Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension.

See related article, pages 1109–1114 Idiopathic pulmonary artery hypertension (IPH) is a rare illness with a poor prognosis. Whereas chronic intravenous prostacyclin relieves some of the symptoms of progressive dyspnea and prolongs survival, most patients ultimately require a lung transplant.1 Newer therapies such as nonintravenously administered prostacyclin derivatives,2,3,4 endothelin receptor blockers,5,6 and, to some extent, phosphodiesterase inhibitors,7 hold some promise as alternatives for intravenous prostacyclin, but current expectation is that, like prostacyclin, they will, at best, retard disease progression, serving as a bridge to transplant rather than as an alternative. The pathological features of IPH are loss of small distal precapillary pulmonary arteries, obliterative changes (plexogenic lesions) in more proximal pulmonary arteries associated with migration and proliferation of smooth muscle cells, and increased extracellular matrix deposition. There is also dysregulation of endothelial cells associated with increased proliferation.8 The mechanism underlying the evolution of these changes is unknown, so there was great interest when 2 groups independently identified a mutation in bone morphogenetic protein receptor 11 (BMP-RII) in 60% of families with IPH.9,10 A BMP-RII mutation also occurs in 20% of sporadic cases of IPH,11 but the biological connection between the mutation and the pathobiology of IPH has been relatively elusive. Recent studies using pulmonary artery smooth muscle cells from patients with IPH, including those with and without a BMP-RII mutation, showed similar abnormal proliferation in response to agents such as transforming growth factor-β (TGF-β) or BMP-2.12 In other studies, pulmonary artery smooth muscle cells were transfected with constructs encoding different mutant forms of BMP-RII expressing aberrant kinase or cytoplasmic domains, and impaired signaling was observed related to alterations in the induction of Smads and p38.13 Specifically, suppression of Smad1/5 and activation of p38 were related to smooth muscle cell proliferation. It …

Pulmonary arterial hypertension (PAH) is a devastating and life-threatening clinical syndrome characterized by elevated pulmonary artery pressures leading to progressive symptoms, including shortness of breath, fatigue, and a decline in functional ability. The hemodynamic definition of PAH is a mean pulmonary artery pressure of >25 mm Hg at rest or >30 mm Hg during exercise, with a normal pulmonary arterial wedge pressure ≤15 mm Hg.1 Recent data from a French registry suggests that the prevalence of PAH is about 15 cases per 1 million,1 and the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL) demonstrates that the mean age at diagnosis is 48 years and that mostly women (80%) are affected.2 The pathophysiology of PAH includes endothelial dysfunction, vascular remodeling with progressive obstruction and obliteration of pulmonary arteries, and ultimately, high right atrial pressure, right ventricular hypertrophy, right ventricular failure, and death.1 Endothelial dysfunction is believed to be an early event in PAH, and is characterized by overproduction of vasoconstrictor/mitogenic compounds, such as endothelin and thromboxane A2, and by insufficient production of vasodilators, such as prostacyclin and nitric oxide (NO). These observations led to the development of 3 different classes of therapies that are currently in clinics, either alone or in combination: prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase (PDE) inhibitors. Although current therapies can improve symptoms and reduce severity of the hemodynamic disorders, gradual deterioration of pulmonary and cardiac functions often necessitates lung transplantation. The prognosis of PAH is reportedly poor, with ≈15% mortality within 1 year of modern therapy.1 Therefore, a renewed interest has been focused on the mechanisms of PAH pathogenesis to identify a novel therapeutic target. Article see p 1194 It is now well established that disruption or dysfunction of the endothelium promotes vascular lesion formation. This …

pulmonary arterial hypertension (PAH) is a rare human disease displaying a very poor prognosis of survival. The chronic increase in pulmonary vascular resistance associated with PAH eventually leads to right ventricular hypertrophy and failure, and ultimately death. It is well accepted that the

Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1, and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124, leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA or its targets could represent a novel therapeutic approach for the treatment of PAH.

Introduction Idiopathic pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vascular occlusion and eventually right heart failure. Causal genetic mutations are implicated in both familial (>80%) and sporadic (30%) forms of idiopathic PAH. From a recent whole genome sequencing study of 1038 PAH patients, we identified ATP13A3 mutations in a subset of patients. ATP13A3 is a poorly characterized P-type ATPase. The aim of this study is to understand the impact of ATP13A3 loss of function on pulmonary vascular cell function and provide insights into the pathogenesis of PAH in these patients. Method ATP13A3 mRNA expression was compared in human pulmonary vascular cells using qPCR. ATP13A3 knockdown was achieved using siRNA transfection. Pulmonary artery endothelial cell (PAEC) proliferation was determined by cell counting. PAEC Apoptosis was assessed by Annexin V/PI staining and Caspase-Glo 3/7 assay. Endothelial permeability was assayed by measuring the transit of horseradish peroxidase across PAEC monolayers in the presence or absence of thrombin. Results ATP13A3 mRNA expression was similar between human PAECs, pulmonary artery smooth muscle cells and blood outgrowth endothelial cells, although immunohistochemistry in human lung indicated a predominant endothelial expression pattern. ATP13A3 was expressed at higher levels in PAECs than other P-type ATPase family members. Knockdown of ATP13A3 in PAECs resulted in reduced cell proliferation, associated with reduced expression of cyclins. Loss of ATP13A3 in PAECs under low serum conditions (0.1%FBS) predisposed cells to early apoptosis, confirmed by both Annexin V/PI staining and increased caspase3/7 activity. ATP13A3 deficiency in human PAECs also resulted in an increase of endothelial monolayer permeability and an enhanced response to thrombin. Conclusion ATP13A3 is expressed in PAECs and plays a role in cell proliferation, apoptosis and endothelial permeability. These data provide initial insights into the pathogenicity of ATP13A3 mutations in patients with PAH. This work is produced by Prof. Nick Morrell’s Group on behalf of the UK PAH Cohort Study.

PULMONARY ARTERIAL HYPERTENSION (PAH) remains a clinically vexing problem considering the high morbidity and mortality that plagues this entity despite indication of improved survival after the introduction of more targeted therapy. While this syndrome is well described from a pathological standpoint the mechanisms involved in the pulmonary vascular remodeling are less clear but are thought to involve endothelial dysfunction and proliferation of various cell types that comprise the wall of distal pulmonary vessels (3). The specific mechanisms leading to distal vessel obliteration and increased pulmonary vascular resistance have been particularly elusive for several decades (14), and although vasoconstriction as a primary event was the focus of research and targeted therapy in early studies, the PAH research community has witnessed a paradigm shift in recent years with a strong tendency to now view the PAH lesion as a quasi-cancerous process (12). In this issue of the Journal of Applied Physiology, Schwenke et al. (8) bring a new tool for assessment of the distal pulmonary vasculature and shake an old specter, vasoconstriction. The investigators employ synchrotron radiation microangiography (SRA), a fairly novel and powerful technique, to more directly evaluate vascular tone in a classic animal model of PAH, monocrotaline (MCT)-challenged rats, providing new insight into regional specific responses of the pulmonary circulation in PAH. SRA allows for both the measurement and assessment of control of blood flow in vivo and collection of data on small arteries and arterioles in situ over time (10). Thus it can supply superior information on kinetic events relative to ex vivo techniques. Since SRA has no focal plane, it can visualize both surface and penetrating vessels simultaneously unlike intravital microscopy, which is limited to assessment of vessel caliber at a single vascular plan. In dynamic organs such as the lung and heart, which are subject to motion, temporal

To be EndMT or not to be, that is the question in pulmonary hypertension.

Cell and gene: from transduction to translation

PULMONARY ARTERIAL HYPERTENSION (PAH) is a progressive disease manifested by maladaptation of the pulmonary vasculature. The development of PAH can be influenced by genetic predisposition and/or by diverse endogenous or environmental stimuli. Regardless of the initial pathogenic factors, pulmonary vascular remodeling, sustained pulmonary vasoconstriction, in situ thrombosis, and increased pulmonary vascular wall stiffness are the major contributors to elevated pulmonary vascular resistance (PVR). The increase in PVR can lead to right ventricular failure and death in patients with PAH. While treatments for this disease are improving, it continues to be a life-threatening condition. MicroRNAs (miRNAs) have been implicated in the development and progression of PAH. MiRNAs are small, noncoding RNAs that regulate gene and protein expression by promoting degradation or suppressing translation of target mRNAs. Several studies have demonstrated aberrant expres

Rationale Pulmonary hypertension (PH) associated with chronic lung disease (COPD-PH) is the second most common cause of PH and carries high morbidity and mortality. Patients with COPD-PH exhibit disproportionate right ventricular dysfunction and poor outcomes compared with other PH groups. While endothelial dysfunction is central to PH pathobiology, prior studies have been limited to end-stage tissues. There remains a critical need to characterize pulmonary artery endothelial cells (PAECs) from patients with early-stage PH to uncover mechanistic differences between COPD-PH and pulmonary arterial hypertension (PAH). Methods PAECs were prospectively isolated from right heart catheterization (RHC) tips of patients with PAH and COPD-PH (n = 4 total; 2 per group) using a recently validated technique. Control subjects were those referred for RHC but who did not meet criteria for a PH diagnosis. Endothelial identity was confirmed by immunofluorescence for CD31, VE-cadherin, and von Willebrand factor (vWF). Functional assays assessed angiogenesis, proliferation, and apoptosis. Single-cell RNA sequencing (scRNA-seq) was performed on 22,000 pre-passaged PAECs, and unsupervised clustering was used to define transcriptional subpopulations. Results PAECs demonstrated typical endothelial morphology and marker expression. Functional assays suggested a hypoproliferative and pro-apoptotic phenotype in COPD-PH compared with control. ScRNA-seq identified 14 transcriptionally distinct PAEC clusters, all expressing canonical endothelial genes (PECAM1, VWF, KDR, CDH5). Both PAH and COPD-PH contained all clusters, but cluster composition differed by disease, with clusters 4 and 12 enriched in COPD-PH. These clusters were characterized by upregulation of cell adhesion and apoptosis pathways. In contrast, PAH PAECs demonstrated enrichment of genes related to angiogenesis and metabolic activity. Expression of circulating or progenitor EC markers (PROM1, THY1, CXCR4, ETV2) was low, supporting pulmonary arterial origin. Conclusions This study establishes a reproducible method to isolate PAECs from RHC samples, enabling investigation of early-stage endothelial phenotypes in PH. Preliminary findings suggest that COPD-PH PAECs exhibit a distinct, pro-apoptotic, adhesion-enriched transcriptomic profile compared to PAH, consistent with endothelial loss and vascular rarefaction observed in COPD-associated vascular remodeling. Ongoing studies are expanding scRNA-seq and transcriptomic analyses of both PAECs and blood outgrowth endothelial cells to define circulating-endothelial interplay in PH pathogenesis. This abstract is funded by: Borstein Family Foundation, Reuben M. Cherniack Fellowship, Colorado Pulmonary Vascular Disease Award, NIH 7R01HL144727, VA Merit Review Award 2 I01 BX00204

Introduction: Pulmonary arterial hypertension (PAH) is a morbid vascular disease where mutations of bone morphogenetic protein receptor 2 (BMPR2) control pulmonary endothelial pathophenotypes. Transcriptomic screening from endothelial cells (ECs) derived from hereditary BMPR2-driven disease offers an opportunity to identify novel effectors in PAH pathogenesis. Methods: Public RNA-sequencing data were analyzed from inducible pluripotent stem (iPS) cell-derived endothelial cells with and without BMPR2 mutations. Candidate genes were assessed in cultured pulmonary arterial ECs (PAECs) and in rodent and human PAH. Correlations were assessed of human plasma expression with clinical disease indices. Results: Signal Peptide CUB-EGF-Domain Containing Protein 1 (SCUBE1), a putative binding partner to BMPR2, was differentially expressed in ECs carrying BMPR2 mutations. SCUBE1 was enriched in PAECs, dependent on hypoxia inducible factor-1α, and downregulated by PAH triggers, including BMPR2 knockdown, hypoxia, and IL-1β exposure. In vitro analyses defined SCUBE1 as a pathogenic effector activating BMPR2-associated SMAD1/5/9, thus regulating endothelial angiogenic potential, proliferation, and apoptosis. SCUBE1 was decreased specifically in plasma and lungs in rodents and patients with PAH but not in those with pulmonary hypertension due to left heart disease, or in the patients of other acute and chronic cardiopulmonary pathologies, including pneumonia, acute lung injury, chronic obstructive pulmonary disease, ischemic heart disease, and cardiomyopathy. An optimal plasma SCUBE1 cut point of 5.02 ng/mL was defined to diagnose PAH from control cohort with a sensitivity of 0.65 and a specificity of 0.82. In PAH patients, plasma SCUBE1 levels negatively correlated with pulmonary arterial pressure, pulmonary vascular resistance, and right ventricular dysfunction. Conclusions: Guided by iPSC-EC sequencing, SCUBE1 was identified as a downregulated secreted factor in PAH, controlling endothelial pathophenotypes and correlated with disease indices. Clinically, SCUBE1 is a sensitive and specific PAH diagnostic marker. It may also serve as a therapeutic target given its inherent links to controlling PAH predisposition and severity.

pulmonary hypertension (PH), diagnosed when mean pulmonary arterial pressure exceeds the upper limits of normal (i.e., >25 mmHg) at rest (2), occurs in a variety of clinical situations and is associated with a broad spectrum of histological patterns and abnormalities. PH is currently classified into five distinct World Health Organization (WHO) groups, based on common clinical parameters, potential etiological mechanisms, and responses to treatment (22). Although any form of PH can contribute to increased patient morbidity and mortality, pulmonary arterial hypertension (PAH) (WHO group 1) is a particularly severe and progressive form associated with right heart failure and premature death (1). At present, therapeutic approaches to stabilize or reverse this debilitating condition involve treatment with one or a combination of up to three specific classes of agents, including prostacyclin analogs, endothelin-1 receptor antagonists, and/or phosphodiesterase-5 inhibitors. Retrospective (meta)analyses of these therapeutic strategies have demonstrated a reduction in mortality with their use (7, 12); however, many experts believe that current PAH treatment is inadequate given the persistently high mortality rate and functional hemodynamic impairment in many patients. These observations have led to continued intensive investigation into pathogenetic mechanisms and many proposals for additional alternative new therapies (20, 24). Among the potential new therapies, increasing interest in the role of endothelial progenitor cells (EPCs) as a cell-based therapy has emerged. However, issues remain regarding what group of PH patients are most likely to benefit from treatment, at what point in the disease is treatment most likely to be successful, and what types of cells should be utilized for therapy.

Pulmonary arterial hypertension (PAH) is characterized by dysregulated pulmonary artery endothelial cell (PAEC) proliferation, apoptosis and permeability. Loss-of-function mutations in the bone morphogenetic protein receptor type-II (BMPR-II) are the most common cause of heritable PAH, usually resulting in haploinsufficiency. We previously showed that BMPR-II expression is regulated via a lysosomal degradative pathway. Here, we show that the antimalarial drug, chloroquine, markedly increased cell surface expression of BMPR-II protein independent of transcription in PAECs. Inhibition of protein synthesis experiments revealed a rapid turnover of cell surface BMPR-II, which was inhibited by chloroquine treatment. Chloroquine enhanced PAEC expression of BMPR-II following siRNA knockdown of the BMPR-II transcript. Using blood outgrowth endothelial cells (BOECs), we confirmed that signalling in response to the endothelial BMPR-II ligand, BMP9, is compromised in BOECs from patients harbouring BMPR-II mutations, and in BMPR-II mutant PAECs. Chloroquine significantly increased gene expression of BMP9-BMPR-II signalling targets Id1, miR21 and miR27a in both mutant BMPR-II PAECs and BOECs. These findings provide support for the restoration of cell surface BMPR-II with agents such as chloroquine as a potential therapeutic approach for heritable PAH.

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BACKGROUND Pulmonary Arterial Hypertension (PAH) is an incurable disease and remains a significant cause of morbidity and mortality in children and adults. In PAH, pulmonary arteries narrow in diameter which increases blood flow resistance in the lungs. Eventually the increase in pulmonary blood pressure causes irreversible damage to the heart. There are a variety of reasons why the pulmonary arteries become obstructed, known and unknown; yet, the molecular mechanisms driving this disease remain to be elucidated. Approximately 1000 new cases of PAH are diagnosed each year in the U.S. alone. The average survival rate for patients diagnosed with PAH is 7 years. Therefore, any insight into the molecular mechanisms that promote PAH progression may represent new strategies to treat patients with PAH. OBJECTIVE Up to 20% of PAH patients with no family history of PAH carry a bone morphogenetic protein receptor 2 (BMPR2) mutation, whereas in patients with a family history of PAH up to 82% carry a BMPR2 mutation. Therefore, it appears extremely likely that BMPR2 loss of function promotes the dysfunctional angio‐proliferative phenotype of PAH. In fact, it has recently been shown that in endothelial cells (EC) deficient in BMPR2 exposed to exogenous bone morphogenetic protein 2 (BMP2), a ligand for BMPR2, are migratory compared to control cells. To determine if the loss of BMPR2 promotes the PAEC dysfunctional angiogenesis observed in patients with PAH, we used cells from control and PAH patients to determine the role of pulmonary artery smooth muscle cell (PASMC) secreted BMP2 in promoting the migration of pulmonary artery endothelial cells (PAEC) and angiogenesis. METHODS PASMC and PAEC from control and PAH patients were examined for the expression of BMPR2 and BMP2. Cell migration, cell invasion, and angiogenetic assays were performed using PASMC and PAEC from control and PAH patients to assess differences in migration, invasion, and angiogenesis. RESULTS PAEC from PAH patients have a significant reduction in the expression of BMPR2, consistent with published reports. PASMC from PAH secrete BMP2, while BMP2 is undetectable in control cells. Co‐culturing PAH PASMC with PAH PAEC promotes PAEC migration. This migration is further enhanced in the presence of exogenous BMP2. PAH PASMC co‐cultured with PAH PAEC promote the development of PAEC‐derived angiogenetic masses. This dysfunctional angiogenetic response is further amplified in the presence of exogenous BMP2. In contrast, control PAEC exposed to control PASMC respond normally and do not form disorganized masses of vessels. Control PAEC exposed to exogenous BMP2 further promotes a normal angiogenetic response. CONCLUSIONS PASMC from PAH patients secrete BMP2. BMP2 exposure promotes the migration of PAH PAEC and a dysfunctional angiogenetic response. In contrast, BMP2 promotes a normal angiogenetic response when added to co‐cultures of control PASMC and PAEC. These data suggest that in PAH, angiogenesis becomes dysfunctional in part due to PAEC exposure to BMP2; therefore, inhibitors of BMP2 may represent a therapeutic strategy to pursue in the treatment of PAH.