Endothelial Dysfunction In Pulmonary Arterial Hypertension Research Articles

Panorama of artery endothelial cell dysfunction in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a fatal lung disease characterized by progressive pulmonary vascular remodeling. The initial cause of pulmonary vascular remodeling is the dysfunction of pulmonary arterial endothelial cells (PAECs), manifested by changes in the categorization of cell subtypes, endothelial programmed cell death, such as apoptosis, necroptosis, pyroptosis, ferroptosis, et al., overproliferation, senescence, metabolic reprogramming, endothelial-to-mesenchymal transition, mechanosensitivity, and regulation ability of peripheral cells. Therefore, it is essential to explore the mechanism of endothelial dysfunction in the context of PAH. This review aims to provide a comprehensive understanding of the molecular mechanisms underlying endothelial dysfunction in PAH. We highlight the developmental process of PAECs and changes in PAH and summarise the latest classification of endothelial dysfunction. Our review could offer valuable insights into potential novel EC-specific targets for preventing and treating PAH.

Role of KCNK3 Dysfunction in Dasatinib-associated PAH and Endothelial Cell Dysfunction.

Pulmonary arterial hypertension (PAH) is severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. We discovered a KCNK3 gene (coding for outward K+ channel) variant in a patient with dasatinib-associated PAH, and we investigated the impact of this variant on KCNK3 function. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression. In control-human in pulmonary arterial smooth muscle cells (hPASMCs) and pulmonary endothelial cells (hPECs), we evaluated the consequence of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Patch-clamp revealed that the KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to pulmonary artery constriction by decreasing KCNK3 function and expression. In control-hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in hPECs. Moreover, KCNK3 knockdown in control-hPECs reduced migration, proliferation, and in vitro tubulogenesis. Using proximity labeling and mass spectrometry, we identified the KCNK3 interactome, revealing that KCNK3 interacts with various proteins across different cellular compartments. We identified a novel pathogenic variant in the KCNK3 and showed that dasatinib downregulates KCNK3, emphasizing the relationship between dasatinib-associated PAH and KCNK3 dysfunction. We demonstrated that loss of KCNK3-dependent signaling contributes to endothelial dysfunction in PAH and glycolytic switch of hPASMCs.

Abstract 14496: HIF2a Activation Reduces Pulmonary Angiogenesis and Promotes Vascular Remodeling in Pulmonary Arterial Hypertension by Suppressing Wnt7a Production in Pulmonary Endothelial Cells

Introduction: Dysfunctional angiogenesis and obliterative vasculopathy are hallmarks of pulmonary arterial hypertension (PAH). Inappropriate HIF1a/2a activation and insufficient Wnt7a production are associated with pulmonary artery endothelial cell (PAEC) dysfunction, suggesting that HIF1a/2a and Wnt7a may interact as part of a signaling axis to promote PAEC homeostasis. Hypothesis: HIF1a/2a overactivation suppresses Wnt7a expression in PAH PAECs. Methods: Healthy and PAH PMVECs were obtained from explanted lungs. Cobalt Chloride (CoCl2) was used to activate HIF. PMVECs were transfected with lentiviruses carrying HIF1a/2a constructs, followed by functional and molecular studies. Lungs from mice with endothelial-specific HIF1a/2a activation (Egln1 Tie2Cre ) and HIF2a deletion (EH2) were analyzed via single cell (sc) RNA-seq and confocal imaging. Results: Compared to controls, PAH lung tissue and PAECs demonstrated an inverse correlation between increased HIF1a/2a, and reduced Wnt7a expression. Treatment with CoCl2 for 24 hours resulted in a ~4-fold increase in Wnt7a transcription that was absent in PAH PMVECs. Lentivirus transfection of healthy PAEC demonstrated that HIF1a increased Wnt7a transcription while HIF2a significantly reduced Wnt7a transcription. In silico analysis of the Wnt7a gene locus demonstrated HIF binding sites at promoter-enhancer sequences suggesting that Wnt7a is a gene target of HIF signaling. In addition, ATAC-seq and ChIP-seq analysis of the Wnt7a gene locus in PAH PMVECs indicated a closed chromatin configuration, suggesting epigenetic repression. Lung scRNA-seq and RNAScope analysis of Egln1 Tie2Cre mice demonstrated a reduction of capillary EC, which was absent in EH2 mice, suggesting a HIF2a-dependent mechanism. Confocal and WB of lung tissues demonstrate that Wnt7a expression was significantly reduced in Egln1 Tie2Cre mice but preserved in EH2 mice. Conclusions: HIF2a and epigenetic repression contribute to PAEC dysfunction by suppressing Wnt7a-dependent angiogenesis in PAH. We speculate that Wnt7a may also be required to maintain the capillary EC phenotype in the lung and that restoring Wnt7a production could compensate for HIF-driven endothelial dysfunction in PAH.

Pulmonary arterial hypertension drugs can partially restore altered angiogenic capacities in bmpr2-silenced human lung microvascular endothelial cells.

Mutations in the bone morphogenetic protein receptor type 2 (bmpr2) gene and signaling pathway impairment are observed in heritable and idiopathic pulmonary arterial hypertension (PAH). In PAH, endothelial dysfunction is currently handled by drugs targeting the endothelin-1 (ET-1), nitric oxide (NO), and prostacyclin (PGI2) pathways. The role of angiogenesis in the disease process and the effect of PAH therapies on dysregulated angiogenesis remain inconclusive. We aim to investigate in vitro whether (i) bmpr2 silencing can impair angiogenic capacity of human lung microvascular endothelial cells (HLMVECs) and (ii) PAH therapies can restore them. The effects of macitentan (ET-1), tadalafil (NO), and selexipag (PGI2), on BMPRII pathway activation, endothelial barrier function, and angiogenesis were investigated in bmpr2-silenced HLMVECs. Stable bmpr2 silencing resulted in impaired migration and tube formation in vitro capacity. Inhibition of ET-1 pathway was able to partially restore tube formation in bmpr2-silenced HLMVECs, whereas none of the therapies was able to restore endothelial barrier function, no deleterious effects were observed. Our findings highlight the potential role of BMPRII signaling pathway in driving pulmonary endothelial cell angiogenesis. In addition, PAH drugs display limited effects on endothelial function when BMPRII is impaired, suggesting that innovative therapeutic strategies targeting BMPRII signaling are needed to better rescue endothelial dysfunction in PAH.

Pathogenic Mechanisms of Pulmonary Arterial Hypertension: Homeostasis Imbalance of Endothelium-Derived Relaxing and Contracting Factors.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease. Sustained pulmonary vasoconstriction and concentric pulmonary vascular remodeling contribute to the elevated pulmonary vascular resistance and pulmonary artery pressure in PAH. Endothelial cells regulate vascular tension by producing endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs). Homeostasis of EDRF and EDCF production has been identified as a marker of the endothelium integrity. Impaired synthesis or release of EDRFs induces persistent vascular contraction and pulmonary artery remodeling, which subsequently leads to the development and progression of PAH. In this review, the authors summarize how EDRFs and EDCFs affect pulmonary vascular homeostasis, with special attention to the recently published novel mechanisms related to endothelial dysfunction in PAH and drugs associated with EDRFs and EDCFs.

Abstract 13476: ROR2 Deficiency is Associated With Endothelial Dysfunction and Reduced Angiogenesis in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction and progressive loss of lung microvessels. Our group has shown that Wnt pathways are critical for pulmonary angiogenesis but the role of the Wnt/planar cell polarity specific receptor ROR2 in lung endothelial angiogenic responses is unknown. Hypothesis: We hypothesized that ROR2 is required for proper angiogenic responses in pulmonary microvascular endothelial cells (PMVECs) and its loss contributes to endothelial dysfunction in PAH. Methods and Results: Analysis of PAH lung tissues demonstrated reduced ROR2 expression within vascular lesions and in explanted PMVECs. ROR2 deficiency was associated with reduced proliferation and tube formation in response to VEGF-A, a major inducer of angiogenesis. We found that ROR2 promotes VEGF receptor 2 activation via direct phosphorylation of cytoplasmic residues (Y1175/Y951) and that restoring ROR2 expression in PAH PMVECs led to improved angiogenic responses. Besides angiogenic sprouting, we found that ROR2 is required for lung endothelial barrier formation by promoting the assembly of mature focal adhesions and adherens junctions through activation of Src and focal adhesion kinase (FAK). Compared to wild-type controls, endothelial-specific ROR2 knockout (ROR2 ECKO) mice demonstrated more severe hemodynamics, small vessel loss, and muscularization in chronic hypoxia. Moreover, ROR2 ECKO demonstrated severe RV dysfunction associated with reduced capillary density. We validated these findings in samples of decompensated RV tissue from PAH patients, monocrotaline, and Sugen-hypoxia rats which also demonstrated a significant reduction of ROR2 expression that correlated with RV capillary loss. Conclusion: ROR2 plays a protective role in the pulmonary and RV circulations and its loss prevents vessel repair in PAH. Targeting ROR2 could promote recovery of small vessel loss and RV adaptation in PAH.

Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

Endothelial dysfunction is one of the hallmarks of different vascular diseases, including pulmonary arterial hypertension (PAH). Ion channelome changes have long been connected to vascular remodeling in PAH, yet only recently has the focus shifted towards Ca2+-activated Cl− channels (CaCC). The most prominent member of the CaCC TMEM16A has been shown to contribute to the pathogenesis of idiopathic PAH (IPAH) in pulmonary arterial smooth muscle cells, however its role in the homeostasis of healthy human pulmonary arterial endothelial cells (PAECs) and in the development of endothelial dysfunction remains underrepresented. Here we report enhanced TMEM16A activity in IPAH PAECs by whole-cell patch-clamp recordings. Using adenoviral-mediated TMEM16A increase in healthy primary human PAECs in vitro and in human pulmonary arteries ex vivo, we demonstrate the functional consequences of the augmented TMEM16A activity: alterations of Ca2+ dynamics and eNOS activity as well as decreased NO production, PAECs proliferation, wound healing, tube formation and acetylcholine-mediated relaxation of human pulmonary arteries. We propose that the ERK1/2 pathway is specifically affected by elevated TMEM16A activity, leading to these pathological changes. With this work we introduce increased TMEM16A activity in the cell membrane of human PAECs for the development of endothelial dysfunction in PAH.

Endothelial TRPV4 Channel Function is Impaired in Pulmonary Arterial Hypertension

Endothelial dysfunction contributes to the pathogenesis of pulmonary arterial hypertension (PAH) by reducing arterial vasodilation. Endothelium‐dependent vasodilation is an important regulator of pulmonary vascular resistance (PVR) and overall pulmonary arterial pressure (PAP). We recently identified endothelial TRPV4 (transient receptor potential vanilloid 4) channels as important regulators of vasodilation in pulmonary arteries (PAs), and showed that unitary Ca2+ influx events through TRPV4 channels, termed TRPV4 sparklets, activate endothelial nitric oxide synthase (eNOS) to cause vasodilation. Reduced NO bioavailability is a hallmark of endothelial dysfunction in PAH. However, the mechanisms underlying reduced NO bioavailability in PAH are not well understood. We hypothesized that impairment of TRPV4 channel function attenuates NO release in PAH. PAH was induced in mice using three‐week chronic hypoxia (CH) and Sugen 5416 + CH models. Mouse right ventricular pressures (RVP) were measured as an indicator of PAP. RVP was significantly higher in TRPV4−/− mice when compared to the control mice after three weeks of CH. High‐speed Ca2+ imaging was used to analyze TRPV4 sparklets in the native endothelium from resistance‐sized, fourth‐order PAs. Vascular diameter studies were performed on isolated, pressurized (15 mmHg), small PAs. Ca2+ imaging studies were also performed in PAs obtained from control and PAH patients. In CH and Sugen 5416 + CH models, TRPV4 sparklet activity and TRPV4 channel‐dependent vasodilation were significantly lower compared to normoxic control mice. However, TRPV4 mRNA expression and immunofluorescence were not altered in both mouse models of PAH, suggesting an impairment in the regulation of channel function rather than channel expression. TRPV4 sparklet activity was also consistently lower in PAs from PAH patients, when compared to control patients. Elevated levels of superoxide radicals have been shown to contribute to the development of PAH. Superoxides can react with NO to form peroxynitrite (PN). A PN scavenger, uric acid, restored TRPV4 channel activity in CH mice. We, therefore, hypothesized that PN‐induced impairment of TRPV4‐dependent Ca2+ signaling inhibits endothelial function in PAH. Indeed, PN attenuated TRPV4 sparklet activity in a reversible manner. Our results support the concept that PN‐induced regulation of TRPV4‐dependent Ca2+ signaling may contribute to the development of PAH. Moreover, altered TRPV4 channel activity may contribute to the reduced NO bioavailability in PAH.Support or Funding Information1R56HL138496, 5R00HL121484, 17PRE33660762This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial dysfunction in pulmonary arterial hypertension: loss of cilia length regulation upon cytokine stimulation.

Pulmonary arterial hypertension (PAH) is a syndrome characterized by progressive lung vascular remodelling, endothelial cell (EC) dysfunction, and excessive inflammation. The primary cilium is a sensory antenna that integrates signalling and fine tunes EC responses to various stimuli. Yet, cilia function in the context of deregulated immunity in PAH remains obscure. We hypothesized that cilia function is impaired in ECs from patients with PAH due to their inflammatory status and tested whether cilia length changes in response to cytokines. Primary human pulmonary and mouse embryonic EC were exposed to pro- (TNFα, IL1β, and IFNγ) and/or anti-inflammatory (IL-10) cytokines and cilia length was quantified. Chronic treatment with all tested inflammatory cytokines led to a significant elongation of cilia in both control human and mouse EC (by ∼1 µm, P < 0.001). This structural response was PKA/PKC dependent. Intriguingly, withdrawal of the inflammatory stimulus did not reduce cilia length. IL-10, on the other hand, blocked and reversed the pro-inflammatory cytokine-induced cilia elongation in healthy ECs, but did not influence basal length. Conversely, primary cilia of ECs from PAH patients were significantly longer under basal conditions compared to controls (1.86 ± 0.02 vs. 2.43 ± 0.08 µm, P = 0.002). These cilia did not elongate further upon pro-inflammatory stimulation and anti-inflammatory treatment did not impact cilia length. The missing length modulation was specific to cytokine stimulation, as application of fluid shear stress led to increased cilia length in the PAH endothelium. We identified loss of cilia length regulation upon cytokine stimulation as part of the endothelial dysfunction in PAH.

Rescuing BMPR2-driven endothelial dysfunction in PAH: a novel treatment strategy for the future?

Pulmonary arterial hypertension (PAH) may be idiopathic and corresponds to sporadic disease without any familial history or identified risk factors, or heritable when it occurs in the hereditary context; germline mutations in the bone morphogenetic protein receptor type 2 ( BMPR2 ) gene, are detected in 70% of heritable PAH cases (1). Even in sporadic cases, PAH is associated with impaired BMPRII signalling, occurring through small mothers against decapentaplegic (SMAD) proteins (2).

S35 Bmp9 And Bmp10 Mediate Connexin Expression In Endothelial Cells: Implications For Pah And Hht

Background Germ-line mutations in the bone morphogenetic protein type-II receptor, BMPR-II, underlie 80% of heritable pulmonary arterial hypertension (PAH) cases and approximately 25% of idiopathic PAH cases. PAH may arise due to endothelial dysfunction as mice with BMPR-II deficiency exhibit increased pulmonary vascular permeability. BMP9 is an endothelial quiescence factor and is thought to maintain the integrity of the endothelium. We previously reported that BMPR-II and ALK1 are the key receptors through which BMP9 inhibits the proliferation of human pulmonary artery endothelial cells (hPAECs). We hypothesised that BMPR-II deficiency impacts on endothelial cell connectivity and may contribute to endothelial dysfunction in PAH. Methods Human pulmonary artery endothelial cells were obtained from Lonza and blood outgrowth endothelial cells (BOECs) were isolated from peripheral blood of unaffected controls or PAH patients with identified BMPR-II mutations. Cells were transfected with siRNAs targeting BMPR-II followed by stimulation with BMP9. RNA was extracted and the expression of candidate genes determined by quantitative PCR. Further siRNA studies were performed for ALK1 and endoglin siRNAs. The promotion of gap junction assembly by BMP9 and BMP10 were assessed by immunofluorescence, Western blotting and functionally using parachute assays. Results Screening of candidate BMP9-induced junctional and structural proteins highlighted a subset of endothelial connexins that are BMP9 and BMP10-responsive and dependent on BMPR-II and ALK1. BMP9 and BMP10 increased the expression of the connexins, assessed by Western blotting and immunostaining. In addition, BMP9 and BMP10 significantly increased the transfer of calcein from labelled donor cells to unlabelled acceptor cells, indicating a promotion of endothelial cell connectivity. Conclusion In addition to their roles promoting endothelial quiescence, BMP9 and BMP10 directly promote endothelial cell connectivity by increasing connexin expression and assembly. The central contributions of BMPR-II and ALK1 to this process may implicate impaired endothelial connectivity as a pathological component of PAH and HHT.

Circulating Endothelial Cells in Refractory Pulmonary Hypertension in Children: Markers of Treatment Efficacy and Clinical Worsening

BackgroundPulmonary vasodilators in general and prostacyclin analogues in particular have improved the outcome of patients with pulmonary arterial hypertension (PAH). Endothelial dysfunction is a key feature of PAH and we previously described that circulating endothelial cell (CEC) level could be used as a biomarker of endothelial dysfunction in PAH. We now hypothesized that an efficient PAH-specific vasodilator therapy might decrease CEC level.Methods/ResultsCECs were prospectively quantified by immunomagnetic separation with mAb CD146-coated beads in peripheral blood from children with idiopathic PAH (iPAH, n = 30) or PAH secondary to congenital heart disease (PAH-CHD, n = 30): before, after treatment and during follow up. Controls were 23 children with reversible PAH. Oral treatment with endothelin receptor antagonists (ERA) and/or phosphodiesterase 5 inhibitors (PDE5) significantly reduced CEC counts in children. In 10 children with refractory PAH despite oral combination therapy, subcutaneous (SC) treprostinil was added and we observed a significant decrease in CEC counts during the first month of such treatment. CECs were quantified during a 6 to 36 month-follow-up after initiation of SC treprostinil and we found that CEC counts changed over time, with rising counts always preceding clinical deterioration.ConclusionCECs might be useful as a biomarker during follow-up of pediatric iPAH and PAH-CHD to assess response to treatment and to anticipate clinical worsening.

280: Circulating endothelial cell levels decrease after vasodilator therapy and are a biomarker of deterioration in pediatric pulmonary hypertension

Pulmonary vasodilators in general and prostacyclin therapy in particular have markedly improved the outcome of patients with pulmonary arterial hypertension (PAH). Endothelial dysfunction is a key feature of PAH and we previously described that circulating endothelial cells (CECs) could be used as a biomarker of endothelial dysfunction in PAH. We now hypothesized that PAH-specific vasodilator therapy might decrease CEC numbers. We quantified CECs in peripheral blood from children with idiopathic PAH (iPAH, n =30) or PAH secondary to congenital heart disease (PAH-CHD, n =30), before and after treatment and during follow up. CEC were enumerated by immunomagnetic separation with mAb CD146-coated beads. CEC counts were significantly decreased in children after treatment with oral endothelin antagonists and/or PDE5 inhibitors. In 10 children with refractory PAH despite combination oral therapies, SC treprostinil was added and we found a further significant decrease in CEC count during the first month of treatment in every patient. We quantified CEC during 6 to 36 months follow-up after initiation of SC treprostinil and found that CEC count is modified according to clinical status. CEC counts fall with vasodilatator therapy in PAH and could also be used as biomarker of deterioration in refractory pediatric pulmonary hypertension treated with SC treprostinil.

Role of Rho-kinase signaling and endothelial dysfunction in modulating blood flow distribution in pulmonary hypertension

Rho-kinase-mediated vasoconstriction and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). However, their contribution to the adverse changes in pulmonary blood flow distribution associated with PAH has not been addressed. This study utilizes synchrotron radiation microangiography to assess the specific role, and contribution of, Rho-kinase-mediated vasoconstriction and endothelial dysfunction in PAH. Male adult Sprague-Dawley rats were injected with saline (Cont-rats) or monocrotaline (MCT-rats) 3 wk before microangiography was performed on the left lung. We assessed dynamic changes in vessel internal diameter (ID) in response to 1) the Rho-kinase inhibitor fasudil (10 mg/kg iv); or 2) ACh (3 μg · kg⁻¹ · min⁻¹), sodium nitroprusside (SNP, 5 μg · kg⁻¹ · min⁻¹), and N(ω)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg iv). We observed that MCT-rats had fewer vessels of the microcirculation compared with Cont-rats. The fundamental result of this study is that fasudil improved pulmonary blood flow distribution and reduced pulmonary pressure in PAH rats, not only by dilating already-perfused vessels (ID > 100 μm), but also by restoring blood flow to vessels that had previously been constricted closed (ID < 100 μm). Endothelium-dependent vasodilation was impaired in MCT-rats primarily in vessels with an ID < 200 μm. Moreover the vasoconstrictor response to l-NAME was accentuated in MCT-rats, but only in the 200- to 300-μm vessels. These results highlight the importance of Rho-kinase-mediated control and endothelial control of pulmonary vascular tone in PAH. Indeed, an effective therapeutic strategy for treating PAH should target both the smooth muscle Rho-kinase and endothelial pathways.

Therapeutic Challenges for Systemic Sclerosis

Pulmonary arterial hypertension (PAH) is an important cause of death in systemic sclerosis (SSc), despite the improvement of therapies. An early diagnosis and the use of drugs interfering with the main pathogenic pathways of PAH is pivotal for the improvement of prognosis in primary PAH and PAH secondary to autoimmune rheumatic diseases, mainly SSc. Lately, new specific therapies have been developed targeting prostacyclin, endothelin, and nitric oxide pathways, the major pathogenic pathways leading to endothelial dysfunction in PAH. Epoprostenol improved life expectancy of patients with primary and secondary PAH, but its continuous intravenous administration requires experienced centers. More stable analogues of prostacyclin, administrated by intravenous (iloprost, treprostinil), subcutaneous, inhalatory (treprostinil, iloprost), and oral route (Beraprost) have shown efficacy in PAH. Bosentan, the first oral endothelin receptor antagonist (with affinity for endothelin A and B receptors) improves exercise function and survival in PAH, both primary and secondary to autoimmune rheumatic diseases. This is confirmed also for Sitaxsentan and Ambrisentan, selective A receptor antagonists. Because of its short half-life and systemic side effects, short-term NO inhalation is used only in short-term management of PAH in critically ill adults. Inhibitors of NO degradation, such as sildenafil, a phosphodiesterase (PDE) type 5 inhibitor, improved functional and hemodynamic parameters without significant side effects. Vardenafil and taladafil, longer-acting PDE inhibitors, also have vascular pulmonary selectivity. All these drugs may be used in combination, to maximize their clinical benefit not only in patients unresponsive to single drugs, but also potentially as initial therapy of PAH.