Pulmonary arterial hypertension (PAH) is a complex and devastating disease with a poor long-term prognosis. While women are at increased risk for developing PAH, they exhibit superior right heart function and higher survival rates than men. Susceptibility to disease risk in PAH has been attributed, in part, to estrogen signaling. In contrast to potential pathological influences of estrogen in patients, studies of animal models reveal estrogen demonstrates protective effects in PAH. Consistent with this latter observation, an ovariectomy in female rats appears to aggravate the condition. This discrepancy between observations from patients and animal models is often called the “estrogen paradox.” Further, the tissue-specific interactions between estrogen, its metabolites and receptors in PAH and right heart function remain complex; nonetheless, these relationships are essential to characterize to better understand PAH pathophysiology and to potentially develop novel therapeutic and curative targets. In this review, we explore estrogen-mediated mechanisms that may further explain this paradox by summarizing published literature related to: (1) the synthesis and catabolism of estrogen; (2) activity and functions of the various estrogen receptors; (3) the multiple modalities of estrogen signaling in cells; and (4) the role of estrogen and its diverse metabolites on the susceptibility to, and progression of, PAH as well as their impact on right heart function.

Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension (PAH) severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of PAH cases versus controls, highlighting potentially novel candidate genes involved in PAH development.

Rationale Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2- Sox17 ( Sox17 EC−/− ) deletion and attenuated by transgenic Tie2- Sox17 overexpression ( Sox17 Tg ). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17 EC−/− and reduced in those from Sox17 Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)–mediated repression of SOX17 promoter activity, Sox17 Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations ( n = 1,326). Conclusions Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

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Introduction: Sexual dimorphism in pulmonary arterial hypertension (PAH) is attributed, in part, to estrogen signaling. 16α-hydroxyestrone (16αOHE) is considered a major contributor to PAH pathogenesis. Recent genetic studies have also suggested that deficiency of SOX17, an endothelial cell (EC)-specific transcription factor, contributes to PAH risk. While functional studies of SOX17 are absent, we hypothesized that 16αOHE contributes to PAH, in part, via, SOX17 downregulation. Methods/Results: Sox17 expression was reduced in 3 animal PAH models and in human pulmonary artery ECs (HPAECs) isolated from patients with PAH (vs controls). Inducible Tie2-specific Sox17 knockout ( Sox17 EC-/- ) mice exhibited increased right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and PA wall thickness (PAWT) after chronic hypoxia (CH) (Fig 1A). Inducible Tie2- Sox17 transgenic overexpressing ( Sox17 Tg ) mice attenuated CH-induced PH (Fig 1B). While not evident across murine sex, Sox17 expression was increased in baseline lungs from male compared to female rats. Supporting in silico evidence of estrogen response elements (ERE) on the SOX17 promoter, HPAECs exposed to 16αOHE reduced SOX17 expression and promoter luciferase activity via ERα, which was partly negated by serial ERE mutagenesis (Fig 1C ) . Lungs from ERα loss-of-function mutant rats (vs control) confirmed in vivo reductions of Sox17 expression. Sox17 Tg mice attenuated 16αOHE-mediated PH after CH (Fig 1D). To translate these data, we identified a functional coding SNP in the ESR1 gene (encoding ERα), rs746432, previously shown to reduce transcriptional activity of ERα. The SNP was associated with reduced pulmonary vascular resistance in patients with PAH (n=702, Fig 1E) in adjusted analyses. Conclusion: Validating genetic studies, SOX17 deficiency augments preclinical PAH. 16αOHE mediates PH development via downregulation of SOX17, linking SOX17 genetics with the observed sexual dimorphism.

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