Increasing the level of miR-204 by nebulization reverses MCT-PAH. (A) miR-204 restoration decreases mean PA pressure. Mean PA pressure measured by right catheterization in closed chest rats (n = 5 rats per group) is shown. miR-204 antagomir (Inh) and mimic along with appropriate controls (Ctrl) as indicated were intratracheally nebulized after 2 wk of MCT injection. (B) miR-204 restoration decreases PA wall thickness. PA remodeling was measured by the percentage of media wall thickness on lung sections stained by hematoxylin and eosin (n = 5 measurements/rat in 10 rats). The pictures shown illustrate representative distal arteries, and the graph represents mean values. (C) By decreasing SHP2, STAT3, and NFAT activation, miR-204 restoration increases apoptosis and decreases proliferation in MCT-rat PASMCs. SHP2 protein expression (left) was quantified in distal PAs by immunofluorescence (F.U., fluorescent unit) on lung sections; STAT3 and NFAT activation (middle) measured by the percentage of cells presenting p-STAT3 and NFAT nuclear localization, respectively, in distal PAs of rats; apoptosis and proliferation measured by the percentage of cells presenting TUNEL and PCNA nuclear localization, respectively, in distal PAs of rats. Control and miR-204 mimic were intratracheally nebulized after 2 wk of MCT injection as indicated (n = 5 measurements by rat in five rats per group). Graphs represent means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).