Abstract Body (Do not enter title and authors here): Background: Right ventricular failure (RVF) determines survival in patients with pulmonary hypertension (PH). Insufficient angiogenesis and impaired RV endothelial cell (RVEC) function are key contributors of RVF development. Female patients with PH have better RV function compared to males. 17β-estradiol (E2), the most abundant female sex hormone, exerts RV-protective effects, including stimulation of angiogenesis. However, the mechanisms underlying E2’s pro-angiogenic function are unknown.

Hypothesis: Estrogen receptor (ER)α promotes RVEC angiogenic function in vitro and RV angiogenesis in vivo.

Objective: To identify the role of ERα in RVEC angiogenic function and RV angiogenesis.

Methods: ERα loss-of-function mutant (ERα^Mut) rats were generated using CRISPR-Cas9. RVF was induced in rats with monocrotaline (MCT; 60 mg/kg) or pulmonary artery banding (PAB; 11 weeks). RV systolic pressure (RVSP) was measured, and Fulton index and cardiomyocyte area were used as measures of RV hypertrophy. RV capillary density was assessed by Isolectin B4 staining. RVECs were isolated from male and female WT and ERα^Mut rats. Cell migration was assessed using transwell migration assay. Angiogenic gene expression in RVECs was measured using Qiagen RT2 Profiler PCR array. p<0.05 by ANOVA or in Pearson’s correlation analyses was considered significant.

Results: RV capillary loss was observed in male and female WT rats and female ERα^Mut PAB rats, compared to sham (p<0.05). Capillary density correlated negatively with RVSP and Fulton index (p<0.001, r²=0.1386 and 0.4029, respectively). Time course experiments in MCT-rats demonstrated RV hypertrophy and capillary rarefaction in female ERα^Mut rats (but not in WT) 10 days after MCT injection (p<0.05). Microarray indicated reduced expression of pro-angiogenic angiogenin and leptin and increased expression of anti-angiogenic TIMP-1 in female ERα^Mut vs WT RVECs. Pathway analysis reaveled a decrease in mediators of cell migration and blood vessel development in ERα^Mut vs WT RVECs. Transwell migration assay revealed reduced migration in male and female ERα^Mut vs WTs RVECs at 24h (p<0.05).

Conclusions: ERα regulates angiogenic processes in vivo and in RVECs in a sexually dimorphic manner. Functioning ERα protects females against capillary rarefaction during early stages of RV remodeling. Targeting ERα could pave the way for sex-specific treatment strategies aimed at bolstering RV adaptation in PH.