Estrogen Receptor-alpha Effects on Right Ventricular Vascular Density and Angiogenesis in Pulmonary Hypertension

Va Eastern Colorado Health Care System, Aurora CO

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Abstract

Pulmonary hypertension (PH) and right ventricular (RV) dysfunction are extremely common in veterans. Up to 80% of veterans with chronic obstructive pulmonary disease, pulmonary fibrosis, sleep disordered breathing or LV dysfunction (either systolic or diastolic) suffer from PH. Better RV function and female sex have been linked to improved survival in PH, and female patients exhibit better RV function than their male counterparts. This proposal builds on two successful funding periods and the scientific premise that even though RV function and female sex are major determinants of survival in PH, no RV-specific or sex steroid-directed therapies exist. RV endothelial cell (RVEC) dysfunction and impaired angiogenesis play a major role in the development of RV failure, and data obtained in the previous funding period demonstrate that the female sex steroid 17-estradiol (E2) increases capillary density in the RV and stimulates angiogenesis in cultured RVECs. The goal of this proposal is to identify novel and therapeutically targetable mechanisms by which E2 exerts protective effects on RVEC function in PH. We provide evidence that in RVECs, E2 increases expression of the enzyme carnitine palmitoyltransferase 1a (CPT1a), a rate-controlling enzyme of fatty acid oxidation (FAO) responsible for importing fatty acids into the mitochondria. We suggest a new mechanism by which E2, via its receptor ERα, enhances FAO in RVECs to increase their angiogenic function, thus leading to increased RV resilience and better adaptation to increased afterload. Based on these findings, we now put forward the novel hypothesis that E2 improves RV function in PH by ERα- dependent up-regulation of RVEC CPT1 and FAO. We propose the following specific aims: 1) To determine whether ERα is necessary for E2 to increase CPT1a expression and activity and to stimulate FAO in RVECs, 2) To identify transcriptional regulators employed by E2 to regulate FAO in RVECs, and 3) To establish that the ERα-BMPR2-apelin axis in RV cardiomyocytes (RVCMs) enhances CPT1a expression, FAO, and angiogenesis in RVECs. In the prior funding period, we generated a novel ERα loss-of-function rat model that we will now employ to study the role of this receptor in modulating FAO in RV failure. We will complement in vivo studies in clinically relevant models of RV failure with mechanistic experiments in RVECs isolated from rodents with RV failure and from patients with compensated (adaptive) or decompensated (maladaptive) RV hypertrophy. Endpoints investigated will include RV function and structure (by pressure volume loops and echocardiography), exercise capacity (measured as VO2 max via treadmill running), RV capillary density (quantified using unbiased stereology and lectin staining), angiogenesis assays (matrigel tube formation and transwell migration), comprehensive metabolic assessments with interrogation of all major steps of FAO and use of high-throughput metabolomics, and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) followed by transcription factor enrichment analysis (TFEA). The proposed studies are significant, since they will 1) identify the E2-ERα axis as a critical modulator of RVEC metabolic function, 2) identify novel transcription factors engaged by E2-ERα, and 3) establish a novel and therapeutically targetable E2-ERα-CPT1a-FAO axis in RVECs. The proposed studies are innovative, since they, for the first time, will identify FAO modulation as a molecular basis for E2’s RV- and RVEC-protective effects in PH, and since they will identify E2- ERα-signaling in RVCMs as a novel modifier of RVEC metabolism and angiogenic function. In addition, they provide technical innovation through use of high-throughput, mass spectrometry-based metabolomics and ATAC-seq with TFEA. Upon completion of the proposed studies, we will have identified E2- ERα signaling and its targets as novel mediators of FAO and adaptive signaling in RVECs. This may ultimately allow for the development of new RV-directed, non-hormonal treatments for both female and male veterans with PH and RV failure.