Recent studies have revealed that vascular cells can produce reactive oxygen species (ROS) through NAD(P)H oxidase, which may be involved in vascular injury. However, the pathological role of vascular NAD(P)H oxidase in diabetes or in the insulin-resistant state remains unknown. In this study, we examined the effect of high glucose level and free fatty acid (FFA) (palmitate) on ROS production in cultured aortic smooth muscle cells (SMCs) and endothelial cells (ECs) using electron spin resonance spectroscopy. Exposure of cultured SMCs or ECs to a high glucose level (400 mg/dl) for 72 h significantly increased the free radical production compared with low glucose level exposure (100 mg/dl). Treatment of the cells for 3 h with phorbol myristic acid (PMA), a protein kinase C (PKC) activator, also increased free radical production. This increase was restored to the control value by diphenylene iodonium, a NAD(P)H oxidase inhibitor, suggesting ROS production through PKC-dependent activation of NAD(P)H oxidase. The increase in free radical production by high glucose level exposure was completely restored by both diphenylene iodonium and GF109203X, a PKC-specific inhibitor. Exposure to palmitate (200 µmol/l) also increased free radical production, which was concomitant with increases in diacylglycerol level and PKC activity. Again, this increase was restored to the control value by both diphenylene iodonium and GF109203X. The present results suggest that both high glucose level and palmitate may stimulate ROS production through PKC-dependent activation of NAD(P)H oxidase in both vascular SMCs and ECs. This finding may be involved in the excessive acceleration of atherosclerosis in patients with diabetes and insulin resistance syndrome.

Mitochondria are one of the enzymatic sources of reactive oxygen species (ROS) and could also be a major target for ROS-mediated damage. We hypothesized that ROS may induce mitochondrial DNA (mtDNA) damage, which leads to defects of mtDNA-encoded gene expression and respiratory chain complex enzymes and thus may contribute to the progression of left ventricular (LV) remodeling and failure after myocardial infarction (MI). In a murine model of MI and remodeling created by the left anterior descending coronary artery ligation for 4 weeks, the LV was dilated and contractility was diminished. Hydroxyl radicals, which originated from the superoxide anion, and lipid peroxide formation in the mitochondria were both increased in the noninfarcted LV from MI mice. The mtDNA copy number relative to the nuclear gene (18S rRNA) preferentially decreased by 44% in MI by a Southern blot analysis, associated with a parallel decrease (30% to 50% of sham) in the mtDNA-encoded gene transcripts, including the subunits of complex I (ND1, 2, 3, 4, 4L, and 5), complex III (cytochrome b), complex IV (cytochrome c oxidase), and rRNA (12S and 16S). Consistent with these molecular changes, the enzymatic activity of complexes I, III, and IV decreased in MI, whereas, in contrast, complex II and citrate synthase, encoded only by nuclear DNA, both remained at normal levels. An intimate link among ROS, mtDNA damage, and defects in the electron transport function, which may lead to an additional generation of ROS, might play an important role in the development and progression of LV remodeling and failure.

Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (27413 versus 1369 nmol min 1 mg 1 protein, P0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium. (Circ Res. 1999;85:357-363.) Key Words: antioxidant free radical heart failure myocardial contraction reactive oxygen species C ongestive heart failure (HF) is an important cause of morbidity and mortality in patients with various heart diseases. Despite extensive studies, the fundamental mechanisms responsible for the development and progression of left ventricular (LV) failure have not yet been fully elucidated. Reactive oxygen species (ROS) such as superoxide anions (O 2) and hydroxy radicals (OH) cause the oxidation of membrane phospholipids, proteins, and DNAs, and they have been implicated in a wide range of pathological conditions including ischemia-reperfusion injury, neurodegenerative diseases , and aging. Under physiological conditions, their toxic effects are prevented by such scavenging enzymes as super-oxide dismutase (SOD), glutathione peroxidase, and catalase as well as by other nonenzymatic antioxidants. However, when the production of ROS becomes excessive, oxidative stress might have a harmful effect on the functional integrity of biological tissue. Oxygen free radicals have been shown to cause contractile failure and structural damage in the myo-cardium. 1,2 However, their significance has been demonstrated to limited subsets of cardiac diseases, which include ischemic heart disease 3 and adriamycin-induced cardiac toxicity. 4 Recent investigations have suggested the generation of ROS to increase in chronic HF. Lipid peroxides and 8-iso-prostaglandin F 2 , which are the major biochemical consequences of RO...

Background-Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood. Methods and Results-Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro. Conclusions-These

Abstract. Hyperglycemia seems to be an important causative factor in the development of micro-and macrovascular complications in patients with diabetes. Several hypotheses have been proposed to explain the adverse effects of hyperglycemia on vascular cells. Both protein kinase C (PKC) activation and oxidative stress theories have increasingly received attention in recent years. This article shows a PKC-dependent increase in oxidative stress in diabetic vascular tissues. High glucose level stimulated reactive oxygen species (ROS) production via a PKC-dependent activation of NAD(P)H oxidase in cultured aortic endothelial cells, smooth muscle cells, and renal mesangial cells. In addition, expression of NAD(P)H oxidase components were shown to be upregulated in vascular tissues and kidney from animal models of diabetes. Furthermore, several agents that were expected to block the mechanism of a PKCdependent activation of NAD(P)H oxidase clearly inhibited the increased oxidative stress in diabetic animals, as assessed by in vivo electron spin resonance method. Taken together, these findings strongly suggest that the PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased oxidative stress in diabetes.Hyperglycemia seems to be an important causative factor in the development of micro-and macrovascular complications in patients with diabetes (1,2). Various pathophysiological and biochemical mechanisms have been proposed to explain the adverse effects of hyperglycemia on vascular cells (3-6). Among various possible mechanisms, it is widely accepted that high glucose level and a diabetic state induce the persistent activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway in micro-and macrovascular tissues of diabetic animals and of patients with diabetes (7-12). Because PKC is a critical intracellular signaling molecule that can regulate many vascular functions, it is to be expected that activation of PKC may cause alteration in various vascular functions in diabetes. However, accumulating evidence has shown that oxidative stress also may play a role in the development of diabetic vascular complications. A number of in vitro and in vivo studies suggest that the production of reactive oxygen species (ROS) is increased in diabetes (13-16). It has been postulated that ROS production in diabetes may be enhanced by hyperglycemia through various mechanisms such as enhanced formation of glycation products (17), altered polyol pathway activity (18), and increased superoxide release from mitochondria (19). In contrast, attention is increasingly focused on NAD(P)H oxidase as the most important source of ROS production in blood vessels (20 -23). Recent reports have implicated that this oxidase may be involved in the pathophysiology of various vascular diseases, including hypercholesterolemia (24), atherosclerosis (25-27), and hypertension (28). In this review, we show that a PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased ROS ...