Pulmonary arterial hypertension drugs can partially restore altered angiogenic capacities in bmpr2‐silenced human lung microvascular endothelial cells

Figures

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  Figure 1. Effect of macitentan, selexipag, and tadalafil on BMPRII canonical signaling pathway in HLMVECs. Representative Western blots of phosphorylated (P‐Smad) and total Smad (1/5/8) proteins in control (a) and bmpr2‐silenced HLMVECs (b) incubated with vehicle (VEH), macitentan (MACI), selexipag (SELEX) and tadalafil (TADA) for 1 h. Quantitative expression of phosphorylated versus total Smad1/5/8 proteins in control (c) and bmpr2‐silenced HLMVECs (d). Results are presented as ratio of phosphorylated over total Smad (1/5/8) protein expression. Independent experiments were performed in four replicates for HLMVECs between passages 5 and 8 and expressed as median (25th–75th interquartile range). Comparisons by one‐way ANOVA and post‐hoc Tukey's multiple tests (c, d).

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  Figure 2. Effect of macitentan, selexipag, and tadalafil on BMPRII noncanonical signaling pathway in HLMVECs. Representative Western blots of phosphorylated (P‐p38 MAPK) and total p38MAPK in control (a) and bmpr2‐silenced HLMVECs (b) incubated with vehicle (VEH), macitentan (MACI), selexipag (SELEX) and tadalafil (TADA) for 1 h. Quantitative expression of phosphorylated versus total p38MAPK in control (c) and bmpr2‐silenced HLMVECs (d) in bmpr2‐silenced HLMVECs. Results are presented as ratio of phosphorylated over total p38 MAPK protein expression. Independent experiments were performed in four replicates for HLMVECs between passages 5 and 8 and expressed as median (25th–75th interquartile range). Comparisons by one‐way ANOVA and post‐hoc Tukey's multiple tests (c, d).

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  Figure 3. Effect of macitentan, selexipag, and tadalafil on endothelial barrier function of control (a) and bmpr2‐silenced (b) HLMVECs. Results are expressed as BSA‐FITC leakage (ng/mL/h), measured in four independent experiments performed in triplicate in HLMVECs between passages 5 and 8 and expressed as median (25th–75th interquartile range. Comparisons by one‐way ANOVA and post‐hoc Tukey's multiple tests (a, b). MACI, macitentan; SELEX, selexipag; TADA, tadalafil; VEH, vehicle.

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  Figure 4. Effect of bmpr2 silencing on VE‐cadherin activation. Representative Western blots of phosphorylated VE‐cadherin (P‐VE‐cadh) and VE‐cadherin (VE‐cadh) protein expression. β‐actin was used as loading control (a). Quantification of VE‐cadherin expression (b) and phosphorylated over total VE‐cadherin expression (c) in control and bmpr2‐silenced (bmpr2 ‐) HLMVECs; results are presented as ratio of total VE‐cadherin over β‐actin protein expression (b) or phosphorylated over total VE‐cadherin protein expression (c), measured in four independent experiments. Representative pictures of P‐VE‐cadherin (P‐VE‐cadh) and total VE‐cadherin (VE‐cadh) immunofluorescent staining in control and bmpr2‐silenced (bmpr2 ‐) HLMVECs; nuclei were counterstained using 4’,6‐diamino‐2‐phenylindole (DAPI); Scale = 100 µm (d). Quantification of VE‐cadherin (e) and phosphorylated over total VE‐cadherin (f) immunofluorescent staining in tree independent experiments. Data are expressed as median (25th–75th interquartile range). Comparisons by unpaired Student's t test (b–f).

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  Figure 5. Effect of bmpr2 silencing on in vitro angiogenic capacities of HLMVECs. Migration capacities (a) expressed as migration velocity (µm/h) was evaluated in control and bmpr2‐silenced (bmpr2 ‐) HLMVECs. For tube forming capacity, the number of tubes (b) nodes (c), and meshes (d) was measured in control and bmpr2‐silenced (bmpr2 ‐) HLMVECs. Five independent experiments including one to two replicates were performed in HLMVECs between passages 5 and 8 and expressed as median (25th–75th interquartile range). Comparisons by paired Student's t test (a–d).

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  Figure 6. Effect of macitentan, selexipag, and tadalafil on migration capacities of control and bmpr2‐silenced (bmpr2 ‐) HLMVECs. Representative pictures of wound closure after 8 h are shown.

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  Figure 7. Effect of macitentan, selexipag, and tadalafil on tube forming capacity of control and bmpr2‐silenced (bmpr2 ‐) HLMVECs. Representative pictures of the tubular network formed 4 h after seeding cells on Matrigel© are shown.

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  Figure 8. Effect of macitentan, selexipag, and tadalafil on in vitro angiogenic capacities of control and bmpr2‐silenced HLMVECs. Migration capacities (a, e) was expressed as migration velocity (µm/h). For tube forming capacity, the number of tubes (b, f) nodes (c, g), and meshes (d, h) was measured. Four independent experiments including at least two replicates were performed in bmpr2‐silenced HLMVECs between passages 5 and 8 and expressed as median (25th–75th interquartile range) and whiskers to min and max values. Comparisons by one‐way ANOVA and post‐hoc Tukey's multiple tests (a–h). MACI, macitentan; SELEX, selexipag; TADA, tadalafil; VEH, vehicle.