Kensuke Noma

Effect of Different Intensities of Exercise on Endothelium-Dependent Vasodilation in Humans Role of Endothelium-Dependent Nitric Oxide and Oxidative Stress

Background—Aerobic exercise enhances endothelium-dependent vasodilation in hypertensive patients, patients with chronic heart failure, and healthy individuals. However, it is unclear how the intensity of exercise affects endothelial function in humans. The purpose of the present study was to determine the effects of different intensities of exercise on endothelium-dependent vasodilation in humans. Methods and Results—We evaluated the forearm blood flow responses to acetylcholine, an endothelium-dependent vasodilator, and isosorbide dinitrate, an endothelium-independent vasodilator, before and after different intensities of exercise (mild, 25% &OV0312;o2max; moderate, 50% &OV0312;o2max; and high, 75% &OV0312;o2max; bicycle ergometers, 30 minutes, 5 to 7 times per week for 12 weeks) in 26 healthy young men. Forearm blood flow was measured using a mercury-filled Silastic strain-gauge plethysmograph. Twelve weeks of moderate-intensity exercise, but not mild- or high-intensity exercise, significantly augmented acetylcholine-induced vasodilation (7.5±2.4 to 11.4±5.8 mL/min per 100 mL tissue; P <0.05). No intensity of aerobic exercise altered isosorbide dinitrate–induced vasodilation. The administration of NG-monomethyl-l-arginine, a nitric oxide synthase inhibitor, abolished the moderate-intensity exercise-induced augmentation of the forearm blood flow response to acetylcholine. High-intensity exercise increases plasma concentrations of 8-hydroxy-2′-deoxyguanosine (from 6.7±1.1 to 9.2±2.3 ng/mL; P <0.05) and serum concentrations of malondialdehyde-modified low-density lipoprotein (from 69.0±19.5 to 82.4±21.5 U/L; P <0.05), whereas moderate exercise tended to decrease both indices of oxidative stress. Conclusions—These findings suggest that moderate-intensity aerobic exercise augments endothelium-dependent vasodilation in humans through the increased production of nitric oxide and that high-intensity exercise possibly increases oxidative stress.

Rapid nongenomic actions of thyroid hormone

The binding of thyroid hormone to the thyroid hormone receptor (TR) mediates important physiological effects. However, the transcriptional effects of TR mediated by the thyroid response element (TRE) cannot explain many actions of thyroid hormone. We postulate that TR can initiate rapid, non-TRE-mediated effects in the cardiovascular system through cross-coupling to the phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase Akt pathway. In vascular endothelial cells, the predominant TR isoform is TRα1. Treatment of endothelial cells with l-3,5,3′-triiodothyronine (T3) increased the association of TRα1 with the p85α subunit of PI3-kinase, leading to the phosphorylation and activation of Akt and endothelial nitric oxide synthase (eNOS). The activation of Akt and eNOS by T3 was abolished by the PI3-kinase inhibitors, LY294002 and wortmannin, but not by the transcriptional inhibitor, actinomycin D. To determine the physiological relevance of this PI3-kinase/Akt pathway, we administered T3 to mice undergoing transient focal cerebral ischemia. Compared with vehicle, a single bolus infusion of T3 rapidly increased Akt activity in the brain, decreased mean blood pressure, reduced cerebral infarct volume, and improved neurological deficit score. These neuroprotective effects of T3 were greatly attenuated or absent in eNOS−/− and TRα1−/−β−/− mice and were completely abolished in WT mice pretreated with LY294002 or a T3 antagonist, NH-3. These findings indicate that the activation of PI3-kinase/Akt pathways can mediate some of the rapid, non-TRE effects of TR and suggest that the activation of Akt and eNOS contributes to some of the acute vasodilatory and neuroprotective effects of thyroid hormone.

Rho kinase (ROCK) inhibitors.

The Rho kinase (ROCK) isoforms, ROCK1 and ROCK2, were initially discovered as downstream targets of the small GTP-binding protein Rho. Because ROCKs mediate various important cellular functions such as cell shape, motility, secretion, proliferation, and gene expression, it is likely that this pathway will intersect with other signaling pathways known to contribute to cardiovascular disease. Indeed, ROCKs have already been implicated in the regulation of vascular tone, proliferation, inflammation, and oxidative stress. However, it is not entirely clear how ROCKs are regulated, what some of their downstream targets are, and whether ROCK1 and ROCK2 mediate different cellular functions. Clinically, inhibition of ROCK pathway is believed to contribute to some of the cardiovascular benefits of statin therapy that are independent of lipid lowering (ie, pleiotropic effects). To what extent ROCK activity is inhibited in patients on statin therapy is not known, but it may have important clinical implications. Indeed, several pharmaceutical companies are already actively engaged in the development of ROCK inhibitors as the next generation of therapeutic agents for cardiovascular disease because evidence from animal studies suggests the potential involvement of ROCK in hypertension and atherosclerosis.

Decreased Perivascular Fibrosis but Not Cardiac Hypertrophy in ROCK1 / Haploinsufficient Mice

Background— Rho GTPase and its downstream target, Rho-associated kinase (ROCK), have been implicated in diverse cardiovascular diseases such as cardiac hypertrophy. However, pharmacological inhibitors of ROCK are not entirely specific, nor can they discriminate between the ROCK isoforms ROCK1 and ROCK2. To determine the specific role of ROCK1 in the development of cardiac hypertrophy, we generated ROCK1+/− haploinsufficient mice and determined whether cardiac hypertrophy and remodeling are decreased in these mice. Methods and Results— Litters of ROCK1−/− mice on C57Bl/6 background were markedly underrepresented, suggesting lethality in utero or postnatally. ROCK1+/− mice, however, are viable and fertile with no obvious phenotypic abnormalities. Basal blood pressure, heart rate, and cardiac dimension and function in ROCK1+/− mice were similar to those in wild-type (WT) littermates. Infusion of angiotensin II (400 ng · kg−1 · min−1 for 28 days) or treatment with NG-nitro-l-arginine methyl ester (1 mg/mL in drinking water for 28 days) caused similar increases in systolic blood pressure, left ventricular wall thickness, left ventricular mass, ratio of heart weight to tibial length, and cardiomyocyte size in ROCK1+/− mice and WT littermates. In contrast, perivascular fibrosis in hearts was increased to a lesser extent in ROCK1+/− mice compared with WT littermates. This was associated with decreased expression of transforming growth factor-β, connective tissue growth factor, and type III collagen. In addition, perivascular fibrosis induced by transaortic constriction or myocardial infarction was decreased in ROCK1+/− mice compared with WT littermates. Conclusions— These findings indicate ROCK1 is critical for the development of cardiac fibrosis, but not hypertrophy, in response to various pathological conditions and suggest that signaling pathways leading to the hypertrophic and profibrotic response of the heart are distinct.

ROCK1 mediates leukocyte recruitment and neointima formation following vascular injury.

Although Rho-associated kinase (ROCK) activity has been implicated in cardiovascular diseases, the tissue- and isoform-specific roles of ROCKs in the vascular response to injury are not known. To address the role of ROCKs in this process, we generated haploinsufficient Rock1 (Rock1(+/-)) and Rock2 (Rock2(+/-)) mice and performed carotid artery ligations. Following this intervention, we found reduced neointima formation in Rock1(+/-) mice compared with that of WT or Rock2(+/-) mice. This correlated with decreased vascular smooth muscle cell proliferation and survival, decreased levels proinflammatory adhesion molecule expression, and reduced leukocyte infiltration. In addition, thioglycollate-induced peritoneal leukocyte recruitment and accumulation were substantially reduced in Rock1(+/-) mice compared with those of WT and Rock2(+/-) mice. To determine the role of leukocyte-derived ROCK1 in neointima formation, we performed reciprocal bone marrow transplantation (BMT) in WT and Rock1(+/-) mice. Rock1(+/-) to WT BMT led to reduced neointima formation and leukocyte infiltration following carotid ligation compared with those of WT to WT BMT. In contrast, WT to Rock1(+/-) BMT resulted in increased neointima formation. These findings indicate that ROCK1 in BM-derived cells mediates neointima formation following vascular injury and suggest that ROCK1 may represent a promising therapeutic target in vascular inflammatory diseases.

Rho Kinase Inhibition Improves Endothelial Function in Human Subjects With Coronary Artery Disease

Investigations from basic biology suggest that activation of the Rho/Rho kinase pathway reduces the bioavailability of nitric oxide (NO) and thereby promotes atherosclerosis and its clinical complications. Yet, little information is available about the relationship of the Rho/Rho kinase pathway to NO bioavailability in humans with atherosclerosis. Accordingly, we determined whether inhibition of Rho kinase augments NO bioavailability and improves endothelial function in human subjects with coronary artery disease (CAD). Thirteen CAD subjects and 16 age- and sex-matched healthy controls were randomly assigned to receive the Rho kinase inhibitor, fasudil, or placebo for 1 month each in a double-blind crossover trial. Flow-mediated, endothelium-dependent and nitroglycerin-induced, endothelium-independent vasodilation were assessed by brachial artery ultrasonography. Rho kinase activity was measured in peripheral leukocytes. Fasudil increased endothelium-dependent vasodilation in CAD subjects from 9.4±1.9% to 13.4±1.9% (P=0.001) but not in healthy controls (from 11.3±1.4% to 7.7±1.1%; P=0.07). Endothelium-independent vasodilation was not affected by fasudil in either CAD or healthy subjects. Fasudil reduced Rho kinase activity by 59±18% in CAD subjects (P=0.001) but not in healthy subjects (by 3±6%; P=0.60). The change in endothelium-dependent vasodilation achieved with fasudil relative to placebo was inversely proportional to Rho kinase inhibition (ie, greater Rho kinase inhibition was associated with larger improvement in endothelium-dependent vasodilation) (r=−0.48; P=0.01). These findings suggest that Rho/Rho kinase activation promotes endothelial dysfunction in humans with atherosclerosis. Inhibition of the Rho/Rho kinase pathway should provide a useful strategy to restore NO bioavailability in humans with atherosclerosis.

Circadian Variation of Blood Pressure and Endothelial Function in Patients With Essential Hypertension: A Comparison of Dippers and Non-Dippers

OBJECTIVES The purpose of this study was to evaluate the relationship between the circadian blood pressure (BP) rhythm and endothelial function in patients with essential hypertension. BACKGROUND Hypertension is associated with alterations in resistance artery endothelial function. Patients with a non-dipper circadian pattern of BP have a greater risk of cerebrovascular and cardiovascular complications than do patients with a dipper circadian pattern. METHODS We evaluated the forearm blood flow (FBF) response to intra-arterial acetylcholine (ACh), an endothelium-dependent vasodilator, and isosorbide dinitrate (ISDN), an endothelium-independent vasodilator, infusion in 20 patients with non-dipper hypertension and 20 age- and gender-matched patients with dipper hypertension. The FBF was measured using a mercury-filled Silastic strain-gauge plethysmograph. RESULTS The 24-h systolic BP, as well as nocturnal systolic and diastolic BPs were higher in non-dipper patients than in dipper patients. The 24-h urinary excretion of nitrite/nitrate and cyclic guanosine monophosphate was lower in non-dippers than in dippers. The response of FBF to ACh was smaller in non-dippers than in dippers (25.1 +/- 3.1 vs. 20.2 +/- 3.0 ml/min/100 ml tissue, p < 0.05). The FBF response to ISDN was similar in dippers and non-dippers. The FBF response to ACh was similar in the two groups following intra-arterial infusion of the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine. CONCLUSIONS These findings suggest that endothelium-dependent vasodilation is blunted through a decrease in NO release in non-dippers compared with patients who have dipper hypertension.

A low-calorie diet improves endothelium-dependent vasodilation in obese patients with essential hypertension*

BACKGROUND Both obesity and hypertension are associated with endothelial dysfunction. The purpose of this study was to investigate the effects of a low-calorie diet on endothelial function in obese patients with essential hypertension. METHODS We measured forearm blood flow (FBF) during intra-arterial infusion of acetylcholine (ACh; 7.5, 15, 30 microg/min), an index of endothelium-dependent vasodilation, and isosorbide dinitrate (ISDN; 0.75, 1.5, 3.0 microg/min), an index of endothelium-independent vasodilation, in obese patients with essential hypertension before and after 2 weeks on a low-calorie diet (800 kcal/d). The study included 11 obese hypertensive Japanese patients (mean body mass index, 30.8 +/- 3.6 kg/m2). Fifteen healthy Japanese normotensive individuals were recruited as a control group. RESULTS In obese patients with hypertension, the response of FBF to ACh was attenuated compared to healthy individuals (P < .001). Caloric restriction reduced body weight from 77.5 +/- 15.0 to 73.2 +/- 13.5 kg (P < .01), the mean blood pressure from 118.4 +/- 8.7 to 105.7 +/- 8.5 mm Hg (P < .01), fasting plasma insulin from 85.8 +/- 22.8 to 64.8 +/- 27.0 pmol/L (P < .05), serum total cholesterol from 5.30 +/- 0.76 to 4.67 +/- 0.58 mmol/L (P < .05), and low density lipoprotein cholesterol from 3.80 +/- 0.48 to 3.29 +/- 0.44 mmol/L (P < .05). Basal FBF was similar before and after weight reduction. Caloric restriction enhanced the response of FBF to ACh (P < .05), but did not alter the response to ISDN. The intra-arterial infusion of NG-monomethyl-L-arginine (8 micromol/min), a nitric oxide synthase inhibitor, decreased the enhanced ACh-induced blood flow response induced by caloric restriction. CONCLUSIONS The present findings suggest that the caloric restriction improves endothelial-dependent vasodilation through an increased release of nitric oxide in obese hypertensive patients.

Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endothelial mechanisms

Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS−/–) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS−/– mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS−/– mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke.

Beneficial effect of prodromal angina pectoris is lost in elderly patients with acute myocardial infarction

BACKGROUND Prodromal angina pectoris occurring shortly before the onset of acute myocardial infarction is associated with a favorable outcome by the mechanism of ischemic preconditioning. Recent experiments have reported that the beneficial effect of ischemic preconditioning are reversed in the aged heart. METHODS We studied 990 patients who underwent coronary angiography within 12 hours after the onset of acute myocardial infarction. Patients were divided into 2 groups: those aged <70 years (nonelderly patients, n = 722) and those aged >/=70 years (elderly patients, n = 268). Prodromal angina in the 24 hours before infarction was found in 190 of 722 nonelderly patients and in 66 of 268 elderly patients (26% vs 25%, P =.61). RESULTS In nonelderly patients, prodromal angina was associated with lower peak creatine kinase levels (2438 +/- 1939 IU/L vs 2837 +/- 2341 IU/L, P =.04), lower in-hospital mortality rates (3.7% vs 8.8%, P =.02), and better 5-year survival rates (P =. 007). On the contrary, in elderly patients there was no significant difference in peak creatine kinase levels (2427 +/- 2142 IU/L vs 2256 +/- 1551 IU/L, P =.51), in-hospital mortality rate (21.2% vs 17. 4%, P =.49), and 5-year survival rates (P =.47). A multivariate analysis showed that prodromal angina in the 24 hours before infarction was associated with 5-year survival rate in nonelderly patients (odds ratio 0.49, P =.009) but not in elderly patients (odds ratio l.12, P =.65). CONCLUSIONS In nonelderly patients, prodromal angina in the 24 hours before infarction was associated with a smaller infarct size and better short- and long-term survival, suggesting a relation to ischemic preconditioning. However, such a beneficial effect was not observed in elderly patients.