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Introduction:Pulmonary arterial hypertension (PAH) is a disaster 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.Hypothesis:We hypothesis that endothelial fatty acid metabolism is critical for obstructive vascular remodeling in the pathogenesis of PAH.Methods:A severe mouse model of PH Egln1Tie2Cremice were bred with Fabp45-/-mice to generate Egln1Tie2Cre/Fabp45-/-mice. Single-cell RNA sequencing (scRNA-seq) analysis and metabolomic analysis were used to profile the pulmonary cells in Egln1Tie2Cremice and Egln1Tie2Cre/Fabp45-/-mice. Human hPAEC from idiopathic PAH patients and healthy donors, monocrotaline (MCT)-induced and Sugen5416/hypoxia (SuHx)-induced PH rats were used to measure fatty acid-binding protein 4 and 5 (FABP4 and FABP5) expression. siRNA mediated knockdown of FABP4 and FABP5 and lentivirus mediated FABP4 and 5 overexpression were performed to study cell proliferation, apoptosis, glycolysis, fatty acid oxidation. Echocardiography, hemodynamics, histological and immunostaining assay were performed to evaluate the PH phenotypes.Results:Both FABP4 and 5 were highly induced in the ECs of Egln1Tie2Cremice and PAECs from IPAH patients, as well as the whole lungs of MCT and SuHx-induced PH rats. Knockdown or overexpression of FABP4-5 reduced or enhanced EC proliferation, starvation-induced Caspase 3/7 activity, glycolysis and fatty acid oxidation. Genetic deletion of Fabp4and 5 in Egln1Tie2Cremice exhibited a reduction of right ventricular systolic pressure (RVSP), RV hypertrophy, and attenuation of pulmonary vascular remodeling, prevention of right heart failure. Fabp4-5 deletion also normalized EC glycolysis gene programming, reduced HIF-2a expression and endothelial proliferation in Egln1Tie2Cremice.Conclusions:FABP4 and 5 control EC glycolysis and contribute to the development of severe PH.

Introduction:Pulmonary arterial hypertension (PAH) is a disaster 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.Hypothesis:Genetic SOX17 mutations are associated with PAH. We hypothesized that endothelial SOX17 mutations or loss of function contributes to endothelial dysfunction and the pathogenesis of PAH.Methods:Endothelial-specific deletion of Sox17 (Sox17iCKO) mice were generated. Sox17iCKOand Sox17f/f(WT) mice after tamoxifen injection were incubated without or with hypoxia (10% O2) for 3 weeks to induce PH. EC proliferation, apoptosis, cell junction assay, Western blotting, QRT-PCR, RNA-sequencing analysis, and luciferase assays were performed to understand the molecular mechanisms of SOX17 deficiency and mutation in ECs. siRNA and inhibitors were used to determine the involvement of E2F1/BRD4 signaling in SOX17 deficiency ECs.Results:Sox17iCKOmice developed spontaneous mild PH and exaggerated hypoxia-induced PH evidenced by the increase of RVSP and RV hypertrophy compared to WT mice. SOX17 knockdown in human PVECs induced cell proliferation, anti-apoptosis, and impaired cell integrity. SOX17 knockdown reduced BMPR2 signaling. SOX17 knockdown and mutations at the HMG domain lost transcriptional activities and its ability to repress -catenin. RNA-seq analysis demonstrated enrichment of cell cycle pathway after the loss of SOX17 in PVECs. iRegulon analysis revealed the involvement of transcriptional factor E2F1. siRNA-mediated E2F1 knockdown normalized SOX17 deficiency-induced PVECs dysfunction. Knockdown SOX17 significantly upregulated E2F1 promoter activity, which was dependent on the binding site in the E2F1 promoter by luciferase and mutagenesis assay. E2F1 or BRD4 inhibitors attenuated SOX17 deficiency-induced PH in mice.Conclusions:Our study demonstrated that endothelial loss of SOX17 induces PH in mice. Pharmacological inhibition of E2F1/BRD4 signaling attenuated PH development in Sox17iCKOmice.

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Introduction:Pulmonary arterial hypertension (PAH) is a disaster 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.Hypothesis:We hypothesis that endothelial plasticity or distinct cell populations are critical for obstructive vascular remodeling in the pathogenesis of PAH.Methods:Here we applied single-cell RNA sequencing (ScRNA-seq) to profile the pulmonary cells in a severe mouse model (Tie2Cre-mediated deletion of Egln1[encoding Prolyl-4 Hydroxylase 2 (PHD2)], designated Egln1Tie2Cremice) of PAH.Results:ScRNA-seq revealed 20 discrete cell populations from pooled mouse lung single cells from WT and Egln1Tie2Cremice. We identified five distinct EC subpopulations in both WT and Egln1Tie2Cremice, which expressed classical EC markers Emcn,Pecam1and Cdh5. Unexpectedly, there were markedly difference in second abundant EC Cluster (EC2) between WT and Egln1Tie2Crelung. The number of Cluster (EC2) was markedly increased in CKO lung compared with from WT lung. EC2 cluster (mainly from Egln1Tie2Crelung) was characterized by little expression of Cldn5, Tmem100, Tspan7, Calcrland Foxf1. Analysis of genes related to the pathogenesis of PH showed that angiocrine factor genes Pdgfb, Cxcl12, Mifand Edn1are significantly increased in all EC subpopulations from Egln1Tie2Cremice compared to WT mice. We also analyzed genes which were found to be mutated in human PAH patients and found that some of these genes (Sox17, Atp13a3and Smad4) were ubiquitously upregulated in all EC subpopulations, some of these genes were selectively downregulated or upregulated in specific EC subpopulation(s) [down: Bmpr2, Acvrl1, Aqp1, Ptgis, Cav1; Up: Eif2ak4and Smad1] in Egln1Tie2Cremice compared to that from WT mice.Conclusions:ScRNA-seq analysis identifies unique endothelial population only highly enriched in the lung of severe PAH mice.

Background:Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and pulmonary vascular remodeling that result in right heart hypertrophy and failure. Owing to the poor understanding of the underlying mechanisms of obliterative vascular remodeling, current therapies result in only modest improvement in morbidity and mortality. Bi-allelic mutations of the EIF2AK4 gene (encoding GCN2) are linked to heritable pulmonary veno-occlusive disease and PAH. However, the role of GCN2 in the pathogenesis of PAH is not clear.Methods and Results:Employing mice with genetic loss of GCN2 (GCN2-/-), we found that loss of function in GCN2 did not spontaneously develop PAH or PVOD under normoxia condition. However, GCN2-/-mice were protected from Sugen5416/Hypoxia-induced PAH, as evident by reduced right ventricular systolic pressure (RVSP) and lower weight ratio of right ventricle over left ventricle plus septum RV/(LV+S), indicator of right ventricle hypertrophy. Histological and immunofluorescent staining showed that media layer of pulmonary arteries had less cell proliferation and reduced pulmonary vascular remodeling including wall thickness as well as distal pulmonary arterial muscularization in GCN2-/-mice compared with WT mice. QRT-PCR analysis demonstrated that GCN2 deletion normalized expression of molecules related to cell proliferation in mouse lungs. In cultured human pulmonary arterial smooth muscle cells (hPASMCs), GCN2 deficiency inhibits PDGF-BB-induced cell proliferation. To evaluate the translational potential of targeting GCN2 in PAH in vivo, rats were exposed to monocrotaline to induce pulmonary hypertension and then were treated with GCN2 inhibitor A-92. RVSP and RV/(LV+S) ratio were measured upon completion of treatment and histological assessments were performed subsequently, showing attenuated PAH phenotype as well as pulmonary vascular remodeling with pharmacological inhibition of GCN2.Conclusions:These studies demonstrate that GCN2 orchestrates hypoxia and growth factor signaling to mediate pulmonary vascular remodeling. Thus, targeting GCN2 is a promising therapeutic strategy for effective treatment of PAH and thereby promoting survival.