Andrea L. Frump

BACKGROUND: Right ventricular (RV) adaptation to increased afterload determines survival in pulmonary hypertension (PH) and is sexually dimorphic and more preserved in females, mediated in part by protective ERα (estrogen receptor α) signaling in cardiomyocytes. However, effects of ERα on RV endothelial cells (RVECs), a critical mediator of RV adaptation, are unknown. We hypothesized that ERα exerts sexually dimorphic proangiogenic effects on RVECs in vitro and in vivo. METHODS: We isolated RVECs from male and female rats with an ERα ^Mut (ERα loss-of-function mutation) or wild-type controls and assessed angiogenesis end points. Male and female ERα ^Mut or wild-type rats were treated with monocrotaline or underwent pulmonary arterial banding. Single-nucleus RNA sequencing was performed to investigate RVEC transcriptomic changes. RESULTS: Compared with cells isolated from wild-type animals, RVECs from male and female ERα ^Mut rats exhibited reduced ability to form pseudovascular networks, sprout, and migrate. RVECs from female ERα ^Mut rats demonstrated increased apoptosis. Restoring migration rescued the impaired angiogenic phenotype in female ERα ^Mut RVECs. Female monocrotaline-PH ERα ^Mut rats exhibited increased RV hypertrophy and reduced RV capillary density before (10 days) and at the time of established PH (28 days). Female ERα ^Mut pulmonary arterial banding rats also exhibited decreased RV capillary density. Capillary rarefaction was associated with decreased tip cell abundance and increased RVEC apoptosis, and a net loss of the endocardial RVEC subpopulation. Differentially expressed gene analysis and pathway analysis identified decreased expression of migration pathways and increased expression of apoptosis pathways in capillary and endocardial RVECs from female but not male monocrotaline-PH ERα ^Mut rats. CONCLUSIONS: ERα exhibits a sex-specific endothelial-intrinsic role that is essential for RV angiogenesis under both homeostatic and pathological conditions. This effect appears to stem from enhanced survival and migration capacity of capillary and endocardial RVECs. ERα may be a potential target for sex-specific RV-directed therapies in PH.

BACKGROUND: Although pulmonary arterial hypertension (PAH) is a rare and fatal disease that is well-characterized, vasodilator-responsive PAH accounts for a minority of cases, with little mechanistic knowledge, but with dramatically improved survival. METHODS: By assembling national cohorts, we evaluated genetic influences on acute vasodilator drug response, a key determinant of the presence of vasodilator-responsive PAH. Differences between hemodynamics at rest and after a PAH-specific vasodilator were tested in a genome-wide association study. Validated loci were functionally tested in cell culture and in a hypoxic mouse model of pulmonary hypertension. RESULTS: Rs8057488 in the sorting nexin 29 ( SNX29 ) gene reached genome-wide significance in the discovery cohort ( P =4.00×10 ⁻⁸ ) and was nominally replicated ( P =0.027). Consistent with its predicted function, SNX29 demonstrated an endosomal distribution in PA smooth muscle cells. Silencing SNX29 redistributed stromal interaction molecule proteins to the cell membrane and enhanced store-operated calcium entry. Over-expression of SNX29, in vivo, attenuated hypoxic vasoconstriction in isolated perfused murine lung models. CONCLUSIONS: The data cumulatively suggest SNX29 may contribute to vasodilation partly through reduced store-operated calcium entry and endosomal trafficking of store-operated calcium entry proteins, advancing our understanding of vasodilator-responsive PAH.

Rationale: RV adaptation in pulmonary hypertension is sexually dimorphic and more preserved in women. NLRP3 inflammasome activation contributes to RV failure (RVF) development. However, regulators and downstream effects of NLRP3 activation in the RV remain unknown. Objectives: We investigated whether NLRP3 inflammasome activation in RVF is sexually dimorphic, whether NLRP3 is active in RV cardiomyocytes (RVCMs) and causes RVCM contractile dysfunction, and whether 17β-estradiol (E2) and its receptor ERα attenuate this process. Methods: We studied RV tissues from PAH patients with RVF, RV tissues and RVCMs isolated from wild-type and ERα loss-of-function mutant rats with RVF, isolated perfused rat hearts, and human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. NLRP3 activation was assessed via RNA-sequencing, proteomics, immunostaining, and downstream target quantification. RV contractility was assessed via pressure-volume loops, perfused heart studies, and contractility and calcium assessments in isolated RVCMs. Measurements and Main Results: NLRP3 was upregulated in RVCMs during RVF and resulted in altered RVCM calcium handling and RVCM contractile dysfunction. In human RVs, hiPSC-cardiomyocytes and rat RVs, NLRP3 activation and NLRP3-induced RVCM contractile dysfunction were sexually dimorphic and male-biased. Ovariectomy and loss of ERα in females eliminated this sex bias. E2, via ERα, prevented RVCM NLRP3 activation and NLRP3-induced RVCM contractile dysfunction in males and ovariectomized females during both acute and chronic RV pressure overload. ERα directly interacted with NLRP3. Conclusions: NLRP3-driven RVCM contractile dysfunction is male-biased. E2 inhibits NLRP3 through ERα to preserve RVCM contractility. Targeting E2-ERα-NLRP3 signaling may offer novel therapeutic strategies for RVF in low estrogen states. Impact: This is the first study to define a novel estradiol-estrogen receptor α-NLRP3 axis that modulates RV cardiomyocyte function and RV adaptation in pulmonary hypertension. We demonstrate for the first time that NLRP3 activation is therapeutically targetable in low estrogen states via NLRP3 inhibitors or 17β-estradiol. These findings have direct implications for therapeutic strategies aimed at preserving or restoring RV contractile function in pulmonary hypertension, a current area of unmet clinical need.

BACKGROUND: Early apoptosis of pulmonary artery endothelial cells (PAECs) is a driver of vascular remodeling and pulmonary hypertension (PH), but its regulation is poorly defined. Adenosine deaminase acting on RNA 1 (ADAR1, gene name ADAR ) is an RNA editing enzyme that converts adenosine to inosine (A-to-I) in RNA transcripts and participates in RNA metabolism. While deficiency in ADAR1-mediated RNA editing stimulates cellular innate immunity signaling and can promote apoptosis, the exact ADAR1 RNA editing targets and downstream mechanisms regulating PAEC survival are unknown. We sought to define the functions and targets of ADAR1-dependent RNA editing that control pulmonary endothelial pathophenotypes in PH. METHODS: ADAR1 or Nocturnin (NOCT) expression and A-to-I RNA editing levels were evaluated in human PAH lungs by immunofluorescent staining and single cell RNA sequencing, respectively. Mice carrying a human missense ADAR mutation and genetic deletion of Noct with interleukin-6 (il6) transgene were studied in chronic hypoxia-induced PH in vivo models. RESULTS: ADAR1 expression was downregulated in the pulmonary vascular endothelium and in lung tissue of human and mouse PH. Global A-to-I RNA editing was decreased in lungs from PAH patients and hypoxic PH mice. In vitro, hypoxia, a PH trigger, downregulated ADAR1 in PAECs. Circadian gene NOCT was identified as a direct ADAR1 target which carries two active A-to-I RNA editing sites in the 3’UTR. In human PAH lungs, NOCT editing levels were reduced, while NOCT protein level increased. Correspondingly, in vitro, ADAR silencing increased NOCT mRNA levels, thus inducing dsRNA-MDA5 sensing interferon signaling and PAEC apoptosis. Importantly, silencing of NOCT reversed these changes. Forced NOCT expression phenocopied the effect of ADAR1 knockdown, upregulating interferon signaling molecules and increasing apoptosis. Chronically hypoxic PH mice carrying human ADAR mutation displayed worsened PH. Forced adeno-associated virus (AAV) expression of Adar improved monocrotaline-induced PH in rats. Genetic deletion of Noct mitigated PH in hypoxic il6-expressing transgenic PH mice, emphasizing the crucial role of NOCT in PH pathogenesis. CONCLUSIONS: Hypoxia-induced ADAR1 deficiency upregulates NOCT expression to induce PAEC interferon signaling activation, PAEC apoptosis, and PH. This study provides impetus to target the ADAR1-NOCT axis for more effective diagnostics and therapeutics for PH.

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