Upregulation of Nox4 by TGF{beta}1 oxidizes SERCA and inhibits NO 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 diabetes mellitus.


OBJECTIVE
To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied.


RESULTS
In culture, ZO aortic SMCs showed a significant increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL). The sarco-/endoplasmic reticulum Ca(2+) ATPase (SERCA) nitrotyrosine-294,295 and cysteine-674 (C674)-SO(3)H were increased in ZO SMCs, indicating oxidant stress. Unlike ZL SMC, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO. Transfection of Nox4 small interference RNA or overexpression of SERCA2b wild type, but not C674S mutant SERCA, restored the response to NO. Knockdown of Nox4 also decreased SERCA oxidation in ZO SMCs. In addition, transforming growth factor-β1 via Smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the antimigratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO(3)H were significantly increased in ZO aorta. After common carotid artery injury, knockdown of Nox4 by adenoviral Nox4 short hairpin RNA decreased Nox4 and SERCA C674-SO(3)H staining and significantly decreased injury-induced neointima.


CONCLUSIONS
These studies indicate that the upregulation of Nox4 by transforming growth factor-β1 in ZO SMCs is responsible for the impaired response to NO by a mechanism involving the oxidation of SERCA C674. Knockdown of Nox4 inhibits oxidation of SERCA, as well as neointima formation, after ZO common carotid artery injury.
Tong
Xiaoyong
X
Vascular Biology Unit, X720, 650 Albany St, Boston, MA 02118, USA. xytong@bu.edu
Hou
Xiuyun
X
Jourd'heuil
David
D
Weisbrod
Robert M
RM
Cohen
Richard A
RA
D000818:Animals; D001011:Aorta / Q000166:cytology; D020212:Carotid Artery Injuries / Q000378:metabolism / Q000503:physiopathology*; D017536:Carotid Artery, Common / Q000503:physiopathology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009765:Obesity / Q000378:metabolism / Q000503:physiopathology; D018384:Oxidative Stress / Q000502:physiology; D011236:Prediabetic State / Q000378:metabolism / Q000503:physiopathology*; D051381:Rats; D011924:Rats, Zucker; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology*; D017539:Tunica Intima / Q000166:cytology / Q000502:physiology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
C506948:Atp2a2 protein, rat; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D053773:Transforming Growth Factor beta1; D009569:Nitric Oxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases