Mining the Plasma Proteome for Insights into the Molecular Pathology of Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by structural remodeling of pulmonary arteries and arterioles. Underlying biological processes are likely reflected in a perturbation of circulating proteins. Objectives: To quantify and analyze the plasma proteome of patients with PAH using inherited genetic variation to inform on underlying molecular drivers. Methods: An aptamer-based assay was used to measure plasma proteins in 357 patients with idiopathic or heritable PAH, 103 healthy volunteers, and 23 relatives of patients with PAH. In discovery and replication subgroups, the plasma proteomes of PAH and healthy individuals were compared, and the relationship to transplantation-free survival in PAH was determined. To examine causal relationships to PAH, protein quantitative trait loci (pQTL) that influenced protein levels in the patient population were used as instruments for Mendelian randomization (MR) analysis. Measurements and Main Results: From 4,152 annotated plasma proteins, levels of 208 differed between patients with PAH and healthy subjects, and 49 predicted long-term survival. MR based on cis-pQTL located in proximity to the encoding gene for proteins that were prognostic and distinguished PAH from health estimated an adverse effect for higher levels of netrin-4 (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.16-2.08) and a protective effect for higher levels of thrombospondin-2 (OR, 0.83; 95% CI, 0.74-0.94) on PAH. Both proteins tracked the development of PAH in previously healthy relatives and changes in thrombospondin-2 associated with pulmonary arterial pressure at disease onset. Conclusions: Integrated analysis of the plasma proteome and genome implicates two secreted matrix-binding proteins, netrin-4 and thrombospondin-2, in the pathobiology of PAH.

Pulmonary Hypertension: New Insights and Recent Advances from Basic Science to Translational Approaches.

This Special Issue, "Molecular Research on Pulmonary Hypertension 3 [...].

Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease characterised by pro-proliferative and anti-apoptotic phenotype in vascular cells, leading to pulmonary vascular remodelling and right heart failure. Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1), a highly conserved enzyme, which binds to and catalyses the isomerisation of specific phosphorylated Ser/Thr-Pro motifs, acts as a molecular switch in multiple coordinated cellular processes. We hypothesised that Pin1 plays a substantial role in PAH, and its inhibition with a natural organic compound, Juglone, would reverse experimental pulmonary hypertension. RESULTS: We demonstrated that the expression of Pin1 was markedly elevated in experimental pulmonary hypertension (i.e. hypoxia-induced mouse and Sugen/hypoxia-induced rat models) and pulmonary arterial smooth muscle cells of patients with clinical PAH. In vitro Pin1 inhibition by either Juglone treatment or short interfering RNA knockdown resulted in an induction of apoptosis and decrease in proliferation of human pulmonary vascular cells. Stimulation with growth factors induced Pin1 expression, while its inhibition reduced the activity of numerous PAH-related transcription factors, such as hypoxia-inducible factor (HIF)-α and signal transducer and activator of transcription (STAT). Juglone administration lowered pulmonary vascular resistance, enhanced right ventribular function, improved pulmonary vascular and cardiac remodelling in the Sugen/hypoxia rat model of PAH and the chronic hypoxia-induced pulmonary hypertension model in mice. CONCLUSION: Our study demonstrates that targeting of Pin1 with small molecule inhibitor, Juglone, might be an attractive future therapeutic strategy for PAH and right heart disease secondary to PAH.

Genetic Delivery and Gene Therapy in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive complex fatal disease of multiple etiologies. Hyperproliferation and resistance to apoptosis of vascular cells of intimal, medial, and adventitial layers of pulmonary vessels trigger excessive pulmonary vascular remodeling and vasoconstriction in the course of pulmonary arterial hypertension (PAH), a subgroup of PH. Multiple gene mutation/s or dysregulated gene expression contribute to the pathogenesis of PAH by endorsing the proliferation and promoting the resistance to apoptosis of pulmonary vascular cells. Given the vital role of these cells in PAH progression, the development of safe and efficient-gene therapeutic approaches that lead to restoration or down-regulation of gene expression, generally involved in the etiology of the disease is the need of the hour. Currently, none of the FDA-approved drugs provides a cure against PH, hence innovative tools may offer a novel treatment paradigm for this progressive and lethal disorder by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications. Here, we review the effectiveness and limitations of the presently available gene therapy approaches for PH. We provide a brief survey of commonly existing and currently applicable gene transfer methods for pulmonary vascular cells in vitro and describe some more recent developments for gene delivery existing in the field of PH in vivo.

Therapeutic Potential of Regorafenib-A Multikinase Inhibitor in Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by a progressive elevation of mean arterial pressure followed by right ventricular failure and death. Previous studies have indicated that numerous inhibitors of receptor tyrosine kinase signaling could be either beneficial or detrimental for the treatment of PH. Here we investigated the therapeutic potential of the multi-kinase inhibitor regorafenib (BAY 73-4506) for the treatment of PH. A peptide-based kinase activity assay was performed using the PamStation®12 platform. The 5-bromo-2'-deoxyuridine proliferation and transwell migration assays were utilized in pulmonary arterial smooth muscle cells (PASMCs). Regorafenib was administered to monocrotaline- and hypoxia-induced PH in rats and mice, respectively. Functional parameters were analyzed by hemodynamic and echocardiographic measurements. The kinase activity assay revealed upregulation of twenty-nine kinases in PASMCs from patients with idiopathic PAH (IPAH), of which fifteen were established as potential targets of regorafenib. Regorafenib showed strong anti-proliferative and anti-migratory effects in IPAH-PASMCs compared to the control PASMCs. Both experimental models indicated improved cardiac function and reduced pulmonary vascular remodeling upon regorafenib treatment. In lungs from monocrotaline (MCT) rats, regorafenib reduced the phosphorylation of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2. Overall, our data indicated that regorafenib plays a beneficial role in experimental PH.

Adenylate Kinase 4-A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways.

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.

Evidence for the Fucoidan/P-Selectin Axis as a Therapeutic Target in Hypoxia-induced Pulmonary Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). Fucoidan, a polysaccharidic ligand of the adhesion molecule P-selectin, exhibits antiproliferative properties. The effects of the fucoidan/P-selectin axis on vascular remodeling and pulmonary hypertension (PH) after hypoxia remain unexplored. Objectives: We aimed to evaluate the therapeutic potential of targeting the fucoidan/P-selectin axis in PH. Methods: Mice with PH induced by chronic hypoxia (35 d) were given either fucoidan (from Fucus vesiculosus) or anti-P-selectin antibody (Rb40.34) during Days 21-35. Right ventricular (RV) function was determined by echocardiography. Vascular morphometry was assessed by immunohistochemistry. Human and experimental PH lungs and PASMCs were used for assessment of P-selectin expression and function. Measurements and Main Results: Fucoidan attenuated chronic hypoxia-induced PH in mice, reducing pulmonary vascular remodeling and restoring RV function. In vitro, fucoidan inhibited hypoxia and growth factor-stimulated PASMC proliferation and migration. Chronic hypoxia caused an upregulation of P-selectin in the medial layer of the small pulmonary arteries. P-selectin was persistently upregulated in PASMCs of human and hypoxia-induced experimental PH. HIF-1α (hypoxia-inducible factor 1α) directly bound to the P-selectin promoter and transcriptionally activated P-selectin in hypoxia. P-selectin blockage resulted in a marked reduction of PASMC proliferation in vitro. Blockage of P-selectin by administration of anti-P-selectin Rb40.34 antibody and P-selectin-deficient mice improved vascular remodeling and restored RV function. Conclusions: Fucoidan is a potent natural adjuvant that represents a promising therapeutic approach for PH. Our data indicate a previously unrecognized role of P-selectin in the proliferative response of PASMCs associated with PH.

Yarsagumba is a Promising Therapeutic Option for Treatment of Pulmonary Hypertension due to the Potent Anti-Proliferative and Vasorelaxant Properties.

Background and objectives: Pulmonary hypertension (PH) is characterized by the vasoconstriction and abnormally proliferative vascular cells. The available allopathic treatment options for PH are still not able to cure the disease. Alternative medicine is becoming popular and drawing the attention of the general public and scientific communities. The entomogenous fungus Yarsagumba (Cordyceps sinensis) and its biologically active ingredient cordycepin may represent the therapeutic option for this incurable disease, owing to their anti-inflammatory, vasodilatory and anti-oxidative effects. Methods: In this study, we investigated whether Yarsagumba extract and cordycepin possess anti-proliferative and vasorelaxant properties in the context of PH, using 5-bromo-2'-deoxyuridine assay and isolated mice lungs, respectively. Results: Our results revealed that Yarsagumba extract and its bioactive compound cordycepin significantly attenuated the proliferation of human pulmonary artery smooth muscle cells derived from donor and PH subjects. In isolated murine lungs, only Yarsagumba extract, but not cordycepin, resulted in vasodilatation, indicating the probable existence of other bioactive metabolites present in Yarsagumba that may be responsible for this outcome. Conclusion: Future comprehensive in vivo and in vitro research is crucially needed to discover the profound mechanistic insights with regard to this promising therapeutic potency of Yarsagumba extract and to provide further evidence as to whether it can be used as a strategy for the treatment of PH.

Nitric Oxide Synthase 2 Induction Promotes Right Ventricular Fibrosis.

The ability of the right ventricle to compensate pressure overload determines survival in pulmonary arterial hypertension (PAH). Nitric oxide (NO) reduces the right ventricular afterload through pulmonary vasodilation, but excessive NO amounts cause oxidative stress. Oxidative stress drives remodeling of pulmonary arteries and the right ventricle. In the present study, we hypothesized that nitric oxide synthase 2 (NOS2) induction leads to excessive NO amounts that contribute to oxidative stress and impair right ventricular adaptation to PAH. We used a surgical pulmonary artery banding (PAB) mouse model in which right ventricular dysfunction and remodeling occur independently of changes in the pulmonary vasculature. Three weeks after PAB, NOS2 expression was increased twofold in the hypertrophied right ventricle on transcript and protein levels together with increased NO production. Histomorphology localized NOS2 in interstitial and perivascular cardiac fibroblasts after PAB, which was confirmed by cell isolation experiments. In the hypertrophied right ventricle, NOS2 induction was accompanied by an increased formation of reactive oxidants blocked by ex vivo NOS inhibition. We show that reactive oxidant formation in the hypertrophied right ventricle is in part NOS2 dependent (in NOS2-deficient mice [NOS2-/-]). Lack of NOS2 induction prevented superoxide scavenging and decreased reactive oxidant formation. Functional measures of cardiac function by noninvasive echocardiography together with intracardiac catheterization revealed no differences in heart function between both genotypes after PAB. However, reduced NO and reactive oxidant formation in the hypertrophied right ventricle of NOS2-/- mice was linked to reduced collagen accumulation through reduced collagen deposition from the cardiac fibroblast. Together, our data demonstrate a profibrotic role for NOS2 induction in the hypertrophied right ventricle.

ASK1 Inhibition Halts Disease Progression in Preclinical Models of Pulmonary Arterial Hypertension.

RATIONALE: Progression of pulmonary arterial hypertension (PAH) is associated with pathological remodeling of the pulmonary vasculature and the right ventricle (RV). Oxidative stress drives the remodeling process through activation of MAPKs (mitogen-activated protein kinases), which stimulate apoptosis, inflammation, and fibrosis. OBJECTIVES: We investigated whether pharmacological inhibition of the redox-sensitive apical MAPK, ASK1 (apoptosis signal-regulating kinase 1), can halt the progression of pulmonary vascular and RV remodeling. METHODS: A selective, orally available ASK1 inhibitor, GS-444217, was administered to two preclinical rat models of PAH (monocrotaline and Sugen/hypoxia), a murine model of RV pressure overload induced by pulmonary artery banding, and cellular models. MEASUREMENTS AND MAIN RESULTS: Oral administration of GS-444217 dose dependently reduced pulmonary arterial pressure and reduced RV hypertrophy in PAH models. The therapeutic efficacy of GS-444217 was associated with reduced ASK1 phosphorylation, reduced muscularization of the pulmonary arteries, and reduced fibrotic gene expression in the RV. Importantly, efficacy was observed when GS-444217 was administered to animals with established disease and also directly reduced cardiac fibrosis and improved cardiac function in a model of isolated RV pressure overload. In cellular models, GS-444217 reduced phosphorylation of p38 and JNK (c-Jun N-terminal kinase) induced by adenoviral overexpression of ASK1 in rat cardiomyocytes and reduced activation/migration of primary mouse cardiac fibroblasts and human pulmonary adventitial fibroblasts derived from patients with PAH. CONCLUSIONS: ASK1 inhibition reduced pathological remodeling of the pulmonary vasculature and the right ventricle and halted progression of pulmonary hypertension in rodent models. These preclinical data inform the first description of a causal role of ASK1 in PAH disease pathogenesis.

Eplerenone attenuates pathological pulmonary vascular rather than right ventricular remodeling in pulmonary arterial hypertension.

BACKGROUND: Aldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulation. Although pharmacological aldosterone antagonism reduces mortality and morbidity among patients with severe left-sided heart failure, the contribution of aldosterone to the pathobiology of pulmonary arterial hypertension (PAH) and right ventricular (RV) heart failure is not fully understood. METHODS: The effects of Eplerenone (0.1% Inspra® mixed in chow) on pulmonary vascular and RV remodeling were evaluated in mice with pulmonary hypertension (PH) caused by Sugen5416 injection with concomitant chronic hypoxia (SuHx) and in a second animal model with established RV dysfunction independent from lung remodeling through surgical pulmonary artery banding. RESULTS: Preventive Eplerenone administration attenuated the development of PH and pathological remodeling of pulmonary arterioles. Therapeutic aldosterone antagonism - starting when RV dysfunction was established - normalized mineralocorticoid receptor gene expression in the right ventricle without direct effects on either RV structure (Cardiomyocyte hypertrophy, Fibrosis) or function (assessed by non-invasive echocardiography along with intra-cardiac pressure volume measurements), but significantly lowered systemic blood pressure. CONCLUSIONS: Our data indicate that aldosterone antagonism with Eplerenone attenuates pulmonary vascular rather than RV remodeling in PAH.

p38 MAPK Inhibition Improves Heart Function in Pressure-Loaded Right Ventricular Hypertrophy.

Although p38 mitogen-activated protein kinase (MAPK) is known to have a role in ischemic heart disease and many other diseases, its contribution to the pathobiology of right ventricular (RV) hypertrophy and failure is unclear. Therefore, we sought to investigate the role of p38 MAPK in the pathophysiology of pressure overload-induced RV hypertrophy and failure. The effects of the p38 MAPK inhibitor PH797804 were investigated in mice with RV hypertrophy/failure caused by exposure to hypoxia or pulmonary artery banding. In addition, the effects of p38 MAPK inhibition or depletion (by small interfering RNA) were studied in isolated mouse RV fibroblasts. Echocardiography, invasive hemodynamic measurements, immunohistochemistry, collagen assays, immunofluorescence staining, and Western blotting were performed. Expression of phosphorylated p38 MAPK was markedly increased in mouse and human hypertrophied/failed RVs. In mice, PH797804 improved RV function and inhibited cardiac fibrosis compared with placebo. In isolated RV fibroblasts, p38 MAPK inhibition reduced transforming growth factor (TGF)-ß-induced collagen production as well as stress fiber formation. Moreover, p38 MAPK inhibition/depletion suppressed TGF-ß-induced SMAD2/3 phosphorylation and myocardin-related transcription factor A (MRTF-A) nuclear translocation, and prevented TGF-ß-induced cardiac fibroblast transdifferentiation. Moreover, p38 MAPK inhibition in mice exposed to pulmonary artery banding led to diminished nuclear levels of MRTF-A and phosphorylated SMAD3 in RV fibroblasts. Together, our data indicate that p38 MAPK inhibition significantly improves RV function and inhibits RV fibrosis. Inhibition of p38 MAPK in RV cardiac fibroblasts, resulting in coordinated attenuation of MRTF-A cytoplasmic-nuclear translocation and SMAD3 deactivation, indicates that p38 MAPK signaling contributes to distinct disease-causing mechanisms.

Organ-specific function of adhesion G protein-coupled receptor GPR126 is domain-dependent.

Despite their abundance and multiple functions in a variety of organ systems, the function and signaling mechanisms of adhesion G protein-coupled receptors (GPCRs) are poorly understood. Adhesion GPCRs possess large N termini containing various functional domains. In addition, many of them are autoproteolytically cleaved at their GPS sites into an N-terminal fragment (NTF) and C-terminal fragment. Here we demonstrate that Gpr126 is expressed in the endocardium during early mouse heart development. Gpr126 knockout in mice and knockdown in zebrafish caused hypotrabeculation and affected mitochondrial function. Ectopic expression of Gpr126-NTF that lacks the GPS motif (NTF(ΔGPS)) in zebrafish rescued the trabeculation but not the previously described myelination phenotype in the peripheral nervous system. These data support a model in which the NTF of Gpr126, in contrast to the C-terminal fragment, plays an important role in heart development. Collectively, our analysis provides a unique example of the versatile function and signaling properties of adhesion GPCRs in vertebrates.

Lysine methyltransferase Smyd2 suppresses p53-dependent cardiomyocyte apoptosis.

Apoptosis, or programmed cell death, is an essential physiological process for proper embryogenesis as well as for homeostasis during aging. In addition, apoptosis is one of the major mechanisms causing cell loss in pathophysiological conditions such as heart failure. Thus, inhibition of apoptosis is an important approach for preventive and therapeutic strategies. Here we show that the histone 3 lysine 4- and lysine 36-specific methyltransferase Smyd2 acts as an endogenous antagonistic player of p53-dependent cardiomyocyte apoptosis. Smyd2 protein levels were significantly decreased in cardiomyocytes upon cobalt chloride-induced apoptosis or myocardial infarction, while p53 expression was enhanced. siRNA-mediated knockdown of Smyd2 in cultured cardiomyocytes further enhanced cobalt chloride-induced cardiomyocyte apoptosis. In contrast, Smyd2 overexpression resulted in marked methylation of p53 and prevented its accumulation as well as apoptotic cell death in an Hsp90-independent manner. Moreover, overexpression, of Smyd2, but not Smyd2Y240F lacking a methyl transferase activity, significantly rescued CoCl2-induced apoptosis in H9c2 cardioblasts. Finally, Smyd2 cardiomyocyte-specific deletion in vivo promoted apoptotic cell death upon myocardial infarction, which correlated with enhanced expression of p53 and pro-apoptotic Bax. Collectively, our data indicate Smyd2 as a cardioprotective protein by methylating p53.

FGF1-mediated cardiomyocyte cell cycle reentry depends on the interaction of FGFR-1 and Fn14.

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs) mediating a broad range of cellular functions during embryonic development, as well as disease and regeneration during adulthood. Thus, it is important to understand the underlying molecular mechanisms that modulate this system. Here, we show that FGFR-1 can interact with the TNF receptor superfamily member fibroblast growth factor-inducible molecule 14 (Fn14) resulting in cardiomyocyte cell cycle reentry. FGF1-induced cell cycle reentry in neonatal cardiomyocytes could be blocked by Fn14 inhibition, while TWEAK-induced cell cycle activation was inhibited by blocking FGFR-1 signaling. In addition, costimulation experiments revealed a synergistic effect of FGF1 and TWEAK in regard to cardiomyocyte cell cycle induction via PI3K/Akt signaling. Overexpression of Fn14 with either FGFR-1 long [FGFR-1(L)] or FGFR-1 short [FGFR-1(S)] isoforms resulted after FGF1/TWEAK stimulation in cell cycle reentry of >40% adult cardiomyocytes. Finally, coimmunoprecipitation and proximity ligation assays indicated that endogenous FGFR-1 and Fn14 interact with each other in cardiomyocytes. This interaction was strongly enhanced in the presence of their corresponding ligands, FGF1 and TWEAK. Taken together, our data suggest that FGFR-1/Fn14 interaction may represent a novel endogenous mechanism to modulate the action of these receptors and their ligands and to control cardiomyocyte cell cycle reentry.

Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish.

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.

C-type natriuretic peptide/cGMP/FoxO3 signaling attenuates hyperproliferation of pericytes from patients with pulmonary arterial hypertension.

Pericyte dysfunction, with excessive migration, hyperproliferation, and differentiation into smooth muscle-like cells contributes to vascular remodeling in Pulmonary Arterial Hypertension (PAH). Augmented expression and action of growth factors trigger these pathological changes. Endogenous factors opposing such alterations are barely known. Here, we examine whether and how the endothelial hormone C-type natriuretic peptide (CNP), signaling through the cyclic guanosine monophosphate (cGMP) -producing guanylyl cyclase B (GC-B) receptor, attenuates the pericyte dysfunction observed in PAH. The results demonstrate that CNP/GC-B/cGMP signaling is preserved in lung pericytes from patients with PAH and prevents their growth factor-induced proliferation, migration, and transdifferentiation. The anti-proliferative effect of CNP is mediated by cGMP-dependent protein kinase I and inhibition of the Phosphoinositide 3-kinase (PI3K)/AKT pathway, ultimately leading to the nuclear stabilization and activation of the Forkhead Box O 3 (FoxO3) transcription factor. Augmentation of the CNP/GC-B/cGMP/FoxO3 signaling pathway might be a target for novel therapeutics in the field of PAH.