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BACKGROUND: Pulmonary arterial hypertension (PAH) ultimately leads to right ventricular failure and premature death. The identification of circulating biomarkers with prognostic utility is considered a priority. As chronic inflammation is recognized as key pathogenic driver, we sought to identify inflammation-related circulating proteins that add incremental value to current risk stratification models for long-term survival in patients with PAH. METHODS AND RESULTS: Plasma levels of 384 inflammatory proteins were measured with the proximity extension assay technology in patients with PAH (n=60) and controls with normal hemodynamics (n=28). Among these, 51 analytes were significantly overexpressed in the plasma of patients with PAH compared with controls. Cox proportional hazard analyses and C-statistics were performed to assess the prognostic value and the incremental prognostic value of differentially expressed proteins. A panel of 6 proteins (CRIM1 [cysteine rich transmembrane bone morphogenetic protein regulator 1], HGF [hepatocyte growth factor], FSTL3 [follistatin-like 3], PLAUR [plasminogen activator, urokinase receptor], CLSTN2 [calsyntenin 2], SPON1 [spondin 1]) were independently associated with death/lung transplantation at the time of PAH diagnosis after adjustment for the 2015 European Society of Cardiology/European Respiratory Society guidelines, the REVEAL (Registry to Evaluate Early and Long-Term PAH Disease Management) 2.0 risk scores, and the refined 4-strata risk assessment. CRIM1, PLAUR, FSTL3, and SPON1 showed incremental prognostic value on top of the predictive models. As determined by Western blot, FSTL3 and SPON1 were significantly upregulated in the right ventricle of patients with PAH and animal models (monocrotaline-injected and pulmonary artery banding-subjected rats). CONCLUSIONS: In addition to revealing new actors likely involved in cardiopulmonary remodeling in PAH, our screening identified promising circulating biomarkers to improve risk prediction in PAH, which should be externally confirmed.
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Biomarcadores , Proteômica , Hipertensão Arterial Pulmonar , Humanos , Masculino , Feminino , Biomarcadores/sangue , Proteômica/métodos , Pessoa de Meia-Idade , Prognóstico , Hipertensão Arterial Pulmonar/sangue , Hipertensão Arterial Pulmonar/mortalidade , Hipertensão Arterial Pulmonar/diagnóstico , Hipertensão Arterial Pulmonar/fisiopatologia , Adulto , Animais , Medição de Risco , Estudos de Casos e Controles , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Proteínas Relacionadas à Folistatina/sangue , Modelos Animais de Doenças , Valor Preditivo dos Testes , Inflamação/sangue , Mediadores da Inflamação/sangue , Fatores de Risco , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/sangue , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Artéria Pulmonar/fisiopatologiaRESUMO
Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.
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BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.
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Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt , Ubiquitina Tiolesterase , Animais , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/metabolismo , Humanos , Camundongos , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Masculino , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/enzimologia , Ratos Sprague-Dawley , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/etiologia , Remodelação Vascular , Células Cultivadas , Proliferação de Células , Camundongos Endogâmicos C57BL , Indóis , OximasRESUMO
Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Single-cell RNA sequencing (scRNAseq) analysis found that both FABP4 and FABP5 were highly induced in endothelial cells (ECs) of Egln1Tie2Cre (CKO) mice, which was also observed in pulmonary arterial ECs (PAECs) from idiopathic PAH (IPAH) patients, and in whole lungs of pulmonary hypertension (PH) rats. Plasma levels of FABP4/5 were upregulated in IPAH patients and directly correlated with severity of hemodynamics and biochemical parameters using plasma proteome analysis. Genetic deletion of both Fabp4 and 5 in CKO mice (Egln1Tie2Cre/Fabp4-5-/- ,TKO) caused a reduction of right ventricular systolic pressure (RVSP) and RV hypertrophy, attenuated pulmonary vascular remodeling and prevented the right heart failure assessed by echocardiography, hemodynamic and histological analysis. Employing bulk RNA-seq and scRNA-seq, and spatial transcriptomic analysis, we showed that Fabp4/5 deletion also inhibited EC glycolysis and distal arterial programming, reduced ROS and HIF-2α expression in PH lungs. Thus, PH causes aberrant expression of FABP4/5 in pulmonary ECs which leads to enhanced ECs glycolysis and distal arterial programming, contributing to the accumulation of arterial ECs and vascular remodeling and exacerbating the disease.
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Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.
Assuntos
Hipertensão Arterial Pulmonar , Transdução de Sinais , Remodelação Vascular , Animais , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/fisiologia , Ratos , Humanos , Hipertensão Arterial Pulmonar/fisiopatologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/metabolismo , Masculino , Modelos Animais de Doenças , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Camundongos , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A , Proliferação de Células/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/metabolismo , Lisina/análogos & derivadosRESUMO
There is an increasing recognition of the crucial role of the right ventricle (RV) in determining the functional status and prognosis in multiple conditions. In the past decade, the epigenetic regulation (DNA methylation, histone modification, and non-coding RNAs) of gene expression has been raised as a critical determinant of RV development, RV physiological function, and RV pathological dysfunction. We thus aimed to perform an up-to-date review of the literature, gathering knowledge on the epigenetic modifications associated with RV function/dysfunction. Therefore, we conducted a systematic review of studies assessing the contribution of epigenetic modifications to RV development and/or the progression of RV dysfunction regardless of the causal pathology. English literature published on PubMed, between the inception of the study and 1 January 2023, was evaluated. Two authors independently evaluated whether studies met eligibility criteria before study results were extracted. Amongst the 817 studies screened, 109 studies were included in this review, including 69 that used human samples (e.g., RV myocardium, blood). While 37 proposed an epigenetic-based therapeutic intervention to improve RV function, none involved a clinical trial and 70 are descriptive. Surprisingly, we observed a substantial discrepancy between studies investigating the expression (up or down) and/or the contribution of the same epigenetic modifications on RV function or development. This exhaustive review of the literature summarizes the relevant epigenetic studies focusing on RV in human or preclinical setting.
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Ventrículos do Coração , Disfunção Ventricular Direita , Humanos , Ventrículos do Coração/patologia , Epigênese Genética , Miocárdio/patologia , Função Ventricular Direita/fisiologiaRESUMO
Pulmonary hypertension due to left heart disease (PH-LHD) or group 2 PH is the most common and lethal form of PH, occurring secondary to left ventricular systolic or diastolic heart failure (HF), left-sided valvular diseases, and congenital abnormalities. It is subdivided into isolated postcapillary PH (IpcPH) and combined pre- and post-capillary PH (CpcPH), with the latter sharing many similarities with group 1 PH. CpcPH is associated with worse outcomes and increased morbidity and mortality when compared to IpcPH. Although IpcPH can be improved by treatment of the underlying LHD, CpcPH is an incurable disease for which no specific treatment exists, likely due to the lack of understanding of its underlying mechanisms. Furthermore, drugs approved for PAH are not recommended for group 2 PH, as they are either ineffective or even deleterious. With this major unmet medical need, a better understanding of mechanisms and the identification of effective treatment strategies for this deadly condition are urgently needed. This review presents relevant background of the molecular mechanisms underlying PH-LHD that could translate into innovative therapeutic targets and explores novel targets currently being evaluated in clinical trials.
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Insuficiência Cardíaca , Hipertensão Pulmonar , Disfunção Ventricular Esquerda , Humanos , Resultado do TratamentoRESUMO
Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling of small pulmonary arteries (PAs) causing sustained elevation of PA pressure, right ventricular failure, and death. Similar to cancer cells, PA smooth muscle cells (PASMCs), which play a key role in pulmonary vascular remodeling, have adopted multiple mechanisms to sustain their survival and proliferation in the presence of stress. The histone methyltransferase G9a and its partner protein GLP (G9a-like protein) have been shown to exert oncogenic effects and to serve as a buffer against an exaggerated transcriptional response. Therefore, we hypothesized that upregulation of G9a and GLP in PAH plays a pivotal role in pulmonary vascular remodeling by maintaining the abnormal phenotype of PAH-PASMCs. We found that G9a is increased in PASMCs from patients with PAH as well as in remodeled PAs from animal models. Pharmacological inhibition of G9a/GLP activity using BIX01294 and UNC0642 significantly reduced the prosurvival and proproliferative potentials of cultured PAH-PASMCs. Using RNA sequencing, further exploration revealed that G9a/GLP promotes extracellular matrix production and affords protection against the negative effects of an overactive stress response. Finally, we found that therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling and lowered mean PA pressure in fawn-hooded rats. Treatment of Sugen/hypoxia-challenged mice with BIX01294 also improved pulmonary hemodynamics and right ventricular function. In conclusion, these findings indicate that G9a/GLP inhibition may represent a new therapeutic approach in PAH.
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Hipertensão Arterial Pulmonar , Ratos , Camundongos , Animais , Hipertensão Arterial Pulmonar/tratamento farmacológico , Remodelação Vascular , Proliferação de Células , Hipertensão Pulmonar Primária Familiar , Modelos Animais de Doenças , Miócitos de Músculo Liso , Artéria PulmonarRESUMO
Pulmonary arterial hypertension (PAH) is a severe and progressive cardiopulmonary disease characterized by pathological remodeling of the resistance pulmonary arteries (PA), ultimately leading to right ventricular (RV) failure and death. Animal models have been particularly useful for unraveling the pathogenesis of PAH by providing incisive experimental strategies that were impossible in human studies. Over the past decade, gene therapy has been making considerable progress as an alternative strategy for treating PAH disease. Animal models mimicking PAH disease are essential at preclinical stages for assessing the therapeutic potential of gene therapy and determining genome viral vectors transduction, safety, dosage, and localization of transgene expression. The most commonly used PAH rat models in gene therapy studies are the monocrotaline (MCT), the chronic hypoxia-Sugen 5416, and the pneumonectomy (PNT)-MCT models. Here, we provide detailed protocols for creating these preclinical rodent models of PAH commonly used to assess the efficiency of lung gene therapy in PAH.
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Insuficiência Cardíaca , Hipertensão Pulmonar , Animais , Modelos Animais de Doenças , Terapia Genética , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/terapia , Monocrotalina , Artéria Pulmonar , Ratos , RoedoresRESUMO
Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.
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Hipertensão Pulmonar , Animais , Cromatina/metabolismo , Feto/metabolismo , Humanos , Pulmão/patologia , Camundongos , Artéria Pulmonar/patologia , Interferência de RNA , Fatores de Transcrição/metabolismo , Remodelação Vascular/genéticaRESUMO
The authors show that increased poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) and pyruvate kinase muscle isozyme 2 (PKM2) expression is a common feature of a decompensated right ventricle in patients with pulmonary arterial hypertension and animal models. The authors find in vitro that overactivated PARP1 promotes cardiomyocyte dysfunction by favoring PKM2 expression and nuclear function, glycolytic gene expression, activation of nuclear factor κB-dependent proinflammatory factors. Pharmacologic and genetic inhibition of PARP1 or enforced tetramerization of PKM2 attenuates maladaptive remodeling improving right ventricular (RV) function in multiple rodent models. Taken together, these data implicate the PARP1/PKM2 axis as a critical driver of maladaptive RV remodeling and a new promising target to directly sustain RV function in patients with pulmonary arterial hypertension.
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Pulmonary hypertension (PH) describes heterogeneous population of patients with a mean pulmonary arterial pressure >20 mm Hg. Rarely, PH presents as a primary disorder but is more commonly part of a complex phenotype associated with comorbidities. Regardless of the cause, PH reduces life expectancy and impacts quality of life. The current clinical classification divides PH into 1 of 5 diagnostic groups to assign treatment. There are currently no pharmacological cures for any form of PH. Animal models are essential to help decipher the molecular mechanisms underlying the disease, to assign genotype-phenotype relationships to help identify new therapeutic targets, and for clinical translation to assess the mechanism of action and putative efficacy of new therapies. However, limitations inherent of all animal models of disease limit the ability of any single model to fully recapitulate complex human disease. Within the PH community, we are often critical of animal models due to the perceived low success upon clinical translation of new drugs. In this review, we describe the characteristics, advantages, and disadvantages of existing animal models developed to gain insight into the molecular and pathological mechanisms and test new therapeutics, focusing on adult forms of PH from groups 1 to 3. We also discuss areas of improvement for animal models with approaches combining several hits to better reflect the clinical situation and elevate their translational value.
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Insuficiência Cardíaca , Hipertensão Pulmonar , Animais , Modelos Animais de Doenças , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/genética , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Qualidade de VidaRESUMO
RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.