Abstract-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.

Angiotensin II is an important effector molecule controlling blood pressure and volume in the cardiovascular system. Its importance is manifested by the efficacy of angiotensin-converting enzyme inhibitors in the treatment of hypertension and congestive heart failure. Angiotensin II interacts with two pharmacologically distinct subtypes of cell-surface receptors, AT1 and AT2. AT1 receptors seem to mediate the major cardiovascular effects of angiotensin II. Here we report the isolation by expression cloning of a complementary DNA encoding a unique protein with the pharmacological specificity of a vascular AT1 receptor. Hydropathic modelling of the deduced protein suggests that it shares the seven-transmembrane-region motif with the G protein-coupled receptor superfamily. Knowledge of the AT1 receptor primary sequence should now permit structural analysis, definition of the angiotensin II receptor gene family and delineation of the contribution of AT receptors to the genetic component of hypertension.

Abstract-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.

The inducible cyclooxygenase, COX-2, has been associated with vascular inflammation and cellular proliferation. We have discovered that hypoxia increases expression of the COX-2 gene in human vascular endothelial cells in culture independent of other stimuli. Western analysis of human umbilical vein endothelial cells (HUVEC) revealed a greater than 4-fold induction of protein by hypoxia (1% O2). The steady-state level of COX-2 mRNA was correspondingly elevated by both Northern blot and reverse transcriptase-polymerase chain reaction analysis. Using electrophoretic mobility shift assays with antibody supershifting, we also found that hypoxia causes increased binding of NF-kappaB p65 (Rel A) to the one out of the two NF-kappaB consensus elements in the COX-2 promoter which is closest to the transcription start site of the COX-2 gene. Transfection of an immortalized human microvascular endothelial cell line (HMEC-1) with mutation reporter gene constructs and HUVEC with both mutation and deletion reporter gene constructs suggested that transcription of the COX-2 gene was enhanced by hypoxia. In transcription factor decoy experiments, hypoxic HUVEC were exposed in culture to 20 microM of the same NF-kappaB element found to bind NF-kappaB protein. The wild type transcription factor decoy prevented hypoxic induction of COX-2, presumably by binding with cytoplasmic p65; however, mutated or scrambled oligonucleotides did not prevent the increase in COX-2 protein expression by hypoxia. Thus, the intracellular signaling mechanism that leads to induction of COX-2 by hypoxia includes binding of p65 to the relatively 3' NF-kappaB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.

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 Ϫ/Ϫ mice but also preceded atherogenesis in young apolipoprotein E Ϫ/Ϫ mice. Apolipoprotein E Ϫ/Ϫ 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. Key Words: atherosclerosis Ⅲ muscle, smooth Ⅲ antioxidants R eactive species (RS) define a collective grouping of reactive oxygen and nitrogen species that can alter the biological functions of essential molecules such as lipids, proteins, and DNA. Numerous studies have linked excess RS generation with vascular lesion formation and functional defects. [1][2][3] This association has been reported for various RS models and species. 4 -6 A role for RS in atherogenesis is supported by epidemiological evidence of links between common risk factors for coronary artery disease and increased levels of RS. [7][8][9] Among the extensively studied intracellular systems capable of generating RS in vascular cells are the NADH/NADPH oxidase, xanthine oxidase, lipoxygenase, and cyclooxygenase systems. 6,10 -12 Mitochondria are biologically important sources and targets for RS. 13,14 However, their role as mediators of oxidative disease processes such as atherogenesis has not been examined. We recently reported that exposure of vascular cells to RS in vitro results in preferential mitochondrial DNA (mtDNA) damage and dysfunction and that mtDNA damage is a very sensitive marker for RS-mediated cellular effects. 15 In addition to the potential role of mtDNA damage as a marker of ambient oxidative stress, oxidative damage to the mitochondrion can lead to decreased oxidative energetic capacity (via impaired oxidative phosphorylation) and increased generation of intracellular RS. 15-17 Thus, we hypothesized that mitochondrial dysfunction accentuates atherosclerosis by modulating the phenotype of vascular cells and that measurements of mtDNA damage reflect RS-mediated atherosclerosis risk. Conclusions-MitochondrialUsing human aortic specimens and a murine model of early atherogenesis (the apolipoprotein E null, apoE Ϫ/Ϫ ), we examined the correlation between mtDNA damage and atherogenesis and sought to determine whether mtDNA damage is a cause or an effect in this pr...

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.

—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 Sar 1 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, Sar 1 Ang II treatment induced IL-6 transcription in a manner completely dependent on the nuclear factor-κB (NF-κB) motif. Sar 1 Ang II induced cytoplasmic-to-nuclear translocation of the NF-κB subunits Rel A and NF-κB1 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-κB translocation was dependent on weak simultaneous proteolysis of the IκBα and β inhibitors, whereas later translocation was associated with enhanced processing of the p105 precursor into the mature 50-kDa NF-κB1 form. Pretreatment with a potent inhibitor of IκBα proteolysis, TPCK, completely blocked Sar 1 Ang IIAng II-induced NF-κB activation and induction of endogenous IL-6 gene expression, which indicated the essential role of NF-κB in mediating IL-6 expression. We conclude that Ang II is a pleiotropic regulator of the NF-κB transcription factor family and may be responsible for activating the expression of cytokine gene networks in VSMCs.

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