Abstract 74: Crucial Role of ROCK2 in Vascular Smooth Muscle Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

Abstract

Background We have previously demonstrated that Rho-kinase (ROCK) pathway in vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of cardiovascular disease including pulmonary arterial hypertension (PAH). Rho-kinase has 2 isoforms; ROCK1 for inflammatory cells and ROCK2 for VSMC. We thus examined the specific role of ROCK2 in VSMC in the pathogenesis of PAH. Methods and Results In patients with PAH as compared with controls, the immunoreactivity of ROCK2 and phosphorylated ERK1/2 co-expressed with αSMA in pulmonary arterial media were markedly increased. Thus, we developed VSMC-specific ROCK2-deficient (ROCK2 +/- ) and VSMC-specific ROCK2-overexpressing (ROCK2-Tg) mice. The extent of hypoxia-induced pulmonary hypertension (10% O 2 for 4 weeks), as evaluated by pulmonary vascular remodeling, right ventricular (RV) systolic pressure and RV hypertrophy, was significantly reduced in ROCK2 +/- mice and was enhanced in ROCK2-Tg mice compared with the littermates (n=10 each, all P<0.05). The expression of ROCK activity and phosphorylated ERK1/2 in lung tissues analyzed by Western blotting and the numbers of Ki67 + proliferating VSMC and CD45 + inflammatory cells in pulmonary arterioles revealed by immunostaining were significantly reduced in the ROCK2 +/- mice and were increased in the ROCK2-Tg mice compared with the littermates (n=4 each, all P<0.05). In cultured murine aortic VSMC, both scratch assay and Boyden chamber assay showed that migration activity was significantly reduced in the ROCK2 +/- VSMC and was increased in the ROCK2-Tg VSMC compared with littermates (n=5 each, both P<0.01). In addition, in primary pulmonary arterial VSMC (PASMC) cultured from patients with PAH, siRNA knockdown (-81%) of ROCK2 expression (ROCK2-siRNA) significantly suppressed the expression of ROCK activity and PDGF-induced phosphorylated ERK1/2 (n=4 each, both P<0.05). Compared with control-siRNA, PASMC transfected with ROCK2-siRNA exhibited significant reduction in both migration activity by scratch assay and proliferation activity by growth curve (n=3 each, both P<0.01). Conclusions ROCK2 in VSMC plays a crucial role in the pathogenesis of PH through enhanced VSMC migration and proliferation in mice in vivo.