Fig. 7: Delivering Foxf1 to pulmonary endothelial cells of EC-Bmpr2-/- mice restores angiogenesis and reverses persistent pulmonary hypertension.
From: Reduced FOXF1 links unrepaired DNA damage to pulmonary arterial hypertension
a Experimental design: AAV carrying Foxf1 (AAV-Foxf1) or luciferase (AAV-Con) as control were administered via tail vein injection to EC-Bmpr2-/- or control mice following 3 weeks in hypoxia (10% O2). The mice were then returned to room air for 4 weeks. Schema created with BioRender.com. b In vivo luciferase activity was measured in EC-Bmpr2-/- and control mice by LagoX in vivo imaging tool as described in the “Methods”, at 4 and 7 weeks (1 and 4 weeks after AAV injection). c Immunohistochemistry showing luciferase expression colocalized with MECA, a pan-EC marker, and with tdTomato-positive cells in a pulmonary artery of EC-Bmpr2-/- mice treated with AAV-luciferase for four weeks. Three lung sections were analyzed in n = 2 mice/group, with approximately 20 vessels observed. Scale bar, 20 µm. d Right ventricular systolic pressure (RVSP), right ventricular hypertrophy, the weight of right ventricle relative to the left ventricle+septum (RV/LV + S), number of vessels per 100 alveoli and the percent of fully or partially muscularized pulmonary arteries in male EC-Bmpr2-/- and control mice treated with AAV-Con or AAV-Foxf1. Control mice with AAV-Con, n = 7; EC-Bmpr2-/- mice with AAV-Con, control mice with AAV-Foxf1, and EC-Bmpr2-/- mice with AAV-Foxf1, n = 8. e, f FOXF1 (e) and γH2AX (f) immunohistochemistry of pulmonary arteries of EC-Bmpr2-/- and control mice treated with AAV-Con or AAV-Foxf1 following reoxygenation. The average FOXF1 intensity and number of γH2AX foci were quantified in 5 vessels per mouse, in n = 4 mice per group. In EC-Bmpr2-/- mice treated with AAV-Foxf1, FOXF1 levels were restored to those of control mice, and γH2AX immunofluorescence foci number decreased to the level in control mice. Scale bar, 20 µm. The bottom panels in (f) show a magnified merged image of the area delineated by the dotted line (f). Scale bar, 5 µm. g Immunoblot and densitometry of FOXF1, CLDN5, VEGFR2, P53 and ATM in pulmonary EC in EC-Bmpr2-/- and control mice after AAV-Foxf1 or AAV-Con treatment. n = 3 biological replicates. d–g Bars represent mean ± S.E.M. P values determined by 2-way ANOVA with Holm-Sidak posthoc test. ns, not significant. Source data are provided as a Source Data file.
