Nitric oxide may inhibit neointimal hyperplasia by decreasing isopeptidase T levels and activity in the vasculature

Abstract

Objective Isopeptidase T is a cysteine protease deubiquitinating enzyme that hydrolyzes unanchored polyubiquitin chains to free monoubiquitin. Nitric oxide (NO) decreases 26S proteasome activity in vascular smooth muscle cells (VSMCs) and inhibits neointimal hyperplasia in animal models. As NO can cause S-nitrosylation of active-site cysteines, we hypothesize that NO inhibits isopeptidase T activity through S-nitrosylation. Because accumulation of polyubiquitin chains inhibits the 26S proteasome, this may be one mechanism through which NO prevents neointimal hyperplasia. Methods To investigate our hypothesis, we examined the effect of NO on isopeptidase T activity, levels, and localization in VSMCs in vitro and in a rat carotid balloon injury model in vivo. Results NO inhibited recombinant isopeptidase T activity by 82.8% (t = 60 minutes, P < .001 vs control). Dithiothreitol and glutathione (5 mmol/L) both significantly reversed NO-mediated inhibition of isopeptidase T activity (P < .001). NO caused a time-dependent increase in S-nitrosylated isopeptidase T levels in VSMCs, which was reversible with dithiothreitol, indicating that isopeptidase T undergoes reversible S-nitrosylation on exposure to NO in vitro. Although NO did not affect isopeptidase T levels or subcellular localization in VSMCs in vitro, it decreased isopeptidase T levels and increased ubiquitinated proteins after balloon injury in vivo. Conclusions Local administration of NO may prevent neointimal hyperplasia by inhibiting isopeptidase T levels and activity in the vasculature, thereby inhibiting the 26S proteasome in VSMCs. These data provide additional mechanistic insights into the ability of NO to prevent neointimal hyperplasia after vascular interventions. (J Vasc Surg 2013;■:1-8.) Clinical Relevance The 26S proteasome is responsible for degrading polyubiquitinated proteins. Isopeptidase T is a deubiquitinating enzyme that recycles polyubiquitin chains to monoubiquitin. Nitric oxide (NO) decreases formation of neointimal hyperplasia in animal models and decreases 26S proteasome activity in vascular smooth muscle cells. We investigated the effects of NO on isopeptidase T and showed that NO inhibits recombinant isopeptidase T activity, increases S-nitrosylated isopeptidase T levels in vascular smooth muscle cells, and, after balloon injury in vivo, decreases isopeptidase T levels and increases ubiquitinated proteins. Local administration of NO may prevent neointimal hyperplasia by targeting isopeptidase T and inhibiting the 26S proteasome.

Nitric Oxide Prevents Aortic Neointimal Hyperplasia by Controlling Macrophage Polarization

“…26,27 However, no preclinical findings have been successfully implemented to date. Recent contributions elegantly describe the effect of NO in the ubiquitin-proteasome system [28][29][30] and the inhibition of the PDGF-surviving pathway during flowdependent vascular remodeling. 31 Our work is the first to describe the role of NOS3 in the resolution of the immune response elicited in the vessel as result of endothelial denudation, identifying MMP-13 as a new target.…”