEA is a cholesterol-derived steroid hormone secreted mainly from the adrenal cortex. It is a precursor for the synthesis of estrogen and testosterone ²⁰³ and has been found to inhibit hypoxia-induced vasoconstriction by stimulating potassium channels via sGC activation ²⁰⁴ and to improve endothelial function by increased NO synthesis. ²⁰⁵ Administration of DHEA has been observed to partially reverse PAH in both preclinical and clinical studies. The DHEA treatment protects against monocrotaline-induced PH in pneumonectomized rats ²⁰⁶ and ameliorates severe PAH in SU5416-hypoxia rats by protecting the RV against fibrosis and apoptosis. ²⁰⁷ A recent small clinical trial demonstrated an improvement of 6MWD as well as pulmonary hemodynamics in patients associated with chronic obstructive lung disease. ²⁰⁸ Taken together, DHEA is advancing as a possible new drug for the treatment of PH; it remains to be seen if male patients in particular may derive benefits from DHEA hormone therapy.

Other metabolic processes

There continue to be advancing insights into the profound metabolic reprogramming and dysregulation seen in PH. Iron deficiency in patients with PH has been observed, ^{209 -212} accompanied by some mechanistic insights into the role of iron in PH. ^{213 -215} Moreover, deficiency of iron–sulfur clusters, critical for function of oxidative phosphorylation and electron transport, has been causatively linked to PH via both genetic and acquired triggers. ^216,217 Clinical trials are currently ongoing to evaluate whether iron supplementation can improve manifestations of PAH (NCT03371173). However, these trials will need to tread cautiously, given other data in sickle cell–induced PH that iron overload can also worsen PH. ²¹⁸ Furthermore, dysregulation of transcriptional factors such as the Yes-associated protein (YAP) and TAZ has been linked to alterations of glycolysis and glutamine metabolism, driving a pathogenic and hyperproliferative state. ^219,220 Inhibitors of YAP/TAZ and glutaminolysis have been examined in rat models of PH and have shown promise.