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.

Increased pulmonary vascular pressures due to vascular remodeling, elevated vascular resistance, and vasoconstriction characterize Pulmonary Arterial Hypertension (PAH). The narrowing of the pulmonary arteries and obstruction of blood flow increase the Right Ventricular (RV) afterload, forcing the RV to undergo structural and functional changes. While adaptive remodeling leads to RV compensation by maintaining function, maladaptive remodeling leads to RV decompensation, characterized by worsening function and eventual failure. At present, there is no effective treatment for these patients as therapies for left ventricular failure are ineffectual, and there are no therapies specifically targeting the RV. Therefore, there is a clear need to understand the pathophysiology of RV failure and to identify the differences between adaptive and maladaptive RV remodeling. This study analyzes changes in polyadenylation site usage, a process known as alternative polyadenylation (APA), in RV failure. APA is a mechanism used to regulate mRNA maturation that can result in either shortening or elongation of the mRNA 3’UTR. By analyzing APA patterns in RV tissue from donor controls and patients with compensated and decompensated RV failure, we demonstrate a pattern of 3’UTR elongation that is present in decompensated RV failure and not in compensated or control RVs. Further, altered APA was also detected in 3 distinct rat models of PH, where 15 transcripts had shared APA alterations across both rat models and human disease. Our study provides an unbiased approach to identifying the molecular changes leading to RV dysfunction while pinpointing novel therapeutic targets that can be leveraged for intervention. These APA signatures may serve as biomarkers to distinguish adaptive from maladaptive RV remodeling. In addition, the RNA-processing machinery that regulates APA, such as NUDT21 and CPSF6, represents potential therapeutic targets for RNA-based interventions. Together, our findings link RNA processing to diagnostic and therapeutic opportunities in right heart failure.

Right ventricular (RV) function and adaptation to afterload increase determine survival in pulmonary hypertension (PH). RV adaptation in PH is sexually dimorphic and more preserved in females, mediated by protective estrogen receptor α (ERα) signaling in cardiomyocytes. However, the effects of ERα on RV endothelial cells (RVECs), a critical mediator of RV homeostasis and adaptation, are unknown. We hypothesized that ERα exerts sexually dimorphic pro-angiogenic effects on RVECs in vitro and promotes RV vascularization in vivo. Compared to cells isolated from wild-type animals, RVECs from male and female rats with an ERα loss-of-function mutation (ERαMut) showed reduced ability to form pseudo-vascular networks and migrate. RVECs from female ERαMut rats demonstrated increased apoptosis. In a PH model induced by monocrotaline (MCT), female ERαMut rats exhibited increased RV hypertrophy and reduced RV capillary density before (10 days) and at the time of established PH (28 days). Capillary rarefaction was associated with increased RVEC apoptosis, and, as identified by single-nucleus RNA-sequencing, by a net loss of the endocardial RVEC sub-population. Differentially expressed gene analysis and pathway analysis identified that capillary and endocardial RVECs from female MCT-PH ERαMut rats demonstrated decreased expression of migration pathways and increased expression of apoptosis pathways. These findings reveal a sex-specific endothelial-intrinsic role of ERα that is essential for angiogenesis in the RV under both homeostatic and pathological conditions. This effect appears to stem from the enhanced survival and migration capacity of capillary and endocardial RVEC. Collectively, our results identify ERα as a potential target for developing sex-specific RV-directed therapies in PH.

Spatial transcriptomics technologies have emerged as powerful tools for understanding cellular identity and function within the natural spatial context of tissues. Traditional transcriptomics techniques, such as bulk and single-cell RNA sequencing, lose this spatial information, which is critical for addressing many biological questions. Here, we present a protocol for high-resolution spatial transcriptomics using fixed frozen mouse lung sections mounted on 10X Genomics Xenium slides. This method integrates multiplexed fluorescent in situ hybridization (FISH) with high-throughput imaging to reveal the spatial distribution of mRNA molecules in lung tissue sections, allowing detailed analysis of gene expression changes in a mouse model of pulmonary hypertension (PH). We compared two tissue preparation methods, fixed frozen and fresh frozen, for compatibility with the Xenium platform. Our fixed frozen approach, utilizing a free-floating technique to mount thin lung sections onto Xenium slides at room temperature, preserved tissue integrity and maximized the imaging area, resulting in high-fidelity spatial transcriptomics data. Using a predesigned 379-gene mouse panel, we identified 40 major lung cell types. We detected key cellular changes in PH, including an increase in arterial endothelial cells (AECs) and fibroblasts, alongside a reduction in capillary endothelial cells (CAP1 and CAP2). Through differential gene expression analysis, we observed markers of endothelial-to-mesenchymal transition and fibroblast activation in PH lungs. High-resolution spatial mapping further confirmed increased arterialization in the distal microvasculature. These findings underscore the utility of spatial transcriptomics in preserving the native tissue architecture and enhancing our understanding of cellular heterogeneity in disease. Our protocol provides a reliable method for integrating spatial and transcriptomic data using fixed frozen lung tissues, offering significant potential for future studies in complex diseases such as PH.

Rationale: Lack of coordinated angiogenesis as well as reductions in fatty acid oxidation (FAO) and mitochondrial metabolism in the right ventricle (RV) promote RV failure development in PAH. 17β-estradiol (E2), the predominant female sex steroid, improves RV function in PAH, but the mechanisms are incompletely understood. Hypothesis: E2 exerts pro-angiogenic effects in the RV by promoting FAO in RV endothelial cells (RVECs). Methods: Published human RV RNA-sequencing data were interrogated for angiogenesis and FAO regulators. Ovariectomized female sugen/hypoxia (SuHx)-PH rats were treated with E2 (75 mcg/kg/d). RV capillary density was quantified via the capillary/cardiomyocyte ratio, accompanied by RNA-sequencing with a focus on angiogenesis and FAO regulator transcripts. In vitro, we assessed effects of E2 (0.1-100nM; 24h) on processes that regulate angiogenesis including migration (scratch assay), proliferation (CCK8 assay), tube formation (matrigel assay), and angiogenic marker expression (Western-blot). We measured endothelial metabolites, focusing on FAO (mass spectrometry, Western-blot, lipid droplets, FAO activity) and mitochondrial function (MitoSox, mitotracker, Western-blot). P<0.05 was considered statistically significant. Results: Angiogenesis and FAO regulator transcripts were reduced in human RV failure. In SuHx-RVs, E2 prevented capillary rarefaction and increased transcripts of pro-angiogenic and FAO mediators. In RVECs from PAH patients (PAH-RVECs), E2, via estrogen receptor-α (ERα), stimulated tube formation. E2, via ERα, increased PAH-RVEC abundance of carnitine palmitoyltransferase 1 (CPT1), a master FAO regulator. In PAH-RVECs, E2 prevented PAH-induced decreases in FAO metabolites, reduced intracellular lipid accumulation, and increased FAO activity. Stimulatory effects of E2 on PAH-RVEC tube formation and angiogenic mediator expression were CPT1-dependent. E2 also reduced mitochondrial ROS production and expanded the mitochondrial network in vitro. Conclusion: E2 stimulates angiogenesis and increases FAO in vivo and in vitro. E2 increases angiogenesis in PAH-RVECs in an ERα- and CPT1-dependent manner. Harnessing the RVEC E2-ERα-CPT1 axis may be an innovative approach to improve RV function in PAH.

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.

Right ventricular (RV) failure is the major cause of mortality in pulmonary hypertension (PH). Adaptive angiogenesis and RV endothelial cell (RVEC) function are major modifiers of RV adaptation in PH, but the underlying mechanisms and their regulators remain incompletely understood. RV adaptation in PH is sexually dimorphic, and 17β-estradiol (E2) exerts protective effects on RV cardiomyocytes. Whether E2 modifies angiogenesis and RVEC function in RV failure remains unknown. We hypothesized that E2 and estrogen receptor α (ERα) promote RV angiogenesis and RVEC homeostasis in PH and aimed to identify underlying mechanisms. We assessed E2 angiogenic effects using cultured human cardiac microvascular endothelial cells (hCMVECs), RVECs from PH patients with RV failure, and RVECs from sugen/hypoxia (SuHx) and monocrotaline (MCT) rat models. In vivo, we evaluated RV capillary density in PH rats treated with E2 or ERα-selective agonist. Apelin signaling was evaluated via apelin receptor blockade. E2 enhanced angiogenesis in male hCMVECs and RV capillary density in female SuHx- PH rats. E2 reversed angiogenic alterations in RVECs from SuHx-PH rats via apelin receptor signaling. In RVECs from PH patients with RV failure, E2 stimulated vascular network formation. In rat and human PH-RVECs, ERα was necessary and sufficient to mediate E2-induced angiogenesis. Activation of ERα with ERα-specific agonist restored RV capillary density in vivo. ERα-mediated angiogenesis required apelin signaling. These data indicate that E2 promotes RV angiogenesis via ERα and apelin signaling and identify a novel ERα-apelin axis in RVECs as a potential therapeutic target to restore RV vascular integrity in PH.

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, r 2 =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.

Introduction: Estrogen receptor α (ERα) promotes cardioprotective signaling in right ventricle cardiomyocytes (RVCMs). NLRP3 inflammasome activation in macrophages contributes to RV failure (RVF) development in pulmonary hypertension (PH). However, NLRP3 signaling in RVCMs has not been studied. We hypothesized that NLRP3 inflammasome activation mediates RVCM contractile dysfunction and is attenuated by 17β-estradiol (E2) via ERα. Methods: RVs from male or female PH patients with RVF were assessed for NLRP3 activation by analyzing RNA-seq and proteomics data, plus staining for NLRP3 and its binding partner ASC. Male or female human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were treated with endothelin-1 (ET1) ± E2. RVCMs were isolated from male or female monocrotaline (MCT) or pulmonary artery banding (PAB) rats. RVCMs were also isolated from healthy rats and treated with ET1 ± E2 in vitro. Male or female ERα loss-of-function mutant (ERα mut )] rats were employed to study effects of ERα. NLRP3 activation in RVCMs and iPSC-CMs was assessed by NLRP3-ASC colocalization and activation of downstream targets. RVCM contractility and cytosolic calcium (c-Ca 2+ ) were evaluated via IONOPTIX system. P<0.05 was considered significant. Results: RNA-seq, proteomics, and immunofluorescence studies revealed that upregulation of NLRP3 activity and signaling in human RVF are more pronounced in males (p<0.05 for all comparisons). In male patients, NLRP3 activation was inversely correlated with cardiac index (p<0.05). In pilot studies, NLRP3 was more activated in male vs female ET1-treated iPSC-CMs. RVCMs from male, but not female, MCT- or PAB-rats demonstrated increased NLRP3-ASC colocalization (p<0.05). RVCMs treated with ET1 exhibited more NLRP3 activation and contractile dysfunction than female RVCMs (p<0.05). E2 treatment in male WT RVCMs reduced NLRP3-ASC co-localization and increased Ca 2+ dependent contractility (p<0.05). E2 treatment in male iPSC-CMs trended to prevent ET1-induced NLRP3 activation. E2’s inhibitory effects on NLRP3 activation, NLRP3-induced contractile dysfunction, and c-Ca 2+ in male WT RVCMs were abrogated in ERα mut RVCMs (p<0.05). Conclusion: NLRP3 activation in human and rat RVF as well as NLRP3-induced contractile dysfunction in human iPSC-CMs and rat RVCMs exhibit a male bias. E2, via ERα, prevents NLRP3 activation and NLRP3-induced contractile dysfunction. Inhibiting NLRP3 via E2-ERα may be a novel treatment strategy for RVF.

Supplementary Figure S8 - PDF file 1084K, Figure S8: Effects of SSTC-104 on Wnt-regulated tissues

The pulmonary circulation is, under physiologic conditions, a low-pressure, low-resistance system. Pulmonary hypertension (PH) is diagnosed when the mean pulmonary arterial pressure rises above 20mmHg (1). PH is an etiologically heterogeneous condition affecting the pulmonary circulation. PH can be divided into five major groups on the basis of etiology and clinical characteristics: 1) pulmonary arterial hypertension (PAH); 2) PH because of left heart disease; 3) PH because of chronic lung diseases and/or hypoxia; 4) PH because of pulmonary obstructions; and 5) PH with unclear and/or multifactorial origin, including hematologic and systemic disorders (1). Despite differences in etiology, all PH groups exhibit some degree of vasoconstriction and vascular remodeling that contributes to the elevated mean pulmonary arterial pressure. This includes stiffening of the proximal arteries, thickening of the muscularized arteries, andmuscularization of the precapillary arterioles. These structural changes are thought to be driven by endothelial and smooth muscle cell dysfunction leading to abnormal proliferation and recruitment of immune cells and fibroblasts to the vasculature, although the initiation and/or sequence of these events is still under investigation (2). A hallmark of PAH is the occurrence of progressive and severe vaso-occlusive lesions thought to arise from a population of vascular wall cells that become abnormally proliferative and migratory and are also resistant to apoptosis (3). Evidence suggests the highly proliferative andmigratory cells found in occlusive lesions are endothelial or myofibroblasts of endothelial origin, as these cells express endothelial markers (4). In all forms of PH, pulmonary vascular remodeling leads to an increase in pulmonary vascular resistance, which in turn increases right ventricular afterload. Initially, this increase in afterload causes adaptive right ventricular hypertrophy, but this often progresses to maladaptive right ventricular hypertrophy, dilation, and fibrosis, eventually leading to right ventricular failure. Despite decades of research andmultiple therapies approved for clinical use, PAH remains an incurable disease, with a 5-year survival rate of 60% (5). Furthermore, despite being more common than PAH, other groups of PH have limited available therapeutic options to directly treat the disease. There are no approved therapeutics affecting pulmonary vascular remodeling. Although finding a targetable pathway to reverse or prevent pulmonary vascular remodeling has long been a goal, most available PH therapies target pathways regulating vascular tone, stimulating vasodilatory signaling. It is with this goal in mind that Huang and colleagues put forward the RNA-binding protein Quaking as a candidate for reversing pulmonary vascular remodeling in this issue of the Journal (pp. 159–171) (6). RNA binding proteins (RBPs) are critical regulators of pre-mRNA posttranscriptional processing, splicing, localization, and stability. Quaking (QKI) is a member of the STAR (signal transduction and activation of RNA) family. There are three isoforms of QKI: QKI-5, QKI-6, and QKI-7. Each isoform has been reported to regulate pre-mRNA splicing, transportation, and stability distinctly and in a cell-specific manner. Although QKI plays a role in neurological development and has been implicated in neurological disorders, recent work has further demonstrated a role for QKI in other biological systems, including cardiovascular development, immune cell differentiation, bone metabolism, cancer progression (7), and more recently, in the regulation of vascular smooth muscle cell phenotypic switch to a fibroproliferative response after vascular injury (8). Huang and colleagues describe for the first time a role for QKI in the pulmonary vasculature and PH pathogenesis (6). They found total QKI, primarily isoforms QKI5 and QKI6, were increased in the lungs of patients with PH and rodent models of experimental PH. Overexpressing QKI in control cells increased proliferation in PASMCs, corroborating the findings in patients, while overexpressing it in vivo exacerbated experimental PH. They also identified one of the target mRNAs of QKI in pulmonary vascular smooth muscle cells (PASMCs), signal transducer and activator of transcription 3 (STAT3), showing QKI was able to directly bind to STAT3mRNA and regulate its expression. The upregulation of QKI appeared to be sensitive to hypoxia, increasing in cells after exposure to hypoxia or hypoxia inducible factor (HIF)-stabilizing agents. In addition, they describe a mechanism in which STAT3 regulates the expression of microRNAmi146b, STAT1, and Tet methylcytosine dioxygenase 2 (TET2) to induce proliferation in human PASMCs. It remains unknown whether inhibiting QKI in PH patient PASMCs would affect proliferation and STAT3 signaling or if targeting QKI in established PHwould reverse pulmonary vascular remodeling in vivo. It is clear, however, that QKI is a regulator of STAT3 signaling in PASMCs, which may be of therapeutic interest. Given that numerous pathways have been identified as key regulators of pulmonary vascular remodeling, shown promising therapeutic effects in preclinical PHmodels, and yet have not translated to or yielded clinical results underscores the need to deepen our knowledge of drivers of pulmonary vascular remodeling. The current study by Huang and colleagues does this by describing a novel role for RBPs in PH pathogenesis, but it also raises multiple questions: How far up do we go? Is therapeutically targeting a broader regulatory network like RBPs effective and safe, or will it have too many offtarget effects? Is it possible to target injured cells involved in pulmonary

Introduction: NLRP3 inflammasome activation promotes right ventricle (RV) contractile dysfunction in pulmonary hypertension (PH). However, the role of NLRP3 signaling in RVCMs has not been studied. 17β-estradiol (E2) improves RVCM function in PH. Hypothesis: E2, via estrogen receptor-α (ERα), prevents RVCM NLRP3 inflammasome activation and contractile dysfunction in experimental PH. Methods: RV failure in male and female Sprague Dawley rats were induced with monocrotaline (MCT). Co-localization of NLRP3 and its partner ASC was assessed. RVCMs isolated from healthy male or female wildtype (WT) or ERα loss of function mutant (ERα mut ) rats were treated with NLRP3 activators lipopolysaccharide (LPS 1 μg/mL, 4 h) and ATP (2 mM, 10 min) ± E2 (1 nM, 24 h) or NLRP3 inhibitor MCC950 (1 μM, 30 min). RVCM contractility and Ca 2+ levels were evaluated via IONOPTIX system. Protein levels of NLRP3 and its targets were assessed. ERα binding to the NLRP3 promoter was assessed by immunoprecipitation. RNA-seq was performed in RV tissues from male and intact or ovariectomized female rats with RV failure. Results: RNA-seq demonstrated that transcripts involved in NLRP3 activation are E2-regulated. RVCMs from male, but not female, MCT rats showed increased NLRP3 and ASC co-localization/activation (p<0.05). In addition, male MCT rats RVs exhibited increased activation of NLRP3 downstream mediators caspase-1 and IL-1β (p<0.05). Cultured male RVCMs treated with LPS+ATP demonstrated more pronounced NLRP3 activation and decreased contractility and Ca 2+ levels than female RVCMs (p<0.05). NLRP3 activation in RVCMs from male rats resulted in reduced contractility; this was abolished with NLRP3 inhibition (p<0.05). E2 treatment in male RVCMs reduced NLRP3 and ASC interaction and increased contractility Ca 2+ levels in male WT but not in ERα mut RVCMs (p<0.05). IP demonstrated ERα binding to the NLRP3 promoter. Conclusion: NLRP3 signaling in RVCMs from MCT-PH rats is sexually dimorphic, with more pronounced activation and contractile dysfunction in males. E2 prevents RVCM NLRP3 activation and contractile dysfunction in an ERα-dependent manner, likely mediated by ERα binding to the NLRP3 promoter. NLRP3 activation may be therapeutically targetable in PH patients of either sex.

Supplementary Figure S10 4882K, Figure S10a and S10b: Nuclear beta-catenin accumulation in human synovial sarcoma tumors

Supplementary Methods and Legends - PDF file 130K, Methods:Additional information on experimental techniques Legends: describing supplementary figures

Supplementary Figure S9 - PDF file 867K,Figure S9: The SYT-SSX2 domain SSXRD executes -catenin activation and myogenic differentiation

Supplementary Figures S2 and S3 - PDF file 2332K, Figure S2:SG3 tumors are positive for beta-catenin and Myf5 Figure S3:Normal development of Myf5 myoblasts in beta-catenin knockout mice