Fig. 1. Global DNA methylation and Dnmt3b are elevated in lungs of PH rat models and pathological up-regulation of Dnmt3b in PAH.

(A) Lungs of rats at week 3 after MCT injection (n = 6) or (B) under hypobaric hypoxia (HX) (n = 6) had higher levels of global DNA methylation [5-methylcytosine% (5-mC%)] than those of saline controls (n = 6) or normobaric normoxic controls (NOR) (n = 3). (C to F) Western blot analysis of Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l relative to Gapdh from PH rat lungs at week 3 after MCT injection or after hypobaric hypoxic exposure compared to its corresponding controls (n = 7 to 8 per group). (G and H) Representative images and quantification analysis of Dnmt3b-positive staining cell percentage showed increased Dnmt3b (brown) expression in the media and endothelium of pulmonary arteries of rats after MCT administration (n = 5) or under hypobaric hypoxic conditions (n = 5) compared to that of control rats (n = 5). Scale bars, 100 μm. (I and J) Quantification analysis and representative images of Dnmt3b-positive staining of four control patients (top three rows) and eight PAH lungs (bottom three rows). Scale bars, 100 μm. (K) DNMT3B mRNA expression in PASMCs from PAH patients or control subjects (n = 5 per group). (L) DNMT3B mRNA expression in PAECs from PAH patients or control subjects (n = 3 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 versus corresponding controls, Student’s t test (A, C, D, F, and I), Mann-Whitney test (K and L), and one-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons (H). Data are presented as the mean ± SEM (D, F, K, and L) or box-and-whisker plots with scatter (A, B, H, and I). n.s., not significant.