Estrogen receptor-alpha effects on right ventricular vascular density and angiogenesis in pulmonary hypertension

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 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. Endothelial cell (EC) 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 17β-estradiol (E2) increases capillary density in the RV and stimulates angiogenesis in cultured cardiac ECs. The goal of this proposal is to identify novel and therapeutically targetable mechanisms by which E2 exerts protective effects on RV EC function in PH. We provide evidence that E2 exerts its RV EC-protective effects via its receptor ERα, and suggest a new mechanism by which ERα activates bone morphogenetic protein receptor 2 (BMPR2) signaling to upregulate apelin, a potent angiogenesis mediator and EC survival factor, whose regulation in the RV is not yet known. Based on these findings, we now put forward the novel hypothesis that E2 improves RV function in PH by ERα- and BMPR2-dependent up-regulation of EC apelin. We propose the following specific aims: 1) To establish that E2 increases capillary density in the RV via BMPR2-dependent increases in EC apelin, and 2) To identify the contribution of ERα to increasing capillary density in the RV. We generated a novel ERα knockout rat that will enable us to study the role of ERα in the rat pulmonary artery banding model, thus avoiding the pitfalls of prior studies of sex hormone signaling performed in PH models without RV failure. These studies will be complemented by studies of BMPR2-deficient rats and apelin-deficient mice. These loss- of-function studies will be accompanied by studies in which we interrogate the therapeutic potential of ERα agonists, BMPR2 activators and apelin receptor agonists. We will complement these in vivo studies with experiments in RV ECs 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), BMPR2 and apelin signaling pathways, as well as mediators of angiogenesis and EC survival and apoptosis. The proposed studies are significant, since they will 1) identify ERα as a critical modulator of RV function and 2) establish a novel and therapeutically targetable E2-ERα- BMPR2-apelin axis in RV ECs. The proposed studies are innovative, since they, for the first time, will provide a molecular basis for E2's RV- and EC-protective effects in PH. In addition, they provide technical innovation through use of a newly generated ERα knockout rat model, through use of a new highly selective ERα agonist that will allow for mechanistic dissection of ERα’s role in ECs from failing RVs, and through use of unbiased stereology. Upon completion of the proposed studies, we will have identified ERα as a novel mediator of adaptive signaling in RV ECs. 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.

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 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. Endothelial cell (EC) 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 17β-estradiol (E2) increases capillary density in the RV and stimulates angiogenesis in cultured cardiac ECs. The goal of this proposal is to identify novel and therapeutically targetable mechanisms by which E2 exerts protective effects on RV EC function in PH. We provide evidence that E2 exerts its RV EC-protective effects via its receptor ERα, and suggest a new mechanism by which ERα activates bone morphogenetic protein receptor 2 (BMPR2) signaling to upregulate apelin, a potent angiogenesis mediator and EC survival factor, whose regulation in the RV is not yet known. Based on these findings, we now put forward the novel hypothesis that E2 improves RV function in PH by ERα- and BMPR2-dependent up-regulation of EC apelin. We propose the following specific aims: 1) To establish that E2 increases capillary density in the RV via BMPR2-dependent increases in EC apelin, and 2) To identify the contribution of ERα to increasing capillary density in the RV. We generated a novel ERα knockout rat that will enable us to study the role of ERα in the rat pulmonary artery banding model, thus avoiding the pitfalls of prior studies of sex hormone signaling performed in PH models without RV failure. These studies will be complemented by studies of BMPR2-deficient rats and apelin-deficient mice. These loss- of-function studies will be accompanied by studies in which we interrogate the therapeutic potential of ERα agonists, BMPR2 activators and apelin receptor agonists. We will complement these in vivo studies with experiments in RV ECs 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), BMPR2 and apelin signaling pathways, as well as mediators of angiogenesis and EC survival and apoptosis. The proposed studies are significant, since they will 1) identify ERα as a critical modulator of RV function and 2) establish a novel and therapeutically targetable E2-ERα- BMPR2-apelin axis in RV ECs. The proposed studies are innovative, since they, for the first time, will provide a molecular basis for E2's RV- and EC-protective effects in PH. In addition, they provide technical innovation through use of a newly generated ERα knockout rat model, through use of a new highly selective ERα agonist that will allow for mechanistic dissection of ERα’s role in ECs from failing RVs, and through use of unbiased stereology. Upon completion of the proposed studies, we will have identified ERα as a novel mediator of adaptive signaling in RV ECs. 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.