Human Pulmonary Artery Endothelial Cells Research Articles

Specificity Protein 1-Mediated Promotion of CXCL12 Advances Endothelial Cell Metabolism and Proliferation in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a rare yet devastating and incurable disease with few treatment options. The underlying mechanisms of PAH appear to involve substantial cellular proliferation and vascular remodeling, causing right ventricular overload and eventual heart failure. Recent evidence suggests a significant seminal role of the pulmonary endothelium in the initiation and promotion of PAH. Our previous work identified elevated reactive oxygen species (ROS)-producing enzyme NADPH oxidase 1 (NOX1) in human pulmonary artery endothelial cells (HPAECs) of PAH patients promoting endothelial cell proliferation in vitro. In this study, we interrogated chemokine CXCL12's (aka SDF-1) role in EC proliferation under the control of NOX1 and specificity protein 1 (Sp1). We report here that NOX1 can drive hypoxia-induced endothelial CXCL12 expression via the transcription factor Sp1 leading to HPAEC proliferation and migration. Indeed, NOX1 drove hypoxia-induced Sp1 activation, along with an increased capacity of Sp1 to bind cognate promoter regions in the CXCL12 promoter. Sp1 activation induced elevated expression of CXCL12 in hypoxic HPAECs, supporting downstream induction of expression at the CXCL12 promoter via NOX1 activity. Pathological levels of CXCL12 mimicking those reported in human PAH patient serum restored EC proliferation impeded by specific NOX1 inhibitor. The translational relevance of our findings is highlighted by elevated NOX1 activity, Sp1 activation, and CXCL12 expression in explanted lung samples from PAH patients compared to non-PAH controls. Analysis of phosphofructokinase, glucose-6-phosphate dehydrogenase, and glutaminase activity revealed that CXCL12 induces glutamine and glucose metabolism, which are foundational to EC cell proliferation. Indeed, in explanted human PAH lungs, demonstrably higher glutaminase activity was detected compared to healthy controls. Finally, infusion of recombinant CXCL12 into healthy mice amplified pulmonary arterial pressure, right ventricle remodeling, and elevated glucose and glutamine metabolism. Together these data suggest a central role for a novel NOX1-Sp1-CXCL12 pathway in mediating PAH phenotype in the lung endothelium.

Divergent Roles of Ephrin-B2/EphB4 Guidance System in Pulmonary Hypertension.

Smooth muscle cell (SMC) expansion is one key morphological hallmark of pathologically altered vasculature and a characteristic feature of pulmonary vascular remodeling in pulmonary hypertension. Normal embryonal vessel maturation requires successful coverage of endothelial tubes with SMC, which is dependent on ephrin-B2 and EphB4 ligand-receptor guidance system. In this study, we investigated the potential role of ephrin-B2 and EphB4 on neomuscularization in adult pulmonary vascular disease. Ephrin-B2 and EphB4 expression is preserved in smooth muscle and endothelial cells of remodeled pulmonary arteries. Chronic hypoxia-induced pulmonary hypertension was not ameliorated in mice with SMC-specific conditional ephrin-B2 knockout. In mice with global inducible ephrin-B2 knockout, pulmonary vascular remodeling and right ventricular hypertrophy upon chronic hypoxia exposure were significantly diminished compared to hypoxic controls, while right ventricular systolic pressure was unaffected. In contrast, EphB4 receptor kinase activity inhibition reduced right ventricular systolic pressure in hypoxia-induced pulmonary hypertension without affecting pulmonary vascular remodeling. Genetic deletion of ephrin-B2 in murine pulmonary artery SMC, and pharmacological inhibition of EphB4 in human pulmonary artery smooth muscle cells, blunted mitogen-induced cell proliferation. Loss of EphB4 signaling additionally reduced RhoA expression and weakened the interaction between human pulmonary artery smooth muscle cells and endothelial cells in a three-dimensional coculture model. In sum, pulmonary vascular remodeling was dependent on ephrin-B2-induced Eph receptor (erythropoietin-producing hepatocellular carcinoma receptor) forward signaling in SMC, while EphB4 receptor activity was necessary for RhoA expression in SMC, interaction with endothelial cells and vasoconstrictive components of pulmonary hypertension.

The Role of TSP-1, TGF-B1 and Galectin-3 in the Pathogenesis of ACS in Sickle Cell Disease

Sickle cell disease (SCD) is a hereditary blood disorder characterized by painful vaso-occlusive episodes (VOE), acute chest syndrome (ACS), stroke and other clinical complications. Seventy-six percent of the primary and secondary complications of SCD are pulmonary and are strongly associated with hemolysis. ACS is the main acute pulmonary complication of SCD and is characterized by critical lung injury from vaso-occlusion in the pulmonary vasculature, release of inflammatory mediators, and alveolar edema decreasing gas exchange. Endothelial dysfunction, characterized by increased production of inflammatory mediators, increased oxidative stress, and aberrant cellular proliferation via platelet-derived thrombospondin-1 (TSP-1) and transforming growth factor-1 (TGF-B1) is increasingly implicated in pulmonary vascular pathology in other diseases. In addition, a mediator of TGF-B1, galectin-3, has been shown to be induce tissue-specific fibrosis via fibroblast activation in animal disease models. However, the mechanisms by which human pulmonary artery endothelial cell (HPAEC) dysfunction is instigated remains unclear. We hypothesized a novel theory that TSP-1, TGF-B1, and galectin-3 play a causal role in the pathogenesis of ACS in SCD. We set out to test our hypothesis by measuring TSP-1, TGF-B1 and galectin-3 plasma levels in a cohort of patients with SCD with (n=10) and without history of ACS (n=20), and healthy, race and age-matched controls (n=10). Patients with ACS had significantly higher plasma levels of active TGF-B1 (11.34 ng/mL) than those of patients without ACS (5.2 ng/mL) and healthy controls (3.4 ng/mL). Similarly, we found that SCD patients with a history of ACS had significantly higher levels of TSP-1 (642.7 ng/mL) and galectin-3 (8.87 ng/mL) as compared to patients without ACS (285.9 ng/mL and 6.02 ng/mL, respectively) and controls (133.36 ng/mL and 5.2 ng/mL, respectively). We also found a significant correlation between TSP-1 and TGF-B1 levels in non-ACS SCD patients and patients with ACS as well as galectin-3 and TGF-B1 levels in the plasma of all SCD patients combined. Given that platelets are a significant source of TSP-1 and TGF-B1, we next tested whether platelets mechanistically potentiate endothelial cell dysfunction in SCD. HPAECs were co-cultured with platelets isolated from healthy vs steady-state SCD individuals. Platelets in co-culture were quiescent or activated with the agonists 0.1 U/mL thrombin, 25 ug/mL collagen, and 2.5 uM free heme for 6 hours, and HPAEC proliferation was measured via crystal violet staining 48 hours after exposure. We found HPAEC proliferation upon control and SCD platelet exposure to free heme, but SCD platelets exposed to heme induced a more robust response in HPAEC proliferation. In addition, we found that collagen only significantly promoted HPAEC proliferation in the presence of SCD platelets and not control platelets. Activated platelets, in the presence of hemolysis, release large amounts of TSP1 and TGF-B1 that may lead to enhanced endothelial cell proliferation and fibrosis in SCD, possibly via increased response to collagen by activated platelets. We will conduct further experiments to determine whether blockers of TSP-1 or its canonical CD47 endothelial receptor may abrogate aberrant cellular proliferation in SCD. Overall, the roles of TSP-1 and galectin-3 in pulmonary fibrotic injury via the TGF-B1 signaling pathway in SCD need to be further elucidated for therapeutic targeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

BackgroundPulmonary hypertension (PH) associated to idiopathic pulmonary fibrosis (IPF) portends a poor prognosis. IL-11 has been implicated in fibrotic diseases, but their role on pulmonary vessels is unknown. Here we analyzed the contribution of IL-11 to PH in patients with IPF and the potential mechanism implicated.MethodsPulmonary arteries, lung tissue and serum of control subjects (n = 20), IPF (n = 20) and PH associated to IPF (n = 20) were used to study the expression and localization of IL-11 and IL-11Rα. Two models of IL-11 and bleomycin-induced lung fibrosis associated to PH were used in Tie2-GFP transgenic mice to evaluate the contribution of IL-11 and endothelial cells to pulmonary artery remodeling. The effect of IL-11 and soluble IL-11Rα on human pulmonary artery endothelial cells and smooth muscle cell transformations and proliferation were analyzed.ResultsIL-11 and IL-11Rα were over-expressed in pulmonary arteries and serum of patients with PH associated to IPF vs IPF patients without PH. Recombinant mice (rm)IL-11 induced lung fibrosis and PH in Tie2-GFP mice, activating in vivo EnMT as a contributor of pulmonary artery remodeling and lung fibrosis. Transient transfection of siRNA-IL-11 reduced lung fibrosis and PH in Tie2-GFP bleomycin model. Human (h)rIL-11 and soluble hrIL-11Rα induced endothelial to mesenchymal transition (EnMT) and pulmonary artery smooth muscle cell to myofibroblast-like transformation, cell proliferation and senescence in vitro.ConclusionsIL-11 and IL-11Rα are overexpressed in pulmonary arteries of PH associated to IPF patients, and contributes to pulmonary artery remodeling and PH.

Abstract 12755: Role of Micriobiome-Derived Short-Chain Fatty Acid Butyrate in Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is a progressive, severe disease characterized by high blood pressure in the pulmonary circulation, inflammatory cells infiltration and excessive pulmonary vascular remodeling. Endothelial cell (EC) dysfunction is increasingly recognized as a precipitating event for this remodeling. Butyrate, a microbiome-derived molecule with histone deacetylation activity and present in circulating blood, has been associated with benefit in atherosclerosis and associated cardiovascular diseases. Due to a dearth of knowledge on the role of microbiome-derived molecules in PH-EC, we aimed to test whether butyrate plays a protective role in EC under PH. Results: Mice supplementation with butyrate (sodium butyrate, NaB) attenuated hypoxia-induced right ventricular (RV) pressure (43±2 to 35±3 mmHg, p<0.05) and RV hypertrophy (RV/LV+Septum= 0.43±0.008 to 0.38±0.01, p<0.05) in vivo. NaB also lowered hypoxia-induced circulating monocyte numbers and reduced gene expression of inflammatory adhesion molecule VCAM-1 in hypoxic lung-derived CD31+ ECs, as well as increased expression of ERM binding Phosphoprotein 50 (EBP50), a protein we have shown to modulate endothelial reprogramming in PH. In vitro, NaB attenuated PH-related inflammatory cytokine IL1β-induced human pulmonary arterial EC (HPAEC) migration at 48 h. Consistently, NaB also reversed IL1β-induced increase in VCAM-1 expression at the mRNA and protein level in HPAEC. Moreover, NaB rescued the expression of IL1β-induced reduction in cell surface protein integrin α3, EBP50, and Vangl2 (Van Gogh-like) protein, a member of planar cell polarity core whose downregulation has been linked to PH. Conclusions: Collectively our results demonstrate that butyrate attenuates PH potentially via protecting against myeloid cell induction and pathophysiological proinflammatory, promigratory pulmonary vascular EC activation. Butyrate may also reduce EC reprogramming through its effects on EBP50, and may promote pulmonary vascular homeostasis through Vangl2 induction. These findings support the potential for therapeutic benefits of butyrate in PH and the need for further investigation of the role of microbiome-derived metabolites in this devastating disease.

Abstract 12700: Post-GWAS Functional SNP Analysis to Define Genetic and Acquired Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is an enigmatic and morbid disease where insights are emerging regarding genetic susceptibility. Genome-wide Association Studies (GWAS) have identified genomic SNPs associated with increased PAH risk. It has been challenging to define the mechanisms underlying the contribution of non-coding PAH-associated functional SNPs (fSNPs) to the pathogenesis of this disease. Methods: We developed a post-GWAS functional genomics strategy to define the causative fSNPs and the associated biological mechanisms. It includes Reel-seq (Regulatory element-sequencing), an EMSA-based technique to identify fSNPs in a synthetic SNP library; SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify proteins that specifically bind to fSNPs; and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to show allele-imbalanced binding of these proteins to fSNPs. The regulation of risk gene expression by fSNP-binding proteins and their pathogenicity were determined in human pulmonary arterial endothelial cells (PAECs) and confirmed in PAH animal models and patients. Results: An intergenic SNP rs4738801 was identified as a fSNP by Reel-seq. This non-coding fSNP resides in a remote upstream enhancer region of SOX17, an endothelial effector increasingly associated with PAH pathogenesis. Using SDCP-MS and AIDP-Wb, we found that transcription factor FUBP1 binds to rs4738801 risk allele C with lower affinity than non-risk allele G, resulting in a decrease in SOX17 expression. FUBP1 and target gene SOX17 controlled PAH-associated pathophenotypes in PAECs, including proliferation, apoptosis, and angiogenesis. Downregulated by major acquired PAH trigger hypoxia, FUBP1 and SOX17 were decreased in lungs and pulmonary ECs isolated from PAH patients and mouse models. A 3.8-fold enrichment of risk allele C was found in patients with PAH induced by hypoxia, but not in PAH associated with connective tissue disease or other etiology. CONCLUSION: Our functional genomics findings identify a novel role of FUBP1 in the functional regulation of SOX17 locus and elucidate a pathogenic mechanism that combines the acquired PAH-triggering factors and altered genetic susceptibility.

Abstract 15215: Autophagy Inhibition Attenuate the Development and Progression of Pulmonary Hypertension

Introduction: Autophagy plays an important role in the pathogenesis of pulmonary hypertension. However, the potential therapeutic benefit of autophagy inhibition remains undetermined. Hypothesis: ROC-325 was developed as a novel small molecule lysosomal autophagy inhibitor that has demonstrated significantly more potent anticancer activity than the antimalarial drug hydroxychloroquine (HCQ). Methods: Rats with monocrotaline-induced PH (MCT-PH) and hypoxia/Sugen-induced severe PH were treated with ROC-325 and the related effects on echocardiography, hemodynamics, right ventricle hypertrophy, and pulmonary vascular remodeling were assessed. Pulmonary arterial pressure (PAP) and isometric tension were measured in isolated perfused/ventilated mouse lungs and rat PA rings. The in vitro effects on ROC-325 on autophagy, proliferation, viability, apoptosis and endothelial nitric oxide (NO) synthase (eNOS)-NO signaling were tested in human and rat pulmonary artery smooth muscle cells (PASMCs) as well as human pulmonary artery endothelial cells (PAECs) and rat pulmonary microvascular endothelial cells (PMVECs). Results and Conclusions: The therapeutic benefits of a novel lysosomal autophagy inhibitor, ROC-325, in experimental pulmonary hypertension (PH) models. Specifically, ROC-325 treatment prevents the development of monocrotaline (MCT)-induced PH, right ventricular dysfunction, and pulmonary vascular remodeling, and reverses the progression of established MCT-PH. Meanwhile, ROC-325 treatment attenuates high K + or alveolar hypoxia-induced pulmonary vasoconstriction. Mechanistically, in PASMC and PAEC, ROC-325 inhibits autophagic pathways, reduces hypoxia induced-hypoxia inducible factor (HIF) accumulation and blocks endothelial nitric oxide (NO) synthase (eNOS)-NO signaling activation. These studies illustrate the discovery of ROC-325 as a novel promising agent for PH therapy.

Abstract 10523: Involvement of Sur2/Kir6.1 In the Physiopathology of Pulmonary Arterial Hypertension

Introduction: The ATP-sensitive K+ channels (KATP) regulatory subunit ABCC8 may be considered a new therapeutic target for pulmonary arterial hypertension (PAH). Hypothesis: We hypothesised that the second KATP regulatory subunit (ABCC9) contributes to PAH pathogenesis. ABCC9 gene encodes for two regulatory subunits of KATP channels: the SUR2A and SURB proteins. In the KATP channel, the SUR2 subunits are associated with the K+ channel Kir6.1. We investigated how the SUR2/Kir6.1 channel contributes to PAH pathogenesis and its potential as a therapeutic target against PAH. Methods: Using in vitro, ex vivo, and in vivo approaches, we analysed the localisation and expression of SUR2A, SUR2B, and Kir6.1 in pulmonary vasculature of controls and patients with PAH as in experimental PH rat models and its contribution to PAH physiopathology. Finally, we deciphered the consequences of in vivo activation of SUR2/Kir6.1 in the monocrotaline (MCT)-induced PH model. Results: We demonstrated that SUR2A, SUR2B, and Kir6.1 were expressed in the lungs of controls and patients with PAH and MCT-induced PH rat model. Myograph experiments showed that activation of SUR2 by pinacidil induced pulmonary arterial relaxation. In vitro experiments on human pulmonary arterial smooth muscle cells and endothelial cells (hPASMCs and hPAECs) in controls and PAH patients showed decreased cell proliferation and migration after SUR2 activation. Using patch-clamp, we demonstrated that SUR2 activation in rat right ventricular (RV) cardiomyocytes reduced RV action potential duration. Chronic pinacidil administration in control rats increased heart rate without changes in hemodynamic parameters. Finally, in vivo pharmacological activation of SUR2 on MCT-induced-PH rats showed an improvement of PH during the preventive and curative protocol. Conclusions: We demonstrated that SUR2A, SUR2B, and Kir6.1 were expressed in hPASMCs and hPAECs of controls and PAH patients. In vivo SUR2 activation reduced MCT-induced PH phenotype, suggesting that SUR2 activation should be considered for treatment of PAH.

The Novel Lysosomal Autophagy Inhibitor (ROC-325) Ameliorates Experimental Pulmonary Hypertension.

Autophagy plays an important role in the pathogenesis of pulmonary hypertension (PH). ROC-325 is a novel small molecule lysosomal autophagy inhibitor that has more potent anticancer activity than the antimalarial drug hydroxychloroquine, the latter has been prevalently used to inhibit autophagy. Here, we sought to determine the therapeutic benefit and mechanism of action of ROC-325 in experimental PH models. Hemodynamics, echocardiography, and histology measurement showed that ROC-325 treatment prevented the development of PH, right ventricular hypertrophy, fibrosis, dysfunction, and vascular remodeling after monocrotaline and Sugen5416/hypoxia administration. ROC-325 attenuated high K+ or alveolar hypoxia-induced pulmonary vasoconstriction and enhanced endothelial-dependent relaxation in isolated pulmonary artery rings. ROC-325 treatment inhibited autophagy and enhanced endothelial nitric oxide synthase activity in lung tissues of monocrotaline-PH rats. In cultured human and rat pulmonary arterial smooth muscle cell and pulmonary arterial endothelial cell under hypoxia exposure, ROC-325 increased LC3B (light chain 3 beta) and p62 accumulation, endothelial cell nitric oxide production via phosphorylation of endothelial nitric oxide synthase (Ser1177) and dephosphorylation of endothelial nitric oxide synthase (Thr495) as well as decreased HIF (hypoxia-inducible factor)-1α and HIF-2α stabilization. These data indicate that ROC-325 is a promising novel agent for the treatment of PH that inhibits autophagy, downregulates HIF levels, and increases nitric oxide production.

Iron metabolism disorder regulated by BMP signaling in hypoxic pulmonary hypertension

Backgrounds and aimsUnexplained iron deficiency is associated with poorer survival in patients with pulmonary hypertension (PH). Bone morphogenetic protein (BMP) signaling and BMP protein type II receptor (BMPR2) expression are important in the pathogenesis of PH. BMP6 in hepatocytes is a central transcriptional regulator of the iron hormone hepcidin that controls systemic iron balance. This study aimed to investigate the effects of BMP signaling on iron metabolism and its implication in hypoxia-induced PH. Methods and resultsPH was induced in Sprague–Dawley Rats under hypoxia for 4 weeks. Compared with the control group, right ventricular systolic pressure and right ventricle hypertrophy index were both markedly increased, and serum iron level was significantly decreased with iron metabolic disorder in the hypoxia group. In cultured human pulmonary artery endothelial cells (HPAECs), hypoxia increased oxidative stress and apoptosis, which were reversed by supplementation with Fe agent. Meanwhile, iron chelator deferoxamine triggered oxidative stress and apoptosis in HPAECs, and treatment with antioxidant alleviated iron-deficiency-induced apoptosis by reducing reactive oxygen species production. Expression of hepcidin, BMP6 and hypoxia-inducible factor (HIF)-1α were significantly upregulated, while expression of BMPR2 was downregulated in hepatocytes in the hypoxia group, both in vivo and in vitro. Expression of hepcidin and HIF-1α were significantly increased by BMP6, while pretreatment with siRNA-BMPR2 augmented the enhanced expression of hepcidin and HIF-1α induced by BMP6. ConclusionsIron deficiency promoted oxidative stress and apoptosis in HPAECs in hypoxia-induced PH, and enhanced expression of hepcidin regulated by BMP6/BMPR2 signaling may contribute to iron metabolic disorder.

Impaired Interleukin-15 Signaling via BMPR2 Loss Drives Natural Killer Cell Deficiency and Pulmonary Hypertension.

Natural killer (NK) cell impairment is a feature of pulmonary arterial hypertension (PAH) and contributes to vascular remodeling in animal models of disease. Although mutations in BMPR2, the gene encoding the BMP (bone morphogenetic protein) type-II receptor, are strongly associated with PAH, the contribution of BMPR2 loss to NK cell impairment remains unknown. We explored the impairment of IL (interleukin)-15 signaling, a central mediator of NK cell homeostasis, as both a downstream target of BMPR2 loss and a contributor to the pathogenesis of PAH. The expression, trafficking, and secretion of IL-15 and IL-15Rα (interleukin 15 α-type receptor) were assessed in human pulmonary artery endothelial cells, with or without BMPR2 silencing. NK cell development and IL-15/IL-15Rα levels were quantified in mice bearing a heterozygous knock-in of the R899X-BMPR2 mutation (bmpr2+/R899X). NK-deficient Il15-/- rats were exposed to the Sugen/hypoxia and monocrotaline models of PAH to assess the impact of impaired IL-15 signaling on disease severity. BMPR2 loss reduced IL-15Rα surface presentation and secretion in human pulmonary artery endothelial cells via impaired trafficking through the trans-Golgi network. bmpr2+/R899X mice exhibited a decrease in NK cells, which was not attributable to impaired hematopoietic development but was instead associated with reduced IL-15/IL-15Rα levels in these animals. Il15-/- rats of both sexes exhibited enhanced disease severity in the Sugen/hypoxia model, with only male Il15-/- rats developing more severe PAH in response to monocrotaline. This work identifies the loss of IL-15 signaling as a novel BMPR2-dependent contributor to NK cell impairment and pulmonary vascular disease.

Cytotoxicity of Particulate Matter PM10 Samples from Ouagadougou, Burkina Faso.

Particulate matter (PM) is one of the main air pollutants with 257,000 deaths per year in Africa. Studying their toxic mechanisms of action could provide a better understanding of their effects on the population health. The objective of this study was to describe the PM10 toxic mechanism of action collected in 3 districts of Ouagadougou. Once per month and per site between November 2015 and February 2016, PM10 was sampled for 24 hours using the MiniVol TAS (AirMetrics, Eugene, USA). The collected filters were then stored in Petri dishes at room temperature for in vitro toxicological studies using human pulmonary artery endothelial cells (HPAEC) at the Bordeaux INSERM-U1045 Cardio-thoracic Research Center. The three study districts were classified based on PM10 level (high, intermediate, and low, respectively, for districts 2, 3, and 4). PM10 induced a concentration-dependent decrease in cell viability. A significant decrease in cell viability was observed at 1 µg/cm2, 10 µg/cm2, and 25 µg/cm2 for, respectively, districts 2, 3, and 4. A significant increase in the production of reactive oxygen species (ROS) was observed at 10 µg/cm2 for district 2 versus 5 µg/cm2 and 1 µg/cm2 for districts 3 and 4, respectively. Finally, a significant production of IL-6 was recorded from 5 µg/cm2 for district 4 versus 10 µg/cm2 for districts 2 and 3. Consequently, Ouagadougou is subjected to PM10 pollution, which can induce a significant production of ROS and IL-6 to cause adverse effects on the health of the population.

Intracellular Chloride Channels Regulate Endothelial Metabolic Reprogramming in Pulmonary Arterial Hypertension.

Mitochondrial fission and a metabolic switch from oxidative phosphorylation to glycolysis are key features of vascular pathology in pulmonary arterial hypertension (PAH) and are associated with exuberant endothelial proliferation and apoptosis. The underlying mechanisms are poorly understood. We describe the contribution of two intracellular chloride channel proteins, CLIC1 and CLIC4, both highly expressed in PAH and cancer, to mitochondrial dysfunction and energy metabolism in PAH endothelium. Pathological overexpression of CLIC proteins induces mitochondrial fragmentation, inhibits mitochondrial cristae formation, and induces metabolic shift toward glycolysis in human pulmonary artery endothelial cells, consistent with changes observed in patient-derived cells. Interactions of CLIC proteins with structural components of the inner mitochondrial membrane offer mechanistic insights. Endothelial CLIC4 excision and mitofusin 2 supplementation have protective effects in human PAH cells and preclinical PAH. This study is the first to demonstrate the key role of endothelial intracellular chloride channels in the regulation of mitochondrial structure, biogenesis, and metabolic reprogramming in expression of the PAH phenotype.

MircoRNA in Extracellular Vesicles from Patients with Pulmonary Arterial Hypertension Alters Endothelial Angiogenic Response.

Pulmonary arterial hypertension (PAH) is characterized by a progressive elevation of pulmonary pressure leading to right ventricular dysfunction and is associated with a poor prognosis. Patients with PAH have increased numbers of circulating extracellular vesicles (EVs) and altered expression of circulating microRNAs (miRs). The study aimed to evaluate the miR profile contained within purified EVs derived from the plasma of PAH patients as compared to healthy controls (HC). Circulating EVs, purified from platelet-free plasma were analyzed using flow cytometry, western blot, and electron microscopy. Total RNA isolated from EVs was subjected to Microarray analysis using GeneChip miRNA 4.0 Array and bioinformatics tools. Overexpression and inhibition of miRs were conducted in human pulmonary artery endothelial cells (hPAECs) that had been incubated previously with either PAH- or HC-derived EVs. Cell proliferation (MTT assay) and angiogenesis (tube formation assay) were tested in hPAECs to determine miR functionality. MiR profiling revealed 370 heats while comparing PAH and HC groups, 22 of which were found to be down-regulated and 6 were up-regulated in the PAH EVs. Among the altered miRs, miR-486-5p was overexpressed, while miR-26a-5p was downregulated in PAH EVs compared to HC EVs. Inhibition of mir-486-5p or overexpression of miR-26a-5p in hPAECs post-exposure of PAH EVs abrogated proangiogenic and proliferative effects posed by PAH EVs contrary to HC EVs. The angiogenic and proliferative effects of the miRs from PAH EVs were observed to be mediated through nuclear factor (NF)-κB activation. PAH EVs carry and present an altered miR profile that can be targeted to restrict angiogenesis and reduce pulmonary endothelium activation. Further studies concerning miRs from circulating heterogeneous EVs in PAH patients are warranted to understand their potential as targets for treatment in PAH.

Commercial human pulmonary artery endothelial cells have in-vitro behavior that varies by sex.

It is unknown whether biological sex influences phenotypes of commercially available human pulmonary artery endothelial cells (HPAECs). Ten lots of commercial HPAECs were used (Lonza Biologics; PromoCell). Five (50%) were confirmed to be genotypically male (SRY+) and five (50%) were confirmed to be female (SRY-). Experiments were conducted between passages five andeight. HPAEC phenotype was confirmed with a panel of cell expression markers. Standard assays for proliferation, migration and tube formation were performed in triplicate with technical replicates, under three treatment conditions (EndoGRO; Sigma-Aldrich). Apoptosis was assessed by exposing cells treated with complete media or low serum media to hypoxic (1% oxygen) or normoxic (20% oxygen) conditions. Laboratory staff was blinded. The median (range) age of male and female donors from whom the HPAECs were derived was 58 (48-60) and 56 (33-67), respectively. Our results suggest decreased proliferation in genotypically female cells compared with male cells (p = 0.09). With increasing donor age, female cells were less proliferative and male cells were more proliferative (p = 0.001). Female cells were significantly more apoptotic than male cells by condition (p = 0.001). Female cells were significantly more migratory than male cells in complete media but less migratory than male cells under vascular endothelial growth factorenriched conditions (p = 0.001). There are subtle sex-based differences in the behavior of HPAECs that depend on donor sex and, less so, age. These differences may undermine rigor and reproducibility. Future studies should define whether biological sex is an important regulator of HPAEC function in health and disease.

Neuroblastoma Suppressor of Tumorigenicity 1 Mediates Endothelial-to-Mesenchymal Transition in Pulmonary Arterial Hypertension Related to Congenital Heart Disease.

Endothelial-to-mesenchymal transition (EndMT) plays a critical role in the flow-induced vascular remodeling process, such as pulmonary arterial hypertension (PAH) related to congenital heart disease (CHD). NBL1 (neuroblastoma suppressor of tumorigenicity 1) is a secreted glycoprotein that has been implicated in CHD-PAH by aggravating the phenotypic transformation of smooth muscle cells. However, the underlying mechanisms regarding the interplay between NBL1 and endothelial cells in CHD-PAH remain to be fully elucidated. Thus, we aimed to identify the potential effect of NBL1 on EndMT using a novel flow-associated PAH model with Nbl1 knockout rats. The phenotype of EndMT was detected using RNA sequencing and further examined using western blotting and immunostaining of pulmonary arteries. Our observations demonstrated that the novel strategy of Nbl1 knockout effectively attenuated flow-associated PAH through downregulation of EndMT to some extent. Mechanistic experiments were established on human pulmonary artery endothelial cells to confirm that EndMT was induced by NBL1 invitro. After 7 days' stimulation with NBL1, concentrations of EndMT-related biomarkers and downstream transcription factors were quantified using RNA sequencing, western blotting, and immunocytochemistry. Both invitro and invivo experiments supported the imbalance of increased TGF-β (transforming growth factor-β) and dysregulation of BMP (bone morphogenetic protein) signaling by NBL1. Blocking the canonical TGF-β pathway efficiently preserved endothelial function upon NBL1 stimulation. These data suggested that NBL1 aggravated flow-associated PAH by inducing EndMT via the TGF-β and BMP signaling pathway. Thus, antagonizing NBL1 and rebalancing TGF-β and BMP signaling may be a suitable therapeutic target for CHD-PAH.

Circulating Microparticles Are Differentially Increased in Lowlanders and Highlanders with High Altitude Induced Pulmonary Hypertension during the Cold Season.

The role of microparticles (MPs) and cold in high altitude pulmonary hypertension (HAPH) remains unexplored. We investigated the impact of long-term cold exposure on the pulmonary circulation in lowlanders and high-altitude natives and the role of MPs. Pulmonary hemodynamics were evaluated using Doppler echocardiography at the end of the colder and warmer seasons. We further examined the miRNA content of MPs isolated from the study participants and studied their effects on human pulmonary artery smooth muscle (hPASMCs) and endothelial cells (hPAECs). Long-term exposure to cold environment was associated with an enhanced pulmonary artery pressure in highlanders. Plasma levels of CD62E-positive and CD68-positive MPs increased in response to cold in lowlanders and HAPH highlanders. The miRNA-210 expression contained in MPs differentially changed in response to cold in lowlanders and highlanders. MPs isolated from lowlanders and highlanders increased proliferation and reduced apoptosis of hPASMCs. Further, MPs isolated from warm-exposed HAPH highlanders and cold-exposed highlanders exerted the most pronounced effects on VEGF expression in hPAECs. We demonstrated that prolonged exposure to cold is associated with elevated pulmonary artery pressures, which are most pronounced in high-altitude residents. Further, the numbers of circulating MPs are differentially increased in lowlanders and HAPH highlanders during the colder season.

Deoxyelephantopin alleviates lipopolysaccharide-induced septic lung injury through inhibiting NF-ĸB/STAT3 axis.

Sepsis induces multiple organ dysfunction syndromes, such as acute kidney, liver, or lung injury. Septic lung injury is associated with excessive apoptosis and inflammatory responses in hepatocytes. Deoxyelephantopin is a sesquiterpene lactone found in Elephantopus scaber L, and has immunomodulatory, antibacterial, anti-inflammatory, and antifungal properties. The role of deoxyelephantopin in sepsis-associated lung injury was investigated. First, human bronchial epithelial cells (BEAS-2B) and human pulmonary artery endothelial cells (HPAEC) were treated with lipopolysaccharide to induce cytotoxicity. Treatment with lipopolysaccharide reduced cell viability of BEAS-2B and HPAEC, and promoted cell apoptosis through down-regulation of poly (ADP-ribose) polymerase (PARP) and B-cell lymphoma 2 (Bcl-2), and up-regulation of cleaved PARP and B-cell lymphoma-associated X protein (Bax). Second, lipopolysaccharide-treated BEAS-2B and HPAEC were incubated with increasing concentrations of deoxyelephantopin, that is, 1, 5, or 10 μM. Deoxyelephantopin enhanced cell viability and reduced cell apoptosis of lipopolysaccharide-treated BEAS-2B and HPAEC. Third, deoxyelephantopin attenuated lipopolysaccharide-induced decrease of superoxide dismutase and glutathione, and increase of malondialdehyde and myeloperoxidase in BEAS-2B and HPAEC. Moreover, deoxyelephantopin also weakened lipopolysaccharide-induced increase of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Finally, deoxyelephantopin decreased protein expression of p-p65 and p-signal transducer and activator of transcription 3 (STAT3) in lipopolysaccharide-treated BEAS-2B and HPAEC. In conclusion, deoxyelephantopin exhibited anti-oxidative and anti-inflammatory effects against lipopolysaccharide-treated BEAS-2B and HPAEC through inactivation of nuclear factor kappa B/STAT3 signaling.

Pyrroloquinoline quinone (PQQ) improves pulmonary hypertension by regulating mitochondrial and metabolic functions

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs), inflammation, as well as mitochondrial and metabolic dysregulation, contributes to the development of pulmonary hypertension (PH). Pyrroloquinoline quinone (PQQ), a potent natural antioxidant with anti-diabetic, neuroprotective, and cardioprotective properties, is known to promote mitochondrial biogenesis. However, its effect on cellular proliferation, apoptosis resistance, mitochondrial and metabolic alterations associated with PH remains unexplored. The current study was designed to investigate the effect of PQQ in the treatment of PH. Human pulmonary artery smooth muscle cells (HPASMCs), endothelial cells (PAECs), and primary cultured cardiomyocytes were subjected to hypoxia to induce PH-like phenotype. Furthermore, Sprague Dawley (SD) rats injected with monocrotaline (MCT) (60 mg/kg, SC, once) progressively developed pulmonary hypertension. PQQ treatment (2 mg/kg, PO, for 35 days) attenuated cellular proliferation and promoted apoptosis via a mitochondrial-dependent pathway. Furthermore, PQQ treatment in HPASMCs prevented mitochondrial and metabolic dysfunctions, improved mitochondrial bioenergetics while preserving respiratory complexes, and reduced insulin resistance. In addition, PQQ treatment (preventive and curative) significantly attenuated the increase in right ventricle pressure and hypertrophy as well as reduced endothelial dysfunction and pulmonary artery remodeling in MCT-treated rats. PQQ also prevented cardiac fibrosis and improved cardiac functions as well as reduced inflammation in MCT-treated rats. Altogether, the above findings demonstrate that PQQ can attenuate mitochondrial as well as metabolic abnormalities in PASMCs and also prevent the development of PH in MCT treated rats; hence PQQ may act as a potential therapeutic agent for the treatment of PH.

Mechanosensitive cation currents through TRPC6 and Piezo1 channels in human pulmonary arterial endothelial cells.

Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.