Nitric oxide inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by increasing the ubiquitination and degradation of UbcH10

Abstract

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.

Figures

Fig. 1

Nitric oxide (NO) reduced levels of ubiquitin-conjugating (E2) enzymes important for cell cycle progression. Vascular smooth muscle cells (VSMC) were treated with the NO donor diethylenetriamine NONOate (DETA/NO, 500–1000 μM), then subjected to western blot analysis using antibodies to the E2 enzymes shown on the left. Equal loading was verified by staining for β-actin. NO caused decreased levels of UbcH3, UbcH10, and UbcH12 (arrows), with the greatest decrease from baseline seen in UbcH10. t = 24 h. Images are representative of three separate experiments

Fig. 2

NO decreased VSMC proliferation and levels of UbcH10 in a concentration-dependent manner. a VSMC treated with DETA/NO (62.5–1000 μM) were subjected to western blot analysis for UbcH10. As seen in Fig. 1, 500 and 1000 μM DETA/NO caused the greatest reduction in UbcH10 levels. NO was also noted to increase polyubiquitinated UbcH10 (Ub-UbcH10). b Proliferation of VSMC was assessed via [3H]-thymidine incorporation. NO treatment decreased VSMC proliferation 59% compared to control, * P = 0.001. c Immunofluorescent staining of VSMC for UbcH10 (red) showed a distinct localization to the cytoplasm in treated and untreated cells. While NO did not alter the subcellular localization of UbcH10, it markedly decreased UbcH10 levels. Nuclei were stained with DAPI (blue). Data shown are representative of three separate experiments. t = 24 h, n = 3/treatment group

Fig. 3

Knockdown of UbcH10 levels decreased VSMC proliferation. a VSMC were transfected with siRNA against UbcH10, a scrambled control, transfection reagent only (Dharmafect), or treated with DETA/NO (500 μM), then subjected to western blot analysis using antibodies to UbcH10 or β-actin. UbcH10 knockdown using siRNA was similar to that observed with DETA/NO. b Proliferation of VSMC transfected as in a was assessed via [3H]-thymidine incorporation. NO treatment and siRNA transfection and NO treatment caused similar decreases in VSMC proliferation (50% and 58% versus control, respectively, * P < 0.001). t = 48 h. Data are representative of three separate experiments

Fig. 4

Increased UbcH10 levels increased VSMC proliferation. a VSMC transfected with a plasmid bearing the gene for UbcH10 (pUbcH10) were subjected to western blot analysis using antibodies to UbcH10 or β-actin. Transfection caused a significant increase in UbcH10 expression over baseline. b Proliferation of VSMC transfected as in a was assessed via [3H]-thymidine incorporation, and showed that pUbcH10 increased proliferation to 150% of control. * P = 0.008, t = 24 h. Data are representative of three separate experiments

Fig. 5

NO inhibited angiotensin II-stimulated VSMC proliferation and increased UbcH10 levels. a VSMC treated with angiotensin II (AngII, 1000 or 5000 nM) to stimulate proliferation were treated with DETA/NO (500 or 1000 μM) and subjected to western blot analysis using antibodies to UbcH10 or β-actin. AngII treatment caused a significant increase in UbcH10 levels, which was abrogated by treatment with NO. b Proliferation of VSMC treated as in a was assessed via [3H]-thymidine incorporation. AngII increased VSMC proliferation to 212% of control, which was substantially decreased upon exposure to DETA/NO. * P = 0.002 vs. control, t = 24 h. Data are representative of three separate experiments

Fig. 6

NO decreased the injury-mediated increase of UbcH10 levels in vivo. Following balloon injury and treatment with or without the nitric oxide donor proline NONOate (PROLI/NO, 20 mg, t = 14 days), rat carotid artery cross sections underwent immunofluorescent staining for UbcH10 levels (red). UbcH10 levels increased following arterial injury, and was significantly decreased by the addition of NO. Nuclei were stained with DAPI (blue). Auto-fluorescence of the internal elastic lamina is also shown (green). Data are representative of three separate stains

Fig. 7

NO reversed the injury-mediated increase in UbcH10 levels and increased polyubiquitinated UbcH10 levels in the carotid artery in vivo. Following balloon injury and treatment with or without PROLI/NO (20 mg, t = 3 days), rat carotid arteries were homogenized and subjected to western blot analysis using antibodies to UbcH10 or β-actin. As seen in VSMC culture, baseline levels of UbcH10 were low (uninjured group), but increased dramatically following balloon injury. Also similar to VSMC culture, treatment with NO almost completely reversed the increase in UbcH10 levels. Interestingly, balloon injury alone increased levels of polyubiquitinated UbcH10, but the addition of NO treatment significantly increased these higher molecular weight bands, indicating that NO may be causing the breakdown of UbcH10 by increasing the polyubiquitination of UbcH10. n = 3 rats/group. Data are representative of three replicates