Journal: Science translational medicine

Article Title: ACTRIIA-Fc rebalances activin/GDF versus BMP signaling in pulmonary hypertension

doi: 10.1126/scitranslmed.aaz5660

Figure Lengend Snippet: Immunohistochemical staining for activin A, GDF8, and GDF11 along with α-SMA costain performed on paraffin-embedded lung sections from healthy controls and patients with IPAH or HPAH (n = 5 each) and lung sections from control, MCT-exposed, or SU-Hx–exposed rats (n = 3 each). Horseradish peroxidase (HRP)–conjugated secondary antibody (3,3′-diaminobenzidine staining, as indicated by label HRP in brown) and α-smooth muscle actin (α-SMA) AP-conjugated primary antibody (as indicated by label SMA in blue; right) are shown. Scale bars, 50 μm.

Article Snippet: Measurement of GDF8 in human serum was performed using a monospecific anti-human GDF8 antibody lacking cross-reactivity with GDF11 on a multiplex ELISA system (Meso Scale Discovery) using the standard manufacturer’s protocol.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Science translational medicine

Article Title: ACTRIIA-Fc rebalances activin/GDF versus BMP signaling in pulmonary hypertension

doi: 10.1126/scitranslmed.aaz5660

Figure Lengend Snippet: (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.

Article Snippet: Measurement of GDF8 in human serum was performed using a monospecific anti-human GDF8 antibody lacking cross-reactivity with GDF11 on a multiplex ELISA system (Meso Scale Discovery) using the standard manufacturer’s protocol.

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Activation Assay, In-Cell ELISA, Control, Derivative Assay