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The presence of an earlobe crease (ELC) may be a simple sign to predict atherosclerosis. We evaluated the relation between ELC and vascular function. We measured flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID) and observed bilateral earlobes in 400 consecutive subjects. At first, the subjects were divided into 3 groups: non-ELC group, unilateral ELC group, and bilateral ELC group. FMD and NID were significantly lower in the unilateral and bilateral ELC groups than those in the non-ELC group. After adjustment of cardiovascular risk factors, bilateral ELC, but not unilateral ELC, was associated with lower FMD and lower NID. We also investigated whether an increase in the number of ELCs worsens endothelial function, whether the difference in ELC structure (cross stripes and/or ramification) affects endothelial function, and whether endothelial function is impaired in subjects with superficial wrinkles depending on age. The number of ELCs, shape of the ELC, and superficial wrinkles were not associated with endothelial dysfunction. In conclusion, these findings suggest that the presence of bilateral ELCs is associated with vascular dysfunction.

OBJECTIVE Advanced glycation end products (AGEs) and their specific receptor, the receptor for AGEs (RAGE), play an important role in atherosclerosis. Recently, a soluble form of RAGE (sRAGE) has been identified in human serum. However, the role of sRAGE in cardiovascular disease is still controversial. There is no information on the association between simultaneous measurements of AGEs and sRAGE and vascular function. In this study, we evaluated the associations between serum levels of AGEs and sRAGE, ratio of AGEs to sRAGE, and vascular function. RESEARCH DESIGN AND METHODS We measured serum levels of AGEs and sRAGE and assessed vascular function by measurement of flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation in 110 subjects who underwent health examinations. Multivariate regression analyses were performed to identify factors associated with vascular function. RESULTS Univariate regression analysis revealed that FMD correlated with age, BMI, systolic blood pressure, diastolic blood pressure, heart rate, triglycerides, HDL cholesterol, glucose, smoking pack-years, nitroglycerine-induced vasodilation, serum levels of AGEs and sRAGE, and ratio of AGEs to sRAGE. Multivariate analysis revealed that the ratio of AGEs to sRAGE remained an independent predictor of FMD, while serum level of AGEs alone or sRAGE alone was not associated with FMD. CONCLUSIONS These findings suggest that sRAGE may have a counterregulatory mechanism that is activated to counteract the vasotoxic effect of the AGE–RAGE axis. The ratio of AGEs to sRAGE may be a new chemical biomarker of endothelial function.

An imbalance of nitric oxide (NO) and reactive oxygen species (ROS), so-called "oxidative stress," may promote endothelial dysfunction, leading to cardiovascular complications. Activation of nicotinamide-adenine dinucleotide phosphate oxidase, xanthine oxidase, cyclooxygenase, and mitochondrial electron transport, inactivation of the antioxidant system, and uncoupling of endothelial NO synthase lead to oxidative stress along with an increase in ROS production and decrease in ROS degradation. Although experimental studies, both in vitro and in vivo, have shown a critical role of oxidative stress in endothelial dysfunction under the condition of excessive oxidative stress, there is little information on whether oxidative stress is really involved in endothelial function in humans. In a clinical setting, we showed an association between oxidative stress and endothelial function, especially in patients with renovascular hypertension as a model of increased oxidative stress and in patients with Gilbert syndrome as a model of decreased oxidative stress, through an increase in the antioxidant property of unconjugated bilirubin.

Background: An ankle-brachial index (ABI) value of 0.91–0.99 is considered borderline and associated with an increased risk of cardiovascular events. However, there is no information on the relationship between borderline ABI and endothelial function. Methods and Results: We measured ABI and assessed vascular function by flow-mediated vasodilation (FMD) and nitroglycerin-induced vasodilation in 389 subjects who underwent health examinations. Subjects were divided into 3 groups according to ABI (normal group: 1.00–1.40, borderline group: 0.91–0.99, abnormal group: ≤0.90 or >1.40). FMD was significantly smaller in both the borderline and the abnormal group than in the normal group. There was no significant difference in the vascular responses to nitroglycerin between the normal and borderline groups. Vascular response to nitroglycerin was significantly higher in the normal group than in the abnormal group. Borderline and abnormal ABI values were significantly associated with an increased odds ratio of low tertile of FMD levels, using the normal ABI group as the reference. Multiple logistic regression analysis for FMD revealed that age, sex, hypertension, diabetes mellitus, and borderline ABI independently remained associated with FMD. Conclusions: ABI of 0.91–0.99 is associated with endothelial dysfunction. ABI examination is a simple and cost-effective method for obtaining the additional information on the initial step of atherosclerosis beyond the assessment of peripheral artery disease. (Circ J 2014; 78: 1740–1745)

Background A new device for automatic measurement of flow‐mediated vasodilation ( FMD ) using an oscillometric method has been developed to solve technical problems of conventional FMD measurement. This device measures enclosed zone FMD (ez FMD ). The purpose of this study was to evaluate the prognostic value of endothelial function assessed by ez FMD for future cardiovascular events. Methods and Results We measured ez FMD in 272 participants who underwent health‐screening examinations. First, we investigated cross‐sectional associations between ez FMD and cardiovascular risk factors, and then we assessed the associations between ez FMD and first major cardiovascular events (death from cardiovascular causes, stroke, and coronary revascularization). Univariate regression analysis revealed that ez FMD was significantly correlated with age, triglycerides, glucose, smoking pack‐years, estimated glomerular filtration rate, high‐sensitivity C‐reactive protein, and Framingham risk score. During a median follow‐up period of 36.1 months (interquartile range 18.8–40.1 months), 12 participants died (6 from cardiovascular causes), 3 had stroke, 8 had coronary revascularization, and 10 were hospitalized for heart failure. There was no episode of acute coronary syndrome during the study period. Participants were divided into tertiles (low, intermediate, and high) based on ez FMD . Kaplan–Meier curves for first major cardiovascular events among the 3 groups were significantly different ( P =0.004). After adjustment for cardiovascular risk factors, the low group was significantly associated with an increased risk of first major cardiovascular events compared with the high group (hazard ratio 6.47; 95% CI 1.09–125.55; P =0.038). Conclusions These findings suggest that endothelial function assessed by ez FMD may be useful as a surrogate marker of future cardiovascular events. Clinical Trial Registration URL : https://upload.umin.ac.jp . Unique identifier: UMIN 000004902.

The dipeptidyl peptidase-4 inhibitor saxagliptin is a widely used antihyperglycemic agent in patients with type 2 diabetes. The purpose of this study was to evaluate the effects of saxagliptin on endothelial function in patients with type 2 diabetes. This was a prospective, multicenter, interventional study. A total of 34 patients with type 2 diabetes were enrolled at four university hospitals in Japan. Treatment of patients was initially started with saxagliptin at a dose of 5 mg daily. Assessment of endothelial function assessed by flow-mediated vasodilation (FMD) and measurement of stromal cell-derived factor-1α (SDF-1α) were conducted at baseline and at 3 months after treatment with saxagliptin. A total of 31 patients with type 2 diabetes were included in the analysis. Saxagliptin significantly increased FMD from 3.1 ± 3.1% to 4.2 ± 2.4% (P = 0.032) and significantly decreased total cholesterol from 190 ± 24 mg/dL to 181 ± 25 mg/dL (P = 0.002), glucose from 160 ± 53 mg/dL to 133 ± 25 mg/dL (P

BackgroundIt is clinically important to estimate the degree of endothelial dysfunction. Several methods have been used to assess endothelial function in humans. Recently, we developed a new noninvasive method for measurement of vascular response to reactive hyperemia in the brachial artery, named enclosed zone flow-mediated vasodilation (ezFMD). The purpose of this study was to determine the validity of ezFMD for assessment of endothelial function.Methods and resultsWe measured ezFMD by a new device using an oscillometric method and conventional FMD using ultrasonography in 306 subjects, including patients with hypertension, dyslipidemia, and diabetes mellitus (218 men and 88 women, 30 ± 16 yr). Univariate regression analysis revealed that ezFMD significantly correlated with age (r = −0.42, P

The Rho-associated coiled-coil containing kinases, ROCK1 and ROCK2, are important regulators of cell shape, migration, and proliferation through effects on the actin cytoskeleton. However, it is not known whether ROCK2 plays an important role in the development of cardiac hypertrophy. To determine whether the loss of ROCK2 could prevent cardiac hypertrophy, cardiomyocyte-specific ROCK2-null (c-ROCK2−/−) were generated using conditional ROCK2flox/flox mice and α-myosin heavy-chain promoter-driven Cre recombinase transgenic mice. Cardiac hypertrophy was induced by Ang II infusion (400 ng/kg/min, 28 d) or transverse aortic constriction (TAC). Under basal conditions, hemodynamic parameters, cardiac anatomy, and function of c-ROCK2−/− mice were comparable to wild-type (WT) mice. However, following Ang II infusion or TAC, c-ROCK2−/− mice exhibited a substantially smaller increase in heart-to-body weight ratio, left ventricular mass, myocyte cross-sectional area, hypertrophy-related fetal gene expression, intraventricular fibrosis, cardiac apoptosis, and oxidative stress compared to control mice. Deletion of ROCK2 in cardiomyocytes leads to increased expression of four-and-a-half LIM-only protein-2 (FHL2) and FHL2-mediated inhibition of serum response factor (SRF) and extracellular signal-regulated mitogen-activated protein kinase (ERK). Knockdown of FHL2 expression in ROCK2-deficient cardiomyocytes or placing ROCK2-haploinsufficient (ROCK2+/−) mice on FHL2+/−-haploinsufficient background restored the hypertrophic response to Ang II. These results indicate that cardiomyocyte ROCK2 is essential for the development of cardiac hypertrophy and that up-regulation of FHL2 may contribute to the antihypertrophic phenotype that is observed in cardiac-specific ROCK2-deficient mice.—Okamoto, R., Li, Y., Noma, K., Hiroi, Y., Liu, P.-Y., Taniguchi, M., Ito, M., Liao, J. K. FHL2 prevents cardiac hypertrophy in mice with cardiac-specific deletion of ROCK2.

Endothelial dysfunction and abnormal vascular structure may be involved in the pathogenesis of chronic heart failure (HF). The purpose of this study was to evaluate simultaneously vascular function and vascular structure in patients with heart failure with preserved ejection fraction (HFpEF).We measured flow-mediated vasodilatation (FMD) and nitroglycerine-induced vasodilation as indices of vascular function and intima-media thickness (IMT) as an index of vascular structure of the brachial artery in 41 patients with HFpEF (23 men and 18 women; mean age, 66±12yr) and 165 patients without HF (95 men and 70 women; mean age, 54±16yr).FMD was significantly smaller in patients with HFpEF than in patients without HF (2.9±2.1% versus 4.6±2.7%, P=0.0002). Nitroglycerine-induced vasodilation was significantly smaller in patients with HFpEF than in patients without HF (9.3±4.1% versus 12.9±4.9%, P0.0001). Brachial artery IMT was significantly larger in patients with HFpEF than in patients without HF (0.35±0.06mm versus 0.31±0.07mm, P=0.0002). After adjustment for age, sex, hypertension, dyslipidemia, and diabetes mellitus, the associations remained significant between HFpEF and FMD (odds ratio, 0.79; 95% confidence interval, 0.66-0.92; P=0.0032), nitroglycerine-induced vasodilation (odds ratio, 0.88; 95% confidence interval, 0.80-0.96; P=0.0039), and brachial artery IMT (odds ratio, 1.08; 95% confidence interval, 1.01-1.17; P=0.033).These findings suggest that both endothelial dysfunction and abnormal vascular structure may contribute to the pathogenesis and maintenance of HFpEF. Endothelial function and vascular structure may be potential therapeutic targets for HFpEF.

Background Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors. It is thought that PEDF plays a protective role against atherosclerosis. Clinical studies have shown that serum levels of PEDF are increased in subjects with cardiovascular risk factors. The role of PEDF in cardiovascular disease is still controversial. The purpose of this study was to evaluate the associations between serum levels of PEDF and vascular function and structure. Methods We measured serum levels of PEDF, assessed vascular function by measurements of flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation in the brachial artery, and measured brachial artery intima-media thickness (IMT) in 150 subjects who underwent health examinations. Results and conclusions Univariate regression analysis revealed that serum level of PEDF was significantly correlated with body mass index, high-density lipoprotein cholesterol, glucose, FMD, nitroglycerine-induced vasodilation, and brachial artery IMT. Multivariate analysis revealed that serum levels of PEDF remained an independent predictor of nitroglycerine-induced vasodilation (β=−0.20, P=0.02) and brachial artery IMT (β=0.14, P=0.03) after adjustment of cardiovascular risk factors, while serum level of PEDF was not associated with FMD (β=−0.02, P=0.79). These findings suggest that PEDF may be a factor directly associated with atherosclerosis. The serum level of PEDF may be a new biochemical marker of atherosclerosis.

It is established that low-density lipoprotein cholesterol is an independent risk factor for cardiovascular events. Recently, circulating triglycerides level has been focused on as a risk factor for cardiovascular events. In this study, we evaluated the associations between triglycerides and endothelial function in a general population.We analyzed data for 4887 subjects who were enrolled in the FMD-Japan registry. We investigated cross-sectional associations between serum triglyceride levels and endothelial function assessed by measurement of flow-mediated vasodilation (FMD).Serum triglyceride levels were correlated with FMD (r = -0.12, p 0.001). Subjects were divided into six groups based on serum triglyceride levels. FMD was significantly decreased with an increase in serum triglyceride levels (≤0.71 mmol/L, 7.0 ± 3.5%; 0.72-0.94 mmol/L, 6.3 ± 3.5%; 0.95-1.19 mmol/L, 6.0 ± 3.1%; 1.20-1.48 mmol/L, 5.8 ± 3.2%; 1.49-2.02 mmol/L, 5.7 ± 3.1%; ≥2.03 mmol/L, 5.5 ± 3.0%; p for trend0.001). After adjustment for age, sex, and cardiovascular risk factors, including high-density lipoprotein cholesterol, serum triglyceride levels of more than 1.20 mmol/L were independently associated with the low quartile of FMD (1.20-1.48 mmol/L, odds ratio (OR) 1.41, 95% confidence interval (CI) 1.09 to 1.82; 1.49-2.02 mmol/L, OR 1.31, 95% CI 1.00 to 1.70; ≥2.03 mmol/L, OR 1.48, 95% CI 1.13 to 1.95) using serum triglyceride levels of less than 0.71 mmol/L group as the reference.These findings suggest that triglycerides are an independent predictor of endothelial function. Lowering circulating triglyceride levels may improve endothelial function, leading to a decrease in cardiovascular events.URL for Clinical Trial: http://UMIN; Registration Number for Clinical Trial: UMIN000003409.

Hypertension is one of the common diseases in the elderly. The prevalence of hypertension markedly increases with advancing age. Both aging and hypertension have a critical role in cardiovascular and cerebrovascular complications. Although aging and hypertension, either independently or collectively, impair endothelial function, aging and hypertension may have similar cascades for the pathogenesis and development of endothelial dysfunction. Nitric oxide (NO) has an important role in regulation of vascular tone. Decrease in NO bioavailability by endothelial dysfunction would lead to elevation of blood pressure. An imbalance of reduced production of NO or increased production of reactive oxygen species, mainly superoxide, may promote endothelial dysfunction. One possible mechanism by which the prevalence of hypertension is increased in relation to aging may be advancing endothelial dysfunction associated with aging through an increase in oxidative stress. In addition, endothelial cell senescence is also involved in aging-related endothelial dysfunction. In this review, we focus on recent findings and interactions between endothelial function, oxidative stress and hypertension in aging.

Objective— Geranylgeranylacetone (GGA) induces expression of heat shock protein 90 (Hsp90), an adaptor molecule for assembly of endothelial nitric oxide synthase (eNOS) phosphorylation complex. The purpose of this study was to determine whether GGA enhances Hsp90 expression and augments endothelium-dependent vasodilation via upregulation of eNOS in humans. Methods and Results— We evaluated the effects of GGA on human umbilical vein endothelial cells (HUVECs) and on forearm blood flow (FBF) responses to acetylcholine and sodium nitroprusside in 40 healthy young men. Hsp90, eNOS, AMP-activated protein kinase (AMPK), and Akt expression in HUVECs and peripheral blood mononuclear cells was detected by Western blot analysis. GGA increased Hsp90 expression and phosphorylation of eNOS and AMPK but not Akt in HUVECs and increased Hsp90 expression in peripheral blood mononuclear cells. Oral administration of GGA (600 mg) augmented the FBF response to acetylcholine. Infusion of N G -monomethyl-l-arginine, an NO synthase inhibitor, completely abolished GGA-induced augmentation of the FBF response to acetylcholine. GGA also augmented the acetylcholine-stimulated NO release in smokers. Conclusion— These findings suggest that GGA-induced activation of Hsp90/AMPK significantly increased NO-mediated vasodilation in healthy subjects, as well as in smokers. The use of GGA may be a new therapeutic approach for improving endothelial dysfunction.

Measurement of nitroglycerine-induced vasodilation has been performed to differentiate endothelium-dependent vasodilation from endothelium-independent vasodilation as a control test for flow-mediated vasodilation (FMD). Recently, nitroglycerine-induced vasodilation per se has been reported to be a useful marker of the grade of atherosclerosis. The present study aimed to evaluate the prognostic value of FMD combined with nitroglycerine-induced vasodilation for future cardiovascular events. We measured FMD and nitroglycerine-induced vasodilation in 402 subjects, including patients with cardiovascular diseases. During a median follow-up period of 32.3 months, 38 first major cardiovascular events (death from cardiovascular causes, acute myocardial infarction, stroke, and coronary revascularization) occurred. Receiver-operator characteristic curve analysis revealed that FMD alone and nitroglycerine-induced vasodilation alone can predict cardiovascular events with areas under the curve of 0.671 (cutoff 3.3%) and 0.692 (cutoff 11.6%), respectively. FMD combined with nitroglycerine-induced vasodilation predicts cardiovascular events with an area under the curve of 0.701. After adjustment for age, sex, and cardiovascular risk factors, above cutoff FMD (≥3.3%) and below cutoff nitroglycerine-induced vasodilation (P =0.006) and below cutoff FMD (P Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: UMIN000001167.

Background: Primary aldosteronism (PA) is a most common cause of secondary hypertension and patients are divided into two subtypes in clinically, aldosterone producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). Hypertension is associated with endothelial dysfunction. It is well known that endothelial dysfunction is an independent predictor of cardiovascular events. Rho-associated kinases (ROCKs), one of the first downstream targets of the small GTP-binding protein Rho A, mediate various cellular physiologic functions. It has been reported that an increase in ROCK activity is associated with cardiovascular diseases, including hypertension. However, there is little information on the relationship between subtype of PA and the grade of atherosclerosis. The purpose of this study was to evaluate the vascular function and ROCK activity in patients with PA. Methods: Vascular function, including peripheral arterial tonometry (PAT) and ROCK activity in peripheral leukocytes were evaluated in 18 patients with APA group (49.5±15.5 years, 7 males), 14 patients with IHA group (56.1±11.0 years, 8 males), and 23 age-, gender-, and blood pressure-matched EH group (55.6 ± 9.9 years, 13 males). Results: PAT ratio was significantly lower in the APA and IHA groups than in the EH group (0.62±0.23 vs. 0.62±0.24 and 0.79±0.28, P Conclusions: APA and IHA were associated with endothelial dysfunction and APA increased ROCK activity compared with those in IHA and EH. These findings suggest that APA may have a higher risk of future cardiovascular events.

Introduction: Advanced glycation end products (AGEs) and their specific receptor, the receptor for AGEs (RAGE), play an important role in atherosclerosis. Recently, a soluble form of RAGE (sRAGE) has been identified in human serum. However, the role of sRAGE in cardiovascular disease is still controversial. There is no information on the association between simultaneous measurements of AGEs and sRAGE and vascular function. In this study, we evaluated the associations between serum levels of AGEs and sRAGE, ratio of AGEs to sRAGE and vascular function. Methods: We measured serum levels of AGEs and sRAGE and assessed vascular function by measurement of flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation in 110 subjects who underwent health examinations. Multivariate regression analyses were performed to identify factors associated with vascular function. Results: Univariate regression analysis revealed that FMD correlated with age (ρ=-0.38, P Conclusions: These findings suggest that sRAGE may have a counter-regulatory mechanism that is activated to counteract the vasotoxic effect of the AGEs-RAGE axis. The ratio of AGEs to sRAGE may be a new chemical biomarker of endothelial function.

Introduction: Endothelial function is impaired in heavy or binge drinking. Heavy drinking should be a predictor of endothelial dysfunction. However, there is little information on the effects of dose-dependent alcohol consumption on endothelial function. Therefore, we evaluated the relationship between alcohol consumption and endothelial function in a large general population. Methods and Results: We measured flow-mediated vasodilation (FMD) in 2734 men who provided self-report about habitual alcohol intake. The subjects were divided into five groups by alcohol consumption: none (0 g/week), light (0 g/week< to ≤140 g/week), moderate (140 g/week< to ≤280 g/week), heavy (280 g/week< to ≤420 g/week), and excessive (420 g/week Conclusions: These findings suggest that even light alcohol consumption impair the endothelial function. Alcohol drinking may be harmful for vascular function.

Objective: In contrast to low-density lipoprotein cholesterol, and high density lipoprotein cholesterol, which are well-established independent risk factors for cardiovascular disease, the importance of triglycerides remains controversial. In this study, we evaluated the associations between triglycerides and endothelial function in the general population. Methods and Results: We enrolled 4,908 subjects (3842 men and 1066 women; mean age, 48±12 years) who were enrolled in FMD-Japan registry. And, we investigated cross-sectional associations between triglycerides and endothelial function by measurement of flow-mediated vasodilation (FMD). The triglycerides levels were negatively associated with FMD (r=-0.10, P Conclusions: These findings supported an independent association between triglycerides and cardiovascular disease.

Hypertension is associated with endothelial dysfunction and activated Rho-associated kinases (ROCKs). The purpose of this study was to evaluate the effects of the selective mineralocorticoid receptor blocker, eplerenone, on endothelial function and ROCK activity in patients with hypertension. The study was carried out over 48 weeks in 60 untreated patients with hypertension who were randomly assigned to eplerenone, nifedipine, and losartan groups. We evaluated the effects of each treatment on flow-mediated vasodilation (FMD) and ROCK activity in peripheral leukocytes. Eplerenone increased FMD and decreased leukocyte ROCK activity. Nifedipine decreased ROCK activity but did not alter FMD. Losartan increased FMD but did not alter ROCK activity. Hypotensive effects were similar in the three groups, as was nitroglycerin-induced vasodilation during the follow-up period. There were no significant differences between the groups with respect to other parameters. The study results show that eplerenone improves endothelial function and inhibits ROCK activity in patients with essential hypertension.

BackgroundA healthy endothelium maintains vascular tone and structure. The purpose of this study was to evaluate endothelial function in corkscrew collateral arteries in Buerger disease.MethodsWe measured flow-mediated vasodilation (FMD) in corkscrew arteries in 26 patients with Buerger disease, in control arteries in 26 healthy subjects, and in native arteries in 16 patients with Buerger disease.ResultsHyperemic flow was lower in corkscrew arteries than in native arteries in patients with Buerger disease and in control arteries in healthy subjects. There was no significant difference between hyperemic flow in patients with Buerger disease in whom measurements were performed in native arteries and that in healthy subjects. FMD was lower in corkscrew arteries and native arteries in patients with Buerger disease than in control arteries in healthy subjects. There was no significant difference between FMD in corkscrew arteries in patients with Buerger disease and in that in native arteries. The ratio of FMD to hyperemic flow was significantly smaller in native arteries in patients with Buerger disease than in corkscrew arteries and in control arteries in healthy subjects (5.5 ± 6.2 vs 8.8 ± 8.9 and 9.6 ± 7.6 mL/min, P < .001, respectively). There was no significant difference in the ratio of FMD to hyperemic flow between corkscrew arteries in Buerger disease and control arteries in healthy subjects. Nitroglycerin-induced vasodilation was similar in all leg arteries.ConclusionsEndothelial function of a corkscrew collateral artery in patients with Buerger disease is maintained, while endothelial function is impaired in a native artery in Buerger disease.

Objective— Cardiovascular diseases are associated with chronic activation of Rho-associated kinases (ROCKs) and endothelial dysfunction. Both increased ROCK activity and endothelial dysfunction are thought to be closely associated with conventional cardiovascular risk factors. The purpose of this study was to determine the relationships between ROCK activity, endothelial function, and cardiovascular risk factors. Methods and Results— We evaluated ROCK activity in peripheral leukocytes by Western blot analysis and flow-mediated vasodilation by ultrasonography in 242 men who had no cardiovascular or cerebrovascular diseases (mean age, 40±10 years; range, 20 to 73 years). ROCK activity was defined as the ratio of phospho myosin-binding subunit on myosin light chain phosphatase to total myosin-binding subunit. Univariate regression analysis revealed that leukocyte ROCK activity significantly correlated with body mass index ( r =0.29, P =0.003); systolic blood pressure ( r =0.25, P =0.01); low-density lipoprotein cholesterol level ( r =0.21, P =0.04); and Framingham risk factor score, a cumulative cardiovascular risk index for heart attack ( r =0.31, P r =−0.23, P =0.02), body mass index ( r =0.19, P =0.05), systolic blood pressure ( r =−0.22, P =0.03), total cholesterol level ( r =−0.21, P =0.04), low-density lipoprotein cholesterol level ( r =−0.22, P =0.04), glucose level ( r =−0.20, P =0.04), and Framingham risk factor score ( r =−0.37, P r =−0.41, P Conclusion— These findings suggest that cumulative cardiovascular risk may enhance ROCK activity and endothelial dysfunction, leading to progression of cardiovascular diseases and outcomes.

Background— The signaling mechanisms that regulate the recruitment of bone marrow (BM)–derived cells to the injured heart are not well known. Notch receptors mediate binary cell fate determination and may regulate the function of BM-derived cells. However, it is not known whether Notch1 signaling in BM-derived cells mediates cardiac repair after myocardial injury. Methods and Results— Mice with postnatal cardiac-specific deletion of Notch1 exhibit infarct size and heart function after ischemic injury that is similar to that of control mice. However, mice with global hemizygous deletion of Notch1 (N1 ± ) developed larger infarct size and worsening heart function. When the BM of N1 ± mice were transplanted into wild-type (WT) mice, infarct size and heart function were worsened and neovascularization in the infarct border area was reduced compared with WT mice transplanted with WT BM. In contrast, transplantation of WT BM into N1 ± mice lessened the myocardial injury observed in N1 ± mice. Indeed, hemizygous deletion of Notch1 in BM-derived cells leads to decreased recruitment, proliferation, and survival of mesenchymal stem cells (MSC). Compared with WT MSC, injection of N1 ± MSC into the infarcted heart leads to increased myocardial injury whereas injection of MSC overexpressing Notch intracellular domain leads to decreased infarct size and improved cardiac function. Conclusions— These findings indicate that Notch1 signaling in BM-derived cells is critical for cardiac repair and suggest that strategies that increase Notch1 signaling in BM-derived MSC could have therapeutic benefits in patients with ischemic heart disease.

Rho-associated kinases (ROCKs) play an important role in Ca sensitization and vascular resistance. Activation of ROCKs is associated with hypertension. The purpose of this study was to evaluate the effect of the calcium channel blocker amlodipine on ROCKs activity in patients with hypertension.We evaluated ROCK activity in peripheral leukocytes by Western blot analysis in 651 patients with hypertension treated with antihypertensive agents, 28 untreated hypertensive patients and 28 healthy individuals, and the effects of treatment with amlodipine or losartan for 12 weeks on ROCK activity in 28 untreated hypertensive patients who were randomly divided into an amlodipine group (n = 14) and a losartan group (n = 14). ROCK activity was defined as the ratio of phospho myosin-binding subunit (MBS) on myosin light-chain phosphatase to total MBS.Leukocyte ROCK activity was greater in untreated patients with essential hypertension than in the healthy individuals (0.84 ± 0.24 vs. 0.61 ± 0.18, P = 0.03). In 651 patients with hypertension treated with antihypertensive agents, ROCK activity was significantly lower in the calcium channel blocker-treated group than in the groups treated with renin-angiotensin system inhibitors, diuretics, and β-blockers (0.70 ± 0.24 vs. 0.85 ± 0.29, 0.83 ± 0.24, and 0.86 ± 0.31, P0.05, respectively). ROCK activity after 4 and 12 weeks of treatment was significantly decreased in the amlodipine group (0 weeks: 0.85 ± 0.25, 4 weeks: 0.66 ± 0.16, 12 weeks: 0.64 ± 0.15, P0.05, respectively) but not in the losartan group, whereas the antihypertensive effects were similar in the two groups.These findings suggest that calcium channel blocker amlodipine inhibits ROCK activity in patients with hypertension.

Rho-associated kinases (ROCKs) have been shown to be involved in the pathogenesis of atherosclerosis. It is clinically important to estimate the degree of ROCK activity in humans. The purpose of this study was to confirm the validity of a leukocyte ROCK parameter as an index of ROCK activity in comparison with vascular response to a ROCK inhibitor.We evaluated the ratio of phospho myosin-binding subunit (p-MBS) on myosin light-chain phosphatase to total MBS in peripheral leukocytes by Western blot analysis and forearm blood flow (FBF) response to the ROCK inhibitor fasudil using strain-gauge plethysmography in 36 healthy subjects and 39 patients with cardiovascular diseases. Fasudil (3, 10, 30μg/min) was infused intra-arterially for 5min at each dose. Leukocyte p-MBS/total-MBS was higher in cardiovascular diseases than in healthy subjects (0.97±0.37 vs. 0.51±0.14; P=0.002). Fasudil increased FBF from 4.9±1.2 to 14.5±5.7mL/min/100mL tissue (P0.0001) in patients with cardiovascular diseases, while fasudil did not alter FBF in healthy subjects. There was a significant relationship between leukocyte p-MBS/total-MBS and maximal FBF response to fasudil in all subjects (r=0.72, P0.0001). There was also a significant correlation between p-MBS/total-MBS and maximal FBF response to fasudil in patients with cardiovascular diseases (r=0.59, P0.0001). In healthy subjects, there was no significant correlation between the two parameters.These findings suggest that assessment of leukocyte ROCK activity is minimally invasive and does not require pharmacologic intervention using ROCK inhibitors. Leukocyte p-MBS/total-MBS may be useful for evaluating ROCK activity in a clinical setting.

Thromboangitis obliterans (TAO; Buerger's disease) and atherosclerosis obliterans (ASO) are associated with endothelial dysfunction. The purpose of this study was to evaluate the role of circulating progenitor cells (CPCs) in endothelial function in patients with TAO and ASO. We measured flow-mediated vasodilation (FMD), nitroglycerine-induced vasodilation, and circulating CPCs in 30 patients with TAO and 30 age- and sex-matched healthy subjects and in 40 patients with ASO. FMD was smaller in both the TAO group and ASO group than in the control group (6.6±2.7%, 5.7±3.3% versus 9.5±3.1%, P P r =0.43 and r =0.40, P

We have shown recently that repetition of ischemic preconditioning stimulus augments endothelium-dependent vasodilation in forearm circulation of healthy subjects through increases in NO production and the number of circulating progenitor cells under a local condition. The purpose of this study was to evaluate the “late” effect of ischemic preconditioning on endothelial function in smokers. Ischemic preconditioning was induced by upper-limb ischemia 6 times a day for 1 month. We evaluated forearm blood flow responses to acetylcholine and sodium nitroprusside before and after ischemic preconditioning stimulus in 15 male smokers (27±7 years) and 15 male nonsmokers (26±5 years). Forearm blood flow was measured by using a strain-gauge plethysmography. The ischemic preconditioning stimulus resulted in significant increases in the circulating level of circulating progenitor cells from 1029±261 to 1232±341 mL ( P =0.02), cell migration response to vascular endothelial growth factor from 38±16 to 52±17 per high-power field ( P =0.02), and forearm blood flow response to acetylcholine from 25.1±5.2 to 32.4±6.6 mL/min per 100 mL of tissue ( P =0.002) in nonsmokers, but these did not change in the smoker group. The forearm blood flow responses to sodium nitroprusside before and after the ischemic preconditioning stimulus were similar. Intra-arterial infusion of N G -monomethyl- l -arginine, an NO synthase inhibitor, completely eliminated the ischemic preconditioning stimulus-induced augmentation of forearm blood flow responses to acetylcholine in nonsmokers. These findings suggest that repetition of ischemic preconditioning stimulus may be a simple, safe, and feasible therapeutic technique for endothelial protection of peripheral vessels. However, smoking abolishes ischemic preconditioning stimulus-induced augmentation of endothelium-dependent vasodilation.

The vascular endothelium is involved in the release of various vasodilators, including nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor, as well as vasoconstrictors. NO plays an important role in the regulation of vascular tone, inhibition of platelet aggregation, and suppression of smooth muscle cell proliferation. Endothelial dysfunction is the initial step in the pathogenesis of atherosclerosis. Cardiovascular diseases are associated with endothelial dysfunction. It is well known that the grade of endothelial function is a predictor of cardiovascular outcomes. Oxidative stress plays an important role in the pathogenesis and development of cardiovascular diseases. Several mechanisms contribute to impairment of endothelial function. An imbalance of reduced production of NO or increased production of reactive oxygen species, mainly superoxide, may promote endothelial dysfunction. One mechanism by which endothelium-dependent vasodilation is impaired is an increase in oxidative stress that inactivates NO. This review focuses on recent findings and interaction between endothelial function and oxidative stress in cardiovascular diseases. (Circ J 2009; 73: 411 - 418)

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.

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.

Muscle tightness (MT) is believed to be an important cause of injury for runners. This study evaluated the change of serum creatine kinase (CK), MT in the leg muscles, and delayed onset muscle soreness after running.We evaluated 11 college students who completed a full marathon race. Participants completed a questionnaire on the right quadriceps muscle soreness. The CK activity and MT (iliopsoas, rectus femoris, hamstrings, gastrocnemius, and soleus muscles) were measured. The time points for CK measurements were before; immediately after; and at 1, 2, and 5 days after the race. The time points for MT measurements were the same as for CK except MT was not measured one day after the race. The time points for muscle soreness analysis were before the race and then every morning and night for 5 days after the race.Long-distance running led to significant increases in CK, MT, and muscle soreness. The CK levels peaked day 1 after the race. MT of iliopsoas peaked on day 5; of rectus femoris immediately after the race; and of hamstrings, gastrocnemius, and soleus on day 2. muscle soreness peaked at night on day 1. MT did not decrease to the pre-race levels on day 5. There were no significant changes but CK tended to correlate with the peak of MT of the rectus femoris (r=0.55, P=0.082) and hamstrings (r=0.57, P=0.065).Long-distance running may cause muscle fiber microdamage that may consequently increase CK, MT, and muscle soreness.

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.

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.

Deficiency of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide (NO) synthase, decreases NO production and increases reactive oxygen species. The purpose of this study was to elucidate the effects of aging on endothelial function and to determine whether the degree of BH4 deficiency is related to aging and oxidative stress. We evaluated forearm blood flow (FBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and isosorbide dinitrate (ISDN), an endothelium-independent vasodilator, before and after co-infusion of BH4 (500 mg/min) in 37 healthy men (mean age, 41+/-18 yr; range, 19-81 yr). FBF was measured using strain-gauge plethysmograph. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and serum malondialdehyde-modified low-density lipoprotein (MDA-LDL) were measured as indices of oxidative stress. Both ACh and ISDN increased the FBF in a dose-dependent manner in all subjects. Co-infusion of BH4 resulted in a significant increase in ACh-induced vasodilation (from 22.3+/-6.7 to 30.1+/-7.5 mL/min/100 mL tissue, P

Rho-associated kinases (ROCKs), the immediate downstream targets of RhoA, are ubiquitously expressed serine-threonine protein kinases that are involved in diverse cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, and gene expression. Recent studies have shown that ROCKs may play a pivotal role in cardiovascular diseases such as vasospastic angina, ischemic stroke, and heart failure. Indeed, inhibition of ROCKs by statins or other selective inhibitors leads to the upregulation and activation of endothelial nitric oxide synthase (eNOS) and reduction of vascular inflammation and atherosclerosis. Thus inhibition of ROCKs may contribute to some of the cholesterol-independent beneficial effects of statin therapy. Currently, two ROCK isoforms have been identified, ROCK1 and ROCK2. Because ROCK inhibitors are nonselective with respect to ROCK1 and ROCK2 and also, in some cases, may be nonspecific with respect to other ROCK-related kinases such as myristolated alanine-rich C kinase substrate (MARCKS), protein kinase A, and protein kinase C, the precise role of ROCKs in cardiovascular disease remains unknown. However, with the recent development of ROCK1- and ROCK2-knockout mice, further dissection of ROCK signaling pathways is now possible. Herein we review what is known about the physiological role of ROCKs in the cardiovascular system and speculate about how inhibition of ROCKs could provide cardiovascular benefits.

Background: The Rho kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton. We have recently shown that ROCK activity is increased during the onset of ischemic stroke and that the ROCK inhibitor, fasudil, decreased the severity of focal cerebral ischemia, in part, through the upregulation of endothelial nitric oxide synthase (eNOS). However, the precise mechanism by which ROCK regulates eNOS expression is unknown. Methods and Results: Using RNA affinity chromatography with 3’UTR of eNOS mRNA followed by MALDI-TOF-MS analysis, we have identified eukaryotic elongation factor 1 alpha 1 (eEF1A1) as an actin-binding protein, which directly binds to eNOS mRNA and regulates eNOS mRNA stability. Overexpression of eEF1A1 decreased, and knock-down of eEF1A1 by siRNA increased, eNOS expression. Protein kinase assays using eEF1A1 with purified ROCK1 and ROCK2 showed that eEF1A1 is strongly phosphorylated by ROCK2, but not ROCK1, and that eEF1A1 phosphorylation by ROCK2 is required for binding to and destabilization of eNOS mRNA. Indeed, treatment of human endothelial cells (EC) with fasudil decreased eEF1A1 phosphorylation, but not expression. This was associated with increased eNOS mRNA stability and expression, and NO production. Furthermore, compared to that of WT and ROCK1+/- mice, eNOS mRNA stability and expression were increased in ECs from ROCK2+/- mice and endothelium-dependent relaxation was enhanced in both ROCK2+/- and EC-specific ROCK2 KO mice (EC-ROCK2-/-). These results correlated with decreased cerebral infarct size and neurological deficits in both ROCK2+/- and EC-ROCK2-/- mice subjected to transient focal cerebral ischemia. When ROCK2+/- mice were place on eNOS-/- background (double mutant ROCK2+/-/eNOS-/- mice), the neuroprotective effects observed in ROCK2+/- mice were abolished, indicating that eNOS mediates neuroprotective effects in ROCK2-deficient mice. Conclusion: ROCK2 negatively regulates eNOS expression by phosphorylating eEF1A1, which is required for eEF1A1 to bind to eNOS mRNA. The binding of eEF1A1 decreases eNOS mRNA stability and expression. These findings suggest a novel mechanism by which ROCK2 could impair endothelial function and worsen cerebral ischemia.

Introduction: Flow-mediated vasodilation (FMD) as an index of endothelium-dependent vasodilation and nitroglycerine-induced vasodilation as an index of endothelium-independent vasodilation in the brachial artery using high-resolution ultrasound are useful for assessing vascular function. Some studies have shown that endothelial function assessed by FMD can serve as an independent predictor of cardiovascular events. Previously, we reported that nitroglycerine-induced vasodilationcan be used as a marker for identifying patients with advanced atherosclerosis. The purpose of this study was to determine the prognostic impact of cardiovascular events between FMD and FMD combined with nitroglycerine-induced vasodilation. Methods and Results: We measured FMD and nitroglycerine-induced vasodilation in 402 subjects (mean age, 58±17 years), including patients with cardiovascular diseases. We assessed the associations between FMD combined with nitroglycerine-induced vasodilation and cardiovascular events (death from cardiovascular causes, myocardial infarction, stroke, and revascularization). During a median period of 32.3 months (interquartile range, 17.1 to 49.1 months) of follow-up, 15 subjects died (5 from cardiovascular causes), 3 myocardial infarction, 7 stroke, and 26 revascularization. The receiver operating characteristic curve analysis revealed that FMD alone predicts cardiovascular events with an area under curve of 0.67. FMD combined with nitroglycerine-induced vasodilation predicts cardiovascular events with an area under curve of 0.70. FMD < median value of 3.9% and nitroglycerine-induced vasodilation < median value of 12.2% were associated with increased cardiovascular event. After adjustment for age, sex, body mass index, cardiovascular risk factors, below median FMD and nitroglycerine-induced vasodilation group remained a strong independent indicator of cardiovascular events (hazard ratio, 4.95; 95 percent confidence interval, 1.65 to 21.43; P=0.002). Conclusions: These findings suggest that the combination of FMD and nitroglycerine-induced vasodilation measurement could more accurately predict cardiovascular events than FMD alone.

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 N G -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.

Nicorandil, N-(2-hydroxyethyl)-nicotinamide nitrate, exerts its vasodilatory effects by opening ATP-sensitive potassium (K-ATP) channels and by acting as the exogenous nitric oxide (NO). It is not clear, however, whether the actions of other endothelium-dependent vasodilators, such as NO, endothelium-derived hyperpolarizing factor (EDHF), and prostaglandins, contribute to nicorandil-induced vasodilation in the vasculature in humans. We evaluated forearm blood flow (FBF) response to intraarterial infusion of nicorandil alone and in the presence of glibenclamide, a K-ATP channel inhibitor, N(G)-monomethyl-L-arginine, an NO synthase inhibitor, indomethacin, a cyclooxygenase inhibitor, or miconazol, a cytochrome P-450 inhibitor, in 24 healthy male subjects. FBF was measured using strain-gauge plethysmography. Infusion of nicorandil significantly increased the FBF response in a dose-dependent manner. Intraarterial infusion of glibenclamide attenuated nicorandil-induced vasodilation (160.9 +/- 21.2% versus 90.2 +/- 19.4%, P < 0.01), and miconazole also attenuated the FBF response to nicorandil (160.9 +/- 21.2% versus 66.1 +/- 9.2%, P < 0.001). N-monomethyl-L-arginine or indomethacin did not alter the FBF response to nicorandil. These findings suggest that nicorandil causes vasodilation in forearm circulation in humans, at least in part through a pathway that is dependent on K-ATP channels and cytochrome P-450, but not on endogenous NO and prostaglandins. EDHF may contribute to nicorandil-induced vasodilation in humans.

Background— Patients with limb ischemia were associated with endothelial dysfunction. The purpose of this study was to determine whether autologous bone-marrow mononuclear cell (BM-MNC) implantation improves endothelial dysfunction in patients with limb ischemia. Methods and Results— We evaluated the leg blood flow (LBF) response to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, before and after BM-MNC implantation in 7 patients with limb ischemia. LBF was measured with a mercury-filled Silastic strain-gauge plethysmograph. The number of BM-MNCs implanted into ischemic limbs was 1.6×10 9 ±0.3×10 9 . The number of CD34 + cells included in the implanted BM-MNCs was 3.8×10 7 ±1.6×10 7 . BM-MNC implantation improved the ankle-brachial pressure index (0.33±0.21 to 0.39±0.17, P =0.06), transcutaneous oxygen pressure (28.4±11.5 to 36.6±5.2 mm Hg, P =0.03), and pain-free walking time (0.8±0.6 to 2.9±2.2 minutes, P =0.02). After BM-MNC implantation, LBF response to ACh was enhanced (19.3±6.8 versus 29.6±7.1 mL/min per 100 mL; P =0.002). The vasodilatory effect of SNP was similar before and after BM-MNC implantation. Conclusions— These findings suggest that BM-MNC implantation augments endothelium-dependent vasodilation in patients with limb ischemia.

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% V̇ o 2 max; moderate, 50% V̇ o 2 max; and high, 75% V̇ o 2 max; 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 G -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 P 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.

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.Back...

本研究では有酸素運動を行うことで一酸化窒素（NO）産生増加を介する血管内皮機能にどのような影響を及ぼすかを検討した。対象は運動習慣を持たない健常男性8名（平均年齢：27±3歳）とした。血管内皮依存性拡張物質としてアセチルコリン（ACh）を使用し，血管内皮非依存性拡張物質として 硝酸イソソルビド（ISDN）を使用した。また，NO合成酵素阻害薬としてNG-モノメチル-L-アルギニン（L-NMMA）を使用した。運動方法は最大酸素摂取量の50%とし，1日30分，5回／週の頻度で3ヶ月間行った。前腕血流量の変化はプレチスモグラフにて測定した。ACh投与では運動後に有意な増加を認め，NO合成酵素阻害薬であるL-NMMA投与下では消失した。血管内皮非依存性拡張反応では有意な変化を認めなかった。これらより有酸素運動による血管内皮機能の増強は，NO産生増加を介することが示唆された。

BACKGROUND Poor oral health is an independent predictor of cardiovascular outcome. Endothelial dysfunction is the initial step of atherosclerosis, resulting in cardiovascular outcomes; but there is no information on the association between oral health and endothelial function. The purpose of this study was to determine the relationships between oral health and endothelial function. METHODS AND RESULTS A total of 190 subjects who underwent health examinations (mean age, 57±18 years), including patients with cardiovascular disease, completed a questionnaire on oral health and frequency of tooth brushing, and underwent measurement of vascular function, flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation. The subjects were divided into 2 groups according to frequency of tooth brushing (≥twice/day and

Nitroglycerine-induced vasodilation is usually used as a control test for flow-mediated vasodilation (FMD). However, nitroglycerine-induced vasodilation per se has also been reported to be impaired in patients with atherosclerosis. The purpose of this study was to determine the relationship between nitroglycerine-induced vasodilation and the clinical severity of peripheral artery disease (PAD).We measured nitroglycerine-induced vasodilation and FMD in 144 subjects (mean age: 63.8 ± 15.1 years), including 32 PAD patients with critical limb ischemia (CLI group), 28 PAD patients without CLI (non-CLI group), 60 age- and sex-matched patients without established cardiovascular disease (at-risk group), and 24 healthy subjects (healthy group). Nitroglycerine-induced vasodilation was significantly impaired in the CLI group compared to that in the other three groups (healthy group, 16.0 ± 5.3%; at-risk group, 12.9 ± 3.8%; non-CLI group, 10.3 ± 5.1%; CLI group, 6.7 ± 3.9%; P0.05, respectively). Even after multivariate adjustment, the differences remained significant. On the other hand, FMD was significantly impaired in the at-risk, non-CLI, and CLI group compared with that in the healthy group (healthy group, 7.1 ± 2.9%; at-risk group, 3.4 ± 2.3%; non-CLI group, 3.5 ± 2.7%; CLI group, 3.0 ± 2.8%; P0.001, respectively), but the differences among the at-risk, non-CLI, and CLI groups were not significant. Multivariate analysis revealed that nitroglycerine-induced vasodilation (odds ratio: 0.77, 95% confidence interval [CI]: 0.61-0.97) and diabetes mellitus (odds ratio: 8.75, 95% CI: 1.74-44.2) were independent variables for CLI in PAD patients.There was no significant difference in FMD between PAD patients with and those without CLI, but nitroglycerine-induced vasodilation was significantly smaller in PAD patients with CLI compared with those without CLI.

Objective Rho-associated kinase (ROCK) signaling pathway has been shown to mediate various cellular functions including cell proliferation, migration, adhesion, apoptosis, and contraction, all of which may be involved in pathogenesis of atherosclerosis. Endogenous nitric oxide (NO) is well known to have an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Methods VSMCs migration was evaluated using a modified Boyden chamber assay. ROCK activities were measured by Western blot analysis in murine and human VSMCs and aorta of mice treated with or without angiotensin II (Ang II) and/or sodium nitroprusside (SNP), an NO donor. Results Co-treatment with SNP inhibited the Ang II-induced cell migration and increases in ROCK activity in murine and human VSMCs. Similarly, the increased ROCK activity 2 weeks after Ang II infusion in the mouse aorta was substantially inhibited by subcutaneous injection of SNP. Conclusions These findings suggest that administration of exogenous NO can inhibit ROCK activity in VSMCs in vitro and in vivo.

Cardiovascular diseases are associated with chronic activation of Rho-associated kinase. Rho-associated kinase activity is significantly correlated with endothelial function and Framingham risk score. However, there is no information on the prognostic value of Rho-associated kinase activity. We evaluated Rho-associated kinase activity in peripheral leukocytes by Western blot analysis in 633 subjects who underwent health-screening examination at Hiroshima University Hospital. We assessed the associations between Rho-associated kinase activity and first major cardiovascular events (death from cardiovascular causes, myocardial infarction, and stroke), death from cardiovascular causes, acute myocardial infarction, stroke, revascularization (percutaneous coronary intervention, coronary artery bypass grafting), and hospitalization for heart failure. During a median period of 42.0 months (interquartile range, 24.4–56.6 months) of follow-up, 29 subjects died (10 from cardiovascular causes), 2 myocardial infarction, 20 revascularization, 15 stroke, and 17 hospitalization for heart failure. After adjustment for age, sex, cardiovascular risk factors, and other relevant variables, Rho-associated kinase activity remained a strong independent indicator of first major cardiovascular events (hazard ratio, 2.19; 95% confidence interval, 1.35–3.70; P =0.002), death from cardiovascular disease (hazard ratio, 2.57; 95% confidence interval, 1.18–6.60; P =0.002), stroke (hazard ratio, 2.14; 95% confidence interval, 1.24–3.86; P =0.006), and revascularization (hazard ratio, 2.68; 95% confidence interval, 1.60–4.66; P

The purpose of this study was to determine whether a high NaCl intake impairs endothelium-dependent and -independent vasodilation of forearm circulation in salt sensitive (SS) patients with essential hypertension. We evaluated the effects of intra-arterial acetylcholine (ACh) and isosorbide dinitrate (ISDN) on forearm hemodynamics in 29 patients with essential hypertension, while consuming a low NaCl (50 mmol/d) or high Na Cl (340 mmol/d) diet for 1 week. The forearm blood flow (FBF) was measured by strain-gauge plethysmography. Patients were classified as SS (n=12) or salt resistant (SR; n=17) based on salt-induced changes in blood pressures. The FBF responses of ACh and ISDN were similar in the SS and SR patients while on either NaCl diet, and was not altered by salt loading (ACh, SS: low NaCl 22.8+/-4.3 vs. high NaCl 21.1+/-3.6 ml/min per 100 ml, SR: low NaCl 22.5+/-4.0 vs. high NaCl 23.3+/-4.1 ml/min per 100 ml; ISDN, SS: low NaCl 13.9+/-2.1 vs. high NaCl 14.1+/-2.2 ml/min per 100 ml, SR: low NaCl 13.8+/-2.3 vs. high NaCl 14.0+/-2.2 ml/min per 100 ml). There were no significant differences in the vascular responses to ACh and ISDN in the presence of N(G)-monomethyl-L-arginine, a nitric oxide synthase inhibitor, in either group for either NaCl diet. These findings suggest that forearm resistance artery endothelial function may not be influenced by salt loading in either SS patients which finding may play a role in determining salt sensitivity in patients with essential hypertension or SR patients.

Objectives The purpose of this study was to determine the relationships between uric acid, endothelial function and cardiovascular risk factors and to investigate whether menopausal status was associated with the relationship between uric acid and endothelial function in women. Design Cross-sectional study. Setting 3 general hospitals in Japan. Participants 749 Japanese women aged 30–74 years recruited from people who underwent health-screening examinations with agreement for measurement of vascular function. Measures We measured serum concentrations of uric acid and flow-mediated vasodilation (FMD). Percentage of FMD (peak diameter−baseline diameter/baseline diameter) was used for analysis. Endothelial dysfunction was defined as FMD ≤4.90%, division point for the lowest tertile and the middle tertile of FMD. Menopause women were defined as participants without menstruation for over 1 year or participants with a history of hysterectomy or bilateral oophorectomy. Results Of the 749 participants, 368 (49.1%) were premenopausal women and 381 (50.9%) were postmenopausal women. Age, body mass index, systolic blood pressure, total cholesterol, triglycerides, glucose, estimated glomerular filtration rate and Framingham risk score were significantly correlated with serum uric acid level. FMD showed a gradual decrease in accordance with the serum uric acid level in the entire study population (

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.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 aged70 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).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).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.