Fig. 2. ACTRIIA-Fc modulates signaling, gene regulation, and proliferation of human pulmonary vascular cells.

(A) Expression of phosphorylated SMAD1/5/9 and SMAD2/3 in human pulmonary micro-vascular endothelial cells (PMVECs) exposed to BMP9 (100 pg/ml), GDF8 (50 ng/ml), GDF11 (50 ng/ml), activin A (50 ng/ml), or activin B (50 ng/ ml) for 30 min with or without ACTRIIA-Fc (2500 ng/ml), analyzed by Western blot. n = 3 replicates; N = 3 independent experiments. (B) Luciferase activity in telomerase immortalized human microvascular endothelial (TIME) cells transfected with BMP-responsive element transcriptional reporter (BRE-Luc) and exposed to varying concentrations of BMP9 with or without ACTRIIA-Fc (n = 3; N = 3; RLU, relative luminescence units). (C) Activation of SMAD1/5 in human PMVECs treated with BMP9 (below the EC₅₀ 250 ng/ml) with or without varying concentrations of ACTRIIA-Fc, analyzed by In-Cell Western. Data represent means ± SEM. *P < 0.05, **P < 0.01, †P < 0.001, and #P < 0.0001 compared to untreated controls in each group, two-way ANOVA with Dunnett’s test for multiple comparisons (n = 3; N = 3). (D and E) BMP-mediated transcriptional activity in TIME cells treated with BMP9, GDF8, GDF11, and activin A with or without ACTRIIA-Fc (n = 6; N = 3). (F and G) Proliferation of control- and PAH-derived PMVECs in response to BMP9, activin A, and GDF11 with or without ACTRIIA-Fc. (H) Expression of phosphorylated SMAD1/5/9 and SMAD2/3 in human pulmonary artery smooth muscle cells (PASMCs) exposed to TGFβ1 (1 ng/ml), GDF8 (50 ng/ml), GDF11 (50 ng/ml), activin A (50 ng/ml), or BMP4 (50 ng/ml) for 30 min with or without ACTRIIA-Fc (2500 ng/ml), analyzed by Western blot. (I and J) Expression of α-SMA and calponin mRNA at 24 hours after stimulation with TGFβ1 (1 ng/ml), GDF8 (50 ng/ml), GDF11 (50 ng/ml), or activin A (50 ng/ml) in human PASMCs with or without ACTRIIA-Fc (n = 3 to 6). (K and L) Expression of α-SMA and calponin protein in PASMCs at 72 hours after stimulation with TGFβ1, GDF8, GDF11, activin A, or activin B with or without ACTRIIA-Fc, as assessed by In-Cell Western (n = 4; N = 3). (M) Proliferation in PASMCs exposed to GDF8 (100 ng/ml), GDF11 (100 ng/ml), activin A (100 ng/ml), activin B (100 ng/ml), PDGF-BB (1 ng/ml), or complete media for 24 hours with or without ACTRIIA-Fc (10,000 ng/ml) as measured by ³H-thymidine incorporation. (n = 5; N = 3). Data represent means ± SEM. *P < 0.05, **P < 0.01, †P < 0.001, and #P < 0.0001 compared to vehicle controls, one-way ANOVA with Sidak’s test for multiple comparisons.