Objective Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models. Methods and Results Pulse wave velocity, determined using Doppler, increased with age in SOD2+/−, but not in wild-type, p47phox−/− and SOD1+/− mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2+/− vs wild-type (16-month-old) mice. Aortic SMC from aged SOD2+/− mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity and increased sensitivity to staurosporine-induced apoptosis vs aged wild-type and young (4-month-old) SOD2+/− mice. SM α-actin levels were increased with age in SOD2+/− vs wild-type SMC. Aged SOD2+/− SMC had attenuated insulin-like growth factor-1-induced Akt and FoxO3a phosphorylation and prolonged tumornecrosis factor-α-induced Jun N-terminal kinase 1 activation. Aged SOD2+/− SMC had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant negative FoxO3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/− SMC. Conclusion Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part, by inducing vascular wall remodeling, intrinsic changes in SMC stiffness and aortic SMC apoptosis.

Background-Despite a concerted effort by many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homolog of p67phox, in VSMC NADPH oxidase function and atherosclerosis. Methods and Results-The presence of NoxA1 in mouse aortic VSMCs was confirmed by reverse-transcription polymerase chain reaction and sequencing. NoxA1/p47phox interaction after thrombin treatment was observed by immunoprecipitation/ Western analysis of lysates from p47phox Ϫ/Ϫ VSMCs transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with adenoviral NoxA1 significantly enhanced thrombin-induced reactive oxygen species generation in wild-type but not in p47phox Ϫ/Ϫ and Nox1 Ϫ/Ϫ VSMCs. Thrombin-induced reactive oxygen species production and VSMC proliferation were significantly reduced after downregulation of NoxA1 with shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of the redox-sensitive protein kinases (Janus kinase 2, Akt, and p38 mitogen-activated protein kinase) in VSMCs. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMCs and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE Ϫ/Ϫ mice compared with age-matched wild-type mice. Furthermore, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions. Conclusions-NoxA1 is the functional homolog of p67phox in VSMCs that regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases. (Circulation. 2010;121:549-559.)