Update on novel targets and potential treatment avenues in pulmonary hypertension

Abstract

Pulmonary hypertension (PH) is a condition marked by a combination of constriction and remodeling within the pulmonary vasculature. It remains a disease without a cure, as current treatments were developed with a focus on vasodilatory properties but do not reverse the remodeling component. Numerous recent advances have been made in the understanding of cellular processes that drive pathologic remodeling in each layer of the vessel wall as well as the accompanying maladaptive changes in the right ventricle. In particular, the past few years have yielded much improved insight into the pathways that contribute to altered metabolism, mitochondrial function, and reactive oxygen species signaling and how these pathways promote the proproliferative, promigratory, and antiapoptotic phenotype of the vasculature during PH. Additionally, there have been significant advances in numerous other pathways linked to PH pathogenesis, such as sex hormones and perivascular inflammation. Novel insights into cellular pathology have suggested new avenues for the development of both biomarkers and therapies that will hopefully bring us closer to the elusive goal: a therapy leading to reversal of disease.

pulmonary hypertension (PH), defined hemodynamically via right heart catheterization as an increase in pulmonary arterial pressure (P_PA) to ≥ 25 mmHg at rest, is a complex and progressive condition arising from a variety of etiologies. Often fatal, PH can be divided into five major categories based on underlying cause and hemodynamic parameters: 1) pulmonary arterial hypertension (PAH), including idiopathic, heritable, and associated PH; 2) PH due to left heart disease; 3) PH due to interstitial lung diseases and/or hypoxia, including high-altitude and chronic obstructive pulmonary disease (COPD); 4) chronic thromboembolic PH (CTEPH); and 5) PH with unclear and/or multifactorial origin, including hematologic and systemic disorders (14, 83). While most forms of PH are adult onset, PH is also prevalent in the pediatric population. PH in newborns and infants remains a disease with high morbidity and mortality with multiple etiologies (122). Abnormal vascular architecture and function occur, whether due to persistent fetal circulation, secondary to lung hypoplasia, or consequent to premature birth or neonatal infections leading to chronic lung disease.

Sustained vasoconstriction and vascular remodeling contribute to all forms of PH to varying degrees, increasing right ventricular (RV) afterload and inducing RV hypertrophy (RVH) and eventually leading to mortality from RV failure. The structural changes that occur in the vasculature include reduced compliance, or stiffening of the proximal, elastic arteries (85, 210); thickening of the intimal and/or medial layer of muscular, resistance arteries; and muscularization of the precapillary arterioles due to proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and in some cases endothelial-mesenchymal transformation (219). In severe cases, vaso-occlusive lesions arise from exuberant proliferation and reduced apoptosis of PASMCs and endothelial cells (ECs) within the vessels. It should be noted that the source of highly migratory/proliferative ECs found in occlusive lesions is still under investigation, as recent work suggests that cells from the vaso vasorum, the network of vessels that supply blood to the wall of the pulmonary arteries (PAs), undergo significant proliferation in the setting of PH (169). ECs isolated from the vaso vasorum of chronically hypoxic calves contained a high density of colony-forming ECs, which possessed high proliferative capacity and may contribute to vascular proliferation and remodeling elsewhere in the “pan-vasculopathic” PA in PH.

The development of PH can be associated with various pathogenic or genetic causes, and perhaps because of this complexity the cellular mechanisms causing disease are still incompletely understood. Over the past few decades, tremendous progress has been made in identifying contributing factors, including alterations in the production of vasodilator and vasoconstrictor mediators, various ion channels, bone morphogenetic protein (BMP) signaling, and mitochondrial dysfunction. In this review, we highlight advances made within the last 2 yr that have informed our understanding of the basis of this deadly disease, further delineating the molecular pathways in pulmonary ECs, PASMCs, and fibroblasts contributing to alterations in pulmonary vascular structure and identifying new therapeutic uses for medications as well as biomarkers for disease prognostication.

Models of PH

Given the limited availability of patient tissues and inability to perform mechanistic studies in humans, several animal models of PH have been developed to allow investigation into the functional and structural changes that occur during the development of PH and the underlying causes. Unfortunately, to date no single preclinical model perfectly replicates human PAH. Nonetheless, these models provide the opportunity to characterize the development and progression of PH, perform mechanistic studies, and evaluate potential therapeutic treatments.

To model pediatric PH, a variety of approaches have been developed. Exposure based models include short-term neonatal hyperoxia, fetal and/or post-natal hypoxia and a two-hit model of prenatal hypoxia followed by postnatal hyperoxia (reviewed in Ref. 20). All of these approaches recapitulate changes in lung structure, pulmonary vascular dysfunction and PH often observed in humans. For example, antenatal and/or postnatal hypoxic exposure leads to elevated P_PA, impaired relaxation and increased contractility of PAs, PASMC hyperplasia and increased actin polymerization, and adventitial fibroblast proliferation (23, 74, 78, 269, 274, 279). Short-term hyperoxia in mice, which serves as a model of bronchopulmonary dysplasia (BPD), produces rarefaction of the microvasculature and increased smooth muscle actin expression in distal PAs, changes that are associated with downregulation of the BMP signaling pathway in affected lungs (278).

Interestingly, recent findings suggest that short-term neonatal exposure to hyperoxia, even if not directly inducing PH, may predispose adults to PH (95), due to persistent alterations in lung structure. In contrast, neonatal hyperoxia was beneficial for RVH, suggesting that while this exposure can cause vascular abnormalities that predispose the lung to PH later in life, an adaptive mechanism is invoked in the RV that improves tolerance.

While the former approaches are easy to implement and are typically used in small animals, surgical models often used in large animals include congenital diaphragmatic hernia, where a hernia is surgically created and the abdominal bowel is positioned into the chest (reviewed in Ref. 245) and partial ligation of the ductus arteriosus in utero (3). Both of these models result in elevated P_PA after birth, mimicking failure to transition from fetal circulation after birth or persistent PH of the newborn (PPHN) observed in human infants. A large animal model using chronically ventilated lambs also exhibits increased pulmonary vascular resistance (PVR) and pulmonary vascular remodeling (7).

The most widely used model of adult PH remains the chronic hypoxia (CH) model. The phenotype observed in CH models is consistent with PH that develops in humans as a result of high altitude (reviewed in Refs. 219, 228). Both rats and mice are common choices for exposure to CH. Housing rodents at an Fi_O2 of 10% for 2–4 wk produces reliable PH. Rats exhibit substantial increases in P_PA and RV mass, accompanied by increased vascular wall thickness, due to PASMC hypertrophy and hyperplasia (224, 225). Mice also develop PH upon exposure to CH, although elevations in RV systolic pressure (RVSP) and vascular remodeling are milder (18, 225). The most consistent finding in both rats and mice is muscularization of distal vessels and thickening and functional stiffening of proximal, conduit arteries (219, 225). Early studies using contrast media or microspheres to visualize blood flow suggested that rarefaction, or vascular pruning, occurs in this model (192). However, these results may have been contributed to by excessive hypoxic pulmonary vasoconstriction, as acute exposure to Rho kinase (ROCK) inhibitors almost completely normalizes P_PA (166) and vascular remodeling appears to occur in an outward manner and is associated with angiogenesis (116).

The model that perhaps best reflects severe human PAH combines a single dose of the vascular endothelial growth factor (VEGF) receptor inhibitor SU5416, followed by exposure to hypoxia (SuHx), resulting in initial EC apoptosis with subsequent proliferation of a subset of apoptosis-resistant ECs (168, 234). In rats, this model develops robust EC and PASMC proliferation and occlusive lesions reminiscent of PAH in humans (2, 234). PH induced in the SuHx model is irreversible even after return to normoxia. The severity of PH that develops in response to the SuHx exposure is strain-dependent in rats, making strain selection an important consideration (125). A murine model has also been described, with increased arterial muscularization, vaso-occlusive lesions, collagen deposition in the media and adventitia, and RV dysfunction (48); however, mice required multiple injections of SU5416 and PH resolved upon removal of the mice from hypoxia (48, 251).

Debate continues as to the exact cause of endothelial dysfunction, vascular remodeling, and enhanced contractile mechanisms in all models of PH. Recent work using cells exposed to cyclic stretch to mimic hemodynamic forces to which pulmonary cells might be exposed in vivo suggests that mechanical forces alone may be sufficient to trigger cellular dysfunction (259). Similarly, PH induced in the SuHx model is irreversible even after return to normoxia, and ROCK inhibitors only partially reversed P_PA (240), suggesting a significant portion of the increased PVR may be due to remodeling and occlusion of flow in small vessels. Temporal examination of the SuHx model demonstrated the presence of vaso-occlusive lesions early in the course of exposure; however, concentric neointimal, or plexiform, lesions did not form until later time points (2, 240). More recently, PA banding was used to alter blood flow in SuHx rats, resulting in diminished occlusive lesion formation in lobes with reduced blood flow (1). These results would suggest that the plexiform lesions observed in PAH patients, while perhaps contributing to progression/severity of the rise in P_PA, may in fact be a consequence of EC damage and/or increased hemodynamic stresses during PH rather than the inciting cause. Intriguingly, both studies from sequential human lung biopsies as well as evaluation of the temporal sequence of remodeling in the SuHx rat model suggest that medial remodeling may precede intimal remodeling in PH (242), suggesting abnormal PASMC signaling could be a potential primary locus of disease pathology under certain conditions.

It should be noted that in assessing the effect of various pathways on the pathobiology of PH, careful histologic and hemodynamic assessment in animal models of PH is critical. Such in vivo data provide crucial correlates to in vitro study of endothelial function, but methodologic limitations exist. Specifically, assessment of alveolar capillary rarefaction (or angiogenesis) can be difficult using traditional histologic specimens. However, newer stereological assessments of the alveolar capillary network provide more accurate assessment of alveolar capillary volume, including a recent methodology for estimating capillary number (266). Additional areas of concern with respect to morphology include tissue distortion, as the fixative and embedding material used can alter tissue properties, a critical factor when determining structural remodeling. Most studies assessing morphometric analyses have used inflation at a fixed pressure and/or volume with formalin or low-melting point agarose. Recent studies suggest that preservation of lung tissue dimensions can be improved compared with formalin fixation and paraffin embedding by initial fixation with a mixture of glutaraldehyde and formaldehyde followed by embedding the samples in glycol methacrylate and treating them with osmium tetroxide and uranyl acetate (212). Thus, for quantitative histological studies, fixative and embedding protocols should be chosen carefully and adherence to published guidelines for evaluating lung structure (117) will help improve reproducibility and interpretations of results.

Pathways and Targets

The exact mechanisms governing abnormal migration and proliferation of vascular cells in PH are not fully known, but imbalances in normal antimigratory and/or antiproliferative mechanisms, activation of deleterious signaling pathways due to paracrine release of inflammatory substances, as well as increased endogenous and exogenous production of reactive oxygen species (ROS) are all thought to play a role (191). In this section, we will highlight some of the pathways that have recently been shown to contribute to abnormal cell function during PH.

Cell metabolism.

Altered mitochondrial function and metabolism (including fatty acid oxidation, glucose oxidation, and glutaminolysis) in the pulmonary vasculature has been an area of intense recent focus in PH research (reviewed in Ref. 203). A cancer-like metabolic shift known as the Warburg effect, in which glycolysis is uncoupled from glucose oxidation (referred to as aerobic glycolysis), has been noted in both the pulmonary vasculature and the RV in PH. This shift is related to impaired mitochondrial function resulting in inhibited glucose oxidation. There have been numerous recent advances in understanding the mechanisms of mitochondrial dysfunction in PH. Genetic loss of sirtuin 3 (SIRT3), a mitochondrial deacetylase, in mice results in impaired mitochondrial function and spontaneous development of PH, and further a loss-of-function SIRT3 polymorphism is associated with human PAH (179). PASMC-specific knockout of uncoupling protein 2 (UCP2), which conducts Ca²⁺ from the endoplasmic reticulum to the mitochondria, leads to low mitochondrial Ca²⁺ levels and mitochondrial hyperpolarization in addition to the development of pulmonary vascular remodeling and PH (63). Furthermore, the BMP receptor 2 (BMPR2) axis has been linked to mitochondrial function, such that mice with EC-specific deletion of BMPR2 have sustained PH following hypoxia then reoxygenation that is associated with lowered mitochondrial membrane potential and ATP production as well as accumulation of ΔmtDNA⁴⁹⁷⁷, a mitochondrial DNA deletion known to be associated with cancer (59).

Glycolysis represents the initial step in glucose metabolism, in which glucose is converted to pyruvate in a cytosolic pathway involving glucose-6-phosphate (G6P). In the glycolytic pathway, G6P usage results in the generation of pyruvate, which can either be transported into the mitochondria or converted to lactate in the cytosol (Fig. 1). Alternatively, G6P can be utilized in the pentose phosphate pathway, in which G6P dehydrogenase (G6PD) catalyzes the conversion of NADP⁺ to NADPH, which protects against oxidative damage, and results in the subsequent generation of ribose sugars that are used in the synthesis of nucleotides. Recent papers have shed further light on the contribution of G6P and G6PD to pulmonary vascular remodeling.

Fig. 1.

Glucose-6-phosphate (G6P) serves as a substrate that can be used in the glycolytic pathway resulting in generation of pyruvate. Alternatively, it can be used in the pentose phosphate pathway, in which glucose-6-phosphate dehydrogenase (G6PD) catalyzes a reaction resulting in formation of NADPH as well as precursors for nucleotide synthesis (such as ribulose 5-phosphate).

Prior work showed that hypoxic exposure increased G6PD activity within PASMCs, which modulated expression of smooth muscle phenotype proteins such as SM22α (44). Recent work has further unveiled the pathways via which G6PD modulates PASMC function in response to moderate hypoxia. The hypoxia-induced increase in G6PD activity was found to be necessary for the rise in rat PASMC proliferation (42). This G6PD-induced increase in proliferation was associated with increased Sp1 and hypoxia-inducible factor-1α (HIF-1α) expression and a decrease in contractile protein (SM22α and myocardin) expression combined with increased expression of proproliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition in vivo prevented the development of PH in the SuHx rat, with reduced remodeling associated with increased SM22α and decreased HIF-1α expression. Thus these data suggest G6PD serves as an upstream regulator that switches PASMCs from a contractile to a proliferative phenotype in response to hypoxia. Interestingly, although this paper demonstrated a correlation between increased HIF-1α and reduced SM22α levels, it was not directly tested whether SM22α was regulated by HIF-1. Perhaps surprising in the face of these findings, SM22α was initially identified from proteomics analysis of hypoxic PASMCS as a protein that was markedly upregulated (283). In human PASMCs, likely from conduit vessels, hypoxia upregulated SM22α via HIF-dependent induction of trasnforming growth factor-β (TGF-β) (282). In this case, SM22α induction was mediated by HIF-2 rather than HIF-1 (282). Similarly, PASMCs isolated from CH rats showed elevated SM22α levels that were attenuated, along with PH, in animals receiving lentivirus to reduce SM22α levels (282). However, others also observed reduced SM22α expression in hypoxic PASMCs (126), in PAs exposed to hypoxia (44), and in whole lung tissue from hypoxic rats (36). In contrast, SM22α downregulation was not observed in vivo in rats exposed to either CH or SuHx (42). While these results would seem at odds, variations in culture conditions, species (rat vs. human), location in the lung from which cells were derived (proximal vs. conduit), and level of hypoxia (moderate vs. severe) could all contribute to the differences observed.

G6PD also appears to be important in the hypoxia-induced proliferation and differentiation of CD133⁺ cells (43), bone-marrow derived progenitor cells that are recruited to the pulmonary vasculature in response to hypoxia and differentiate into mesenchymal or smooth muscle-like cells. CD133⁺ proliferation in response to hypoxia required G6PD, which modulated HIF-1α, cyclin A, and phospo-histone H3 expression (43). Furthermore, in coculture with PASMCs, differentiation of CD133⁺ cells into smooth muscle-like cells required G6PD-dependent production of H₂O₂ by the PASMCs. In vivo inhibition of G6PD in the SuHx rat decreased the number of CD133⁺ cells accumulating around occlusive lesions, as well as decreased the number of lesions (43). Thus G6PD is important to the function of both PASMCs and CD133⁺ cells, potentially via the stimulation of NADPH oxidase (Nox)-induced ROS production.

Altered cell metabolism is not restricted to the pulmonary vasculature in PH, as the RV has also become an area of recent interest. Under normal conditions, 95% of ATP is produced from oxidative phosphorylation, with 60–90% of the substrate from β-oxidation of fatty acids and the rest from glucose metabolism (222). Several studies have now shown RV mitochondrial and metabolic dysregulation in PH models (reviewed in Refs. 203, 229). In the SuHx model of PH, severe RV dysfunction was associated with a shift in metabolic substrate utilization, measured via imaging for 2-deoxy-2-[¹⁸F]fluoroglucose (glucose analog) and 14-(R,S)-[¹⁸F]fluoro-6-thia-heptadecanoic acid (fatty acid analog), demonstrating increased glucose, and reduced fatty acid, uptake (96). These results were consistent with data in patients indicating increased RV glucose uptake that correlated with RV dysfunction (33, 150, 275).

While it would seem clear that advanced and/or severe disease is associated with a metabolic shift in the RV, exactly when the glycolytic switch is “flipped” is unknown. In the neonatal calf model, where PH can be induced by short-term exposure to hypoxia, elevated P_PA is accompanied by exuberant structural remodeling of the pulmonary vasculature and RV; however, overall hemodynamic function appears compensated with preserved cardiac output (29, 143, 257). Consistent with previous work demonstrating metabolic dysregulation in the left ventricle (LV) during heart failure (19, 204, 215), it was speculated that similar mitochondrial and metabolic abnormalities might be present in the RV. Surprisingly, following 2 wk of hypoxic exposure, mitochondrial function appeared to be largely maintained, with no significant changes in mitochondrial structure or dynamics and only small reductions in mitochondrial number and complex I activity (30). Interestingly, all differences noted in the RV were also observed in the LV, suggesting an overall response to hypoxia rather than chamber-specific adaptation. A small shift in metabolism consistent with an early switch to glycolytic metabolism was also noted, although again this was observed in both ventricles (30), suggesting that at this early stage of PH where RV function is largely compensated, mitochondrial and metabolic pathways do not contribute to the response to pressure overload.

Oxidative stress.

Closely related to dysfunctional mitochondrial metabolism, oxidative stress has also received significant attention with regards to the pathogenesis of pulmonary vascular remodeling (76). Perturbations in ROS levels have a myriad of effects on the pulmonary vasculature, ranging on a spectrum of oxidant-mediated signaling to oxidant-induced injury, depending on the type and concentration of ROS involved. In the case of models of PH in neonatal animals, increases in pulmonary blood flow and impaired mitochondrial respiration trigger ROS production, which in turn causes a vasoconstriction response (118). Recent work has begun to delineate mechanisms whereby altered ROS induces changes in vascular function. In ECs from PH fetal lambs, superoxide dismutase-2 (SOD2) was chaperoned into the mitochondrial matrix in an ATP-dependent manner by inducible heat shock protein 70 (iHSP70) (4). In this model, impaired SOD2 function with resultant increased mitochondrial oxidative stress led to impaired relaxation of PA rings in the presence of the vasodilator, nitric oxide (NO). Restoration of normal mitochondrial function with an oxygen radical scavenger returned relaxation of PA rings to baseline. In normal fetal lamb PASMCs, cyclic stretch, thought to mimic mechanical forces that contribute to PPHN, was found to induce increased Nox4 expression (259), which was mediated by mitochondrial complex III and NF-κB, and resulted in increased ROS levels in the PASMC cytosol as well as increased cyclin D1 expression. Thus ROS generation appears to be a key component of pathways through which fetal vascular function is altered in response to changes in flow.

In addition to pathologic ROS generation intracellularly, elevations in ROS can also be due to circulating inflammatory cells. Smooth muscle-specific deletion of extracellular superoxide dismutase (EC-SOD, or SOD3), the primary defender against elevations of ROS originating in the extracellular space, augmented CH-induced PH (CHPH) in mice, arguing for a protective role for this enzyme (174). While overexpression of EC-SOD attenuated (172), and total body loss of EC-SOD accentuated (271), hypoxic PH, the results from conditional EC-SOD deletion suggest that EC-SOD bound to SMC may be a key regulator of oxidant-induced injury in the CH model of PH. These findings are consistent with recent work demonstrating reduction in EC-SOD expression in lung tissue and PASMCs from PAH patients mediated by histone deacetylation (173).

One downstream effector of changes in ROS homeostasis is a nonselective cation channel, acid-sensing ion channel 1a (ASIC1a), which is a redox-sensitive channel that contributes to hypoxia-induced PH via augmentation of store-operated calcium entry (SOCE) in PASMCs (171). Recent work has shown that CH decreased H₂O₂ levels in rat PAs, likely due to a combination of decreased Cu/Zn superoxide dismutase (SOD1) activity and increased glutathione peroxidase activity (186). H₂0₂ decreases ASIC1a-dependent SOCE, suggesting that inhibition of H₂O₂ in CH could be a mechanism whereby SOCE is augmented, resulting in hypoxic elevations in intracellular calcium.

Peroxisome proliferator-activated receptor-γ.

Peroxisome proliferator-activated receptor-γ (PPARγ), a ligand-activated transcription factor, has been found to regulate adipogenesis and both glucose and lipid metabolism by modulating gene transcription through binding to PPAR response elements. Thiazolidinediones, such as rosiglitazone, are PPARγ agonists that have been widely used in the clinical treatment of diabetes. PPARγ expression is decreased in the pulmonary vasculature of patients with PH (10). The exact mechanisms resulting in PPARγ downregulation in PH are still under investigation, although proinflammatory mediators, such as IL-6 and TNF-α, may play a role (112). Conditional deletion of PPARγ in either SMCs (105) or ECs (100) results in the development of PH, whereas PPARγ agonism attenuates PH and pulmonary vascular remodeling in animal models (50, 170). PPARγ signaling results in antiproliferative effects (22) and is mediated by a number of pathways important to PH, such as miR-21 (98) and NF-κB (149), to list only a couple.

With the use of the model of PH where fetal sheep undergo partial ligation of the ductus arteriosus during late gestation, PPARγ signaling was shown to be crucial to normal endothelial function and angiogenesis in a NO-dependent pathway and was inhibited by endothelin-1 (ET-1), a potent vasoconstrictor known to be elevated in clinical PH (270). ET-1 receptor blockade or PPARγ agonism increased endothelial nitric oxide synthase (eNOS) protein and activity. In PASMCs from the same model, decreased PPARγ levels were associated with increased ROCK activity and cell numbers (91). In these cells, ROCK inhibition or PPARγ agonism was able to normalize cell growth.

p38 MAPK.

p38 kinases are members of the stress-activated kinase family that are induced by an array of stressors including inflammatory cytokines, osmotic changes, and mechanical deformation, controlling adaptive responses as well as driving pathologic signaling in varied disease states. In the case of PH, p38 has been implicated in maladaptive signaling in both the pulmonary vasculature and RV. In ECs, p38 has been found to function as a downstream effector of BMPR2 signaling (13). In pulmonary ECs, short-term BMPR2 silencing with siRNA was sufficient to increase migration and proliferation, providing evidence of the important role of ligand-independent BMPR2 in regulating EC behavior. Furthermore, BMPR2 silencing increased basal levels of ERK1/2 and p38 MAPK and BMPR2 silencing-induced increases in migration and proliferation could be attenuated through inhibition of the p38 MAPK pathway. These results argue a role for BMPR2 as a tonic suppressor of promigratory, proproliferative pathways in a noncanonical, ligand-independent fashion and for p38 MAPK as a downstream component of this signaling pathway.

The significance of p38 signaling to vascular cell function appears to not be limited to the endothelium. In lung sections from PAH patients, increased p38 was observed in all layers of the vessel walls (47), and in PASMCs from PAH patients, abnormal growth is reduced when p38 is inhibited (268). Moreover, migration of PASMCs from PAH patients is associated with increased p38 activation (267). Similar to ECs, downregulation of BMPR2 in PASMCs is associated with upregulation of p38 (157), further solidifying the link between BMPR2 loss of function and p38 activation in PH vascular remodeling.

Altered fibroblast function, both in the pulmonary vasculature and the RV, is an important contributor to PH pathogenesi (226) and also appears to be related to p38 signaling. In pulmonary fibroblasts, activation of p38 promotes fibroblast proliferation under hypoxic conditions (165). p38 can also contribute to release of mitogens from the fibroblast that stimulate adjacent PASMC proliferation and recruit proinflammatory cells (200). Inhibiting p38, in particular the α-isoform, in vitro prevented fibroblast production of IL-6 in response to hypoxia, while in vivo inhibition of p38 attenuated PH, as well as reduced lung and circulating levels of IL-6 (47). A number of studies have demonstrated a role for IL-6 in mediating PH (92, 108, 209, 223), with circulating levels of IL-6 correlating with outcome in PAH patients (124). IL-6 is also known to promote PASMC proliferation, migration, and contractility (112, 209). Together, these data suggest an important role for p38 not only in mediating fibroblast proliferation but also in promoting a proinflammatory milieu that can also contribute to PASMC dysfunction.

In the remodeling RV, p38 signaling appears to be important for hypertrophy. While compensatory hypertrophy is necessary for RV adaptation to increased afterload in PH, persistently elevated afterload eventually leads to RV fibrosis and failure (24). Several reports have indicated that increased RV afterload and hypertrophy are associated with alterations in RV, but not LV, PKC isoform expression, with increased PKCα (26, 244), PKCβ (46), and PKCδ (46, 244). The increase in PKC expression is likely downstream of increased angiotensin II (ANG II) signaling (46, 247), which can exert effects through either AT₁ or AT₂ receptors. In cardiac fibroblasts, ANG II leads to collagen production via inactivation of p38 (46), similar to the role of reduced p38 described in LV hypertrophy (27). AT₂ receptors are abundantly expressed in the neonatal lung and, in a model of BPD, AT₂ activation reduced RVH (254). Interestingly, low levels of the AT₂ antagonist PD123319 provided a similar benefit for RVH, and reduced influx of inflammatory cells into the lung (255). At higher doses, the protective effect of the antagonist was lost, and it blocked the beneficial effects of the agonists as well. Whether the effects of the low dose of PD123319 were due to a partial-agonist effect remains to be determined.

Arginase.

The arginases, of which there are two isoforms (I, localized to the cytosol, and II, localized to the mitochondria), are emerging proteins of interest with regards to PH. These enzymes metabolize l-arginine to l-ornithine and urea, a step in the polyamine and proline synthesis pathways relevant to cell proliferation. Prior work has shown that arginase II protein expression and arginase activity were increased in cultured human PASMCs in response to hypoxia and that arginase II inhibition prevented hypoxia-induced PASMC proliferation (38). Furthermore, arginase activity was shown to be increased in lung tissue from chronically hypoxic mice (128) and in serum from PAH patients relative to controls (272), the latter being linked to decreased NO synthesis in pulmonary arterial endothelial cells (PAECs) from PAH patients. Both NO synthase (NOS) and arginase II share l-arginine as a substrate such that increased activity of one enzyme may result in decreased availability of l-arginine for the other. Arginase II expression, as well as that of arginase I, was increased in lung tissue from neonatal rats exposed to the chemotherapeutic agent bleomycin (97), which induces a robust inflammatory response, lung morphology resembling emphysema, and severe PH (87, 97, 123). In this model, the arginase inhibitor amino-2-borono-6-hexanoic acid enhanced NO production (despite decreased NOS2 expression) and prevented development of PH and pulmonary collagen deposition (97).

More recently, HIF-2α has been shown to control hypoxic upregulation of arginase I and II in PAECs (49). Pulmonary endothelial deletion of either HIF-2α or arginase I in mice resulted in blunted hypoxic increase in RVSP and vascular remodeling. Furthermore, plasma NO levels were increased in the arginase I-deficient mice. This suggests that HIF-2α and arginase I are necessary for hypoxia-induced PH, via regulation of NO production.

Resveratrol, a natural polyphenol found in red grapes and berries that inhibits arginase activity, has been found to prevent the development of pulmonary hypertension in the monocrotaline rat (51). Resveratrol inhibited hypoxia-induced arginase activity as well as proliferation in human PASMCs (39) and in vivo resveratrol treatment attenuated the development of hypoxia-induced RVH in rats. The effect of resveratrol upon arginase II was mediated via inhibition of the PI3K-Akt signaling pathway, findings consistent with other recent reports demonstrating a role for Akt signaling in PASMC proliferation in response to hypoxia (233). The Akt family contains three isoforms, and the use of mice with deficiency for Akt1 or Akt2 revealed that loss of Akt1 prevented hypoxia-induced proliferation in vitro and development of hypoxia-induced PH. Although the exact isoform inhibited by resveratrol is not clear, it is likely that Akt1 was the target.

Further insight into the regulation of arginase revealed the contribution of microRNA (miR) to the upstream and downstream signaling mechanisms of arginase II within human PASMCs (129). Specifically, hypoxic induction of miR-17-5p, which is known to induce PASMC proliferation, was necessary for increased arginase II expression. Conversely, inhibition of arginase also prevented hypoxia-induced increases in miR-17-5p expression, suggesting that hypoxia results in an arginase II/miR-17-5p feedback loop.

Sex hormones.

Female sex is perhaps one of the most well-established risk factors for development of PH. Registries of PAH patients report a female:male ratio of between 1.4–4.1:1 (reviewed in Ref. 139). Paradoxically, those men that develop PAH have worse outcomes and increased risk of mortality than female patients (139), perhaps due to preserved RV ejection fraction and function and better RV response to therapy in females (121, 134). The discrepancy in both risk and severity of disease between males and females has prompted intense research into the sex-based differences in PAH, with recent studies shedding new light into the mechanisms that may underlie both risk and protection.

While both male and female rats develop severe PH in response to SuHx, RV function is better preserved in females (82), consistent with the data obtained in patients. Treatment with E₂ in ovarectomized rats normalized RVSP, RVH, and RV function, likely a consequence of reduced oxidative stress, proapoptotic signaling, metabolic dysfunction, and proinflammatory cytokine expression (82). Interestingly, E₂ therapy attenuated RVH and RV function in male rats but did not impact RVSP, suggesting a cardiac-specific effect.

Estrogen signaling also appears to play a protective role in hypoxia-induced PH, where females are protected (159, 193, 197) and administration of estradiol improves PH (197). However, in other models, female sex clearly confers susceptibility. For example, PAH that developed in association with use of diet medications that increased serotonin availability can be modeled in mice with administration of anorexigens (57) or forced overexpression of the serotonin transporter (SERT), which facilitates both cellular uptake and efflux of serotonin (151). Increased uptake of serotonin into PASMCs is associated with proliferation, and SERT expression is upregulated in PASMCs from PAH patients (151). Female mice exposed to dexfenfluramine or with increased SERT expression (SERT⁺) exhibited vascular remodeling and enhanced PH at baseline or in response to hypoxia (57, 101, 152, 263) that was not observed in male mice (57, 263). Interestingly, in the SuHx rat model, loss of SERT had no effect (55). It should be noted, however, that since SERT overexpression appears to preferentially play a role in females, and the study using SERT deletion in the SuHx model was performed only in male animals, the impact of loss of SERT in female SuHx may be different and needs to be determined.

Recent studies have attempted to elucidate some of the mechanisms by which estrogen may mediate its effects in the patient population, with focus shifting to the enzymes that mediate estrogen metabolism. In particular, expression of the gene encoding cytochrome P450 1B1 (CYP1B1) was markedly downregulated in female PAH patients (12) while genetic polymorphisms in CYP1B1, which catalyzes estrogen to 2-hydroxy-estrogen (2-OHE) (Fig. 2), were associated with reduced disease penetrance in females with BMPR2 mutations (12). BMPR2 mutation carriers with wild-type alleles not only had fourfold higher disease penetrance but had diminished CYP1B1 activity and produced lower levels of 2-OHE, resulting in an imbalance between 2-OHE, a metabolite with protective actions, and 16α-hydroxyoestrone (16α-OHE₁) (12), a highly mitogenic and genotoxic metabolite (130). Further evidence that altering the balance between 2-OHE/16α-OHE₁, with increased 16α-OHE₁, is detrimental can be found in studies where administration of 16α-OHE₁ caused PH in mice (264). Although the exact actions of 16α-OHE₁ are still being uncovered, one of the mechanisms by which 16α-OHE₁ may facilitate PAH is via upregulation of miR-29, which in turn downregulates PPARγ and alters molecular and functional indexes of cellular energy metabolism (40). Perhaps surprisingly, increased expression of CYP1B1 was found in the PAs from animal models and PAH patients in a different registry (264). Consistent with these findings, CYP1B1 was increased in female SERT⁺ mice, and inhibiting CYP1B1 reduced development of PH (130). In these studies, elevated 16α-OHE₁ levels were also observed (130, 264), suggesting that while more work will need to be done to understand the complex regulation of estrogen metabolism, any perturbations that result in increased 16α-OHE₁ are likely to be detrimental.

Fig. 2.

Schematic of simplified estrogen metabolism. Estrone (E₁) and estradiol (E₂) can be metabolized through oxidation pathways by cytochrome P450 (CYP) 1A, 1B, and 3A producing 2-, 4- and 16α-hydroxyestrogen (2-OHE, 4-OHE and 16α-OHE, respectively). 2-OHE and 4-OHE derivatives can be further converted to 2- and 4-methoxyestrogen metabolites (2-MeOE and 4-MeOE, respectively), which can have beneficial, antiproliferative properties. 16α-OHE, on the other hand, exerts proproliferative effects.

Inflammation.

It is now recognized that perivascular inflammation is a common contributing factor in almost all forms of PH (69, 188, 189, 194, 243). Macrophages (81, 237, 239, 248) and mast cells (17, 52, 114) have been shown to play a critical role in promoting vascular dysfunction in preclinical PH models. In samples from PAH patients, accumulation of immune cells, including T cells, B cells, macrophages, dendritic cells, and mast cells has been observed in and around sites of vascular lesions (52, 208, 243) and high levels of procontractile and proproliferative cytokines are observed in both patients and animal models of PH (reviewed in Refs. 69, 188, 194). In addition, anti-endothelial (58, 230), anti-fibroblast (231), and anti-nuclear (199) autoantibodies have been identified in PAH patients, although the extent to which these autoantibodies initiate or facilitate progression of disease is unknown. Interestingly, severe PH developed in rats given SU5416 following sensitization with ovalbumin (162), even in the absence of a hypoxic stimulus, suggesting that an allergic inflammatory phenotype can facilitate development of PH. Taken together these data providing compelling evidence for a link between inflammation and the vascular hallmarks of PH.

While the exact mechanisms by which inflammation might facilitate progression of PH are still under investigation, recent evidence has pointed to a role of inflammasomes. Inflammasome activation and assembly lead to caspase-1-mediated cleavage of pro-IL-18 and pro-IL-1β, leading to the release of activated IL-1β and IL-18. Increased circulating levels of IL-1β and IL-18 in PAH patients (120, 201) and animal models (250) suggest inflammasome activation. Interestingly, conflicting results have been reported with IL-18 loss, with some reporting protection (164) and others finding no effect (31), although it should be noted that in the latter study, control mice were housed at Denver altitude, and both male and female mice were used for the study, which could influence outcomes. Thus the downstream mediator of inflammasome activation, at least with respect to the development of PH, remains to be determined. Nonetheless, recent work demonstrated that loss of the common inflammasome component, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), blunted RVSP and RVH and lowered IL-18 levels in mice exposed to CH (34). Interestingly, although the NLRP3 inflammasome has been shown to be activated in CHPH and antioxidant treatment reduced both NLRP3 activation and PH (250), loss of NLR pyrin domain-containing 3 (NLRP3), which is involved in the NLRP3 inflammasome only, did not recapitulate the effects of loss of ASC, suggesting multiple inflammasomes may participate in the pathogenesis of PH.

Many recent investigations have focused on the role played by exogenous signaling molecules produced by inflammatory cells in driving abnormal vascular cell function in PAH (68, 228, 252). Hypoxia-induced mitogenic factor (HIMF, FIZZ1), a protein secreted by M2 macrophages, promotes a proinflammatory milieu in the lung vasculature. Exogenous administration of HIMF was sufficient to induce lung endothelial apoptosis in vivo and this action of HIMF was dependent on IL-4 (273), suggesting that vascular inflammation in the context of PH may require both endogenous activators of EC apoptosis as well as immune cell cytokine responses to effect initial EC apoptosis and subsequent migration and proliferation of both ECs and SMCs. In addition, a metabolite of the 15-lipoxygenase pathway, 15-HETE, upregulates several cytokines such as IL-6 and TNF-α (37, 262), both of which have been implicated in PH. Macrophages, ECs, and platelets are all sources of 15-HETE, and inhibition of 15-HETE reduced platelet aggregation and thrombosis in SuHx and CH rats (216). In PBMCs from PAH patients, 15-HETE levels correlated with IL-6 and PVR (216). Incubating PAs with IL-6 or TNF-α induces a procontractile phenotype and enhances PASMC migration (112). Treatment with the omega-3 fatty acid-derived E-series resolvin, resolvin E1, reduced markers of inflammation (i.e., NF-κB activation), contractility and migratory capacity (112), suggesting a potential role for resolvins in reducing PASMC dysfunction caused by inflammation.

Whereas inflammation seems to clearly play a role in promoting PH, there is also evidence that immune pathways may be protective as well. For example, it was hypothesized that athymic rats exposed to SU5416 would develop less inflammation and thus diminished PH. Instead, these animals exhibited severe PH, even in the absence of hypoxia, and augmented inflammation (235), which was determined to be due to loss of regulatory T cells, which limit perivascular inflammation and vascular injury (232).

Having gained traction in the cancer field, immunotherapy may represent a promising avenue for treatment of PAH. However, with such a complex balance between detrimental and beneficial immune functions, any future immunomodulatory approaches aimed at blocking immune cell recruitment or function will likely require highly specific targeting of individual cell populations within the lung. The need for precise timing of immune targeting and/or the ability to target individual subpopulations of cells will also likely come into play. For example, lung-specific delivery of clondronate, to deplete macrophages, has been achieved using liposomal formulations (107, 158). However, since macrophages can be injurious or protective/anti-inflammatory, depending on subtype (11, 72, 110, 158), the differential contributions from macrophage subsets may represent a dynamic response to disease or injury, a factor that will need to be considered in any immunomodulatory regimen.

Extracellular matrix.

The extracellular matrix (ECM) is clearly important in the development of the pulmonary vasculature, as neonatal mice haploinsufficient for elastin are known to develop PH as adults (73, 217, 256). When challenged with a course of mechanical ventilation, these mice developed increased collagen and fibrillin deposition, which was associated with decreased respiratory system elastance (111). While the number of alveoli remained stable, the number of pulmonary microvessels was significantly decreased, providing important insight into how changes in neonatal anatomic development, lung growth arrest, and impaired respiration may impact long-term outcomes (Fig. 3). In rat pups with PH induced by chronic bleomycin infusion, increased perivascular collagen content was noted that was abrogated by arginase inhibition (97), suggesting links between the NO pathway and matrix remodeling.

Fig. 3.

Schematic illustrating impaired formation of alveoli and lung capillaries and extracellular matrix (ECM) remodeling in wild-type (Eln^+/+) and elastin haploinsufficient (Eln^+/−) neonatal mice subjected to mechanical ventilation. Eln^+/− mice exhibit attenuated lung elastic fibers, increased collagen-1 and fewer capillaries at baseline. In wild-type mice, mechanical ventilation increases lung elastase activity and elastin degradation, leading to fragmented elastic fibers; however, no significant increase of lung elastase activity occurred in Eln^+/− mice. Instead, Eln^+/− mice exhibit reduced elastin synthesis and increased fibrillin production. In both genotypes, mechanical ventilation reduces abundance of the growth factors, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) and causes apoptosis and reduces the numbers of capillaries and alveoli, resulting in diminished lung growth and abnormal lung function. [Reproduced with permission from Ref. 97.]

Accumulation of extracellular matrix components, including collagen, is a key feature of the vascular remodeling that contributes to RV afterload as increased collagen deposition and fibrosis causes stiffening of the proximal arteries (176, 195, 238, 241). Indeed, inhibiting new collagen synthesis using a proline analog prevented the effects of CH on RV hypertrophy and RVSP and attenuated collagen deposition in both the main PA and the distal vasculature (213). Interestingly, while most studies have found perivascular collagen deposition, examination of PAs from idiopathic pulmonary AH patients revealed collagen accumulation mainly in the intima, which correlated with increased expression of collagens (COL4A5, COL14A1, and COL18A1), matrix metalloproteinase (MMP) 19, and a disintegrin and metalloprotease (ADAM) and reduced expression of MMP10, ADAM17, tissue inhibitor of metalloproteinase 1 (TIMP1), and TIMP3 (113). Interestingly, collagen 4A1 and 4A2 expression was reduced in ECs from PAH patients, secondary to BMPR2 downregulation (198). Reductions in collagen 4A1 and 4A2 were associated with EC dysfunction (198), suggesting the potential for increased susceptibility to vascular injury with loss of BMPR2 signaling.

The mechanisms regulating collagen turnover and deposition during PH are still being defined. Main PAs from a fetal sheep PH model exhibited increased mechanical stiffness in the circumferential and axial orientations (62), likely caused by ECM remodeling due to hemodynamic stress. Mathematical modeling and histologic findings showed collagen fibers contributed a greater role to the ECM than elastin. During hypoxia, collagen synthesis has been shown to be upregulated by aldosterone (156), which interacts with mineral corticosteroid receptors to upregulate connective tissue growth factor (CTGF) and collagen III. Inflammatory pathways are also likely to contribute to fibrosis. In a model of antenatal inflammation due to endotoxin exposure, a strong risk factor for subsequent development of PH, pre- and post-natal vitamin D supplementation decreased PA and RV stiffness by restoring the normal organization of ECM collagen and elastin (154), likely due to preserved lung vascular development secondary to the anti-inflammatory properties of vitamin D. Changes PA stiffness were associated with improved oxygen saturations, postnatal survival, and EC function (155).

In addition to increasing afterload via arterial stiffness, collagen metabolism may also play a direct role in RV function. In PAH patients, RV diastolic stiffness was increased and correlated with RV fibrosis and collagen content (195). Similarly, SuHx rats exhibit increased RV fibrosis, collagen deposition and procollagen expression (94). In these animals, the expression of CTGF was also increased, consistent with the effects of mineralocorticoids in the vasculature. Mutant mice in which collagen type 1 turnover was impaired exhibited preserved RV function in response to PH induced by SuHx (93). In these mice RVSP was similar to wild-type SuHx, suggesting that RV-PA coupling was perhaps better maintained. While arterial stiffness was not measured, exposure to CH alone caused reduced arterial stiffening as well as decreased RVSP (176). PKCβII and -δ protein expression and p38 dephosphorylation were increased in cardiac fibroblasts from CH rats with RV fibrosis (46). In vitro application of angiotensin II recapitulated the effects of CH, causing PKCβII and -δ-dependent inactivation of p38 and collagen deposition (46). Whether mineralocorticoids, PKC, or p38 regulate RV collagen accumulation in PAH patients remains to be determined.

Transglutaminase 2.

Calcium signaling has been a recurrent motif in pathways that contribute to altered PASMC function in PH. Transglutaminase 2 (TG2) is a Ca²⁺-dependent enzyme that catalyzes posttranslational attachment of serotonin to glutamine (Gln) residues. TG2 activity increased in the lungs of hypoxic mice and TG2 inhibition attenuated the development of PH in the SuHx mouse (61). In cultured bovine PASMCs, hypoxia-related elevations in cell proliferation were found to depend on TG2 activity (181). Furthermore, extracellular Ca²⁺-sensing receptor or the transient receptor potential channel V4 was necessary for hypoxic increases in TG2 activity, whereas HIF-1α was necessary for hypoxic induction of TG2 expression. Thus TG2 activation represents one downstream avenue via which Ca²⁺ fluxes effect changes in PASMC function in the setting of hypoxia.

In addition to modifications of Gln, TG2 also catalyzes covalent crosslinking of lysine residues in the ECM, providing stability, rigidity, and protection from degradation (148). TG2 has recently been suggested to be responsive to mechanical stress (119) and, when activated, can facilitate arterial stiffening (207) and narrowing of mesenteric arteries in organ culture (15) and in vivo (16, 185). These results suggest the possibility that increased mechanical loads (i.e., increased shear stress or elevated wall strain) during PH could trigger stiffening and/or narrowing via activation of TG2.

Potential Treatments

ET-1 receptor antagonists.

The vasoactive peptide ET-1 has long been recognized as a contributor to the development of many forms of PH. Research in animal models has revealed many of the cellular mechanisms involved in the vascular actions of ET-1, including inhibition of K⁺ channels, elevation in intracellular calcium, enhancing sensitivity of the contractile apparatus, and production of ROS (184, 220). Widely recognized as one of the most potent vasoconstrictors, ET-1 can also act as a mitogen (281). While primarily derived from ECs, PASMCs and other cells can be a source of ET-1. Indeed, a role for SMC-derived ET-1 in modulating PASMC function has been shown previously, where ET-1 mediated the effects of hypoxia on ion channel expression via upregulation of HIF-1α (184, 265). More recently, SMC-specific loss of ET-1 reduced CH-induced PH and PASMC migration/proliferation (135). Interestingly, inhibition of ET_A or ET_B receptors did not prevent the effects of hypoxia, suggesting a potential intracellular role for ET-1 or signaling via a non-ET_A or ET_B receptor. This is in contrast to other studies, which found blockade of either receptor prevented the ET-1-mediated effects of hypoxia (184, 265).

Because ET-1 levels were found to be elevated in almost all forms of PH and preclinical data suggested beneficial actions, endothelin receptor antagonists (ETAs) were rapidly translated to clinical use and the mixed ET_A/ET_B antagonist bosentan remains one of the frontline therapies for treatment (54). However, recent advances in developing novel chemical analogs of bosentan have provided some new options. In particular, the recently approved drug macitentan appears quite promising. Macitentan differs from other antagonists in its slow receptor dissociation, rendering the compound more pharmacologically active than other ET receptor antagonists (86). Initial clinical studies demonstrated that macitentan significantly improved mortality in PAH patients, with a better safety profile than other ETAs and minimal adverse events (190). Interestingly, unlike most other drugs, macitentan entered clinical trials before being tested in preclinical PH models. It is now known that macitentan is effective at reversing established PH in SuHx (137) and monocrotaline (236) models. In the SuHx model, macitentan was able to partially reverse RVSP and RVH at both early and later time points; in both cases proliferation of cells in occlusive lesions was decreased. Additionally, with early treatment, apoptosis was increased (221). Thus preclinical research continues to improve our understanding of the role of ET-1 in mediating PH and of the effects of novel ETAs that may provide improved ability to target pathways that will allow regression of remodeling with more favorable safety profiles.

K_V7 (KCNQ) Channel Activators

K⁺ channels play a significant role in setting the resting membrane potential. Several lines of evidence indicate that downregulation or inhibition of K_V channels is a likely modulator of pulmonary vascular tone and PH. Recent interest has focused on K_V7 channels as possible targets for modulation of tone (103). PASMCs have been shown to express several KCNQ family members, including K_V7.1, K_V7.4, and K_V7.5 (131). Evidence is accumulating that these channels, especially K_V7.4, play a major role in regulating tone in PAs. For example, in contrast to most other K_V channels, K_V7 channels are activated at much more negative potentials (−80 to −60 mV), suggesting that these channels are likely open at the resting membrane potential in PASMCs (103).

Inhibitor and activator studies also suggest that KCNQ channels may be contributing to PH. K_V7 inhibitors have minimal effect on systemic vessels (131) but cause pulmonary vascular contraction (131, 214) that is lost with exposure to hypoxia (214). On the other hand, flupirtine, an orally active K_V7 activator, reduced P_PA in rats that were exposed to short-term (5 days) hypoxia (214). Marked downregulation of K_V7.4 mRNA expression was observed in this model, but surprisingly, protein levels were not altered. Whether the lack of effect on K_V7.4 protein requires longer duration of exposure to become evident is unknown. K_V7.5 protein expression was also reduced by hypoxia in PASMCs (147). What remains unclear is the mechanism by which hypoxia might decrease K_V7 channel expression; however, KCNQ5 (K_V7.5) was found to be a target of miR190 (147). Since hypoxia downregulates K_V7.5 and upregulates miR190 (147), it is tempting to speculate that miR190 targeting of K_V7.5 might contribute to increased vasoreactivity in CH-induced PH.

Flupirtine also protected against development of PH in CH and SERT⁺ mice (163). The use of drugs targeting these channels are attractive since the KCNQ activators flupirtine and retigabine have completed clinical trials for pain and seizures and appear to be relatively safe when administered to humans, with minimal side effects (146, 187).

cGMP pathway.

It has long been recognized that impaired NO production and/or responsiveness can contribute to PH. NO, produced primarily from ECs, diffuses to the SMCs, where it activates soluble guanylate cyclase (sGC), leading to production of cGMP, activation of protein kinase G (PKG) and relaxation (253). Phosphodiesterases (PDEs) catalyze cGMP breakdown, thus antagonizing the effects of the NO-cGMP-PKG pathway (89). In PH, ample evidence has accumulated that decreased production of NO and reduced sGC activity, coupled with increased PDE activity, may all facilitate reduced effectiveness of the NO pathway to maintain low vasomotor tone. For example, loss of eNOS (71, 227) or PKG isoform 1 (PKG-1) (284) increases P_PA under control conditions. Alternative splicing of the gene encoding PKG-1 results in two isoforms, of which PKG-1α contains a function leucine zipper motif that allows binding to myosin light chain phosphatase and RhoA and mediates relaxation. Selective loss of PKG-1α activity, via a mutation in the leucine zipper domain, increased ROCK activity and resulted in spontaneous development of PH in mice (196).

Reduced NO production has been well documented in PH (132), in some cases due to reduced expression of eNOS (90, 249). In addition, oxidant stress can lead to reduced phosphorylation of eNOS at Ser 1177, which is required for efficient NO production (60). During CH, levels of Ser1177 phosphorylated eNOS are markedly reduced (138) in adult animals. In newborns, oxidative stress also caused persistent downregulation of the NO pathway that resulted in enhanced PA contractility and muscularity and RV hypertrophy that persisted into adulthood (218). Along these lines, lung NOS levels were reduced in an experimental model of congenital diaphragmatic hernia (CDH) in fetal rats (153). Based on observations of high PVR in PPHN, augmentation and/or stimulation of the NO pathway has been an area of intense focus in the treatment of the pediatric population (253). In infants, inhaled NO has been used widely in the clinical setting, and preclinical studies have assessed methods to enhance endogenous NO production. For example, in the hypoxic piglet model, treatment with l-citrulline, the precursor for NO production, helps to recouple eNOS and attenuates PH (77). Similarly, treatment with genestein, a phytoestrogen with tyrosine kinase inhibiting and anti-oxidant properties, restored eNOS phosphorylation, NO production, and cGMP levels and prevented PH (138). The actions of genestein may result from upregulated Akt activity, which has been shown to activate eNOS (60). Finally, antenatal therapy with simvastatin in a CDH model restored NOS levels, normalized pulmonary vessel density and diameter and reduced arteriolar remodeling (153). While the results from the latter study would seem promising, caution should be exercised when interpreting these results, as the effects on P_PA or RVH were not assessed and simvastatin has a number of effects unrelated to NOS.

Although inhaled NO remains the first line defense in infants with PH, it should be noted that only ∼60% of patients in this population are NO responsive. The reason for lack of response in some patients is unknown, but may be due to impairments in downstream signaling. For example, in newborn rats the cGMP pathway appears to desensitize, as vasorelaxation induced by activating sGC or application of an NO donor was lost when the vessels were preincubated with agents that increase cGMP activity (70). These results indicate that other modes of activating the sGC/cGMP pathway or alleviating cGMP desensitization will be needed.

In the adult PH population, the PDE5 inhibitors sildenafil and tadalafil have been used to increase cGMP levels with positive impact on pulmonary hemodynamics and exercise capacity (28, 89, 136). A recent study compared the effect of sildenafil with exercise training in the CH mouse model (261), finding that exercise training reduced RVSP and vascular remodeling to a similar extent as sildenafil, with no additive effect. The beneficial effect of exercise training were similar to results reported in PH patients (35, 99), although whether the effects of exercise are due to increased local or systemic production of NO and whether the effects of exercise and sildenafil are synergistic in patients remains to be determined.

While most attention has focused on PDE5, a novel PDE2 inhibitor, BAY 60–7550, ameliorated CH- and bleomycin-induced PH and reduced proliferation in PASMCs from PAH patients (32), suggesting that other PDEs may also be suitable targets. Interestingly, while PDE5 inhibition is most often associated with vasodilation due to reduced cGMP breakdown, it has also been found to interact with the ET-1 pathway, reducing ET-1-induced contraction (285), suggesting that these agents may have multifactorial benefits.

PDE inhibition would be expected to lead to an accumulation of cGMP due to reduced breakdown; however, increasing cGMP synthesis is another potential approach to stimulate the pathway. Interestingly, sGC expression appears to be upregulated during PH (211), suggesting that reduced activity may reflect oxidation and reduced NO responsiveness of the enzyme (202). A number of compounds have been developed to increase sGC activity. Some, such as BAY 58–2667 (cinaciguat) activate sGC when the heme iron is in its oxidized state or missing (202). BAY 58–2667 reduced P_PA in fetal lambs after ductus arteriosus ligation (41) and PH in CH mouse and monocrotaline rat models (66). Other methods of increasing sGC activity target sGC that is missing a heme moiety. For example, δ-aminolevulinic acid, the rate-limiting step in heme biosynthesis, targets heme-lacking sGC. In this case, increasing protoporphyrin IX, the iron-free form of heme, activates sGC by allowing protoporphyrin IX to bind to the heme site. Consistent with other methods that activate sGC, δ-aminolevulinic acid prevented PH in mice exposed to CH (8).

Similarly, stimulating sGC with the novel, orally available compound BAY 63–2521 (riociguat), which also increases sensitivity of sGC to NO or stimulates sGC in the absence of bound NO, increased NO-stimulated cGMP production and prevented progression of PH in CH, SuHx, and monocrotaline-treated animals (142, 211). In clinical trials, riociguat improved patient outcomes (88) and has now been approved for the treatment of Group 1 and Group 4 PH.

Cell metabolism.

Several pharmacological agents directed at treating altered metabolism in PH are currently under investigation. Metformin, commonly used in the treatment of type 2 diabetes, activates AMP-activated protein kinase (AMPK), which serves as a key regulator of cellular energy metabolism via a plethora of downstream effects (106). Metformin has been found to prevent PH induced by either monocrotaline or CH (5). More recently, both nitrite and metformin were shown to activate a SIRT3-AMPK-GLUT4 signaling pathway in skeletal muscle, enhancing muscle glucose uptake (140). Additionally, in a rat model of metabolic syndrome with PH associated with heart failure with preserved ejection fraction (PH-HFpEF) induced by a combination of leptin receptor defect and SU5416 injection, treatment with metformin at the time of SU5416 injection prevented increased P_PA and vascular remodeling. However, there was no benefit of metformin therapy in older rats with established PH-HFpEF. In the female rat SuHx model of PH, metformin therapy reversed elevated P_PA and vascular remodeling (56). In this study, metformin had several effects on SuHx lungs, including increased AMPK activity, decreased aromatase and estrogen levels and decreased PASMC proliferation, suggesting that one of the downstream effects of metformin-induced AMPK activation is to regulate the sex hormone axis via aromatase inhibition. A clinical trial of metformin in Group 1 PH is now ongoing (NCT01884051).

Dichloroacetate (DCA), which inhibits pyruvate dehydrogenase kinase, resulting in activation of pyruvate dehydrogenase and thereby promoting glucose oxidation, has long been of interest in the study of PH. DCA prevents and reverses CH- and monocrotaline-induced PH in rats via upregulation of K_V1.5 and increased apoptosis, together with decreased proliferation of PASMCs (160, 161). DCA also improves glucose oxidation in the RV, resulting in better function (183). Results from a phase 1 study of DCA in PAH patients are pending (NCT01083524).

Oxidative stress.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a nuclear transcription factor that regulates expression of multiple genes encoding antioxidant enzymes, including NAD phosphate reduced quinone oxidoreductase (NQO1), glutathione S-transferase, and hemoxygenase-1 (HO-1), as well as genes controlling mitochondrial DNA replication, such as the mitochondrial transcription factor A (TFAM). Nrf2 is known to act in a PAEC signaling pathway whereby BMPR2 regulates TFAM and mitochondrial DNA replication (59). Mice treated with the Nrf-2 activator olitpraz showed an attenuated increase in RVH and pulmonary vascular remodeling but surprisingly showed no change in RVSP (67). A pharmacologic Nrf2 activator, bardoxolone methyl, which also inhibits NF-κB, is currently in a phase 2 clinical trial in patients with World Health Organization (WHO) Group I, III, or V PH (NCT02036970).

ASK1/p38 MAPK.

Targeting the p38 MAP kinase signaling pathway has been of interest in a number of disease states. Unfortunately, p38 inhibitors have been marked by poor side-effect profiles (79). Thus there has been interest in targeting other components of the MAPK signaling cascade. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase that lies upstream of both p38 MAPK as well as c-Jun NH₂-terminal kinase (JNK), another member of the MAPK family. Hypoxia increases JNK, as well as p38 MAPK, activity in rat PASMCs (127), while serotonin-induced enhancement of PASMC proliferation and migration are regulated by JNK, which signals through the Akt pathway (260). Additionally, JNK inhibition decreases proliferation of human PAH PASMCs (268). These findings raise the possibility that ASK1 inhibition could therefore lead to beneficial effects in PH via downstream inhibition of both p38 MAPK and JNK. Indeed, a phase 2 clinical trial of the ASK1 inhibitor GS-4997 is underway in WHO Group I PH patients (NCT02234141). Another potential method of inhibiting MAPKs would be to activate MAPK phosphatases. Genetic loss of MAPK phosphatase-1 (MKP-1) in mice resulted in exaggerated increases in RVSP, RVH, and vascular remodeling in response to CH (as well as exaggerated increases in lung arginase I and II), supporting the idea of MAPK phosphatases as potential therapeutic targets (128). Despite the potential for targeting MAPK signaling as a treatment option, given their multiple physiological roles, it seems likely that distal parts of the signaling cascades, rather than more proximal, will provide better therapeutic options with reduced toxicity and less frequent unwanted side effects.

Sex hormones.

Dehydroepiandrosterone (DHEA), a steroid hormone, exhibited protective effects in preclinical models of PH (9, 25, 64) and low levels of DHEA are associated with the development of PAH in men (246). Although DHEA is a precursor for estrogens, and can bind estrogen receptors, the actions of DHEA are likely to be multifold. For example, DHEA inhibits G6PD (102, 104), a key player in altered cellar metabolism. In PASMCs, DHEA also inhibits Src/STAT3 signaling pathways (180), which are involved in both proliferative and contractile mechanisms, and downregulates ROCK signaling (115). In addition to inhibiting pathways that promote contraction, DHEA also increases the expression of K⁺ channels (25) and inhibits Ca²⁺ influx through T-type Ca²⁺ channels (45), perhaps helping to reduce intracellular Ca²⁺ levels. With the effects on calcium influx, coupled with upregulation of sGC (175) and activation of PKG (177), the overall effect of DHEA is to facilitate vasorelaxation. Clinical trials of DHEA in PH patients are ongoing, although encouraging results from an initial pilot study of patients with PH secondary to COPD reported beneficial effects on P_PA, PVR, and 6-min walk distance (65).

Other studies have been launched to investigate hormone therapy in PAH. A double-blind, placebo-controlled randomized phase II clinical trial using anastrozole in PAH patients (NCT01545336) has recently shown that this therapy was well-tolerated and effectively reduced E₂ levels (133). Although limited by the small sample size, preliminary results suggest potential positive benefits on 6 min walk test, laying the foundation for future phase III trials.

Elafin.

Since increased matrix deposition and crosslinking is clearly linked to vascular stiffening, targeting the ECM could provide a potential therapeutic option. The serine elastase inhibitor elafin is endogenously produced primarily in the epithelium and provides protection from inflammatory damage. Mice engineered to genetically overexpress elafin are protected from CH-induced PH (280), with reduced metalloprotease activity. However, recent studies demonstrating that treatment with elafin regressed RVSP and vascular remodeling in the SuHx rat model (167) through a mechanism involving augmentation of endothelial BMPR2 signaling and caveolin-1 interactions indicates a previously unappreciated link to endothelial dysfunction. Similarly, in celiac disease, elafin inhibited TG2 activity (84), suggesting additional cross-over with pathways known to be involved in PH. Clinical trials have been performed or are currently underway in coronary artery bypass graft surgery (6) and esophagectomy and elafin, under the trade name Tiprelestat, has been granted orphan drug status for PAH, with clinical trials likely forthcoming. Given the systemic roles of elastase inhibitors, and potential as yet unidentified actions of elafin, limiting the systemic effects of elafin by lung-directed targeting, perhaps through inhaled versions, deserves consideration.

Circulating Biomarkers

Reliable biomarkers for diagnosis, prognostication, and treatment response would be ideal for the management of PH (80). However, the search for convenient, minimally invasive, specific, and sensitive biomarkers for PH has not revealed any one circulating marker that can be used to accurately diagnose PH or monitor disease progression. While ET-1 and BNP/NH₂-terminal-proBNP have proven useful in certain cases, circulating ET-1 levels only represent “spillover” rather than local concentrations and BNP can be affected by demographic factors and may differ with PH etiology (182). As such, only BNP/NT-proBNP are used clinically and the search for additional biomarkers continues.

Circulating biomarkers that have been examined in the pediatric population include NT-proBNP (21, 141) and circulating ECs (144) and fibrocytes (277). In addition, proteomics analysis of plasma derived from children with PH was able to distinguish responders to long-term vasodilators (276). More recently, prospective investigation of the relationship between two markers specific for pulmonary angiogenesis, vascular endothelial growth factor A (VEGFA) and placental growth factor (PLGF), in a cohort of neonates with CDH undergoing standard of care revealed a relationship between a higher VEGFA:PLGF ratio and echocardiographic measures of PH, including RVSP, diastolic ventricular dysfunction, and oxygenation index (178). Most striking, the ratio was elevated at days 3–4 of life and in the second week in patients who died (178). Thus the relationship between VEGFA:PLGF may provide a promising biomarker in the pediatric patient population.

In the adult PH population, novel biomarkers that may identify those patients that respond to vasodilator therapy have been identified in circulating lymphocytes (109). In particular, combined high mRNA levels of RHOQ, which encodes a cytoskeletal protein involved in insulin-mediated signaling, and TPD52, a protein cancer marker, could correctly distinguish patients that were nonresponsive to vasodilators.

While vascular levels of collagen and collagen metabolism have been shown to increase with PH, circulating biomarkers of collagen metabolism have been shown to predict disease severity in PAH patients, with higher levels of NH₂-terminal pro-peptide of type III procollagen (PIIINP), COOH-terminal telopeptide of collagen type I, MMP-9, and TIMP1 in PAH patients (205). Moreover, levels of PIIINP significantly correlated with severity of PAH (205) and worse quality of life (206). High circulating levels of collagen XVIII, and its cleavage product, endostatin, have also been shown to be positively correlated with poor outcomes in PAH (53, 113), as has galectin-3 (75), a regulator of matrix deposition and PIIINP. While further testing will be needed, panels of biomarkers based on collagen metabolism appear promising.

Finally, metabolic profiling indicates that several pathways are disturbed in PAH patients. Tracking metabolic abnormalities may provide insight into disease progression and offer clues to therapeutic efficacy. Along these lines, several plasma metabolite biomarkers of PH and associated RV-pulmonary vasculature dysfunction were identified, including metabolites of the indoleamine 2,3-dioxygenase (IDO) pathway (145). The IDO pathway was also upregulated in CH mice. Since these metabolites are thought to function as vasodilators (258), these markers are not believed to be pathogenic but rather to reflect a compensatory mechanism.

Because PH is multifactorial with respect to underlying etiology and pathophysiology, it is difficult to envision a single biomarker that will fit all cases. However, only time and additional testing will tell if any of these proposed biomarkers, alone or in combination, will prove useful for early diagnosis or monitoring disease activity.

Conclusion

PH remains a complex and mysterious disease with often fatal outcome. Tremendous progress has been made over the last few years in elucidating novel cellular mechanisms involved in the pathogenesis of disease, developing new therapeutic strategies to treat the patient population, and identifying potential biomarkers that can facilitate diagnosis, predict prognosis and monitor disease progression (Fig. 4). However, as is often the case, with new insights arise new questions. Which biomarkers will ultimately allow physicians to distinguish patients that will respond to a given therapy? Which drug, or combination of drugs, will prove most effective in slowing progression of disease? In addition, perhaps the holy grail of PH research: will any therapy ultimately lead to reversal of disease (i.e., curative) in the patient population? Given the progress made to date, confidence is high that ongoing and future research will provide additional insight toward answering these pressing issues.

Fig. 4.

Overview of mechanisms contributing to pulmonary hypertension. Pulmonary hypertension is characterized by remodeling of all layers of the pulmonary vasculature, including endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts, resulting in vascular wall thickening and occlusion of the lumen, which play roles of varied prominence depending on the etiology of disease. In addition, recruitment of inflammatory cells contributes to the remodeling process. Included here are several of the pathogenic mechanisms that are highlighted in this review of recent progress in the field. AMPK, AMP-activated protein kinase; G6PD, glucose-6-phosphate dehydrogenase; SOD, superoxide dismutase; Nrf2, nuclear factor E2-related factor 2; PPARg, peroxisome proliferator-activated receptor-γ; JNK, c-Jun NH₂-terminal kinase; CYP1B1, cytochrome P450 1B1; DHEA, didehydroepiandrosterone; HIMF, hypoxia-induced mitogenic factor; 15-HETE, 15-hydroxyeicosatetraenoic acid.

GRANTS

This work was funded by National Institutes of Health Grants HL-124727, HD-044355, HL-126514, HL-073859, and HL-124930.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

J.C.H. and L.A.S. prepared figures; J.C.H., K.S., M.B., and L.A.S. drafted manuscript; J.C.H., K.S., M.B., and L.A.S. edited and revised manuscript; J.C.H., K.S., M.B., and L.A.S. approved final version of manuscript.