Increased arterial stiffness and cardiovascular risk prediction in controlled hypertensive patients with coronary artery disease: post hoc analysis of FMD-J (Flow-mediated Dilation Japan) Study A.

The usefulness of brachial-ankle pulse wave velocity (baPWV), an index of arterial stiffness, is not fully known for the management of treated hypertensive patients with a history of coronary artery disease (CAD) who have blood pressure less than 130/80 mmHg, a recommended blood pressure target in the updated major hypertension guidelines. We analyzed data for 447 treated hypertensive patients with CAD enrolled in FMD-J Study A for assessment of the predictive value of baPWV for future cardiovascular events. The primary outcome was a composite of coronary events, stroke, heart failure, and sudden death. During a median follow-up period of 47.6 months, the primary outcome occurred in 64 patients. Blood pressure less than 130/80 mmHg was significantly associated with a lower risk of the composite outcome independent of other cardiovascular risk factors in treated hypertensive patients with CAD (hazard ratio, 0.59; 95% confidence interval (CI), 0.35-0.99; P = 0.04). In treated hypertensive patients with CAD who had blood pressure less than 130/80 mmHg, baPWV above the cutoff value of 1731 cm/s, derived from receiver-operator characteristic curve analysis for the composite outcome was significantly associated with a higher risk of the composite outcome independent of conventional risk factors (hazard ratio, 2.83; 95% CI, 1.02-7.91; P = 0.04). baPWV was an independent predictor of cardiovascular events in treated hypertensive patients with CAD who had blood pressure less than 130/80 mmHg, for whom measurement of baPWV is recommended for cardiovascular risk assessment.

Endothelial Dysfunction, Increased Arterial Stiffness, and Cardiovascular Risk Prediction in Patients With Coronary Artery Disease: FMD-J (Flow-Mediated Dilation Japan) Study A.

BACKGROUND: The usefulness of vascular function tests for management of patients with a history of coronary artery disease is not fully known. METHODS AND RESULTS: We measured flow-mediated vasodilation (FMD) and brachial-ankle pulse wave velocity (baPWV) in 462 patients with coronary artery disease for assessment of the predictive value of FMD and baPWV for future cardiovascular events in a prospective multicenter observational study. The first primary outcome was coronary events, and the second primary outcome was a composite of coronary events, stroke, heart failure, and sudden death. During a median follow-up period of 49.2 months, the first primary outcome occurred in 56 patients and the second primary outcome occurred in 66 patients. FMD above the cutoff value of 7.1%, derived from receiver-operator curve analyses for the first and second primary outcomes, was significantly associated with lower risk of the first (hazard ratio, 0.27; 95% confidence interval, 0.06-0.74; P=0.008) and second (hazard ratio, 0.32; 95% confidence interval, 0.09-0.79; P=0.01) primary outcomes. baPWV above the cutoff value of 1731 cm/s was significantly associated with higher risk of the first (hazard ratio, 1.86; 95% confidence interval, 1.01-3.44; P=0.04) and second (hazard ratio, 2.19; 95% confidence interval, 1.23-3.90; P=0.008) primary outcomes. Among 4 groups stratified according to the combination of cutoff values of FMD and baPWV, stepwise increases in the calculated risk ratio for the first and second primary outcomes were observed. CONCLUSIONS: In patients with coronary artery disease, both FMD and baPWV were significant predictors of cardiovascular events. The combination of FMD and baPWV provided further cardiovascular risk stratification. CLINICAL TRIAL REGISTRATION: URL: www.umin.ac.jp. Unique identifier: UMIN000012950.

Brachial artery diameter as a marker for cardiovascular risk assessment: FMD-J study.

BACKGROUND AND AIMS: Baseline brachial artery (BBA) diameter has been reported to be a potential confounding factor of flow-mediated vasodilation (FMD). The purpose of this study was to evaluate the relationships between BBA diameter and cardiovascular risk factors and compare the diagnostic accuracy of BBA diameter in subjects without cardiovascular risk factors and patients with cardiovascular disease (CVD) with that of FMD. METHODS: We measured BBA diameter and FMD in 5695 male subjects. In addition, we retrospectively investigated the incidence of cardiovascular events using another population sample consisting of 440 male subjects, to compare the accuracy of BBA diameter with that of FMD in predicting cardiovascular events. RESULTS: BBA diameter and FMD significantly correlated with age, body mass index, systolic blood pressure, diastolic blood pressure, triglycerides, high-density lipoprotein cholesterol, and glucose as well as Framingham risk score. The prevalence of cardiovascular risk factors and CVD increased with the increase in BBA diameter and FMD. Area under the curve (AUC) value of the receiver operating characteristic (ROC) curve for BBA diameter to diagnose subjects without cardiovascular risk factors (0.59 vs. 0.62, p = 0.001) or patients with CVD (0.58 vs. 0.64, p < 0.001) was significantly lower than that for FMD. In the retrospective study, the AUC value of the ROC curve for BBA diameter to predict first major cardiovascular events was significantly lower than that of FMD (0.50 vs. 0.62, p = 0.03). CONCLUSIONS: In men, BBA diameter was inferior to FMD for assessment of cardiovascular risk.

Endothelial Function Is Impaired in Patients Receiving Antihypertensive Drug Treatment Regardless of Blood Pressure Level: FMD-J Study (Flow-Mediated Dilation Japan).

Hypertension is associated with endothelial dysfunction. Blood pressure significantly correlates with endothelial function in antihypertensive drug-naive subjects. The purpose of this study was to determine whether treatment status affects the relationship between blood pressure and endothelial function. We measured flow-mediated vasodilation (FMD) in 2297 subjects, including 1822 antihypertensive drug-naive subjects and 475 treated hypertensive patients. FMD significantly decreased in relation to increase in systolic blood pressure (8.2±3.1% in subjects with systolic blood pressure of <120 mm Hg, 7.5±2.8% for 120-129 mm Hg, 7.1±2.8% for 130-139 mm Hg, and 6.7±2.6% for ≥140 mm Hg; P<0.001). Systolic blood pressure was independently associated with FMD in untreated subjects. In contrast, there was no significant relationship between systolic blood pressure and FMD in treated hypertensive patients (4.6±3.1% in treated hypertensives with systolic blood pressure of <120 mm Hg, 4.8±2.7% for 120-129 mm Hg, 4.9±2.8% for 130-139 mm Hg, and 4.5±2.3% for ≥140 mm Hg; P=0.77). Propensity score matching analysis revealed that the prevalence of endothelial dysfunction defined as FMD of less than the division point for the lowest tertile, and the middle tertile of FMD was significantly higher in treated hypertensive patients than in untreated subjects in all systolic blood pressure categories. Endothelial function assessed by FMD was impaired regardless of the level of blood pressure achieved by antihypertensive drug treatment in hypertensive patients.

Exogenous nitric oxide inhibits Rho-associated kinase activity in patients with angina pectoris: a randomized controlled trial.

The RhoA/Rho-associated kinase (ROCK) pathway has a key physiological role in the pathogenesis of atherosclerosis. Increased ROCK activity is associated with cardiovascular diseases. Endogenous nitric oxide (NO) has 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 patients with angina pectoris. This is a prospective, open-label, randomized, controlled study. A total of 30 patients with angina pectoris were randomly assigned to receive 40 mg day(-1) of isosorbide mononitrate (n=15, 12 men and 3 women, mean age of 63±12 years, isosorbide mononitrate group) or conventional treatment (n=15, 13 men and 2 women, mean age of 64±13 years, control group) for 12 weeks. ROCK activity in peripheral leukocytes was measured by western blot analysis. ROCK activities at 4 and 12 weeks after treatment were decreased in the isosorbide mononitrate group (0.82±0.33 at 0 week, 0.62±0.20 at 4 weeks, 0.61±0.19 at 12 weeks, n=15 in each group, P<0.05, respectively) but not altered in the control group. ROCK1 and ROCK2 expression levels were similar in all treatment periods in the two groups. These findings suggest that the administration of exogenous NO can inhibit ROCK activity, indicating that the usage of exogenous NO could have a protective effect in patients with angina pectoris.

Ratio of serum levels of AGEs to soluble form of RAGE is a predictor of endothelial function.

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.

Hyperuricemia is independently associated with endothelial dysfunction in postmenopausal women but not in premenopausal women.

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 (<4 mg/dL, 6.85±3.65%; 4 to <5 mg/dL, 6.79±3.60%; 5 to <6 mg/dL, 6.24±3.58%; ≥6 mg/dL, 5.27±3.18%; p=0.01). Multivariate analysis revealed that uric acid was a significantly independent risk factor for endothelial dysfunction in postmenopausal women (OR 1.23, 95% CI 1.01 to 1.50), but not in premenopausal women. CONCLUSIONS: These findings suggest that uric acid can be used as a risk marker of endothelial dysfunction in a female population, and particularly as an independent risk factor in postmenopausal women but not in premenopausal women. REGISTRATION NUMBER OF THE STUDY: UMIN000003409.

Relationship between flow-mediated vasodilation and cardiovascular risk factors in a large community-based study.

OBJECTIVE: To determine the relationships between flow-mediated vasodilation (FMD) and cardiovascular risk factors, and to evaluate confounding factors for measurement of FMD in a large general population in Japan. METHODS: This was a cross-sectional study. A total of 5314 Japanese adults recruited from people who underwent health screening from 1 April 2010 to 31 August 2012 at 3 general hospitals in Japan. Patients' risk factors (age, Body Mass Index, blood pressure, cholesterol parameters, glucose level and HbA1c level) and prevalence of cardiovascular disease (coronary heart disease and cerebrovascular disease) were investigated. RESULTS: Univariate regression analysis revealed that FMD correlated with age (r=-0.27, p<0.001), Body Mass Index (r=-0.14, p<0.001), systolic blood pressure (r=-0.18, p<0.001), diastolic blood pressure (r=-0.13, p<0.001), total cholesterol (r=-0.07, p<0.001), triglycerides (r=-0.10, p<0.001), high-density lipoprotein cholesterol (r=0.06, p<0.001), low-density lipoprotein cholesterol (r=-0.04, p=0.01), glucose level (r=-0.14, p<0.001), HbA1c (r=-0.14, p<0.001), and baseline brachial artery diameter (r=-0.43, p<0.001) as well as Framingham Risk score (r=-0.29, p<0.001). Multivariate analysis revealed that age (t value=-9.17, p<0.001), sex (t value=9.29, p<0.001), Body Mass Index (t value=4.27, p<0.001), systolic blood pressure (t value=-2.86, p=0.004), diabetes mellitus (t value=-4.19, p<0.001), smoking (t value=-2.56, p=0.01), and baseline brachial artery diameter (t value=-29.4, p<0.001) were independent predictors of FMD. CONCLUSIONS: FMD may be a marker of the grade of atherosclerosis and may be used as a surrogate marker of cardiovascular outcomes. Age, sex, Body Mass Index, systolic blood pressure, diabetes mellitus, smoking and, particularly, baseline brachial artery diameter are potential confounding factors in the measurement of FMD.

A novel noninvasive and simple method for assessment of endothelial function: enclosed zone flow-mediated vasodilation (ezFMD) using an oscillation amplitude measurement.

BACKGROUND: It 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 RESULTS: We 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 < 0.0001), body mass index (r = -0.13, P = 0.028), systolic blood pressure (r = -0.15, P = 0.009), diastolic blood pressure (r = -0.14, P = 0.011), fasting glucose level (r = -0.27, P = 0.006), smoking (r = -0.21, P = 0.007) and baseline pulse wave amplitude (r = -0.51, P < 0.0001). ezFMD significantly correlated with conventional FMD (r = 0.34, P < 0.0001). Multiple regression analysis revealed that age (P = 0.002), body mass index (P = 0.013), systolic blood pressure (P = 0.009), smoking (P = 0.004) and baseline pulse wave amplitude (P < 0.001) were independent predictors of ezFMD. CONCLUSIONS: These findings suggest that measurement of ezFMD, a novel noninvasive and simple method, may be useful for determination of vascular diameter response to reactive hyperemia. Since ezFMD is automatically measured by a device with an oscillometric method, measurement of ezFMD is easier and less biased than that of conventional FMD.

Nitroglycerine-induced vasodilation for assessment of vascular function: a comparison with flow-mediated vasodilation.

OBJECTIVE: Nitroglycerine-induced vasodilation has been used as a control test for flow-mediated vasodilation (FMD) to differentiate endothelium-dependent from endothelium-independent response when evaluating endothelial function in humans. Recently, nitroglycerine-induced vasodilation has also been reported to be impaired in patients with atherosclerosis. The purpose of this study was to determine the relationships between nitroglycerine-induced vasodilation and cardiovascular risk factors. APPROACH AND RESULTS: We measured nitroglycerine-induced vasodilation and FMD in 436 subjects who underwent health examinations (mean age, 53 ± 19 years; age range, 19-86 years), including patients with cardiovascular diseases. There was a significant relationship between nitroglycerine-induced vasodilation and FMD (r=0.42; P<0.001). Univariate regression analysis revealed that nitroglycerine-induced vasodilation correlated with age (r=-0.34; P<0.001), systolic blood pressure (r=-0.32; P<0.001), diastolic blood pressure (r=-0.24; P<0.001), heart rate (r=-0.21; P<0.001), glucose (r=-0.23; P<0.001), and smoking pack-year (r=-0.12; P=0.01), as well as Framingham risk score (r=-0.30; P<0.001). Nitroglycerine-induced vasodilation was significantly smaller in patients with cardiovascular disease than in both subjects with and without cardiovascular risk factors (10.5 ± 5.6% versus 13.7 ± 5.4% and 15.3 ± 4.3%; P<0.001, respectively), whereas there was no significant difference in nitroglycerine-induced vasodilation between subjects with and without cardiovascular risk factors. Multivariate analysis revealed that male sex, body mass index, hypertension, diabetes mellitus, baseline brachial artery diameter, and FMD were independent predictors of nitroglycerine-induced vasodilation. CONCLUSIONS: These findings suggest that nitroglycerine-induced vasodilation may be a marker of the grade of atherosclerosis. FMD should be interpreted as an index of vascular function reflecting both endothelium-dependent vasodilation and endothelium-independent vasodilation in subjects with impaired nitroglycerine-induced vasodilation.

Flow-mediated vasodilation and anatomical variation of the brachial artery (double brachial artery) in healthy subjects and patients with cardiovascular disease.

BACKGROUND: The purpose of this study was to evaluate the effect of anatomical variation of the brachial artery on flow-mediated vasodilation (FMD) in healthy subjects and patients with cardiovascular disease (CVD). METHODS AND RESULTS: There was no significant difference in the prevalence of double brachial artery between healthy subjects (6.1%) and patients with CVD (6.5%). In healthy subjects, FMD was larger in a single brachial artery than in large and small vessels of a double brachial artery (7.2±3.4% vs. 4.7±3.3% and 4.5±2.5%, P<0.01, respectively). In patients with CVD, there were no significant differences in FMD among a single brachial artery, large vessel of a double brachial artery and small vessel of a double brachial artery (3.3±1.4%, 3.1±2.3% and 3.6±2.1%). FMD in a single brachial artery was smaller in patients with CVD than in healthy subjects. There were no significant differences in FMD in the large vessel of a double brachial artery between the 2 groups or in the small vessel of a double brachial artery between the 2 groups. Nitroglycerine-induced vasodilation was similar in all arteries in healthy subjects and patients with CVD. CONCLUSIONS: When measuring FMD, the existence of a double brachial artery should be checked. FMD measured in a double brachial artery may be underestimated in healthy subjects.

Intima-media thickness of brachial artery, vascular function, and cardiovascular risk factors.

OBJECTIVE: Cardiovascular diseases are associated with impaired flow-mediated vasodilation (FMD) and increase in carotid intima-media thickness (IMT). Both FMD and IMT are independent predictors for cardiovascular outcomes. When measuring FMD and nitroglycerine-induced vasodilation in the brachial artery, IMT can also be simultaneously assessed in the same brachial artery. The purpose of this study was to determine the relationships between IMT of the brachial artery, vascular function, and cardiovascular risk factors. METHODS AND RESULTS: We measured brachial IMT, FMD, and nitroglycerine-induced vasodilation by ultrasound in 388 subjects who underwent health examination (mean age, 45±22 years; age range, 19-86), including patients with cardiovascular diseases. Univariate regression analysis revealed that brachial IMT significantly correlated with age (r=0.71; P<0.001), body mass index (r=0.27; P<0.001), systolic blood pressure (r=0.40; P<0.001), diastolic blood pressure (r=0.31; P<0.001), heart rate (r=0.15; P=0.002), glucose level (r=0.18; P=0.01), and smoking pack-years (r=0.42; P<0.001), as well as Framingham risk score, a cumulative cardiovascular risk index for heart attack (r=0.49; P<0.001). FMD and nitroglycerine-induced vasodilation were inversely associated with brachial IMT (r=-0.39, P<0.001; r=-0.32, P<0.001, respectively). In addition, there was a significant relationship between brachial IMT and carotid IMT (r=0.58; P<0.001). Multivariate analysis revealed that age, sex, hypertension, and brachial artery diameter were independent predictors of brachial IMT. CONCLUSIONS: These findings suggest that brachial IMT may be a marker of the grade of atherosclerosis and may be used as a marker of vascular function, providing additive information for stratifying subjects with cardiovascular risk factors.

Hyperbilirubinemia, augmentation of endothelial function, and decrease in oxidative stress in Gilbert syndrome.

BACKGROUND: Patients with Gilbert syndrome have mild unconjugated hyperbilirubinemia. It has been shown that bilirubin is an endogenous antioxidant. We evaluated the role of oxidative stress in endothelial function in patients with Gilbert syndrome under normal conditions without cardiovascular risk factors. METHODS AND RESULTS: A total of 108 young men with Gilbert syndrome without cardiovascular risk factors and 108 age-matched healthy men (normal controls) were enrolled in this study. Serum concentrations of bilirubin were higher in patients with Gilbert syndrome than in control subjects (29.2±11.6 versus 9.4±2.7 µmol/L; P<0.001). Serum concentrations of malondialdehyde-modified low-density lipoprotein and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), as indices of oxidative stress, were lower in patients with Gilbert syndrome than in control subjects (61.8±24.5 versus 72.5±21.8 U/L, P=0.034; 7.8±2.4 versus 10.4±3.2 ng/mg creatinine, P=0.001, respectively). Flow-mediated vasodilation was greater in patients with Gilbert syndrome than in normal control subjects (7.2±2.2% versus 5.9±1.7%; P<0.001). Vascular responses to nitroglycerine were not significantly different between the 2 groups. Flow-mediated vasodilation correlated with serum concentration of bilirubin (r=0.44, P<0.001), malondialdehyde-modified low-density lipoprotein (r=-0.25, P=0.01), and urinary excretion of 8-OHdG (r=-0.27, P=0.004) in patients with Gilbert syndrome but not in control subjects. In addition, serum concentration of bilirubin correlated with malondialdehyde-modified low-density lipoprotein (r=-0.20, P=0.04) and 8-OHdG (r=-0.21, P=0.02) in patients with Gilbert syndrome but not in control subjects. CONCLUSIONS: Patients with Gilbert syndrome had low levels of oxidative stress associated with hyperbilirubinemia and enhancement of endothelium-dependent vasodilation. CLINICAL TRIAL REGISTRATION: URL: http://www.umin.ac.jp. Unique identifier: UMIN000003409.

Deep echo attenuation without calcification increases the risk of periprocedural myonecrosis after elective percutaneous coronary intervention in patients with coronary artery disease.

OBJECTIVE: To investigate the clinical factors, including intravascular ultrasound (IVUS) findings, associated with percutaneous coronary intervention (PCI)-related periprocedural myonecrosis (PM) in patients with stable coronary artery disease (CAD). METHODS: The study included 46 patients (mean age 65±2 y, 37 men) with stable CAD who underwent elective PCI using IVUS and a Doppler flow guidewire. In addition to routine IVUS measurements, the presence of deep echo attenuation (DEA) without calcification was assessed. The sum of all high-intensity transient signals (HITS) after a balloon deflation procedure was calculated using a Doppler flow guidewire. PM was defined as an elevation in troponin T to >0.03 ng/mL 24-hour after PCI. Patients were divided into 2 groups on the basis of the presence or absence of PM. RESULTS: PM was identified in 17 patients (37%). Estimated glomerular filtration ratio was lower in the PM group (p=0.021). Target vessel distribution was different between the groups (p=0.004). Positive remodeling and DEA, as observed on IVUS, were more common in the PM group (p=0.03 and p<0.0001, respectively). The total number of HITS was higher in the PM group (p=0.003). Logistic regression analysis demonstrated that the presence of DEA (p=0.003) was the sole factor associated with the occurrence of PM. CONCLUSION: These results demonstrated that DEA is an important factor associated with PM in patients who underwent an elective PCI. Therefore, IVUS to determine the presence of DEA should be performed before PCI.

Geranylgeranylacetone, heat shock protein 90/AMP-activated protein kinase/endothelial nitric oxide synthase/nitric oxide pathway, and endothelial function in humans.

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. CONCLUSIONS: 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.

Flow-mediated vasodilation is augmented in a corkscrew collateral artery compared with that in a native artery in patients with thromboangiitis obliterans (Buerger disease).

BACKGROUND: A healthy endothelium maintains vascular tone and structure. The purpose of this study was to evaluate endothelial function in corkscrew collateral arteries in Buerger disease. METHODS: We 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. RESULTS: Hyperemic 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. CONCLUSIONS: Endothelial 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.

Rho-associated kinase activity, endothelial function, and cardiovascular risk factors.

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<0.001), and that flow-mediated vasodilation significantly correlated with age (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<0.001). There was a significant correlation between leukocyte ROCK activity and flow-mediated vasodilation (r=-0.41, P<0.001). Multivariate analysis revealed that flow-mediated vasodilation was an independent predictor of leukocyte ROCK activity. CONCLUSIONS: These findings suggest that cumulative cardiovascular risk may enhance ROCK activity and endothelial dysfunction, leading to progression of cardiovascular diseases and outcomes.

Autologous bone-marrow mononuclear cell implantation reduces long-term major amputation risk in patients with critical limb ischemia: a comparison of atherosclerotic peripheral arterial disease and Buerger disease.

BACKGROUND: Bone-marrow mononuclear cell (BM-MNC) implantation improves ischemic symptoms in patients with critical limb ischemia (CLI). The purpose of this study was to evaluate long-term clinical outcomes after autologous BM-MNC implantation in patients with CLI. METHODS AND RESULTS: We assessed long-term clinical outcomes after BM-MNC implantation in 51 patients with CLI, including 25 patients with peripheral arterial disease (PAD) and 26 patients with Buerger disease. Forty-six CLI patients who had no BM-MNC implantation served as control subjects. Median follow-up period was 4.8 years. The 4-year amputation-free rates after BM-MNC implantation were 48% in PAD patients and 95% in Buerger disease, and they were 0% in control PAD patients and 6% in control Buerger disease. The 4-year overall survival rates after BM-MNC implantation were 76% in PAD patients and 100% in Buerger disease, and they were 67% in control PAD patients and 100% in control Buerger disease. Multivariable Cox proportional hazards analysis revealed that BM-MNC implantation correlated with prevention of major amputation and that hemodialysis and diabetes mellitus correlated with major amputation. In Buerger disease, ankle brachial pressure index and transcutaneous oxygen pressure were significantly increased after 1 month and remained high during 3-year follow-up. However, in patients with PAD, ankle brachial pressure index and transcutaneous oxygen pressure significantly increased after 1 month and gradually decreased during 3-year follow-up and returned to baseline levels. CONCLUSIONS: These findings suggest that BM-MNC implantation is safe and effective in patients with CLI, especially in patients with Buerger disease. Clinical Trial Registration- URL: http://home.hiroshima-u.ac.jp/angio/. Unique identifier: 001769.

Measurement of Rho-associated kinase (ROCK) activity in humans: validity of leukocyte p-MBS/t-MBS in comparison with vascular response to fasudil.

BACKGROUND: 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. METHODS AND RESULTS: 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 (P<0.0001) 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, P<0.0001). 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, P<0.0001). In healthy subjects, there was no significant correlation between the two parameters. CONCLUSIONS: 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.