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This study was undertaken to elucidate a novel mechanism underlying angiotensin II-induced cardiac injury, focusing on the role of oxidative stress and myocardial capillary density. Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats), a useful model for hypertensive cardiac remodeling or heart failure, were orally given irbesartan (an AT1 receptor blocker), tempol (a superoxide dismutase mimetic) or hydralazine (a vasodilator). Irbesartan significantly ameliorated left ventricular ischemia and prevented the development of cardiac hypertrophy and fibrosis in DS rats. The benefits were associated with the attenuation of oxidative stress, normalization of myocardial capillary density and inhibition of capillary endothelial apoptosis. Moreover, DS rats with significant cardiac hypertrophy and fibrosis displayed decreased myocardial vascular endothelial growth factor (VEGF) expression and increased cardiac apoptosis signal-regulating kinase 1 (ASK1) activation. Treatment with irbesartan significantly reversed these phenotypes. Tempol treatment of DS rats mimicked all the above-mentioned effects of irbesartan, indicating the critical role of oxidative stress in cardiac injury. We also investigated the role of VEGF and ASK1 in oxidative stress-induced endothelial apoptosis by using cultured endothelial cells from wild-type and ASK1-deficient mice. Oxidative stress-induced ASK1 activation led to endothelial apoptosis, and VEGF treatment prevented oxidative stress-induced endothelial apoptosis by inhibiting ASK1 activation. We obtained the first evidence that oxidative stress-induced cardiac VEGF repression and ASK1 activation caused the enhancement of endothelial apoptosis and contributed to a decrease in myocardial capillary density. These effects resulted in angiotensin II-induced progression of cardiac injury.

Objectives: High-salt diet is closely associated with the increase in cardiovascular events. However, the mechanism of high-salt-induced cardiovascular injury is unknown. The present study was undertaken to test our hypothesis that apoptosis signal-regulating kinase (ASK) 1 may be involved in salt-induced cardiovascular injury., Methods: Wild-type and ASK1-/- mice were fed a low-salt or a high-salt diet for 10 weeks and the effects of high-salt diet on food intake, urinary volume and electrolyte excretion, and cardiovascular injury were compared between both groups of mice., Results: High-salt diet in wild-type and ASK1-/- mice similarly increased food intake, water intake, urine volume, and urinary sodium excretion, and comparably decreased plasma renin activity and aldosterone. Thus, ASK1 appears to play a minor role in the increase in natriuresis and the decrease in plasma renin, and aldosterone caused by high-salt diet. High-salt diet enhanced the phosphorylation of cardiovascular ASK1 in wild-type mice. High-salt diet in wild-type mice enhanced cardiac transforming growth factor-β1, interstitial fibrosis, coronary perivascular fibrosis, and inflammatory cell infiltration, and these changes were associated with the increase in cardiac superoxide and Nox2. ASK1 deficiency abolished the above-mentioned high-salt-induced cardiac injury. High-salt diet also caused the impairment of vascular endothelium-dependent relaxation by acetylcholine and increased vascular superoxide, and Nox2 in wild-type mice, whereas it did not cause vascular injury in ASK1-/- mice., Conclusion: ASK1 is implicated in cardiac inflammation and fibrosis, and vascular endothelial dysfunction caused by high-salt diet, through the enhancement of oxidative stress.

The molecular mechanism underlying aldosterone/salt-induced cardiovascular injury remains to be defined. This work was undertaken to determine the role of apoptosis signal-regulating kinase 1 (ASK1) in the mechanism underlying aldosterone-induced cardiac injury in vivo. We compared the in vivo effects of 4 weeks of aldosterone/salt treatment on wild-type and ASK1-deficient mice. Aldosterone infusion plus high salt intake in wild-type mice significantly increased blood pressure and urinary albumin excretion and decreased plasma potassium concentrations, and these effects of aldosterone/salt were not affected by ASK1 deficiency. Thus, ASK1 seems to play a minor role in aldosterone-induced hypertension and renal injury. ASK1 deficiency also failed to affect aldosterone-induced cardiac hypertrophy. However, ASK1 deficiency markedly ameliorated aldosterone-induced cardiac injury, eg, the enhancement of cardiac macrophage infiltration, monocyte chemotactic protein 1 expression, interstitial fibrosis, perivascular fibrosis, and transforming growth factor-beta1 and collagen type I expressions. Thus, ASK1 participates in aldosterone-induced cardiac inflammation and fibrosis. Furthermore, the enhancement of NADPH oxidase-mediated cardiac oxidative stress caused by aldosterone infusion was markedly lessened by ASK1 deficiency, which was associated with the significant amelioration by ASK1 deficiency of aldosterone-induced cardiac Nox2 upregulation. Furthermore, aldosterone/salt treatment significantly enhanced cardiac expression of the angiotensin-converting enzyme and angiotensin II type 1 receptor in wild-type mice, whereas the enhancement of these proteins by aldosterone/salt was abolished by ASK1 deficiency. Our results demonstrate that ASK1 is implicated in aldosterone/salt-induced cardiac inflammation and fibrosis through the enhancement of NADPH oxidase-mediated oxidative stress and the upregulation of the cardiac renin-angiotensin system.

Dietary obesity is associated with type 2 diabetes and cardiovascular diseases, although the underlying mechanism is unknown. This study was undertaken to elucidate the role of angiotensin II and apoptosis signal regulating kinase-1 (ASK1) in obesity/diabetes-associated cardiovascular complications and hepatic steatosis. Mice fed a high-fat diet were treated with olmesartan, an angiotensin II type 1 receptor blocker, to elucidate the role of angiotensin II in diabetic mice. Treatment of mice fed a high-fat diet with olmesartan markedly suppressed cardiac inflammation and fibrosis, as well as vascular endothelial dysfunction and remodeling, induced by obesity/diabetes. Moreover, olmesartan suppressed the disruption of the vascular endothelial NO synthase dimer in diabetic mice. Olmesartan also significantly prevented hepatic steatosis and fibrosis in diabetic mice. These beneficial effects of olmesartan on diabetic mice were associated with the attenuation of ASK1 activation in these mice. ASK1-deficient mice and wild-type mice were compared, regarding the effects of a high-fat diet. A comparison between ASK1-deficient and wild-type mice showed that ASK1 deficiency attenuated cardiac inflammation and fibrosis, as well as vascular endothelial dysfunction and remodeling induced by obesity/diabetes. The amelioration of vascular endothelial impairment by ASK1 deficiency was attributed to the prevention of endothelial NO synthase dimer disruption. ASK1 deficiency also significantly lessened hepatic steatosis in diabetic mice. In conclusion, our work provided the evidence that ASK1 is significantly activated in diet-induced diabetic mice and contributes to cardiovascular diseases and hepatic steatosis in diabetic mice. Moreover, the beneficial effects of angiotensin II inhibition on dietary diabetic mice seem to be mediated by the inhibition of ASK1 activation.

Objective: The mechanism and role of angiotensin II-induced vascular endothelial injury is unclear. We examined the molecular mechanism of angiotensin (AII)-induced vascular endothelial injury and its significance for hypertensive diastolic heart failure., Methods and Results: We compared the effect of valsartan and amlodipine on Dahl salt-sensitive hypertensive rats (DS rats). Valsartan improved vascular endothelial dysfunction of DS rats more than amlodipine, by inhibiting endothelial apoptosis and eNOS uncoupling more. Moreover, valsartan inhibited vascular apoptosis signal-regulating kinase 1 (ASK1) more than amlodipine. Thus, AT1 receptor contributed to vascular endothelial apoptosis, eNOS uncoupling, and ASK1 activation of DS rats. Using ASK1(-/-) mice, we examined the causative role of ASK1 in endothelial apoptosis and eNOS uncoupling. AII infusion in wild-type mice markedly caused vascular endothelial apoptosis and eNOS uncoupling accompanied by vascular endothelial dysfunction, whereas these effects of AII were absent in ASK1(-/-) mice. Therefore, ASK1 participated in AII-induced vascular endothelial apoptosis and eNOS uncoupling. Using tetrahydrobiopterin, we found that eNOS uncoupling was involved in vascular endothelial dysfunction in DS rats with established diastolic heart failure., Conclusions: AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by ASK1 and contributed to vascular injury in diastolic heart failure of salt-sensitive hypertension.

Long-term treatment with N(omega)-nitro-l-arginine methylester (l-NAME), an NO synthase inhibitor, induces hypertension and cardiovascular injury. However, its precise mechanism is unknown. Using apoptosis signal-regulating kinase-1 (ASK1)-deficient mice, we investigated the role of ASK1 in cardiovascular injury caused by l-NAME treatment. l-NAME was orally administered to ASK1-deficient and C57BL/6J (wild) mice for 8 weeks. l-NAME treatment increased blood pressure of wild and ASK1-deficient mice to a similar extent, indicating no role of ASK1 in NO-deficient hypertension. l-NAME treatment significantly impaired acetylcholine-induced carotid arterial relaxation in wild mice (P<0.01), being associated with the decreased endothelial NO synthase (eNOS) activity (P<0.01) and the increased disruption of eNOS dimer (P<0.01), whereas these changes by l-NAME were substantially attenuated in ASK1-deficient mice. Thus, ASK1 is involved in the impairment of vascular endothelial function by reducing eNOS activity and disrupting eNOS dimer. l-NAME treatment increased vascular reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide in wild mice to a greater extent than in ASK1 deficient mice. l-NAME treatment in wild mice caused cardiac hypertrophy, myocyte apoptosis, macrophage infiltration, coronary arterial remodeling, interstitial fibrosis, and the expression of monocyte chemoattractant protein-1 and transforming growth factor-beta1, whereas these cardiac changes by l-NAME were absent in ASK1-deficient mice. Cardiac reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and superoxide elevation by l-NAME were much less in ASK1-deficient mice than in wild mice. Our work provided the first evidence that ASK1 is implicated in vascular endothelial dysfunction and cardiovascular remodeling induced by NO deficiency by regulating eNOS and reduced nicotinamide-adenine dinucleotide phosphate oxidase.