Abstract 543: cGMP Kinase Phosphorylation of RhoA as a Critical Mechanism for Cardiovascular Fibrosis and Remodeling in Mice
[Introduction] NO/Natriuretic peptides/cGMP and RhoA/Rho-kinase pathways counteract to mediate various cardiovascular functions, such as vascular tone, hypertrophy and fibrosis. We previously showed that cGMP-dependent kinase I (cGK I), the major cGMP effector, prevents RhoA activation by directly phosphorylating it at Ser188. However, the in vivo relevance of this interaction is unknown. We hypothesized that anti-fibrotic and growth inhibitory effect of cGMP pathway is mediated by cGK phosphorylation of RhoA in vivo.
[Methods and Results] Angiotensin II (1500ng/kg/min) provoked a more pronounced effect on cGK I haploinsufficient mice (cGK I(+/−)) than control for coronary artery medial thickening (MT; media/lumen area ratio; 3.67-fold), perivascular fibrosis (PVF; fibrosis/total vascular area; 1.59-fold), and fibrosis-related gene upregulation (CTGF 1.29-fold; fibronectin 1.42-fold; collagen type I 1.48-fold; collagen type III 2.08-fold). Rho-kinase inhibitor Y-27632 (50mg/kg/day) more preferentially inhibited Ang II-induced gene expression in cGK I(+/−) hearts than control without affecting BP. Vascular smooth muscle-specific Tg were generated that express cGK-unphosphorylatable RhoA mutant (RhoA(A188)) or wild type RhoA (RhoA(wt)) by using SM22α promoter. RhoA(wt)-Tg and RhoA(A188)-Tg aortas showed 1.6- and 2.1-fold increase in Rho-kinase activity compared to non-Tg without significant difference in BP. Both Tg showed significantly increased MT (RhoA(wt) 1.67-fold; RhoA(A188) 2.48-fold) and PVF (RhoA (wt) 1.46-fold; RhoA(A188) 1.86-fold) compared to non-Tg. Double Tg for RhoA(wt) and BNP demonstrated substantial reduction in MT (58.1%) and PVF (32.5%) compared to RhoA(wt)-Tg. In contrast, RhoA(A188)/BNP-Tg exhibited less reduction in MT (25.7%) and PVF (18.1%). While RhoA(A188)/BNP-Tg and non-Tg showed 2 to 3-fold upregulation of TGF-β, CTGF, FN, Col I and Col III in response to Ang II, RhoA(wt)/BNP-Tg and BNP-Tg showed almost complete inhibition of this gene induction.
[Conclusion] The present study demonstrates that cGK I phosphorylation and inhibition of RhoA serves as a major mechanism in vivo for the inhibitory effects of NO/natriuretic peptides/cGMP pathway on cardiovascular fibrosis and remodeling.