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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.

Objective— In mice that are heterozygous for mitochondrial superoxide dismutase (SOD2 +/− ) with apoE deficiency (apoE −/− ), mitochondrial DNA damage increases formation of atherosclerotic lesions. The purpose of this study was to determine whether SOD2 provides protection against increased vascular superoxide and endothelial dysfunction in apoE-deficient mice. Methods and Results— Four groups of mice [apoE −/− /SOD2 +/− ( apoe / sod2 ), apoE −/− /SOD2 +/+ ( apoe / SOD2 ), apoE +/+ /SOD2 +/− ( apoE / sod2 ), and apoE +/+ /SOD2 +/+ ( apoE / SOD2 )] were fed normal chow diet, and studied at 15 to 17 months of age. Serum cholesterol levels were similar in apoe / sod2 and apoe / SOD2 mice, and also were similar in apoE / sod2 and apoE / SOD2 mice. Intimal area was increased in aorta, but not carotid artery, of apoe / sod2 and apoe / SOD2 mice. In carotid artery, superoxide was increased (67±5.2 relative fluorescence intensity/vessel area [RI] in apoe/sod2 mice, 31±3.1 RI in apoE / SOD2 mice, P apoe / sod2 mice versus apoe / SOD2, apoE/sod2, apoE/SOD2 mice. Tiron improved relaxation to acetylcholine. In aorta, superoxide levels were increased and relaxation to acetylcholine was impaired in apoe/sod2 and apoe/SOD2 mice, but responses were similar in apoe / sod2 and apoe / SOD2 mice. Conclusion— SOD2 protects against oxidative stress and endothelial dysfunction in carotid artery of apoE-deficient mice.

Growing evidence indicates that chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions is integral in the development of cardiovascular diseases (CVD). These ROS can be released from nicotinamide adenine dinucleotide (phosphate) oxidase, xanthine oxidase, lipoxygenase, mitochondria, or the uncoupling of nitric oxide synthase in vascular cells. ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis: from the initiation of fatty streak development through lesion progress to ultimate plaque rupture. Various animal models of oxidative stress support the notion that ROS have a causal role in atherosclerosis and other cardiovascular diseases. Human investigations also support the oxidative stress hypothesis of atherosclerosis. Oxidative stress is the unifying mechanism for many CVD risk factors, which additionally supports its central role in CVD. Despite the demonstrated role of antioxidants in cellular and animal studies, the ineffectiveness of antioxidants in reducing cardiovascular death and morbidity in clinical trials has led many investigators to question the importance of oxidative stress in human atherosclerosis. Others have argued that the prime factor for the mixed outcomes from using antioxidants to prevent CVD may be the lack of specific and sensitive biomarkers by which to assess the oxidative stress phenotypes underlying CVD. A better understanding of the complexity of cellular redox reactions, development of a new class of antioxidants targeted to specific subcellular locales, and the phenotype-genotype linkage analysis for oxidative stress will likely be avenues for future research in this area as we move toward the broader use of pharmacological and regenerative therapies in the treatment and prevention of CVD.

The widespread use of nucleoside analog reverse transcriptase inhibitors (NRTIs) and HIV protease inhibitors (PIs) in Western countries has substantially reduced morbidity and mortality in patients with HIV infection. Concomitantly, however, adverse effects associated with long-term use of these agents are becoming recognized. A growing body of literature suggests that many adverse effects associated with the use of NRTIs such as lactic acidosis, hepatic steatosis, myopathy, cardiomyopathy, peripheral neuropathy, pancreatitis and lipodystrophy syndrome are due to mitochondrial toxicity.1–4⇓⇓⇓ In contrast, the adverse effects associated with the use of PIs, hyperlipidemia, lipodystrophy,5 and perhaps, the resulting accelerated atherosclerosis,6,7⇓ have not been attributed to mitochondrial toxicity. In the October 2002 issue of Atherosclerosis, Thrombosis and Vascular Biology , Zhong et al8 challenge this paradigm by demonstrating PI-mediated mitochondrial dysfunction in endothelial cells and the resultant apoptosis-independent cytotoxicity and suggest that PI-induced endothelial cell toxicity contributes to accelerated atherosclerosis in HIV patients. See page 1560 Lipoprotein oxidation is a critical step in the initiation of atherosclerosis.9 Lipoproteins undergo oxidation by endothelial cells during transport from the plasma into the arterial wall.10 A clear association has been established between elevated serum LDL cholesterol levels and increased atherosclerotic disease.11 In addition, the extent of oxidation of LDL cholesterol impacts its atherogenic potency.12 Reduction of LDL cholesterol by diet, exercise,13 and/or pharmacologic agents reduces atherosclerotic risk.14 These data suggest that the accelerated atherosclerosis in HIV patients treated with PIs is due to the PI-induced hyperlipidemia. Lipodystrophy and hyperlipidemia can occur under conditions that affect mitochondrial integrity and function.15 Fatty acids are derived from hydrolysis of triglycerides and are removed from blood into mitochondria. In the mitochondrial matrix, fatty acids are degraded by oxidation at the β-carbon atom yielding ATP, in a …

Objective—We previously showed that NAD(P)H oxidase deficiency significantly reduces atherosclerosis in apoE−/−mice. The present study was designed to determine the relative contribution of monocyte/macrophage versus vascular wall cell NAD(P)H oxidase to atherogenesis in this model.Methods and Results—Cell-specific NAD(P)H oxidase inhibition was achieved via allogenic, sex-mismatched bone marrow transplantation. Aortic atherosclerosis and superoxide production in apoE−/−mice (Control) with functional NAD(P)H oxidase in both monocytes/macrophages and vascular wall cells was compared with that in apoE−/−mice with nonfunctional monocyte/macrophage NAD(P)H oxidase (BMO) or nonfunctional vessel wall NAD(P)H oxidase (VWO). A significant decrease in superoxide production and atherosclerotic lesions was observed in BMO and VWO mice compared with control mice. Interestingly, BMO mice had significantly lower plasma oxidized LDL levels compared with control and VWO mice, whereas aortic sections of VWO mice showed decreased expression of cellular adhesion molecules compared with control and BMO mice. NAD(P)H oxidase deficiency also attenuated neointimal hyperplasia and mitogenic protein activation in apoE−/−mice after arterial injury.Conclusions—We conclude that (1) both monocyte/macrophages and vessel wall cells play critical roles in atherogenesis; (2) decrease in atherosclerosis results from attenuated superoxide generation in monocyte/macrophages or vessel wall cells; and (3) superoxide generation may impact atherosclerosis, in part, by activating smooth muscle cell mitogenic signaling pathways.

Objective— Reactive oxygen species (ROS) integrate cellular signaling pathways involved in aortic smooth muscle cell (SMC) proliferation and migration associated with atherosclerosis. However, the effect of subcellular localization of ROS on SMC mitogenic signaling is not yet fully understood. Methods and Results— We used superoxide dismutase (SOD)–deficient mouse aortic SMCs to address the role of subcellular ROS localization on SMC phenotype and mitogenic signaling. Compared with wild-type, a 54% decrease in total SOD activity (≈50% decrease in SOD1 protein levels) and a 42% reduction in SOD2 activity (≈50% decrease in SOD2 protein levels) were observed in SOD1 +/− and SOD2 +/− SMCs, respectively. Consistent with this, basal and thrombin-induced superoxide levels increased in these SMCs. SOD1 +/− and SOD2 +/− SMCs exhibit increased basal proliferation and enhanced [ 3 H]-thymidine and [ 3 H]-leucine incorporation in basal and thrombin-stimulated conditions. Our results indicate preferential activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases in SOD1 +/− and janus kinase/signal transducer and activator of transcriptase (JAK/STAT) pathway in SOD2 +/− SMCs. Pharmacological inhibitors of ERK1/2 p38 and JAK2 confirm the SOD genotype-dependent SMC proliferation. Conclusions— Our results suggest that SOD1 and SOD2 regulate SMC quiescence by suppressing divergent mitogenic signaling pathways, and dysregulation of these enzymes under pathophysiological conditions may lead to SMC hyperplasia and hypertrophy.

Abstract —Reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) activate intracellular signal transduction pathways implicated in the pathogenesis of cardiovascular disease. H 2 O 2 is a mitogen for rat vascular smooth muscle cells (VSMCs), and protein tyrosine phosphorylation is a critical event in VSMC mitogenesis. Therefore, we investigated whether the mitogenic effects of H 2 O 2 , such as stimulation of extracellular signal–regulated kinase (ERK)2, are mediated via activation of cytoplasmic Janus tyrosine kinases (JAKs). JAK2 was activated rapidly in VSMCs treated with H 2 O 2 , and signal transducers and activators of transcription (STAT) STAT1 and STAT3 were tyrosine-phosphorylated and translocated to the nucleus in a JAK2-dependent manner. Inhibition of JAK2 activity with AG-490 partially inhibited H 2 O 2 -induced ERK2 activity, suggesting that JAK2 is upstream of the Ras/Raf/mitogen-activated protein kinase–ERK/ERK mitogenic pathway. Because heat-shock proteins (HSPs) can protect cells from ROS, we investigated the effect of H 2 O 2 on HSP expression. H 2 O 2 stimulated HSP70 expression in a time-dependent manner, and AG-490 abolished H 2 O 2 -induced HSP70 expression. H 2 O 2 activated the HSP70 promoter via enhanced binding of STATs to cognate binding sites in the promoter. Regulation of chaperones such as HSP70 via activation of the JAK/STAT pathway suggests that in addition to its growth-promoting effects, this pathway may help VSMCs adapt to oxidative stress.

Abstract —Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis, restenosis, and hypertension. Although multiple mediators of VSMC growth have been identified, few effective pharmacological tools have been developed to limit such growth. Recent evidence indicating an important role for oxidative stress in cell growth led us to investigate the potential role of aldose reductase (AR) in the proliferation of VSMCs. Because AR catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, we hypothesized that it might be a potential regulator of redox changes that accompany VSMC growth. Herein we report several lines of evidence suggesting that AR facilitates/mediates VSMC growth. Stimulation of human aortic SMCs in culture with mitogenic concentrations of serum, thrombin, basic fibroblast growth factor, and the lipid peroxidation product 4-hydroxy- trans -2-nonenal (HNE) led to a 2- to 4-fold increase in the steady-state levels of AR mRNA, a 4- to 7-fold increase in AR protein, and a 2- to 3-fold increase in its catalytic activity. Inhibition of the enzyme by sorbinil or tolrestat diminished mitogen-induced DNA synthesis and cell proliferation. In parallel experiments, the extent of reduction of the glutathione conjugate of HNE to glutathionyl-1,4-dihydroxynonene in HNE-exposed VSMCs was decreased by serum starvation or sorbinil. Immunohistochemical staining of cross sections from balloon-injured rat carotid arteries showed increased expression of AR protein associated with the neointima. The media of injured or uninjured arteries demonstrated no significant staining. Compared with untreated animals, rats fed sorbinil (40 mg · kg −1 · d −1 ) displayed a 51% and a 58% reduction in the ratio of neointima to the media at 10 and 21 days, respectively, after balloon injury. Taken together, these findings suggest that AR is upregulated during growth and that this upregulation facilitates growth by enhancing the metabolism of secondary products of reactive oxygen species.

Angiotensin II (Ang II) is the dominant effector of the renin-angiotensin system. In addition to its well-known hemodynamic and endocrine effects, Ang II regulates the cardiovascular remodeling associated with hypertension, atherosclerosis, heart failure, and diabetes mellitus. The importance of Ang II in cardiovascular disease states is highlighted by the prominent role that ACE inhibitors and angiotensin receptor blockers play in cardiovascular medicine. These drugs reduce clinical events and improve survival in patients with vascular disease or congestive heart failure and are among the most widely used medications in the world.1 2 There is accumulating evidence that Ang II has direct effects on smooth muscle cells (SMCs) that contribute to abnormalities ranging from subtle vascular dysfunction to severe atherosclerosis, ischemia, and necrosis. Ang II stimulates proliferation, hypertrophy, and migration of cultured vascular SMCs (VSMCs) via binding to the angiotensin type 1 (AT1) receptor.3 This receptor is a member of the superfamily of 7 transmembrane–spanning, G protein–coupled cell surface receptors (GPCRs). Traditionally, these receptors were thought to elicit intracellular signaling solely via activation of heterotrimeric G proteins, …

This issue of Arteriosclerosis, Thrombosis, and Vascular Biology includes an elegant review titled “Vascular Inflammation and the Renin-Angiotensin System” by Brasier et al.1 This is the first in a series of articles on extracellular mediators in atherosclerosis and thrombosis. The articles in this series will focus on both molecular mediators and cellular effectors that modulate vascular function and lead to …