Marschall S. Runge

Oxidative Stress and Vascular Disease

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.

p47phox is required for atherosclerotic lesion progression in ApoE–/– mice

NADPH oxidase is upregulated in smooth muscle cells (SMCs) in response to growth factor stimulation, concomitant with increased reactive oxygen species (ROS) production. We investigated the role of ROS production by NADPH oxidase in SMC responses to growth factors and in atherosclerotic lesion formation in ApoE(-/-) mice. SMCs from wild-type, p47phox(-/-), and gp91phox(-/-) mice differed markedly with respect to growth factor responsiveness and ROS generation. p47phox(-/-) SMCs had diminished superoxide production and a decreased proliferative response to growth factors compared with wild-type cells, whereas the response of gp91phox(-/-) SMCs was indistinguishable from that of wild-type SMCs. The relevance of these in vitro observations was tested by measuring atherosclerotic lesion formation in genetically modified (wild-type, p47phox(-/-), ApoE(-/-), and ApoE(-/-)/p47phox(-/-)) mice. ApoE(-/-)/p47phox(-/-) mice had less total lesion area than ApoE(-/-) mice, regardless of whether mice were fed standard chow or a high-fat diet. Together, these studies provide convincing support for the hypothesis that superoxide generation in general, and NADPH oxidase in particular, have a requisite role in atherosclerotic lesion formation, and they provide a rationale for further studies to dissect the contributions of ROS to vascular lesion formation.

Mitochondrial Dysfunction in Atherosclerosis

Increased production of reactive oxygen species in mitochondria, accumulation of mitochondrial DNA damage, and progressive respiratory chain dysfunction are associated with atherosclerosis or cardiomyopathy in human investigations and animal models of oxidative stress. Moreover, major precursors of atherosclerosis—hypercholesterolemia, hyperglycemia, hypertriglyceridemia, and even the process of aging—all induce mitochondrial dysfunction. Chronic overproduction of mitochondrial reactive oxygen species leads to destruction of pancreatic β-cells, increased oxidation of low-density lipoprotein and dysfunction of endothelial cells—factors that promote atherosclerosis. An additional mechanism by which impaired mitochondrial integrity predisposes to clinical manifestations of vascular diseases relates to vascular cell growth. Mitochondrial function is required for normal vascular cell growth and function. Mitochondrial dysfunction can result in apoptosis, favoring plaque rupture. Subclinical episodes of plaque rupture accelerate the progression of hemodynamically significant atherosclerotic lesions. Flow-limiting plaque rupture can result in myocardial infarction, stroke, and ischemic/reperfusion damage. Much of what is known on reactive oxygen species generation and modulation comes from studies in cultured cells and animal models. In this review, we have focused on linking this large body of literature to the clinical syndromes that predispose humans to atherosclerosis and its complications.

Hydrogen Peroxide– and Peroxynitrite-Induced Mitochondrial DNA Damage and Dysfunction in Vascular Endothelial and Smooth Muscle Cells

The mechanisms by which reactive species (RS) participate in the development of atherosclerosis remain incompletely understood. The present study was designed to test the hypothesis that RS produced in the vascular environment cause mitochondrial damage and dysfunction in vitro and, thus, may contribute to the initiating events of atherogenesis. DNA damage was assessed in vascular cells exposed to superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite. In both vascular endothelial and smooth muscle cells, the mitochondrial DNA (mtDNA) was preferentially damaged relative to the transcriptionally inactive nuclear beta-globin gene. Similarly, a dose-dependent decrease in mtDNA-encoded mRNA transcripts was associated with RS treatment. Mitochondrial protein synthesis was also inhibited in a dose-dependent manner by ONOO(-), resulting in decreased cellular ATP levels and mitochondrial redox function. Overall, endothelial cells were more sensitive to RS-mediated damage than were smooth muscle cells. Together, these data link RS-mediated mtDNA damage, altered gene expression, and mitochondrial dysfunction in cell culture and reveal how RS may mediate vascular cell dysfunction in the setting of atherogenesis.

Mitochondrial Integrity and Function in Atherogenesis

Background—Coronary atherosclerotic disease remains the leading cause of death in the Western world. Although the exact sequence of events in this process is controversial, reactive oxygen and nitrogen species (RS) likely play an important role in vascular cell dysfunction and atherogenesis. Oxidative damage to the mitochondrial genome with resultant mitochondrial dysfunction is an important consequence of increased intracellular RS. Methods and Results—We examined the contribution of mitochondrial oxidant generation and DNA damage to the progression of atherosclerotic lesions in human arterial specimens and atherosclerosis-prone mice. Mitochondrial DNA damage not only correlated with the extent of atherosclerosis in human specimens and aortas from apolipoprotein E−/− mice but also preceded atherogenesis in young apolipoprotein E−/− mice. Apolipoprotein E−/− mice deficient in manganese superoxide dismutase, a mitochondrial antioxidant enzyme, exhibited early increases in mitochondrial DNA damage and a phenotype of accelerated atherogenesis at arterial branch points. Conclusions—Mitochondrial DNA damage may result from RS production in vascular tissues and may in turn be an early event in the initiation of atherosclerotic lesions.

Angiotensin II Induces Interleukin-6 Transcription in Vascular Smooth Muscle Cells Through Pleiotropic Activation of Nuclear Factor-κB Transcription Factors

Interleukin-6 (IL-6) is a multifunctional cytokine expressed by angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs) that functions as an autocrine growth factor. In this study, we analyze the mechanism for Ang II-inducible IL-6 expression in quiescent rat VSMCs. Stimulation with the Ang II agonist Sar1 Ang II (100 nmol/L) induced transcriptional expression of IL-6 mRNA transcripts of 1.8 and 2.4 kb. In transient transfection assays of IL-6 promoter/luciferase reporter plasmids, Sar1 Ang II treatment induced IL-6 transcription in a manner completely dependent on the nuclear factor-kappaB (NF-kappaB) motif. Sar1 Ang II induced cytoplasmic-to-nuclear translocation of the NF-kappaB subunits Rel A and NF-kappaB1 with parallel changes in DNA-binding activity in a biphasic manner, which produced an early peak at 15 minutes followed by a nadir 1 to 6 hours later and a later peak at 24 hours. The early phase of NF-kappaB translocation was dependent on weak simultaneous proteolysis of the IkappaBalpha and beta inhibitors, whereas later translocation was associated with enhanced processing of the p105 precursor into the mature 50-kDa NF-kappaB1 form. Pretreatment with a potent inhibitor of IkappaBalpha proteolysis, TPCK, completely blocked Sar1 Ang IIAng II-induced NF-kappaB activation and induction of endogenous IL-6 gene expression, which indicated the essential role of NF-kappaB in mediating IL-6 expression. We conclude that Ang II is a pleiotropic regulator of the NF-kappaB transcription factor family and may be responsible for activating the expression of cytokine gene networks in VSMCs.

Differential Activation of Mitogenic Signaling Pathways in Aortic Smooth Muscle Cells Deficient in Superoxide Dismutase Isoforms

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 [3H]-thymidine and [3H]-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.

Reactive Oxygen Species Regulate Heat-Shock Protein 70 via the JAK/STAT Pathway

Abstract —Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) activate intracellular signal transduction pathways implicated in the pathogenesis of cardiovascular disease. H2O2 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 H2O2, 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 H2O2, 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 H2O2-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 H2O2 on HSP expression. H2O2 stimulated HSP70 expression in a time-dependent manner, and AG-490 abolished H2O2-induced HSP70 expression. H2O2 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.

Angiotensin II induces gene transcription through cell-type-dependent effects on the nuclear factor-κB (NF-κB) transcription factor

The vasopressor octapeptide, angiotensin II (Ang II), exerts homeostatic responses in cardiovascular tissues, including the heart, blood vessel wall, adrenal cortex and liver (a major source of circulating plasma proteins). One of the effects of Ang II is to induce expression of regulatory, structural and cytokine genes that play important roles in long-term control of blood pressure, vascular remodeling, cardiac hypertrophy and inflammation. The identification of nuclear signaling pathways and target transcription factors has provide important insight into cellular responses and the spectrum of genes controlled by Ang II. Here we will review how Ang II activates the transcription factors, Activator Protein 1 (AP-1), Signal Transducer and Activator of Transcription (STATs ), and Nuclear Factor-κB (NF-κB). NF-κB is of particular interest because it is an important mediator of resynthesis of the Ang II precursor, angiotensinogen AGT. Through this positive feedback loop, long-term changes in the activity of the renin angiotensin system occur. Although NF-κB is ubiquitously expressed, surprisingly the mechanism for Ang II-inducible NF-κB regulation differs between aortic smooth muscle cells (VSMCs) and hepatocytes. In VSMC, Ang II induces nuclear translocation of cytoplasmic transactivatory NF-κB proteins through proteolysis of its inhibitor, IkB. By contrast, in hepatocytes, Ang II induces large nuclear isoforms of NF-κB1 to bind DNA through a mechanism independent of changes in IkB turnover. NF-κB activation depends upon the activity of DAG-sensitive PKC isoforms and ROS signaling pathway. These observations indicate that significant differences exist in Ang II signaling depending upon cell-type involved and suggest the possibility that tissue-selective modulation of Ang II effects can be modulated in cardiac tissues.

OXIDATIVE STRESS IN ATHEROGENESIS AND ARTERIAL THROMBOSIS: THE DISCONNECT BETWEEN CELLULAR STUDIES AND CLINICAL OUTCOMES

Summary.  Atherosclerosis is a multifactorial disease for which the molecular etiology of many of the risk factors is still unknown. As no single genetic marker or test accurately predicts cardiovascular death, phenotyping for markers of inflammation may identify the individuals at risk for vascular diseases. Reactive oxygen species (ROS) are key mediators of signaling pathways that underlie vascular inflammation in atherogenesis, starting from the initiation of fatty streak development through lesion progression to ultimate plaque rupture. Various animal models of atherosclerosis support the notion that ROS released from NAD(P)H oxidases, xanthine oxidase, lipoxygenases, and enhanced ROS production from dysfunctional mitochondrial respiratory chain indeed have a causatory role in atherosclerosis and other vascular diseases. Human investigations also support the oxidative stress hypothesis of atherogenesis. This is further supported by the observed impairment of vascular function and enhanced atherogenesis in animal models that have deficiencies in antioxidant enzymes. The importance of oxidative stress in atherosclerosis is further emphasized because of its role as a unifying mechanism across many vascular diseases. The main contraindicator for the role oxidative stress plays in atherosclerosis is the lack of effectiveness of antioxidants in reducing primary endpoints of cardiovascular death and morbidity. However, this lack of effectiveness by itself does not negate the existence or causatory role of oxidative stress in vascular disease. Lack of proven markers of oxidative stress, which could help to identify a subset of population that can benefit from antioxidant supplementation, and the complexity and subcellular localization of redox reactions, are among the factors responsible for the mixed outcomes in the use of antioxidants for the prevention of cardiovascular diseases. To better understand the role of oxidative stress in vascular diseases, future studies should be aimed at using advances in mouse and human genetics to define oxidative stress phenotypes and link phenotype with genotype.