Fig 2: Activin A induces excessive ET-1 (endothelin-1) production in pulmonary artery endothelial cells (PAECs), reversible by follistatin or bosentan. A and B, Quantitative real-time polymerase chain reaction analysis of ET-1 mRNA expression (A, n=6 biologically independent samples per group) and ELISA measurement of ET-1 concentration in culture medium (B, n=3–4) from PAECs treated for 6 hours with recombinant activin A (100 ng/mL) or vehicle. C and D, ET-1 mRNA expression (C, n=6) and secreted ET-1 concentration (D, n=4-5) in PAECs 48 hours after INHBA (inhibin β-A) overexpression (OE) or GFP (green fluorescent protein) control retroviral transfection. E and F, INHBA mRNA expression (E, n=6) and activin A concentration in culture medium (F, n=4) in PAECs treated for 6 hours with recombinant ET-1 (100 nmol/L) or vehicle. G, ET-1 mRNA expression in PAECs treated for 6 hours with vehicle or recombinant activin A in the presence of vehicle (VEH), FST (follistatin; 100 ng/mL), bosentan (BOS; 10 μM), or both (FST+BOS; n=4). H, ET-1 mRNA expression in PAECs 48 hours after INHBA OE or GFP transfection, followed by 24 hours of treatment with VEH, FST (100 ng/mL), BOS (10 μM), or FST+BOS (n=4). I, ET-1 mRNA expression in PAECs treated for 6 hours with vehicle or recombinant activin A in the presence of VEH or ACTRIIA-Fc (activin receptor type IIa fusion protein; 2500 ng/mL; n=3-4). J, ET-1 mRNA expression in PAECs 48 hours after INHBA OE or GFP transfection, followed by 24 hours of treatment with VEH or ACTRIIA-Fc (2500 ng/mL; n=4). Data are mean±SEM. P<0.05 is deemed statistically significant. Statistical tests: 2-sided Student t test for A through F; 1-way ANOVA with Tukey post hoc test for G and H.

Fig 3: Canonical SMAD2/3 signaling mediates activin A–induced ET-1 (endothelin-1) expression. A, Representative immunoblots and quantification of p-SMAD2/3 (phosphorylated SMAD2/3; small mother against decapentaplegic family member 2/3) and total SMAD2/3 in GFP (green fluorescent protein)- or INHBA (inhibin β-A) overexpression (OE)–pulmonary artery endothelial cells (PAECs) treated with vehicle (VEH), FST (follistatin), bosentan (BOS), or FST+BOS for 24 hours (n=3). B, Immunoblots showing p-SMAD2/3 and total SMAD2/3 in GFP- or INHBA OE–PAECs treated with SB505124 (5 μM) or vehicle for 24 hours. C, ET-1 mRNA expression in GFP- or INHBA OE–PAECs treated with SB505124 or vehicle for 24 hours (left, n=3) and in PAECs treated for 6 hours with activin A±SB505124 (right, n=3–4). D, Immunoblots showing SMAD2/3 and β-actin in PAECs pretreated with SMAD2 siRNA (small interfering RNA; siSMAD2), SMAD3 siRNA (siSMAD3), dual SMAD2-SMAD3 siRNA (dual siSMAD), or control siRNA (siNC [siRNA negative control]). E, ET-1 mRNA expression in GFP- or INHBA OE–PAECs pretreated with siSMAD2, siSMAD3, dual siSMAD, or siNC (n=3–4). F, INHBA and ET-1 mRNA expression in PAECs exposed to normoxia or hypoxia (0.1% O2, 24 hours; n=6). G and H, ET-1 mRNA expression in PAECs under hypoxia treated with FST (G) or SB505124 (H; n=3). Data are mean±SEM. P<0.05 is deemed statistically significant. Tests: 1-way ANOVA with Tukey post hoc test for A, C, E, G, and H; 2-sided Student t test for F.

Fig 4: Activin A–driven ET-1 (endothelin-1) contributes to pulmonary artery endothelial cell (PAEC) dysfunction. A and B, Representative images (A) and quantification (B) of chord length and number of branching points in a Matrigel tube formation assay (n=3–4) using PAECs transfected with GFP (green fluorescent protein) or INHBA (inhibin β-A) overexpression (OE), treated with vehicle (VEH), FST (follistatin; 100 ng/mL), bosentan (BOS; 10 μM), or both (FST+BOS). C and D, Representative images (C) and quantification (D) of apoptotic cells assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining (n=3–4) under serum starvation. Apoptotic cells are indicated by white arrows. E, Cell proliferation measured by WST-1 (water-soluble tetrazolium-1) assay in GFP- or INHBA OE–transfected PAECs treated with VEH, FST, BOS, or FST+BOS (n=3). Data are mean±SEM. P<0.05 is deemed statistically significant. Statistical test: 1-way ANOVA with Tukey post hoc test for B, D, and E. AU indicates absorbance units; and DAPI, 4′,6-diamidino-2-phenylindole.

Fig 5: In vivo activin A inhibition improves pulmonary hypertension (PH) phenotype comparably or more than ET-1 (endothelin-1) blockade. A, Experimental design: wild-type (WT) and VE-cadherin (vascular endothelial cadherin)–INHBA (inhibin β-A)-Tg (TG/transgenic) mice were exposed to hypoxia (10% O2) for 3 weeks, with vehicle (VEH), FST (follistatin; 8.5 μg/kg), bosentan (BOS; 30 mg/kg), or FST+BOS administered during the final 2 weeks. B, Right ventricular systolic pressure (RVSP; n=4–9). C, Fulton index (RV/[LV+S] [right ventricle to left ventricle plus septum] ratio; n=4–8). D, Representative hematoxylin and eosin–stained lung sections. Blue arrows indicate vessels. E, Representative immunofluorescent staining of α-SMA (α-smooth muscle actin protein; green, SMC [smooth muscle cell] marker), vWF (von Willebrand Factor; red, endothelial cell [EC] marker), and DAPI (4′,6-diamidino-2-phenylindole; blue, nuclei). White arrows indicate vessels. F, Quantification of pulmonary artery muscularization (non-, partial-, full; n=12–15 fields from 3–4 mice). PA indicates pulmonary artery. G, Lung ET-1 mRNA expression (n=3–4). H, mRNA expression of INHBA, ET-1, eNOS (endothelial NO synthase), SOD2 (superoxide dismutase 2), fibronectin, SLUG (snail family transcriptional repressor 2), CD31 (cluster of differentiation 31), and BMP4 (bone morphogenetic protein 4) in lung ECs isolated from WT and TG mice (n=3–4). Data are mean±SEM. P<0.05 is deemed statistically significant. Tests: 1-way ANOVA with Tukey post hoc test for B, C, and G; 2-way ANOVA with Tukey post hoc test for F; 2-sided Student t test for H.