Figure 4.

SIRT7 regulates PAEC function via KLF4. (A) Immunoblotting analysis of KLF4 protein levels in PAECs isolated from IPAH patients (IPAH, n = 4) and control individuals (Ctrl, n = 4). (B) PAECs were transfected with siRNA against SIRT7 (siS7, 20 nM) or control siRNA (siNC, 20 nM) for 48 h. Protein levels of SIRT7 and KLF4 were detected by western blotting analysis, and RNA levels of KLF4 were measured by real-time qPCR. (C) PAECs were infected with SIRT7 lentivirus (S7) or control lentivirus (Ctrl) for 48 h. Protein levels of SIRT7 and KLF4 were detected by Western blotting analysis, and KLF4 mRNA level was measured by real-time-qPCR. (D–F) PAECs were transfected with control siRNA (siNC, 20 nM) or siSIRT7 (siS7, 20 nM) for 24 h, then transfected with KLF4 plasmid (KLF4, 2 µg/mL) or vehicle plasmid (2 µg/mL) for 48 h. Proliferation of PAECs was measured by EdU incorporation (D); migration of PAECs was detected by wound healing assays (E); and angiogenesis ability of PAECs was measured by tube formation assays (F). Scale bar = 20 µm (F). (G, H) Eight-week-old Sirt7^f/f; Cdh5-Cre^ERT mice challenged with Tx or vehicle (Veh) were exposed to normoxia (Nor) or SuHx model for 4 weeks. (G) Western blotting analysis of Sirt7, Klf4, eNOS, and Bmpr2 protein levels, and (H) qPCR analysis of Sirt7, Klf4, Ctgf, and Bmpr2 mRNA levels in lung tissues of indicated groups of mice. Data are means ± SEM. Four independent experiments in (B, C) and five independent experiments in (D–F) were performed. Data are of six mice per group (G, H). Data were analysed by Mann–Whitney U test (A–C), and by Kruskal–Wallis test with Dunn post-hoc test in (D–F). Data were analysed by two-way ANOVA with Holm-Šídák's post-hoc test (G, H). *P < 0.05, **P < 0.01, ***P < 0.001 with comparisons indicated by lines.