Sexually dimorphic role of estrogen receptor α in preserving right ventricular endothelial integrity

Sexually dimorphic role of estrogen receptor α in preserving right ventricular endothelial integrity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Sexually dimorphic role of estrogen receptor α in preserving right ventricular endothelial integrity Jiajun Li , Vijaya Karoor , Andrea L. Frump , Hanqiu Zhao , Shunning Liang , Chen-Shan Chen Woodcock , Irina Petrache , Zhiyu Dai , Tim Lahm doi: https://doi.org/10.1101/2025.11.20.689545 Jiajun Li 1 Department of Pharmacology and Molecular Medicine, University of Colorado , Anschutz Medical Campus, Aurora, CO 2 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, National Jewish Health , Denver, CO Find this author on Google Scholar Find this author on PubMed Search for this author on this site Vijaya Karoor 3 Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado , Anschutz Medical Campus, Aurora, CO Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andrea L. Frump 4 Division of Pulmonary, Critical Care, Sleep and Occupational Medicine, Indiana University School of Medicine , Indianapolis, IN Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hanqiu Zhao 5 Division of Pulmonary Critical Care and Medicine, Department of Medicine, Washington University School of Medicine in St. Louis , Saint Louis, MO Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shunning Liang 5 Division of Pulmonary Critical Care and Medicine, Department of Medicine, Washington University School of Medicine in St. Louis , Saint Louis, MO Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chen-Shan Chen Woodcock 2 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, National Jewish Health , Denver, CO Find this author on Google Scholar Find this author on PubMed Search for this author on this site Irina Petrache 2 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, National Jewish Health , Denver, CO 3 Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado , Anschutz Medical Campus, Aurora, CO Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhiyu Dai 5 Division of Pulmonary Critical Care and Medicine, Department of Medicine, Washington University School of Medicine in St. Louis , Saint Louis, MO Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tim Lahm 2 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, National Jewish Health , Denver, CO 3 Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado , Anschutz Medical Campus, Aurora, CO 6 Rocky Mountain Regional VA Medical Center , Aurora, CO Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: lahmt{at}njhealth.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract 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 (ERα Mut ) showed reduced ability to form pseudo-vascular networks and migrate. RVECs from female ERα Mut rats demonstrated increased apoptosis. In a PH model induced by monocrotaline (MCT), female ERα Mut 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 ERα Mut 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. Translational perspective Effects of ERα on vascular function in RV failure induced by PH are poorly understood. We unveiled a novel sexually dimorphic role of ERα in regulating RV vascularization and RVEC function. Single nucleus RNA-Sequencing in female wild-type and ERα loss-of-function rats with PH identified 5 unique RVEC sub-populations under transcriptional control of ERα. Our findings provide insights into previously undescribed pro-angiogenic, pro-migratory and anti-apoptotic roles of ERα in female RVs and RVECs. Promoting RVEC migration or inhibiting RVEC apoptosis to enhance RV angiogenesis may be viable pathways to maintain RV function in PH patients of either sex. These findings offer novel opportunities and potential therapeutic avenues for preventing or treating RV failure. Introduction Pulmonary hypertension (PH) is characterized by adverse vascular remodeling and intravascular pressure elevations in the lung 1 . However, right ventricle (RV) adaptation to the increased afterload is the major determining factor of survival 2 – 5 . Initially, the RV undergoes adaptive RV hypertrophy (RVH) to compensate for the pressure overload 6 , 7 . However, as RV afterload continues to rise, maladaptive RVH develops, characterized by reduced RV ejection fraction as well as RV dilation and fibrosis 8 , 9 . Pulmonary endothelial cell alterations have been one of the focuses in the pathogenesis of multiple types of PH, and pulmonary arterial endothelial cell dysfunction is a major contributor to lung vascular remodeling 10 , 11 . However, the role of endothelial cell function in the RV has been less well studied. While the pathophysiology of RV failure is complex and multi-factorial and involves a myriad of cell types, insufficient angiogenesis 12 – 15 and impaired RV endothelial cell (RVEC) function 16 have been identified as key contributors. Recent research revealed there is increased angiogenesis in the RV at early stages of PH-induced RVH 14 , 17 . This indicates that RVECs and RV capillaries may be able to adapt to RV pressure overload in early stages but fail to do so in later stages of RVH. However, mechanisms of RVEC adaptation and maladaptation to PH-induced RVH remain incompletely understood. Importantly, RV adaptation in response to PH is sexual dimorphic, with female patients demonstrating better RV function 18 and survival 19 . 17β-estradiol (E2), the most abundant female sex steroid, exerts RV-protective effects 16 , 20 – 22 that may include stimulation of angiogenesis 20 , 22 , 23 . Previous studies also suggest that estrogen receptor α (ERα) plays a major role in angiogenesis 23 – 26 . Importantly, ERα expression is reduced in RVECs from patients with PH-induced RV failure 16 . We recently demonstrated that ERα protects RV function in PH through increasing BMPR2 and apelin in RV cardiomyocytes 16 , both of which are known to be proangiogenic factors. However, it remains unknown if ERα regulates RVEC function and RV angiogenesis in the progression from adaptive RVH to RVF. Here, we hypothesize that ERα exerts pro-angiogenic effects in the RV and in RVECs. We report that loss of ERα induces RVEC dysfunction and reduces angiogenesis both in vitro and in vivo. We identified an early onset of RVH and capillary rarefaction as well as increased RVEC apoptosis in female PH rats without functioning ERα. Single-nucleus RNA sequencing revealed newly identified RVEC subtypes in the female failing wild-type RV that exhibit impaired cell survival and migration signaling with the loss of ERα . Our findings provide evidence for a sex-specific role of ERα in RV angiogenesis, with ERα specifically mediating protection against the development of RVH and RV capillary rarefaction. Methods Main methods and techniques are presented here. Please see supplement for additional methods and information. All experiments were performed in accordance with recent recommendations 27 , 28 , including randomization and blinding at the time of measurement and analysis. Monocrotaline (MCT)-PH MCT (10 mg/ml in PBS; C2401, Sigma-Aldrich) was prepared on the day of injection. Male and female Sprague-Dawley rats or in-house bred wild-type (WT) and ERα loss-of-function mutant (ERα Mut ) rats (200–250 g, 6-8 weeks of age) were subcutaneously injected with MCT (60 mg/kg) and maintained for 3, 10 or 28 days, followed by endpoint analysis. Hemodynamic assessment Rats were anaesthetized by inhalation of 5% isoflurane an then orotracheally intubated and mechanically ventilated (100% Fi O2 ) during measurements with supplemental oxygen, after verification of euthermia, normocapnea, and normal pH. RV systolic pressure (RVSP) was measured by right heart cathetherization with 2-F Mikro-Tip® pressure catheter (Millar) via transjugular approach under 2% isoflurane as described previously 16 and recorded with LabChart software (ADInstruments). Once all hemodynamic endpoints were obtained, animals were euthanized by exsanguination under anesthesia via the arterial line, followed by immediate organ harvest. RV hypertrophy assessment Hearts were harvested and the atria were removed. The RV free wall was separated from the left ventricle (LV) and septum. The Fulton index was calculated as: RV weight / (LV + septum weight). Tube formation assay RVECs were serum starved overnight (18h) with low-serum media (EBM phenored-free media with 0.5% charcoal stripped-FBS) prior to experimentation. Flat bottom 96-well plates (Corning) were coated with 40 µl of phenol red-free reduced growth factor Matrigel (Corning) per well, 1h before the experiment at 37°C in a humidified atmosphere containing 5% CO 2 . 10,000 RVECs in low-serum media were plated into each well for 24h. Plates were imaged at 10x magnification using Incucyte® Live-Cell Analysis System at 1h intervals. Images were then exported and uploaded to Wimasis.com for analysis. Transwell migration assay RVECs isolated from WT and ERα Mut rats were fserum-starved for 18h in low-serum media. Cells were then trypsinized and 50,000 RVECs were seeded to the top insert with 8µm pore size (Corning) in low-serum media (EBM phenored-free media with 0.5% charcoal stripped-FBS). Inserts were placed into a 24-well plate containing either low-serum medium (0.5% charcoal stripped-FBS) or low-serum medium supplemented with 10 ng/mL vascular endothelial growth factor A (VEGF-A; PeproTech) in the lower chamber to serve as a chemoattractant. Cells were incubated for 24 h at 37°C in a humidified incubator with 5% CO₂ to allow migration. Following incubation, inserts were washed twice with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. Non-migrated cells remaining on the upper surface of the membrane were gently removed with cotton-tipped applicators. Migrated cells on the lower surface were stained with 0.1% (w/v) crystal violet solution (Ward’s Science) for 20 min, rinsed with distilled water, and air-dried. Membranes were imaged under bright-field microscopy. Migrated cells were quantified in five random fields per insert using ImageJ software (NIH). Each condition was assayed in triplicate and repeated in at least three independent experiments. Wound healing assay 50,000 RVECs were seeded into each well of ImageLock 96-well plate (Sartorius). RVECs were then serum-starved overnight (18h) in low-serum media. A single layer of confluent RVECs was then scratched with Woundmaker Tool (Sartorius) and fresh low-serum media (EBM phenored-free media with 0.5% charcoal stripped-FBS) was added into each well with VEGF-A at a final concentration of 10 ng/ml. The ImageLock 96-well plate was then placed into Incucyte® S3 Live-Cell Analysis System (Sartorius) for imaging. 10x images of each well were taken every 2h. Duplicate samples from 3 different biological replicates were used. Migration was quantified as cells in initial wound area. Growth curve/cell counting assay RVECs were seeded at a density of 2×10⁴ cells per well in 24-well plates containing 1 mL of EGM2-MV medium (Lonza) and allowed to adhere for 24 h at 37°C in a humidified atmosphere with 5% CO₂. After 24h, cells were serum-starved in low-serum medium (EBM phenored-free media with 0.5% charcoal stripped-FBS) for 48h. Following starvation, cells were detached using trypsin–EDTA (Gibco) and counted with a hemocytometer (Hausser Scientific); this time point was designated as day 0. At the same time, cell culture media was changed into either low-serum media or complete EGM2-MV media and cells were counted at days 1, 3, and 5 as described. Each condition was assayed in duplicate and repeated in at least three biological replicates. Caspase 3/7 activity assay 20,000 RVECs were plated into each well of the 96-well plate in 100µl EBM phenored free media (Lonza), and cells were allowed to attach for 24h at 37°C in a humidified atmosphere containing 5% CO 2 . 24h later, Caspase-Glo® 3/7 Assay (Promega) was performed on RVECs according to manufacturer’s protocol. Relative light units (RLU) were measured 30 min after the reagent was added. AnnexinV and PI assay 500,000 RVECs were plated into 10-cm dish in EGM2-MV media (Lonza). When confluency reached 70%, RVECs were serum-starved for 24h before proceeding to AnnexinV and PI assay (ProteinTech) using manufacturer recommened protocol. All stained RVECs were analyzed with LSRFortessa cell analyzer (BD Biosciences). Unstained cells were used as negative controls. Fluorescent and capillary staining RV formalin-fixed, paraffin-embedded (FFPE) slides were rehydrated. Antigens were retrieved with citrate buffer in pressure cooker. Slides were cooled and incubated in PBS+Triton X-100 (0.25%) for 5min and washed with 3x PBST, 5 min each. Griffonia Simplicifolia Lectin (Vector Labs) was diluted in PBST at a final concentration of 5µg/ml. Slides were then incubated in Griffonia Simplicifolia Lectin at 4°C for 1 day. 1 day later, lectin was rinsed off with 3x PBST, 1 min each. Wheat Germ Agglutinin (WGA; Vector Labs) was diluted in PBST at a final concentration of 5 µg/ml. Slides were then incubated in WGA at room temperature for 2h. Lectin was then rinsed off with 3x PBST, 1 min each. 1 µg/ml of DAPI (Thermo Fisher Scientific) was used to stain the slides for 1 min to visualize nuclei and then rinsed off. Alides were dehydrated and mounted with ProLong™ Gold Antifade Mountant (Invitrogen). Slides were imaged with IX83 inverted microscope (Olympus). TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay FFPE RV tissue sections were deparaffinized and rehydrated per the manufacturer’s protocol. Apoptotic cells were detected using In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol. After completion of TUNEL labeling, slides were incubated overnight at 4°C with Griffonia simplicifolia lectin I (GSL I; Vector Laboratories) to visualize capillary structures. Slides were rinsed, mounted with ProLong™ Gold Antifade Mountant (Invitrogen), and imaged using an IX83 inverted fluorescence microscope (Olympus). Western blot analysis Protein concentration was measured using BCA Protein Assay (ThermoFisher). Rat RV tissues (40 µg/sample) and RVECs (20 µg/sample) samples were prepared with 4x Laemmli Sample Buffer (Bio-Rad) and were run on 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) in Tris/Glycine/SDS Running Buffer and transferred to Immobilon-P PVDF membranes (Millipore Sigma) using Tris/Glycine Running Buffer (Bio-Rad). The membranes were blocked with Pierce Protein-Free (TBS) Blocking Buffer (ThermoFisher) or 1% BSA in TBST. Primary antibodies (1:1000) were diluted in Blocking Buffer. Anti-rabbit-HRP and anti-mouse-HRP (Azure Biosystems) secondary antibodies were diluted 1:5000 in 1% BSA with TBST. SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used to image the blots. Densitometry was performed using Image Lab (Bio-Rad). More detailed information on antibodies is listed in the supplement. Quantitative PCR RVECs isolated from WT and ERα Mut were first serum-starved for 18h in low-serum media. Total RNA was isolated from rat RVECs using RNeasy Plus Mini Kit (Qiagen). RNA concentration was measured by NanoDrop (Thermofisher). 1 µg total RNA was reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad). Gene expressions were quantified with TaqMan assays. Changes in mRNA expression were determined by comparative CT (2 -ΔΔC T ) method. Single nucleus RNA-Seq (snRNA-Seq) RV tissues from female WT and ERα Mut rats were collected at 10 days and 4 weeks of MCT treatment. Nuclear extraction was performed on RV frozen tissues using 10x Genomics Chromium Nuclei isolation kit. cDNA libraries were prepared with GEM-X Universal 3’ Gene Expression v4 4-plex kit. Equal number of single cells isolated from WT and ERα Mut rats was loaded on 10X Genomics Chromium Single Cell Controller to generate barcoded single cells for construction of single-cell cDNA libraries. SnRNA-seq libraries were sequenced on NovaSeq X Plus platform at Washington University in St. Louis Genomic Technology Access Center (GTAC). Cell Ranger (v9.0.0) was used for demultiplexing and counting. The rat reference genome Rattus_norvegicus_Rnor_6-0-101 was used as reference genome. R package Seurat (v5.3.0) was used for data preprocessing and visualization. Initially, cells with fewer than 100 or more than 4000 detected genes, or with >15% mitochondrial gene expression were removed. Genes expressed in <5 cells were discarded. Doublets were identified using a 2-layer approach as described previously: First, scDblFinder (v1.22.0) was used to predict potential doublets using default settings in an automated and unbiased fashion. Doublets were additionally identified manually when expressing combinations of marker genes from different cell types. After doublet removal, single cells were normalized with SCTransform and then used this method to integrate ( https://satijalab.org/seurat/reference/integratedata ). For subclustering of ECs, clusters of interests were extracted. Resolution was set at 0.5. SingleR and Azimuth for human heart reference 29 – 31 were then used for annotation. Differential gene expression analyses between WT and ERα Mut rat samples for each cluster of interest were performed using the Wilcoxon Rank-Sum test algorithm with log normalized counts in the RNA assay as input. A threshold of 0.25 for log fold change, 0.05 for the adjusted P value, and 0.1 for minimal fraction of cells was applied for downstream analysis. Pathway analysis was performed with Qiagen Ingenuity Pathway Analysis software. Bar charts of top upregulated and downregulated pathways were selected within diseases and functions analysis. Pathways are ranked by activation Z-score. Data presentation Due to the more pronounced phenotype observed in female rats, data from female rats are shown in the main manuscript. Data from male animals, due to space contraints, are shown in the supplement. Statistical analysis Unless otherwise indicated, data represent mean ± SEM of biological replicates (run in technical triplicates). All statistical analyses were performed with Prism v9.0 (GraphPad Software, La Jolla, CA, USA). Sample sizes were estimated by power calculation. Correlations were determined using Pearson’s coefficient (R). For normally distributed data, comparisons between three or more groups were performed using two-way analysis of variance (ANOVA). For comparisons between two groups, significance was calculated with unpaired Student’s t -test. Statistically significant difference was accepted at p<0.05. Results ERα regulates RVEC angiogenic function and signaling in vitro We first confirmed reduced ERα abundance in ERα Mut rats. As shown previously 16 , female and male ERα Mut rats exhibited an 80% reduction in RVEC expression of full-length Esr1 transcript ( Supplementary Fig. 1A,B ). ERα Mut rats also exhibited higher serum E2 levels than WT ( Supplementary Fig. 1C-F ). We then sought to investigate whether ERα plays a pro-angiogenic role in RVECs. Compred to WT, loss of ERα resulted in reduced pseudo-vascular network forming capability (total tube length, numbers of branching points and rings) in both female ( Fig. 1A-D ) and male RVECs ( Supplementary Fig. 2A-D ). This suggests that ERα regulates pro-angiogenic function in RVECs. We next investigated effects of ERα on other significant contributors to angiogenesis, such as RVEC proliferation and migration 32 – 34 . Interestingly, female ERα Mut RVECs exhibited increased proliferation compared to WT ( Fig. 1E ), whereas male ERα Mut RVECs demonstrated reduced proliferation compared to their WT counterparts ( Supplementary Fig. 2E ). We next focused on RVEC migration. Both transwell migration and scratch assays revealed that loss of ERα resulted in decreased migration in female RVECs ( Fig. 2F-I ). Male ERα Mut RVECs also exhibited decreased migration compared to WT RVECs ( Supplementary Fig. 2F-I ). These results suggest that ERα enhances pseudo-vascular network formation in RVECs in both sexes primarily via enhancing migration. ERα’s effects on RVEC proliferation, on the other hand, are sex-specific and appear to inhibit proliferation in female RVECs, indicating that impaired proliferation is unlikely to explain the reduced RVEC pseudo-vascular network formation found in ERα Mut females. Download figure Open in new tab Figure 1. Loss of ERα reduces angiogenesis and migration in female right ventricle endothelial cells (RVECs). ( A-D ) Tube formation assays in female wild type (WT) and ERα Mut RVECs at 10h after plating cells on Matrigel. Representative images ( A ) and quantification of number of rings ( B ), number of branching points ( C ) and total tube length ( D ). ( E ) Growth curves of female WT and ERα Mut RVECs. ( F, G ) Transwell migration assay representative images and quantification of female WT and ERα Mut RVECs at 24h. ( H, I ) Scratch assay representative images and quantification of female WT and ERα Mut RVECs at 24h. Purple shade represents initial wound. ( J ) GO biological process comparisons between female WT and ERα Mut RVECs from Qiagen rat angiogenesis microarray (n=4 animals/group, pooled). ( K ) qPCR analysis of leptin RNA expression of female WT and ERα Mut RVECs from Taqman gene expression (relative expression level, 2-ΔΔCt calculation). Each data point = one animal. * p<0.05, ** p<0.01; *** p<0.001 by ANOVA with Tukey’s post-hoc analysis. Error bars represent mean ± SEM. Error bars represent mean ± SEM. Scale bars, 200µm Download figure Open in new tab Figure 2. Loss of ERα exerts pro-apoptotic effects in female RVECs. ( A ) Caspase 3/7 activity assay in female WT and ERα Mut RVECs grown for 24h. ( B ) Quantification of Annexin V + and Propidium Iodide (PI) + population in female WT and ERα Mut RVECs grown for 24h. ( C ) Representative images of Western blots and quantification of cleaved/full length PARP1 ( D ) and Bax/Bcl-2 ( E ) grown for 24h. Each data point = one animal. * p<0.05, ** p<0.01; *** p<0.001 by ANOVA with Tukey’s post-hoc analysis. Error bars represent mean ± SEM. Error bars represent mean ± SEM. Scale bars, 50µm To identify pathways and genes regulated by ERα in RVECs, angiogenesis PCR array was performed on female and male ERα Mut versus WT RVECs ( Supplementary Fig. 3A ). The top 5 ranked differentially expressed genes (DEGs; including up-regulated and down-regulated) from each group are listed in Supplementary Fig. 3B . We then compared genes that were altered in female and male WT vs ERα Mut . Gene Ontology Biological Process analysis revealed that cell migration was the process with the most altered DEGs in both male (identified as cell migration, Supplementary Fig. 2J ) and female RVECs (identified as taxis, Fig. 2J ). Of note, leptin, a regulator of metabolism and angiogenesis 35 – 38 , was consistently downregulated with the loss of ERα (increased in WT), both in male ( Supplementary Fig. 2K ) and female RVECs ( Fig. 2K ). To identify mechanisms underlying reduced angiogenic function in female ERα Mut RVECs, we assessed apoptosis-related endpoints. Female ERα Mut RVECs exhibited higher caspase 3/7 activity ( Fig. 2A ) and apoptotic cell percentages by AnnexinV-PI staining ( Fig. 2B ). After serum starvation, female ERα Mut RVECs also exhibited higher cleaved PARP1 levels ( Fig. 2C,D ) and higher Bax/Bcl-2 ratios ( Fig. 2C,E ) than WT, suggesting increased pro-apoptotic signaling with loss of functioning ERα. Together, the data in Fig. 1 &2 indicate that loss of functioning ERα in female RVECs results in increased propensity for apoptosis but also increased proliferation, with the latter potentially being a compensatory response for the increased cell death. These data also identify ERα as a sexually dimorphic pro-angiogenic mediator in RVECs. ERα protects RV capillaries from rarefaction in female MCT-PH rats We next studied whether ERα regulates RVEC angiogenesis to prevent RV capillary rarefaction. Male and female WT and ERα Mut rats were injected with MCT 15 , 39 , 40 ( Fig. 3A ). As expected, and consistent with prior data, female WT rats exhibited only minimal increases in RVSP or Fulton index ( Fig. 3B,C ) 41 – 43 . On the other hand, as reported previously, female ERα Mut rats exhibited a doubling in RVSP and Fulton index ( Fig. 3B,C ), suggesting ERα is protective against RVSP increase and RVH in females. Male WT MCT rats, on the other hand, exhibited expected increases in RVSP and Fulton index after MCT, but these changes were not affected by loss of functioning ERα ( Supplementary Fig. 4B,C ). Download figure Open in new tab Figure 3. ERα deficiency results in PH and decreased RV capillary density in female MCT rats. ( A ) Schematic of experimental design. ( B-E ) Effects of ERα in female rats on RV systolic pressure (RVSP), RV hypertrophy and RV capillary density. ( B ) RVSP and ( C ) Fulton index in female WT and ERα Mut rats treated with MCT or phosphate buffered saline (PBS; vehicle control). ( D ) Representative images of RV lectin staining and ( E ) Quantification of vascularization (as capillary per myocyte area) in female WT and ERα Mut rats. Pearson correlation of vascularization (capillary per myocyte area) and ( F ) RVSP or ( G ) Fulton index. Dark dots indicate WT rats and light dots indicate ERα Mut rats. Each data point represents one animal. WGA (Wheat Germ Agglutinin), cell membrane. GSL (Griffonia Simplicifolia Lectin), endothelial cells. Each data point = one animal. * p<0.05, ** p<0.01; *** p<0.001 by ANOVA with Tukey’s post-hoc analysis. Error bars represent mean ± SEM. Error bars represent mean ± SEM. Scale bars, 50µm Next, we investigated RV capillary density in female PH rats after 4 weeks of MCT ( Fig. 3D,E ). Lectin staining revealed that female WT rats are resistant to MCT-induced RV capillary loss ( Fig. 3E ). Accompanying increases in RVSP and Fulton index, loss of ERα caused severe (∼65%) RV capillary rarefaction ( Fig. 3E ). Both RVSP ( Fig. 3F ) and RVH ( Fig. 3G ) demonstrated an inverse correlation with RV capillary density per myocyte area, suggesting a potential role for sufficient RV capillary density in preventing RVH. Taken together, these data suggest ERα exerts protective and pro-angiogenic effects in the female RV. Interestingly, we did not observe significant RV capillary loss in male WT PH rats, and only male ERα Mut PH rats exhibited RV capillary rarefaction ( Supplementary Fig. 4D,E ). Unlike female rats, RV capillary density per myocyte area only inversely correlated with Fulton index ( Supplementary Fig. 4G ), but not RVSP ( Supplementary Fig. 4F ). ERα protects female rats from early onset of RVH and RV capillary rarefaction after MCT exposure To understand how ERα regulates RV capillary density in early stages of PH-induced RVH, we performed a time course experiment in male and female WT and ERα Mut MCT rats. RVs were harvested at day 3 and day 10 after MCT injection. Interestingly, 10 days after MCT injection, Fulton index as well as RV cardiomyocyte cross-sectional area were significantly increased in female MCT ERα Mut but not WT rats ( Fig. 4B,C ). This was accompanied by a significant decrease in RV capillary density ( Fig. 4D-G ). In contrast, there was no RVH and only a trend for RV capillary loss in male rats, with no difference between WT and ERα Mut ( Supplementary Fig. 5B-G ). These data highlight the importance of ERα’s protective and pro-angiogenic role in the female RV even in early stages of PH and RV remodeling. Download figure Open in new tab Figure 4. Loss of functional ERα results in early RV capillary rarefaction after MCT injection in female rats. ( A ) Schematic of experimental design. ( B-G ) Effects of ERα on RV hypertrophy and RV capillary density in female rats treated with MCT or PBS. ( B ) Fulton index and ( C ) RV cardiomyocyte size in female WT and ERα Mut MCT-PH rats. ( D ) Representative images and ( E-G ) quantification of RV capillary density via lectin staining. WGA (Wheat Germ Agglutinin), cell membrane. GSL (Griffonia Simplicifolia Lectin), endothelial cells. CSA, cross-sectional area. Each data point = one animal. * p<0.05, ** p<0.01; *** p<0.001; **** p<0.0001 by ANOVA with Tukey’s post-hoc analysis. Error bars represent mean ± SEM. Error bars represent mean ± SEM. Scale bars, 50µm Download figure Open in new tab Figure 5. Female ERα Mut rats exhibit increased RVEC apoptosis after MCT treatment. (A-D) Representative images and quantification of Western blot analysis of Bax and Bcl-2 in female WT PBS or ERα Mut RV tissues ten days and four weeks after MCT administration, respectively. ( E ) Representative images of TUNEL + RVECs (RVECs stained with griffonia simplicifolia lectin; GSL). ( F ) Quantification of TUNEL + RVECs (as percentage of total RVEC number). White arrows, TUNEL + RVECs. PBS, phosphate buffered saline. Each data point represents one animal. Each data point = one animal. * p<0.05, ** p<0.01; *** p<0.001 by ANOVA with Tukey’s post-hoc analysis. Error bars represent mean ± SEM. Error bars represent mean ± SEM. Scale bars, 50µm Capillary staining of male and female RVs three days after MCT administration showed similar capillary density as compared to controls and did not reveal a sex bias or ERα regulation ( Supplementary Fig. 6D-G & 7D-G ). Female ERα Mut rats exhibit more severe pulmonary vascular remodeling during early stages of PH development In PH, RVH is induced by increased RV afterload from pulmonary vascular remodeling. We recently reported greater PA muscularization in female ERα Mut rats 44 . Therefore, we examined if the RVH and RV capillary rarefaction noted in female ERα Mut rats at 10 days are associated with more severe pulmonary vascular remodeling. Indeed, female ERα Mut rats demonstrated fewer partially muscularized vessels and more fully muscularized vessels ( Supplementary Fig. 8A,B ). These data indicate that the RVH and RV capillary loss in female ERα Mut rats 10 days after MCT may be due to enhanced pulmonary vascular remodeling. On the other hand, we observed a modest increase in the number of partially muscularized vessels in male ERα Mut rats with MCT treatment at day 10, but no difference in nonmuscularized or fully muscularized PAs ( Supplementary Fig. 9A,B ). These data mirror the RV capillary density data, where female ERα Mut rats exhibit more RVH and RV capillary rarefaction than WT, paralleled by more severe pulmonary vascular remodeling. Male ERα Mut rats on the other hand exhibit no increase in RVH, RV capillarization, and pulmonary vascular remodeling as compared to WT ( Supplementary Fig. 5B-G ). ERα Mut inhibits apoptotic signaling in RVECs from female MCT rats We next examined whether enhanced RVEC apoptosis contributes to the reduced angiogenic capacity and capillary rarefaction observed in female ERα Mut rats at early and late timepoints. Bax/Bcl-2 ratio was not different in the RV of female WT and ERα Mut rats 10 days after MCT ( Fig. 7A,B ). However, at 4 weeks after MCT treatment, female ERα Mut rats demonstrated a higher Bax/Bcl-2 ratio ( Fig. 7C,D ) compared to WT. TUNEL staining revealed a greater number of TUNEL⁺ RVECs in female ERα Mut rats at both 10 days and 4 weeks post-MCT treatment relative to WT rats ( Fig. 7E,F ). Together, these results suggest that functional ERα protects the female RV from endothelial apoptotic cell death during both early and late stages of MCT-induced remodeling. ERα regulates migration and apopotosis pathways in female capillary and endocardial RVECs To identify genes and pathways regulated by ERα in female RVECs, we performed snRNA-seq on RV tissues from female WT and ERα Mut rats at both 10 days and 4 weeks after MCT administration ( Fig. 6A ). We discovered five distinct populations of RVECs: arterial ECs, capillary ECs, endocardial ECs, lymphatic ECs, and venous ECs ( Fig. 6B,C ). Featured genes of each RVEC sub-population are shown in Fig. 6B . At 4 weeks, there was a reduction in the endocardial EC population and an increase in the capillary EC population in female ERα Mut ( Fig. 6D,E ). We identified higher numbers of DEGs between female WT and ERα Mut across all 5 sub-populations of RVECs at 10 days compared to 4 weeks after MCT ( Fig. 7A,B ). This suggests ERα regulates gene expression predominantly at early stages of RVH. In addition, Qiagen Ingenuity® Pathway Analysis uncovered differentially regulated pathways between WT and ERα Mut ( Fig. 7C-F ). At 10 days, migration was higher in female WT capillary and endocardial RVECs than in ERα Mut cells ( Fig. 7C,E ), suggesting that loss of ERα reduces migration in vivo. Moreover, compared to ERα Mut cells, female WT endocardial RVECs demonstrated increased cell survival and a less apoptotic phenotype ( Fig. 7E ), linking loss of functioning ERα to increased apoptotic cell death. At 4 weeks, female WT capillary RVECs displayed increased survival programming compared to ERα Mut cells ( Fig. 7D ), again linking loss of functioning ERα to increased cell death. Other significantly increased pathways in WT included organization of cytoskeleton, microtubule dynamics, and morphogenesis of cellular protrusions. Taken together, snRNA-seq data suggest that ERα plays a fundamental role in maintaining cell viability and RVEC migration during the development of RV failure. Download figure Open in new tab Figure 6. Single nucleus RNA-sequencing of female MCT-PH RVs reveals five distinct populations of RVECs. ( A ) Schematic of snRNA-Seq experimental design. ( B ) Featured gene expressions of each RVEC sub-population. ( C ) Uniform Manifold Approximation and Projection (UMAP) plots of RVEC sub-populations at ten days and four weeks.( D ) Cellular proportion plot of RVEC sub-populations. ( E ) Total numbers and percentages of RVEC sub-populations in relation to total RV cell number (including all RV cell types). n=2 animals per group. Download figure Open in new tab Figure 7. ERα regulates early changes in RVEC gene programs in female MCT-PH RVs. (A, B) Differentially expressed genes (DEGs) of female WT ERα Mut RVECs ten days ( A ) and four weeks ( B ) after MCT administration. (C-F) Qiagen IPA disease and function pathway analysis in female capillary and endocardial RVECs in WT vs ERα Mut cells at ten days ( C, D ) and four weeks ( E,F ) after MCT administration. Pathways are ranked by Z-score. Only top upregulated and downregulated pathways are shown. Discussion We report for the first time that ERα regulates RVEC function and RV angiogenesis in a sexually dimorphic manner. Loss of functioning ERα resulted in reduced RVEC pseudovascular network formation and migration in male and female RVECs at baseline conditions, as well as increased apoptosis in female RVECs. In vivo, loss of functioning ERα resulted in reduced RV capillary density in female MCT-PH rats. In addition, we observed early onset of RVH and capillary loss with the loss of ERα in females. These findings were accompanied by a higher number of apoptotic RVECs in female ERα Mut MCT-PH RVs. snRNA-Seq in female MCT-PH RVs revealed previously undescribed RVEC subtypes that are under transcriptional regulation of ERα. In particular, downregulation of migration and upregulation of apoptosis pathways were identified in female ERα Mut MCT-PH rats. Together, these data indicate that ERα’s effects on RVEC function and RV angiogenesis exhibit a female sex bias. In response to the increased pressure overload caused by PH, the RV adapts via cardiomyocyte hypertrophy and increased cardiac contractility 6 , 7 . In the initial adaptive stage, there is increased capillary density and vascularization 45 . This is achieved by up-regulating pro-angiogenic signaling 46 and RVEC proliferation 45 . The molecular switch that drives the transition from adaptive vascular maintenance to maladaptive capillary loss, however, remains poorly understood. Our findings implicate ERα as a novel regulator of RVEC function and RV vascularization during PH progression. We previously demonstrated beneficial effects of ERα on RV cardiomyocytes 16 . The current study expands those findings by investigating the role of ERα in RVECs (another cell type central to the pathogenesis of RVF 13 , 47 ). This was motivated by the established role of RVEC dysfunction in the pathogenesis of RVF, the known sexual dimorphisms in RV adaptation to PH, and the well-characterized vascular-protective effects of ERα in systemic and left ventricular endothelium. Vascular ECs are known to express all three types of estrogen receptors (ERα, ERβ and GPER), and the function of ERα in the vasculature has been well studied. ERα has EC-protective effects in various types of vascular injury 48 , 49 . For example, ERα is directly linked to eNOS expression 50 , 51 and regulation of vascular tone 52 . Mouse models with ERα deletion and/or mutations have delineated the function of ERα in the vasculature (reviewed in 53 ). For example, ERα is essential for the beneficial effects of E2 on re-endothelialization and injury-induced medial hyperplasia 54 . Expression of ERα in vascular ECs is modulated by estrogen status and strongly related to endothelial-dependent dilation in women 55 . Variations in ESR1 (the gene encoding ERα) have been linked to myocardial infarction in both men and postmenopausal women 56 , 57 , highlighting the protective effect of ERα in the cardiovascular system. ERα promotes EC migration through the c-Src and focal adhesion kinase pathway 58 , as well as the RhoA/Moesin pathway 59 . Our findings expand this observation and, for the first time, demonstrate stimulatory effects of ERα on migration in RVECs. Effects of ERα on apoptosis are less understood. E2 inhibits TNF-α induced EC apoptosis 60 and ERβ protects human umbilical vein ECs against apoptosis 61 . We, for the first time, discovered that loss of ERα causes increased apoptosis in female RVECs, suggesting that ERα-mediated suppression of endothelial apoptosis contributes to the maintenance of RV capillary density in females. This is particularly relevant given clinical evidence showing increased apoptotic vessels in RVs from female PAH patients compared to control RVs 62 . Upregulating RV angiogenesis, inhibiting RVEC apoptosis, or increasing RVEC migration are potential new mechanisms by which ERα maintains RV function. Further research is needed to uncover the downstream pathways that are affected by the loss of ERα. Our research also unveiled a sexual dimorphic role of ERα in protecting RV from failure. In males MCT-PH rats, RVSP elevations, Fulton indices, and capillary density reductions are comparable between WT and ERα Mut , suggesting that ERα does not provide protective benefits in the male RV and is playing a dispensable role in RVH induced by PH. On the other hand, femaleMCT-PH rats are relying upon ERα for resilience against PH-induced RVH and vascularization defects. Previous studies identified sex differences in the timing and severity of PH-induced RV dysfunction 63 , 64 . In male WT rats, significant elevation of mPAP and Fulton index were observed after 2 weeks of MCT injection 64 , as well as reduced RV capillary density 63 . Female rats, on the other hand, demonstrated less pronounced RVH and dilatation 63 . This indicates that female WT rats are more resistant to RVH development caused by MCT at early stages. Given the beneficial effects provided by ERα in the vasculature, we focused on ERα’s pro-angiogenic effects in RV. Our time course experiments suggest that 1) RV remodeling in female rats happens earlier post-MCT than in male rats; 2) RV capillary density in female rats contributes more to RVH and RV dysfunction than in their male counterparts; and 3) ERα is a critical mediator of RV protection and capillary maintenance in females during PH development. The ERα loss-of-function rat model investigated in this study was generated through CRISPR/Cas9 as previously described 16 . Our rats exhibit a host of phenotypes in the pulmonary vasculature and RV when subjected to experimental PH or RV pressure overload. Interestingly, compared to male ERα Mut rats, female ERα Mut rats exhibit a more profound phenotype. When subjected to MCT-PH, female ERα Mut rats exhibit higher RVSP and Fulton index, more pulmonary vascular remodeling and more profound decreases in RV function, whereas male WT and ERα Mut rats have comparable disease phenotypes 44 . At a cellular level, we discovered a novel E2-ERα-BMPR2-apelin axis in RV cardiomyocytes. In light of the focus of our paper, this is of particular interest, since both BMPR2 as well as apelin are known pro-angiogenic mediators 16 . We also found that the RV cardiomyocyte secretome increases RVEC angiogenic ability in vitro, and that the pro-angiogenic function of RV cardiomyocytes is blunted when apelin is silenced 16 . However, in our snRNA-Seq analyses in female MCT RVECs we did not detect BMPR2 nor apelin being differentially expressed between WT and ERα Mut . This may be a reflection of this axis being suppressed in the setting of PH and RV failure or indicate that BMPR2 or apelin are not under transcriptional control of ERα in RVECs. Our snRNA-Seq experiments, for the first time, identified five distinct sub-populations of RVECs. This allows us to better understand RVEC dynamics and functions during the development of RVH. We noticed capillary and endocardial RVECs were the most changed populations among the five subpopulations. Of note, ventricular endocardial cells play an vital role in coronary artery angiogenesis as these cells have the potential to differentiate into arterial or capillary endothelial cells 65 . However, the transition from endocardial to capillary ECs in the context of RVH remains elusive. Not surprisingly, ERα differentially regulates the gene programming of RVEC sub-populations during the development of RVH. The observation that capillary and endocardial RVEC numbers are drastically changed suggests that ERα provides beneficial effects by regulating these cells. The drastic decline of endocardial ECs at 4 weeks after MCT treatment points to a new mechanism of angiogenesis pertubation in RV failure where a loss of endocardial EC could potentially explain the insufficient RV angiogenesis. Maintaining endocardial EC viability could therefore be a novel therapeutic avenue. Furthermore, the DEG analysis indicates that transcriptome changes regulated by ERα happen early in the disease progression. This suggests a need for early intervention and identifies enhancing ERα signaling as a potential novel therapeutic strategy to protect the RV in PH. One limitation of our research is the use of a two-diemsional method for quantifying the three-dimensional capillary structure of the RV. Stereology is considered the gold standard for assessing three-dimensional structures. We did not consistently use stereology due to its limitation of requiring the entire RV to be fixed, which would not allow for isolating RVECs or performing biochemical analyses. Several important prior studies in the field employed two-dimensional analysis methods for quantifying RV vascularization 12 , 13 , 66 , 67 . In addition, we and others 13 demonstrated that findings of decreased vascular density in vivo are accompanied by decreased pseudo-vascular network formation and impaired angiogenic behavior in cultured RVECs, thus supporting the in vivo findings. A recent paper suggests that it may not be the loss of capillaries in RV, but rather, a lack of sufficient contact area between RVECs and RV cardiomyocytes that causes maladaptive RVH 17 . We focused on studying ERα without its ligand E2. This is due to the fact that ERα can elicit downstream signaling even in absence of activation by E2 53 . It has been shown that there is increased susceptibility to PH in women with premature menopause 68 and we found reduced RVEC and RV cardiomyocyte expression levels of ERα in patients with RV failure 16 . This suggests that reduced ERα activity could be a contributor to RV failure. Therefore, we chose to study ERα without the influence of E2 as a first step. Studies employing E2 supplementation are currently ongoing in our laboratory. In summary, our research provides novel insights into how ERα regulates RV angiogenesis and RVEC function and identifies ERα as being essential for preventing RV failure and RV capillary rarefaction in females with PH. Our reseach also sheds light on the sexually dimorphic role of ERα in males and females with PH. Funding This project was supported, in part, with support from NIH R01HL158596, NIH R01HL62794, NIH R01HL169509, and NIH R01HL170096 to Z.D., VA Merit Review Award 2 I01BX002042, NIH R01HL144727, NHLBI P01 HL158507, and Borstein Family Foundation to T.L., NIH 1R01HL16479 to A.L.F.. Disclosures I.P. is a Scientific Co-founder of Allinaire Therapeutics. I.P. has received consulting fees from Ceramedix, Allinaire, and Astra Zeneca. T.L. has received consulting fees from Arrowhead Pharmaceuticals and Allinaire Therapeutics. None of the other authors has any conflicts of interest, financial or otherwise, to disclose. Acknowledgement We thank the Genome Technology Access Center at the McDonnell Genome Institute at Washington University School of Medicine for help with genomic analysis. The Center is partially supported by NCI Cancer Center Support Grant #P30 CA91842 to the Siteman Cancer Center from the National Center for Research Resources (NCRR). This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR or NIH. Footnotes Author’s contributions : JL designed, performed, and analyzed studies and wrote the manuscript; VK and AF designed, performed, and analyzed studies; HZ, SL and ZD performed and analyzed experiments and provided data; CSCW and IP provided input on data analysis and edited the manuscript; TL designed and analyzed studies and wrote and edited the manuscript. 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