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Objective Isopropylamine NONOate (IPA/NO) is a nitroxyl (HNO) donor at physiological pH. HNO is a positive inotrope and vasodilator, but little is known about its effect on neointimal hyperplasia. The aims of this study are to determine the effect of IPA/NO on endothelial and vascular smooth muscle cells (VSMC) in vitro, and to determine if IPA/NO inhibits neointimal hyperplasia in vivo. Methods VSMC were harvested from the abdominal aortas of male Sprague Dawley rats, and human umbilical vein endothelial cells were purchased from ATCC. In vitro, cellular proliferation was assessed by 3H-thymidine incorporation, cell migration was assessed using the scrape assay, and cell death was assessed using Guava Personal Cell Analysis (PCA). Cell cycle analysis was performed using propidium iodide staining and FACS analysis. Protein expression was assessed using Western blot analysis. Phosphorylated proteins were assessed using immunoprecipitation and Western blot analysis. In vivo, the carotid artery injury model was performed on male Sprague Dawley rats treated with (n=12) or without (n=6) periadventitial IPA/NO (10 mg). Arteries harvested at 2 weeks were assessed for morphometrics using ImageJ. Inflammation was assessed using immunohistochemistry. Endothelialization was assessed by Evans blue staining of carotid arteries harvested 7 days after balloon injury from rats treated with (n=6) or without (n=3) periadventitial IPA/NO (10 mg). Results In vitro, 1000 μmol/L IPA/NO inhibited both VSMC (38.7±4.5% inhibition vs. control, P=0.003) and endothelial cell proliferation (54.0±2.9% inhibition vs. control, P=<0.001) without inducing cell death or inhibiting migration. In VSMC, this inhibition was associated with an S-phase cell cycle arrest and increased expression of cyclin A, cyclin D1 and the cyclin-dependent kinase inhibitor p21. No change was noted in the phosphorylation status of cdk2, cdk4, or cdk6 by IPA/NO. In rodents subjected to the carotid artery balloon injury model, IPA/NO caused significant reductions in neointimal area (298±20 vs. 422±30, P=<0.001) and medial area (311±14 vs. 449±16, P=<0.001) compared to injury alone, and reduced macrophage infiltration to 1.7±0.8 from 16.1±3.5 cells per high power field (P=<0.001). IPA/NO also prevented re-endothelialization compared to injury alone (55.9±0.5% non-endothelialized vs. 21±4.4%, respectively, P=0.001). Lastly, a 50% mortality rate was observed in the IPA/NO-treated groups. Conclusions In summary, while IPA/NO modestly inhibited neointimal hyperplasia by inhibiting VSMC proliferation and macrophage infiltration, it also inhibited endothelial cell proliferation and induced significant mortality in our animal model. Since HNO is being investigated as a treatment for congestive heart failure, our results raise some concerns about the use of IPA/NO in the vasculature and suggest that further studies be conducted on the safety of HNO donors in the cardiovascular system.

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.