Daisuke Jitsuiki

Autologous Bone-Marrow Mononuclear Cell Implantation Improves Endothelium-Dependent Vasodilation in Patients With Limb Ischemia

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 1.6×109±0.3×109. The number of CD34+ cells included in the implanted BM-MNCs was 3.8×107±1.6×107. 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.

Periodontal Infection Is Associated With Endothelial Dysfunction in Healthy Subjects and Hypertensive Patients

The purpose of this study was to evaluate endothelial function in patients with periodontitis. We evaluated forearm blood flow responses to acetylcholine and sodium nitroprusside in patients with periodontitis who had no other cardiovascular risk factors (32 men; 25±3 years of age), in a normal control group (20 men; 26±3 years of age), and in hypertensive patients with periodontitis (28 men and 10 women; 56±12 years of age) and without periodontitis (control group; 18 men and 6 women; 54±13 years of age). Forearm blood flow was measured using strain-gauge plethysmography. Circulating levels of C-reactive protein and interleukin-6 were significantly higher in the periodontitis group than in the control group. Both in healthy and hypertensive subjects, forearm blood flow responses to acetylcholine were significantly smaller in the periodontitis group than in the control group. Sodium nitroprusside–stimulated vasodilation was similar in the 2 groups. Periodontal therapy reduced serum concentrations of C-reactive protein and interleukin-6 and augmented acetylcholine-induced vasodilation in periodontitis patients with and without hypertension. After administration of NG-monomethyl-l-arginine, an NO synthase inhibitor, forearm blood flow response to acetylcholine was similar before and after treatment. These findings suggest that periodontitis is associated with endothelial dysfunction in subjects without cardiovascular risk factors, as well as hypertensive patients, through a decrease in NO bioavailability and that systemic inflammation may be, at least in part, a cause of endothelial dysfunction, leading to cardiovascular diseases.

Edaravone (3-Methyl-1-Phenyl-2-Pyrazolin-5-one), A Novel Free Radical Scavenger, for Treatment of Cardiovascular Diseases

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a strong novel free radical scavenger, is used for treatment of patients with acute brain infarction. Edaravone has preventive effects on myocardial injury following ischemia and reperfusion in patients with acute myocardial infarction. Antioxidant actions of edaravone include enhancement of prostacyclin production, inhibition of lipoxygenase metabolism of arachidonic acid by trapping hydroxyl radicals, inhibition of alloxan-induced lipid peroxidation, and quenching of active oxygen, leading to protection of various cells, such as endothelial cells, against damage by reactive oxygen species (ROS). Recently, we have shown that edaravone improves endothelial function through a decrease in ROS in smokers. From a clinical perspective, it is important to select an appropriate drug that is effective in improving endothelial function in patients with cardiovascular diseases. The novel free radical scavenger edaravone may represent a new therapeutic intervention for endothelial dysfunction in the setting of atherosclerosis, chronic heart failure, diabetes mellitus, or hypertension. This review focuses on clinical findings and on putative mechanisms underlying the beneficial effects of the antioxidative agent edaravone on the artherosclerotic process in patients with cardiovascular diseases.

Aging and hypertension are independent risk factors for reduced number of circulating endothelial progenitor cells.

BACKGROUND Recent studies have revealed the existence of bone marrow-derived endothelial progenitor cells (EPCs). The number of circulating EPCs might reflect the pathogenesis of atherosclerosis and progression of cardiovascular diseases (CVDs). The purpose of this study was to evaluate the relationship between the number of EPCs and cardiovascular risk factors. METHODS Flow cytometry analysis was used to quantify the number of EPCs (CD34(+)AC133(+)CD45(low)) in 135 consecutive hospitalized patients with CVD and 25 healthy subjects. RESULTS The number of EPCs was less in the patients than in the healthy subjects (1,047.4 +/- 521.1 vs. 612.8 +/- 461.6/ml, P < 0.0001). The number of EPCs significantly correlated with the number of risk factors (r = 0.424, P < 0.0001). The numbers of EPCs in patients with hypertension and diabetes mellitus were less than those in patients without those diseases (762.6 +/- 579.5 vs. 495.2 +/- 297.7/ml, P < 0.01 and 666.8 +/- 505.5 vs. 477.0 +/- 290.4/ml, P < 0.05, respectively). In healthy subjects a reduced number of EPCs was found in smokers compared with nonsmokers (833.3 +/- 347.5 vs. 1,274.6 +/- 560.9/ml, P < 0.05), whereas smoking did not alter the number of EPCs in the patients group. In multivariate analysis, hypertension and age were independent predictors of reduced number of EPCs. Renin-angiotensin system (RAS) inhibitors increased the number of EPCs (464.7 +/- 252.1/ml vs. 617.5 +/- 343.5/ml, P < 0.05), while calcium antagonists, diuretics, and beta-blockers did not alter the number of EPCs in patients with hypertension. CONCLUSIONS These findings suggest that both aging and hypertension are risk factors for reduced number of EPCs and that RAS inhibitors increase the number of EPCs.

Repetition of Ischemic Preconditioning Augments Endothelium-Dependent Vasodilation in Humans: Role of Endothelium-Derived Nitric Oxide and Endothelial Progenitor Cells

Background—Several studies have shown that both early and late effects of ischemic preconditioning (IPC) protect against myocardial injury after ischemic reperfusion. Methods and Results—The purpose of this study was to evaluate the late effects of IPC on endothelial function in humans. Late phase of IPC was induced by upper limb ischemia (cuff inflation of over 200 mm Hg for 5 minutes) 6 times a day for 1 month. We evaluated forearm blood flow (FBF) responses to acetylcholine (ACh) and to sodium nitroprusside (SNP) before and after IPC stimulus in 30 young healthy men. FBF was measured using a strain-gauge plethysmograph. The IPC stimulus significantly increased plasma concentration of vascular endothelial growth factor (VEGF), circulating level of endothelial progenitor cells (EPCs), and FBF responses to ACh, but these did not change in the control group. The FBF responses to SNP were similar before and after the IPC stimulus. Infusion of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, completely eliminated the IPC stimulus-induced augmentation of FBF responses to ACh. In the cotralateral arms of subjects that received the IPC stimulus, FBF responses to ACh did not change, but levels of VEGF and circulating EPCs increased. Conclusions—These findings suggest that repetition of late IPC stimulus augments endothelium-dependent vasodilation in humans through increases in nitric oxide production and number of EPCs under a local condition. Repetition of IPC stimulus may be a simple, safe, and feasible therapeutic technique for endothelial protection of peripheral vessels.

Smoking, Endothelial Function, and Rho-Kinase in Humans

Objective—Smoking is associated with endothelial dysfunction and activated Rho-kinase in vascular smooth muscle cells (VSMCs) in humans. The purpose of this study was to elucidate the relationship between endothelial function and Rho-kinase activity in forearm VSMCs in healthy young men. Methods and Results—We evaluated the forearm blood flow (FBF) responses to acetylcholine (ACh), fasudil, a Rho-kinase inhibitor, and sodium nitroprusside (SNP) in male smokers (n=10) and nonsmokers (n=14). FBF was measured by using a strain-gauge plethysmography. The vasodilatory effect of ACh was significantly smaller in smokers than that in nonsmokers. The vasodilatory effect of fasudil was significantly greater in smokers than that in nonsmokers. The vasodilatory effects of SNP in the 2 groups were similar. There was a significant correlation between the maximal FBF response to fasudil and that to ACh (r=−0.67; P<0.01). There was no significant correlation between the maximal FBF response to fasudil and that to SNP. The intra-arterial coinfusion of fasudil significantly increased the FBF response to ACh in smokers but not in nonsmokers. There were no significant differences between FBF response to fasudil alone and that in combination with NG-monomethyl-l-arginine in smokers and in nonsmokers. The intra-arterial coinfusion ascorbic acid did not alter the FBF response to fasudil in both groups. Conclusions—These findings suggest that smoking is involved in not only endothelial dysfunction but also activation of Rho-kinase in VSMCs in forearm circulation, and that there is a significant correlation between endothelial function and Rho-kinase activity in VSMCs.

Pycnogenol ® , French Maritime Pine Bark Extract, Augments Endothelium-Dependent Vasodilation in Humans

Pycnogenol®, an extract of bark from the French maritime pine, Pinus pinaster Ait., consists of a concentrate of water-soluble polyphenols. Pycnogenol® contains the bioflavonoids catechin and taxifolin as well as phenolcarbonic acids. Antioxidants, such as bioflavonoids, enhance endothelial nitric oxide (NO) synthase expression and subsequent NO release from endothelial cells. The purpose of this study was to determine Pycnogenol®s effects on endothelium-dependent vasodilation in humans. This was a double-blind, randomized, placebo and active drug study. We evaluated forearm blood flow (FBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and to sodium nitroprusside (SNP), an endothelium-independent vasodilator, in healthy young men before and after 2 weeks of daily oral administration of Pycnogenol® (180 mg/day) (n=8) or placebo (n=8). FBF was measured by using strain-gauge plethysmography. Neither the placebo nor Pycnogenol® altered forearm or systemic hemodynamics. Pycnogenol®, but not placebo, augmented FBF response to ACh, from 13.1±7.0 to 18.5±4.0 mL/min per 100 mL tissue (p<0.05). SNP-stimulated vasodilation was similar before and after 2 weeks of treatment in the control and Pycnogenol® groups. The administration of NG-monomethyl-L-arginine, an NO synthase inhibitor, completely abolished Pycnogenol®-induced augmentation of the FBF response to ACh. These findings suggest that Pycnogenol® augments endothelium-dependent vasodilation by increasing in NO production. Pycnogenol® would be useful for treating various diseases whose pathogeneses involve endothelial dysfunction.

Smoking Activates Rho-Kinase in Smooth Muscle Cells of Forearm Vasculature in Humans

Abstract—Previous studies have shown that smoking is strongly associated with atherosclerosis and coronary vascular disease. Rho-kinase plays an important role in various cellular functions associated with atherosclerosis and hypertension. However, there is no information on the relationship between smoking and Rho-kinase activity in humans. The purpose of this study was to determine the Rho-kinase activity in forearm vascular smooth muscle cells (VSMCs) in healthy young male smokers. We evaluated the forearm blood flow (FBF) responses to fasudil (3, 10, and 30 &mgr;g/min for 5 minutes), a Rho-kinase inhibitor, or sodium nitroprusside (0.75, 1.5, and 3.0 &mgr;g/min for 5 minutes) in current smokers (n=8) and nonsmokers (n=8). FBF was measured with a strain-gauge plethysmograph. The vasodilatory effect of fasudil was significantly greater in smokers than in nonsmokers (14.9±3.5 versus 10.5±3.6 mL/min per 100 mL tissue;P <0.01). The FBF responses to sodium nitroprusside were similar in the 2 groups (34.7±10.4 versus 33.2±10.2 mL/min per 100 mL tissue;P =0.78). These findings suggest that smoking activates Rho-kinase in forearm VSMCs but does not alter the vasodilatory effect induced by exogenous nitric oxide in forearm VSMCs in healthy young men.

Relationship between Augmentation Index and Flow-Mediated Vasodilation in the Brachial Artery

Recent studies have shown that the augmentation index (AI) is a predictor of cardiovascular complications. Endothelial dysfunction is the initial step in the pathogenesis of atherosclerosis, which in turn can lead to cardiovascular complications. Endothelial function assessed by flow-mediated dilation (FMD) can serve as an independent predictor of cardiovascular events. However, there is little information on the relationship between AI and FMD in the human vasculature, and we therefore investigated this relationship in the present study. A total of 100 subjects (71 males and 29 females; age range, 22–88 years; mean age, 59±17 years), including 83 patients with cardiovascular diseases (e.g., atherosclerosis, hypertension, coronary heart disease, stroke and peripheral arterial disease) and 17 healthy subjects were enrolled. High-resolution ultrasonography (automated vessel-diameter measurements; eTRACKING system), a linear array transducer (13 MHz) and an arm holding device were used to measure the arterial diameter response to reactive hyperemia and sublingual nitroglycerine (NTG, 75 μg) in all subjects. AI measured using an automated device was significantly correlated with FMD (r=−0.38, p<0.0001). There was no significant correlation between AI and vascular response to NTG. Multiple regression analysis showed that FMD was a significant independent predictor of AI (p<0.05). These findings suggest that increase in arterial stiffness may be associated with grade of endothelial dysfunction and that AI may be an index of not only arterial stiffness but also endothelial function.

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 NG-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.