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

Figures

• 

  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 ERαMut 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 ERαMut RVECs. (F, G) Transwell migration assay representative images and quantification of female WT and ERαMut RVECs at 24h. (H, I) Scratch assay representative images and quantification of female WT and ERαMut RVECs at 24h. Purple shade represents initial wound. (J) GO biological process comparisons between female WT and ERαMut RVECs from Qiagen rat angiogenesis microarray (n=4 animals/group, pooled). (K) qPCR analysis of leptin RNA expression of female WT and ERαMut 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

• 

  Figure 2.. Loss of ERα exerts pro-apoptotic effects in female RVECs. (A) Caspase 3/7 activity assay in female WT and ERαMut RVECs grown for 24h. (B) Quantification of Annexin V+ and Propidium Iodide (PI)+ population in female WT and ERαMut 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

• 

  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 ERαMut 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 ERαMut 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 ERαMut 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

• 

  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 ERαMut 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

• 

  Figure 5.. Female ERαMut 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 ERαMut 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

• 

  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.

• 

  Figure 7.. ERα regulates early changes in RVEC gene programs in female MCT-PH RVs. (A,B) Differentially expressed genes (DEGs) of female WT ERαMut 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 ERαMut 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.