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Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10

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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.

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Acknowledgments

The authors would like to express their thanks to the Northwestern University Institute for BioNanotechnology in Medicine, the Northwestern University Feinberg Cardiovascular Research Institute, to Lynnette Dangerfield for her administrative support, and to Edwards Lifesciences for providing the Fogarty balloon catheters. This work was supported in part by funding from the National Institutes of Health (1K08HL0842-03 to MRK), the American Vascular Association (Mentored Clinical Scientist Development Award to MRK), the American Heart Association (0725766Z and 09POST2230028 to NDT), and by the generosity of Mrs. Hilda Rosenbloom and Mrs. Eleanor Baldwin. In addition, part of this research was supported with federal funds from the National Cancer Institute, NIH, under Contract N01-CO-2008-00001 with SAIC-Frederick, Inc. and by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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Authors and Affiliations

  1. Division of Vascular Surgery, Northwestern University, 676 N. St Clair, #650, Chicago, IL, 60611, USA

    Nick D. Tsihlis, Chris S. Oustwani, Ashley K. Vavra, Qun Jiang & Melina R. Kibbe

  2. Laboratory for Comparative Carcinogenesis/Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD, USA

    Larry K. Keefer

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  1. Nick D. Tsihlis
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Correspondence to Melina R. Kibbe.

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Tsihlis, N.D., Oustwani, C.S., Vavra, A.K. et al. Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10. Cell Biochem Biophys 60, 89–97 (2011). https://doi.org/10.1007/s12013-011-9179-3

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