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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 superoxide dismutase 2 (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+/- versus wild-type (16-month-old) mice. Aortic SMCs 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 versus aged wild-type and young (4-month-old) SOD2+/- mice. Smooth muscle α-actin levels were increased with age in SOD2+/- versus wild-type SMCs. Aged SOD2+/- SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-α-induced Jun N-terminal kinase 1 activation. Aged SOD2+/- SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/- SMCs., 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.

Atherosclerosis is promoted by a combination of hypercholesterolemia and vascular inflammation. The function of Angiopoietin (Ang)-2, a key regulator of angiogenesis, in the maintenance of large vessels is unknown. A single systemic administration of Ang-2 adenovirus (AdAng-2) to apoE(-/-) mice fed a Western diet significantly reduced atherosclerotic lesion size ( approximately 40%) and oxidized LDL and macrophage content of the plaques. These beneficial effects were abolished by the inhibition of nitric oxide synthase (NOS). In endothelial cells, endothelial NOS activation per se inhibited LDL oxidation and Ang-2 stimulated NO release in a Tie2-dependent manner to decrease LDL oxidation. These findings demonstrate a novel atheroprotective role for Ang-2 when endothelial cell function is compromised and suggest that growth factors, which stimulate NO release without inducing inflammation, could offer atheroprotection.

Objective: Phosphoinositide (PI) 3-kinase promotes vascular smooth muscle cell (VSMC) responses necessary for neointimal hyperplasia. We recently demonstrated that the inositol 3-phosphatase PTEN is expressed in VSMCs and that its overexpression inhibits these cellular responses. The purpose of this study was to determine the effects of adenovirus-mediated overexpression of PTEN on neointimal hyperplasia in vivo in the rat carotid injury model., Methods and Results: Rat carotid arteries were balloon-injured and treated with a recombinant control adenovirus (AdEV) (n=6), an adenovirus encoding wild-type PTEN (AdPTEN) (n=8), or phosphate-buffered saline (sham) (n=5). Injured vessels demonstrated PTEN overexpression by Western blotting and immunohistochemistry after AdPTEN treatment. Neointimal hyperplasia was assessed 2 weeks after balloon injury and adenovirus administration. Compared with controls, AdPTEN treatment significantly decreased neointimal area and percent stenosis. To investigate the mechanisms of action of AdPTEN, vessels were harvested 3 days after balloon injury and virus infection. AdPTEN significantly increased medial cell apoptosis while decreasing proliferation of the remaining viable cells., Conclusions: PTEN overexpression potently inhibits neointimal hyperplasia through induction of apoptosis and inhibition of medial cell proliferation. These findings suggest that modulation of PTEN expression or activity may be a viable approach to treat neointimal hyperplasia. Phosphoinositide (PI) 3-kinase is a critical regulator of neointimal hyperplasia. The inositol 3-phosphatase PTEN modulates PI 3-kinase signaling by hydrolyzing the phospholipid products of PI 3-kinase, and overexpression of PTEN in vascular smooth muscle cells inhibits the cellular processes necessary for neointimal hyperplasia. The effects of adenovirus-mediated PTEN (AdPTEN) overexpression on neointimal hyperplasia were tested in the rat carotid injury model. Compared with control arteries, AdPTEN treatment significantly reduced neointimal area and percent stenosis by enhancing medial cell apoptosis and inhibiting proliferation of the remaining viable cells. Thus, PTEN provides a new target for the treatment of neointimal hyperplasia.

Background: Although endothelial nitric oxide synthase (NOS) is antiatherogenic, the role of inducible NOS (iNOS) in the development of atherosclerosis is not established., Methods and Results: We compared the susceptibility of iNOS knockout (iNOS(-/-)) and wild-type (iNOS(+/+)) mice to the development of atherosclerosis induced by feeding an atherogenic diet for 15 weeks. Plasma lipid level, atherosclerotic lesion size, and cellular density in the lesions were all similar in the 2 strains (lesion size: iNOS(+/+) 285+/-73x10(3) microm(2), iNOS(-/-) 293+/-82x10(3) microm(2), n=10). iNOS mRNA was detected in the lesions of iNOS(+/+) but not iNOS(-/-) mice through RT-PCR. Immunohistochemically, iNOS(+/+) mice showed iNOS staining in macrophages and medial smooth muscle cells in the lesions. Nitrotyrosine staining showed a similar distribution, whereas it was absent in iNOS(-/-) mice. There was no apparent difference in the intensity or distribution of vascular cell adhesion molecule-1 staining in the lesions of the 2 strains. However, the lesions of iNOS(+/+) mice showed a markedly decreased extracellular collagen content compared with those of iNOS(-/-) mice, Conclusions: iNOS induction does not affect the development of atherosclerosis in mice fed an atherogenic diet, but the resulting lesions show decreased levels of extracellular collagen and may be more fragile.