Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.

Introduction Proteins are targeted for degradation by the addition of a polyubiquitin chain. Chains of ubiquitin linked via lysine 48 (K48) are associated with protein degradation while chains linked via lysine 63 (K63) are associated with intracellular signaling. We have previously shown that nitric oxide (NO) inhibits neointimal hyperplasia in association with increasing the ubiquitination and degradation of UbcH10. The aim of this study is to characterize the effect of arterial injury and NO on K48- or K63-linked ubiquitination of cellular proteins. Methods The rat carotid artery balloon injury model was performed. Treatment groups included control, injury, injury+proline NONOate (PROLI/NO), and PROLI/NO alone. Arteries were harvested at designated time points and sectioned for immunohistochemical analysis of K48- and K63-linked ubiquitination or homogenized for protein analysis. Vascular smooth muscle cells (VSMC) harvested from rat aortae were exposed to the NO donor diethylenetriamine NONOate (DETA/NO). Protein expression was determined by Western blot analysis, or immunoprecipitation and Western blot analysis. Results Arterial injury increased K48-linked ubiquitination in vivo. The addition of PROLI/NO following injury caused a further increase in K48-linked ubiquitination at 1 and 3 days, however, levels returned to that of injury alone by 2 weeks. Interestingly, treatment with PROLI/NO alone increased K48-linked ubiquitination in vivo to levels similar to injury alone. There were minimal changes in K63-linked ubiquitination in all three treatment groups. DETA/NO increased K48-linked ubiquitination in VSMC in vitro but had minimal effects on K63-linked ubiquitination. Low doses of DETA/NO decreased K48-linked ubiquitination of cyclin A and B, while high doses of DETA/NO increased K48-linked ubiquitination of cyclin A and B. Minimal changes were seen in K63-linked ubiquitination of cyclin A and B in vitro. Conclusions Arterial injury and NO increased K48-linked ubiquitination in vivo and in vitro. Remarkably, minimal changes were seen in K63-linked ubiquitination. These novel findings provide important insights into the vascular biology of arterial injury and suggest that one mechanism by which NO may prevent neointimal hyperplasia is through regulation of protein ubiquitination.

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