Abstract 120: Protective Role of Vascular Smooth Muscle Rho-Related BTB Domain Containing Protein 1 in Hypertension and Arterial Stiffness

Abstract

Rho-related BTB domain containing protein 1 (RhoBTB1) is an atypical GTPase associated with human hypertension. RhoBTB1 was decreased in aorta from mice expressing a hypertension (HT)-causing PPARγ mutation (P467L) specifically in vascular smooth muscle (VSM, S-P467L mice). Restoration of VSM RhoBTB1 improved vasodilation, reversed arterial stiffness and HT by suppressing phosphodiesterase 5 (PDE5) activity. Here we sought to reveal the molecular function of RhoBTB1 and study its protective role in Angiotensin II (Ang II) induced hypertension. PDE5 and RhoBTB1 reciprocally co-immunoprecipitated in HEK293 cells; and notably, endogenous Cullin3 from HEK293 cells was also present in RhoBTB1/PDE5 immunoprecipitant. Whereas, RhoBTB1 was not sufficient to inhibit PDE5 activity in vitro (1.9±0.2 vs 1.7 ±0.2 nmol/min/mg, n=3), it was required for PDE5 ubiquitination in HEK293 cells. RhoBTB1-mediated PDE5 ubiquitination was blunted by pan-Cullin inhibitor MLN4924 treatment, and MLN4924 increased PDE5 activity in aorta. Thus, RhoBTB1 serves as a substrate adaptor for Cullin3-Ring ubiquitin ligase (CRL3) complex. Like mutation in PPARγ, Ang II infusion also reduced aortic RhoBTB1 expression. We generated mice with VSM specific, tamoxifen inducible RhoBTB1 (S-RhoBTB1) expression. Activation of RhoBTB1 expression with tamoxifen partially prevented Ang II induced HT and vascular dysfunction. However, activation of the RhoBTB1 transgene after 2-week of Ang II infusion did not reverse established Ang II HT and failed to reverse impaired vasodilation to acetylcholine and sodium nitroprusside in aorta and carotid artery. Remarkably however, arterial stiffness as measured by aortic Pulse Wave Velocity was decreased in S-RhoBTB1 compared to non-transgenic mice receiving Ang II infusion (4.6±0.4 vs 3.2±0.3 mm/ms, p<0.05, n=5-7). In conclusion, RhoBTB1 serves as CRL3 substrate adaptor and promotes PDE5 ubiquitination. Pretreatment with RhoBTB1 partially prevents Ang II HT, while restoration of VSM RhoBTB1 was not sufficient to reverse Ang II mediated HT but showed a potential in reducing arterial stiffness.