Abstract 13743: Upregulation of Nox4 by TGFβ1 Oxidizes SERCA the C674 Thiol and Inhibits Nitric Oxide in Arterial Smooth Muscle of the Prediabetic Zucker Rat
Background: Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by Type 2 diabetes mellitus.
Methods and Results: By using a prediabetic model, the obese Zucker rat (ZO), we assessed the mechanism by which increased oxidants cause abnormal vascular SMC migration. In culture, ZO aortic SMCs showed a 2-fold increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL), corresponding to a 2-fold increase in oxidants (n=4–7). Immuno-blotting for oxidation of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) cysteine-674 (C674-SO3H) showed significant site-specific irreversible oxidation of the reactive C674 thiol. Unlike ZL, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO, and overexpression of SERCA2b wild type, but not a C674S mutant SERCA, restored the response to NO (n=10). Knockdown of Nox4 with small interference RNA decreased SERCA oxidation and restored NO response in ZO SMCs (n=4–6). In addition, transforming growth factor β1 via smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the anti-migratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO3H were significantly increased in ZO aorta. After common carotid artery balloon injury, infection of SERCA wild type significantly decreased injury-induced neointima (SERCA: 0.24±0.07 vs lacZ: 1.72±0.39, n=7). In addition, knockdown of Nox4 by adenoviral Nox4 short hairpin (sh) RNA decreased Nox4 and SERCA C674-SO3H and significantly decreased injury-induced neointima in ZO (Nox4 shRNA: 0.15±0.08 vs GFP: 1.22±0.42, n=5).
Conclusions: The upregulation of Nox4 by transforming growth factor β1 is responsible for impaired NO responsiveness in ZO SMCs because of oxidation of SERCA C674. Overexpression of SERCA2b wild type may decrease injury-induced neointima by providing sufficient reduced SERCA C674 thiol to maintain the NO response. Knockdown of Nox4 inhibits oxidation of the SERCA C674 thiol, and may thereby inhibit neointima after ZO common carotid artery injury by maintaining NO responsiveness.
- © 2010 by American Heart Association, Inc.