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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.

The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.

Cyclooxygenase (COX)-derived prostaglandins and cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids are important regulators of inflammation; however, functional interactions between these pathways in the regulation of vascular inflammation in vivo have not been studied. We investigated the relative and additive effects of endothelial CYP2J2 overexpression (Tie2-CYP2J2-Tr), global sEH disruption (Ephx2(-/-)), and pharmacologic COX inhibition with indomethacin on the acute vascular inflammatory response to endotoxin in mice. Compared to vehicle-treated wild-type C57BL/6 controls, induction of myeloperoxidase (MPO) activity in lung and liver was similarly attenuated in Tie2-CYP2J2-Tr mice, Ephx2(-/-) mice and wild-type mice treated with moderate dose indomethacin. Dual modulation of both pathways, however, did not produce an additive anti-inflammatory effect. These findings demonstrate that both COX and CYP epoxygenase-mediated eicosanoid metabolism are important regulators of the acute vascular inflammatory response in vivo, and suggest that the anti-inflammatory effects of modulating each pathway may be mediated, at least in part, by overlapping mechanisms.