Excessive reactive oxygen species Revised abstract, especially superoxide anion (O 2 -), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. Superoxide dismutases (SODs) are the major antioxidant defense systems against O 2 -, which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation. Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of O 2 -H 2 O 2 , which may participate in cell signaling. In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer. Given the essential role of SODs in cardiovascular disease, the concept of antioxidant therapies, that is, reinforcement of endogenous antioxidant defenses to more effectively protect against oxidative stress, is of substantial interest. However, the clinical evidence remains controversial. In this review, we will update the role of each SOD in vascular biologies, physiologies, and pathophysiologies such as atherosclerosis, hypertension, and angiogenesis. Because of the importance of metal cofactors in the activity of SODs, we will also discuss how each SOD obtains catalytic metal in the active sites. Finally, we will discuss the development of future SODdependent therapeutic strategies. Antioxid. Redox Signal. 15, 1583-1606.

Abstract-Emerging evidence indicates that reactive oxygen species, especially superoxide and hydrogen peroxide, are important signaling molecules in cardiovascular cells. Their production is regulated by hormone-sensitive enzymes such as the vascular NAD(P)H oxidases, and their metabolism is coordinated by antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Both of these reactive oxygen species serve as second messengers to activate multiple intracellular proteins and enzymes, including the epidermal growth factor receptor, c-Src, p38 mitogen-activated protein kinase, Ras, and Akt/protein kinase B. Activation of these signaling cascades and redox-sensitive transcription factors leads to induction of many genes with important functional roles in the physiology and pathophysiology of vascular cells. Thus, reactive oxygen species participate in vascular smooth muscle cell growth and migration; modulation of endothelial function, including endothelium-dependent relaxation and expression of a proinflammatory phenotype; and modification of the extracellular matrix. All of these events play important roles in vascular diseases such as hypertension and atherosclerosis, suggesting that the sources of reactive oxygen species and the signaling pathways that they modify may represent important therapeutic targets. Key Words: reactive oxygen species Ⅲ vascular smooth muscle Ⅲ endothelial cells Ⅲ hypertension Ⅲ atherosclerosis R eactive oxygen species (ROS) are some of the newest additions to the family of second-messenger molecules. Although one ROS, nitric oxide (NO⅐), has been known for years to serve as a signaling molecule by activating guanylate cyclase, it has only recently become apparent that other ROS, including superoxide (O 2 Ϫ ⅐) and hydrogen peroxide (H 2 O 2 ), can alter the function of specific proteins and enzymes as well. In most cases, the mechanism by which these agents interact with their molecular targets is still unknown, but it is clear that they can mediate agonist-stimulated signaling. In this review, we will discuss redox-sensitive signaling cascades in vascular cells; their alteration by agonists, with particular attention to angiotensin II (Ang II); and their relevance to cardiovascular disease.

Reactive oxygen species have emerged as important molecules in cardiovascular function. Recent work has shown that NAD(P)H oxidases are major sources of superoxide in vascular cells and myocytes. The biochemical characterization, activation paradigms, structure, and function of this enzyme are now partly understood. Vascular NAD(P)H oxidases share some, but not all, characteristics of the neutrophil enzyme. In response to growth factors and cytokines, they produce superoxide, which is metabolized to hydrogen peroxide, and both of these reactive oxygen species serve as second messengers to activate multiple intracellular signaling pathways. The vascular NAD(P)H oxidases have been found to be essential in the physiological response of vascular cells, including growth, migration, and modification of the extracellular matrix. They have also been linked to hypertension and to pathological states associated with uncontrolled growth and inflammation, such as atherosclerosis.

Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.

Angiotensin II induces an oxidant stress-dependent hypertrophy in cultured vascular smooth muscle cells. To investigate the growth-related molecular targets of H 2 O 2 , we examined the redox sensitivity of agonist-stimulated activation of the mitogen-activated protein kinase (MAPK) family. We show here that angiotensin II elicits a rapid increase in intracellular H 2 O 2 and a rapid and robust phosphorylation of both p42/44MAPK (16-fold) and p38MAPK (15-fold). However, exogenous H 2 O 2 activates only p38MAPK (14-fold), and diphenylene iodonium, an NADH/NADPH oxidase inhibitor, attenuates angiotensin II-stimulated phosphorylation of p38MAPK, but not p42/44MAPK. Furthermore, in cells stably transfected with human catalase, angiotensin II-induced intracellular H 2 O 2 generation is almost completely blocked, resulting in inhibition of phosphorylation of p38MAPK, but not p42/44MAPK, and a subsequent partial decrease in angiotensin II-induced hypertrophy. Specific inhibition of either the p38MAPK pathway with SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) or the p42/44MAPK pathway with PD98059 (2-(2-amino-3-methoxyphenyl)oxanaphthalen-4-one) also partially, but significantly, attenuates angiotensin II-induced hypertrophy; however, simultaneous blockade of both pathways has an additive inhibitory effect, indicating that the hypertrophic response to angiotensin II requires parallel, independent activation of both MAPK pathways. These results provide the first evidence that p38MAPK is a critical component of the oxidant stress (H 2 O 2 )-sensitive signaling pathways activated by angiotensin II in vascular smooth muscle cells and indicate that it plays a crucial role in vascular hypertrophy.