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The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H 2 O 2 ) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H 2 O 2 to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K + channel opener levcromakalim. Exogenous H 2 O 2 elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H 2 O 2 upon stimulation by ACh and that the H 2 O 2 production was markedly reduced in eNOS-KO mice. These results indicate that H 2 O 2 is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.J. Clin. Invest. 106:1521-1530. cular smooth muscle (21,22). In this study, we thus tested our hypothesis that H 2 O 2 is an EDHF and also examined a possible role of eNOS as a major source of the reactive oxygen species, using control and eNOS knockout (eNOS-KO) mice (23). MethodsThis study was reviewed by the Committee on Ethics in Animal Experiments of the Kyushu University and was carried out according to the Guidelines for Animal Experiments of the Kyushu University and of the Japanese government.Animals and tissue preparation. Male eNOS-KO mice and C57BL/6 mice, 10-16 weeks of age, were used. The eNOS-KO mice were originally provided by P. Huang and M. Fishman (Harvard Medical School, Boston, Massachusetts, USA) (23) and maintained in the Laboratory of Animal Experiments in the Kyushu University. The eNOS-KO mice were derived from a cross between SV129J and C57BL/6 mice and were backcrossed to C57BL/6 mice over ten generations. Thus, C57BL/6 mice were used as a wild-type control; they were also maintained in the Laboratory of Animal Experiments in the Kyushu University. Some eNOS-KO mice were treated with hydralazine in order to examine the effect of mildly elevated blood pressure on the EDHF-mediated responses. The treatment with hydralazine hydrochloride (20 mg/kg/d) was performed for 6 weeks from 10-16 weeks of age. Systolic blood pressure was measured by tail-cuff method under conscious conditions before the animals were killed. Th...

Background-Excessive myocardial fibrosis impairs cardiac function in hypertensive hearts. Roles of transforming growth factor (TGF)-␤ in myocardial remodeling and cardiac dysfunction were examined in pressure-overloaded rats. Methods and Results-Pressure overload was induced by a suprarenal aortic constriction in Wistar rats. Fibroblast activation (proliferation and phenotype transition to myofibroblasts) was observed after day 3 and peaked at days 3 to 7. Thereafter, myocyte hypertrophy and myocardial fibrosis developed by day 28. At day 28, echocardiography showed normal left ventricular fractional shortening, but the decreased ratio of early to late filling velocity of the transmitral Doppler velocity and hemodynamic measurement revealed left ventricular end-diastolic pressure elevation, indicating normal systolic but abnormal diastolic function. Myocardial TGF-␤ mRNA expression was induced after day 3, peaked at day 7, and remained modestly increased at day 28. An anti-TGF-␤ neutralizing antibody, which was administered intraperitoneally daily from 1 day before operation, inhibited fibroblast activation and subsequently prevented collagen mRNA induction and myocardial fibrosis, but not myocyte hypertrophy. Neutralizing antibody reversed diastolic dysfunction without affecting blood pressure and systolic function. Conclusions-TGF-␤ plays a causal role in myocardial fibrosis and diastolic dysfunction through fibroblast activation in pressure-overloaded hearts. Our findings may provide an insight into a new therapeutic strategy to prevent myocardial fibrosis and diastolic dysfunction in pressure-overloaded hearts.

Endothelium-dependent relaxations are achieved by a combination of endothelium-derived prostacyclin (PGI2), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). However, it remains to be fully clarified whether the relative contribution of these three mechanisms to endothelium-dependent relaxations varies as a function of the vessel size. This study was designed to clarify this point. Acetylcholine (ACh)-induced endothelium-dependent relaxations were examined in isolated blood vessels taken from the aorta and the proximal and distal mesenteric arteries of the rat. The contributions of PGI2, NO, and EDHF were evaluated by the inhibitory effects of indomethacin, N omega-nitro-L-arginine methyl ester (L-NAME) in the presence of indomethacin, and KCl in the presence of indomethacin and L-NAME, respectively. The membrane potentials were recorded with microelectrodes. The expression of endothelial No synthase (eNOS) was examined by both immunostaining and immunoblotting. The contribution of PGI2 was negligible in three different-sized blood vessels. The contribution of NO was most prominent in the aorta, whereas that of EDHF was most prominent in the distal mesenteric arteries. The resting membrane potential was significantly deeper and the ACh-induced hyperpolarization was greater in the distal mesenteric arteries than those in the aorta. The expression of eNOS was the highest in the aorta and the lowest in the distal mesenteric arteries. These results indicate that the importance of EDHF increases as the vessel size decreases in endothelium-dependent relaxations in the rat mesenteric circulation.

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...

Abstract-Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho. Accumulating evidence has demonstrated that Rho/Rho-kinase pathway plays an important role in various cellular functions, not only in vascular smooth muscle cell (VSMC) contraction but also in actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions, all of which may be involved in the pathogenesis of cardiovascular disease. At molecular level, Rho-kinase upregulates various molecules that accelerate inflammation/oxidative stress, thrombus formation, and fibrosis, whereas it downregulates endothelial nitric oxide synthase. The expression of Rho-kinase itself is mediated by protein kinase C/NF-B pathway with an inhibitory and stimulatory modulation by estrogen and nicotine, respectively. At cellular level, Rho-kinase mediates VSMC hypercontraction, stimulates VSMC proliferation and migration, and enhances inflammatory cell motility. In animal studies, Rho-kinase has been shown to be substantially involved in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, stroke and heart failure, and to enhance central sympathetic nerve activity. Finally, in clinical studies, fasudil, a Rho-kinase inhibitor, is effective for the treatment of a wide range of cardiovascular disease, including cerebral and coronary vasospasm, angina, hypertension, pulmonary hypertension, and heart failure, with a reasonable safety.

These results suggest that extracorporeal cardiac SW therapy is an effective and noninvasive therapeutic strategy for ischemic heart disease.