The endothelial generation of reactive oxygen species (ROS) is important both physiologically and in the pathogenesis of many cardiovascular disorders. ROS generated by endothelial cells include superoxide (O2-*), hydrogen peroxide (H2O2), peroxynitrite (ONOO-*), nitric oxide (NO), and hydroxyl (*OH) radicals. The O2-* radical, the focus of the current review, may have several effects either directly or through the generation of other radicals, e.g., H2O2 and ONOO-*. These effects include 1) rapid inactivation of the potent signaling molecule and endothelium-derived relaxing factor NO, leading to endothelial dysfunction; 2) the mediation of signal transduction leading to altered gene transcription and protein and enzyme activities ("redox signaling"); and 3) oxidative damage. Multiple enzymes can generate O2-*, notably xanthine oxidase, uncoupled NO synthase, and mitochondria. Recent studies indicate that a major source of endothelial O2-* involved in redox signaling is a multicomponent phagocyte-type NADPH oxidase that is subject to specific regulation by stimuli such as oscillatory shear stress, hypoxia, angiotensin II, growth factors, cytokines, and hyperlipidemia. Depending on the level of oxidants generated and the relative balance between pro- and antioxidant pathways, ROS may be involved in cell growth, hypertrophy, apoptosis, endothelial activation, and adhesivity, for example, in diabetes, hypertension, atherosclerosis, heart failure, and ischemia-reperfusion. This article reviews our current knowledge regarding the sources of endothelial ROS generation, their regulation, their involvement in redox signaling, and the relevance of enhanced ROS generation and redox signaling to the pathophysiology of cardiovascular disorders where endothelial activation and dysfunction are implicated.

Abstract-Increased reactive oxygen species (ROS) production is implicated in the pathophysiology of left ventricular (LV) hypertrophy and heart failure. However, the enzymatic sources of myocardial ROS production are unclear. We examined the expression and activity of phagocyte-type NADPH oxidase in LV myocardium in an experimental guinea pig model of progressive pressure-overload LV hypertrophy. Concomitant with the development of LV hypertrophy, NADPH-dependent O 2 Ϫ production in LV homogenates, measured by lucigenin (5 mol/L) chemiluminescence or cytochrome c reduction assays, significantly and progressively increased (by Ϸ40% at the stage of LV decompensation; PϽ0.05). O 2 Ϫ production was fully inhibited by diphenyleneiodonium (100 mol/L). Immunoblotting revealed a progressive increase in expression of the NADPH oxidase subunits p22 phox , gp91 phox , p67 phox , and p47 phox in the LV hypertrophy group, whereas immunolabeling studies indicated the presence of oxidase subunits in cardiomyocytes and endothelial cells. In parallel with the increase in O 2 Ϫ production, there was a significant increase in activation of extracellular signal-regulated kinase 1/2, extracellular signal-regulated kinase 5, c-Jun NH2-terminal kinase 1/2, and p38 mitogen-activated protein kinase. These data indicate that an NADPH oxidase expressed in cardiomyocytes is a major source of ROS generation in pressure overload LV hypertrophy and may contribute to pathophysiological changes such as the activation of redox-sensitive kinases and progression to heart failure. Key Words: hypertrophy Ⅲ free radicals Ⅲ heart failure Ⅲ myocardium Ⅲ reactive oxygen species A n increase in oxidative stress resulting from increased cardiac generation of reactive oxygen species (ROS) is implicated in the pathophysiology of pressure-overload left ventricular hypertrophy (LVH) and congestive heart failure, both experimentally and in clinical studies. 1-3 Increased ROS production is implicated in the development of cellular hypertrophy and remodeling, at least in part through activation of redox-sensitive protein kinases such as the mitogenactivated protein kinase (MAPK) superfamily. The transition from compensated pressure-overload LVH to heart failure is associated with increased oxidative stress, which may promote myocyte apoptosis and necrosis. Several key proteins involved in excitation-contraction coupling, such as sarcolemmal ion channels and exchangers and sarcoplasmic reticulum calcium release channels, can undergo redoxsensitive alterations in activity, which contributes to myocardial contractile dysfunction. ROS also has indirect effects resulting from increased inactivation of NO and consequent generation of peroxynitrite, eg, coronary vascular endothelial dysfunction and peroxynitrite-induced inhibition of myocardial respiration. 4 The sources of ROS generation that contribute to these effects in pressure-overload LVH and heart failure remain poorly defined. Potential sources include the mitochondrial electron transport chain, xanthine oxida...

Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH).A n increase in reactive oxygen species (ROS) production is implicated in left ventricular hypertrophy (LVH) pathophysiology. 1 For example, cardiomyocyte hypertrophy in response to angiotensin II (Ang II), tumor necrosis factor-␣ (TNF-␣), or mechanical stretch involves increased ROS production. 2,3 In vivo LVH induced by aortic banding is also inhibited by antioxidants. 4 However, the sources of ROS generation in the hypertrophying heart remain poorly defined.Recently, phagocyte-type NADPH oxidases have emerged as major ROS sources in the cardiovascular system. 5 NADPH oxidase activity is increased by stimuli such as Ang II, TNF-␣, and cyclical load, and NADPH oxidases are implicated in Ang II-induced vascular smooth muscle (VSM) hypertrophy, endothelial dysfunction, and atherosclerosis. 5 The prototypic NADPH oxidase comprises a membranebound p22 phox /gp91 phox heterodimer and 4 regulatory subunits, p40 phox , p47 phox , p67 phox , and rac1. 6 Several gp91 phox homologues (termed Noxs) have recently been identified 7 ; however, a gp91 phox (or Nox2)-containing oxidase is known to be expressed in endothelium, fibroblasts, and cardiomyocytes. 5,8 We previously reported that NADPH oxidase activity increases during development of pressure-overload LVH in guinea pigs. 8 Materials and MethodsA detailed Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org. Results Male gp91phoxϪ/Ϫ and matched wild-type mice underwent (1) subcutaneous Ang II or saline infusion by osmotic minipump or (2) suprarenal abdominal aortic constriction or sham constriction. See the online data supplement for body weights. Hemodynamics and HypertrophySystolic blood pressure (tail-cuff plethysmography) was lower in conscious gp91 phoxϪ/Ϫ mice than wild-type (123Ϯ3.5 versus 132Ϯ2.2 mm Hg; nϾ6 each) as reported previously. 11 We previously showed that subpressor Ang II infusion increased cardiac mass by 5% in gp91 phoxϪ/Ϫ mice versus 20% in wild-type. 11 To ensure that these differences were not due to the lower basal blood pressure in gp91 phoxϪ/Ϫ mice, the present study used pressor Ang II infusion (1.1 mg · kg), which increased blood pressure to 138Ϯ6.0 mm Hg after 6 days in gp91 phoxϪ/Ϫ mice, a level similar to baseline levels in wild-type mice. Nevertheless, heart/body weight ratio increased by only Ϸ4% in gp91 phoxϪ/Ϫ mice (nϭ6; PϭNS) ( Figure 1A). In wild-types, pressor Ang II increased blood pressure to 162Ϯ9.8 mm Hg (PϽ0.05 versus baseline) and heart/body weight ratio by Ϸ20% (PϽ0.05; nϭ6).Aortic constriction caused similar increases in systolic blood pressure (measured invasively 1 week postoperatively) in anesthetized wild-type mice (134.6Ϯ6.0 versus 90Ϯ5.2 mm Hg; PϽ0.001; nϭ4 each) and gp91 phoxϪ/Ϫ mice (135.1Ϯ8.2 versus 81Ϯ2.3 mm Hg; PϽ0.001; nϭ4 each). It should be noted that these values are not directly comparable to those in Ang II-infused animal...

The phagocyte-type NADPH oxidase expressed in endothelial cells differs from the neutrophil enzyme in that it exhibits low level activity even in the absence of agonist stimulation, and it generates intracellular reactive oxygen species. The mechanisms underlying these differences are unknown. We studied the subcellular location of (a) oxidase subunits and (b) functionally active enzyme in unstimulated endothelial cells. Confocal microscopy revealed co-localization of the major oxidase subunits, i. . production (assessed by lucigenin (5 M) chemiluminescence) was found in the 1475 ؋ g fraction. Co-immunoprecipitation studies and measurement of NADPH-dependent reactive oxygen species production (cytochrome c reduction assay) demonstrated that p22 phox , gp91 phox , p47 phox , p67 phox , and p40 phox existed as a functional complex in the cytoskeletal fraction. These results indicate that, in contrast to the neutrophil enzyme, a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as a preassembled intracellular complex associated with the cytoskeleton.