The protease inhibition with Ritonavir impairs endothelial function and promotes vascular smooth cell proliferation via RANTES/C‐C chemokine receptor type 5 and Nox1‐derived reactive oxygen species pathway.

Abstract

Thanks to the advent of highly active antiretroviral therapy (HAART), the life of people living with HIV has been extended to close to that of healthy people. However, patients with HIV are now presenting with accelerated development of cardiovascular disease (CVD) and CVD is currently the leading cause of death in patients with HIV on HAART. Herein, we tested the hypothesis that the protease inhibitor ritonavir contributes to HAART‐associated CVD by impairing vascular function via C‐C chemokine receptor type 5 (CCR5)‐ and reactive oxygen species‐mediated mechanisms. We treated 8–10‐week‐old male C57BL6/J with ritonavir for 4 consecutive weeks (5mg/kg/day, i.p). At the end of the treatment, blood was collected and aortas excised to measure H2O2 via Amplex Red and vascular function via wire myography. Aorta from ritonavir treated animals exhibited higher H2O2 levels, impaired relaxation to acetylcholine (Ach) and increased contractility to phenylephrine (Phe). Nox1 inhibition with GKT771, (10μM) fully restored endothelial function in rings from ritonavir treated animals. Ritonavir treatment also elevated RANTES (CCR5 ligand) plasma levels, as well as CCR5, NADPH oxidase 1 (Nox1) and NoxA1 transcript levels in the aorta. No difference was observed for others oxidases, such as Nox2 and Nox4. Repetition of the ritonavir treatment in CCR5 deficient mice revealed that CCR5 deficiency protects vessels from ritonavir induced‐endothelial dysfunction and upregulation of Nox1 and NoxA1 enzymes. To analyze the direct effects of RANTES on the vasculature, thoracic aorta, endothelial, and vascular smooth muscle cells were exposed to RANTES in different times (20ng/mL). RANTES elevated endothelial NOX1 expression and impaired ACh‐mediated relaxation. Concomitantly, Nox1 inhibition prevented RANTES‐mediated endothelial dysfunction. In addition, RANTES time‐dependently stimulated rat aortic smooth muscle cell proliferation, as measured by Ki67 expression, which was partially reverted by Nox1 inhibition. Our data suggest that the protease inhibitor ritonavir contributes to vascular dysfunction and cardiovascular disease by increasing RANTES‐mediated CCR5 activation ultimately leading to increases in Nox1 expression, ROS production, endothelial dysfunction and vascular smooth muscle cell proliferation. Taken together, these findings provide potential explanation for the increased risk of cardiovascular disease developed by patients living with HIV and present CCR5 as a potential target for treatment.