- Email: [email protected]
Effect of edaravone, a novel free radical scavenger, on endothelium-dependent vasodilation in smokers
ing would occur earlier, and negative remodeling would be a late event. Therefore, negative remodeling should be observed more often in diffuse lesions with extended stenosis than in focal lesions. Although nitroglycerin was administered before IVUS imaging, the occurrence of vasospasm may not be excluded completely. In the present study, we did not assess the influence of vessel remodeling on outcomes after percutaneous coronary intervention. Further studies are needed to assess whether this finding may lead to stent underexpansion, which may contribute to the increased restenosis in diffuse lesions.
1. Glagov S, Weisenberg E, Zarins CK, Stankunavicius R, Kolettis GJ. Com-
pensatory enlargement of human atherosclerotic coronary arteries. N Engl J Med 1987;316:1371–1375. 2. McPherson DD, Sirna SJ, Hiratzka LF, Thorpe L, Armstrong ML, Marcus ML, Kerber RE. Coronary arterial remodeling studied by high-frequency epicardial echocardiography: an early compensatory mechanism in patients with obstructive coronary atherosclerosis. J Am Coll Cardiol 1991;17:79 – 86. 3. Clarkson TB, Prichard RW, Morgan TM, Petrick GS, Klein KP. Remodeling of coronary arteries in human and nonhuman primates. JAMA 1994;271:289 –294. 4. Mintz GS, Kent KM, Pichard AD, Satler LF, Popma JJ, Leon MB. Contribution of inadequate arterial remodeling to the development of focal coronary artery stenoses. Circulation 1997;95:1791–1798. 5. Hirose M, Kobayashi Y, Mintz GS, Moussa I, Mehran R, Lansky AJ, Dangas G, Kreps EM, Collins MB, Stone GW, et al. Correlation of coronary arterial remodeling determined by intravascular ultrasound with angiographic diameter reduction of 20% to 60%. Am J Cardiol 2003;92:141–145. 6. Kornowski R. Impact of smoking on coronary atherosclerosis and remodeling as determined by intravascular ultrasonic imaging. Am J Cardiol 1999;83:443– 445. 7. Kornowski R, Mintz GS, Lansky AJ, Hong MK, Kent KM, Pichard AD, Satler LF, Popma JJ, Bucher TA, Leon MB. Paradoxic decreases in atherosclerotic plaque mass in insulin-treated diabetic patients. Am J Cardiol 1998;81:1298 –1304. 8. Sabate M, Kay IP, de Feyter PJ, van Domburg RT, Deshpande NV, Ligthart JM, Gijzel AL, Wardeh AJ, Boersma E, Serruys PW. Remodeling of atheroscle-
rotic coronary arteries varies in relation to location and composition of plaque. Am J Cardiol 1999;84:135–140. 9. von Birgelen C, Mintz GS, Sieling C, Bose D, Eggebrecht H, Baumgart D, Neumann T, Herrmann J, Haude M, Erbel R. Relation between plaque composition and vascular remodeling in coronary lesions with different degrees of lumen narrowing as assessed with three-dimensional intravascular ultrasound in patients with stable angina pectoris. Am J Cardiol 2003;91: 1103–1107. 10. Iyisoy A, Schoenhagen P, Balghith M, Tsutsui H, Ziada K, Kapadia S, Nissen S, Tuzcu M. Remodeling pattern within diseased coronary segments as evidenced by intravascular ultrasound. Am J Cardiol 2002;90:636 – 638. 11. Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation 2002; 105:297–303. 12. Schmermund A, Denktas AE, Rumberger JA, Christian TF, Sheedy PF II, Bailey KR, Schwartz RS. Independent and incremental value of coronary artery calcium for predicting the extent of angiographic coronary artery disease: comparison with cardiac risk factors and radionuclide perfusion imaging. J Am Coll Cardiol 1999;34:777–786. 13. Di Mario C, The SH, Madretsma S, van Suylen RJ, Wilson RA, Bom N, Serruys PW, Gussenhoven EJ, Roelandt JR. Detection and characterization of vascular lesions by intravascular ultrasound: an in vitro study correlated with histology. J Am Soc Echocardiogr 1992;5:135–146. 14. Lafont A, Guzman LA, Whitlow PL, Goormastic M, Cornhill JF, Chisolm GM. Restenosis after experimental angioplasty. Intimal, medial, and adventitial changes associated with constrictive remodeling. Circ Res 1995;76:996 – 1002. 15. Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation 2002; 105:297–303. 16. Dortimer AC, Shenoy PN, Shiroff RA, Leaman DM, Babb JD, Liedtke AJ, Zelis R. Diffuse coronary artery disease in diabetic patients: fact or fiction? Circulation 1978;57:133–136. 17. Ledru F, Ducimetiere P, Battaglia S, Courbon D, Beverelli F, Guize L, Guermonprez JL, Diebold B. New diagnostic criteria for diabetes and coronary artery disease: insights from an angiographic study. J Am Coll Cardiol 2001;37: 1543–1550. 18. Johnstone MT, Creager SJ, Scales KM, Cusco JA, Lee BK, Creager MA. Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. Circulation 1993;88:2510 –2516. 19. Davi G, Catalano I, Averna M, Notarbartolo A, Strano A, Ciabattoni G, Patrono C. Thromboxane biosynthesis and platelet function in type II diabetes mellitus. N Engl J Med 1990;322:1769 –1774.
Effect of Edaravone, a Novel Free Radical Scavenger, on Endothelium-Dependent Vasodilation in Smokers Daisuke Jitsuiki, MD, Yukihito Higashi, MD, PhD, Chikara Goto, PhD, Masashi Kimura, MD, Kensuke Noma, MD, Keiko Hara, MD, Keigo Nakagawa, MD, PhD, Tetsuya Oshima, MD, PhD, Kazuaki Chayama, MD, Masao Yoshizumi, MD, PhD The forearm blood flow (FBF) responses to acetylcholine and to sodium nitroprusside were evaluated before and after administration of edaravone in 10 smokers and 10 nonsmokers. FBF response to acetylcholine was lower in smokers than in nonsmokers. The vasodilatory effects of sodium nitroprusside were From the Departments of Cardiovascular Physiology and Medicine, Medicine and Molecular Science, Developmental Biology, and Clinical Laboratory Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. This study was supported in part by grants-in-aid 1559075100 and KOSEI151201 for scientific research from the Ministry of Education, Science and Culture of Japan, Tokyo, Japan. Dr. Higashi’s address is: Department of Cardiovascular Physiology and Medicine, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail: [email protected]. Manuscript received April 5, 2004; revised manuscript received and accepted June 28, 2004.
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©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 October 15, 2004
PhD,
and
similar in both groups. Co-infusion of edaravone increased the FBF response to acetylcholine in smokers, but did not affect the FBF response to acetylcholine in nonsmokers. The administration of NG-monomethylL-arginine abolished edaravone-induced augmentation of the FBF response to acetylcholine in smokers. The antioxidative agent edaravone increases nitric oxide mediated vasodilation through a decrease in oxidative stress. 䊚2004 by Excerpta Medica, Inc. (Am J Cardiol 2004;94:1070 –1073)
daravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a strong novel free radical scavenger, is used in E patients with acute brain infarctions. Edaravone prevents brain edema after ischemia and reperfusion injury.1 Moreover, edaravone has preventive effects on 0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.06.072
the subjects were recruited from healthy volunteers. The study protocol Nonsmokers was approved by the Ethics Commit(n ⫽ 10) tee of the Hiroshima University Faculty of Medicine. Informed consent for 21.5 ⫾ 2.2 118.9 ⫾ 1.3 participation in the study was obtained 62.7 ⫾ 2.2 from all subjects. The definition of 61.0 ⫾ 4.9 smokers was those who fulfilled the 4.03 ⫾ 0.42 prespecified entry criteria: a regular 0.95 ⫾ 0.40 1.26 ⫾ 0.30 smoking history of ⬎5 pack-years. 2.33 ⫾ 0.39 The degree of smoking was measured 4.7 ⫾ 0.4 in pack-years. One pack-year was 43.6 ⫾ 9.8 equivalent to 20 cigarettes smoked per 88.4 ⫾ 17.6 day for 1 year. Twenty-five cigarettes 0.22 ⫾ 0.13 5.4 ⫾ 2.5 smoked per day for 1 year would equal 11.2 ⫾ 1.0 1.25 pack-years. All of the smokers 5.6 ⫾ 0.6 (9.5 ⫾ 2.6 pack-years) had a smoking history of ⬎5 years and abstained from smoking for ⱖ3 hours before the forearm blood flow (FBF) measurements. We defined nonsmokers as those who had never smoked. FBF was measured with a mercury-filled Silastic strain-gauge plethysmograph (EC-5R, D. E. Hokanson, Inc., Bellevue, Washington) as previously described.5,6 The study began at 8:30 A.M. Subjects fasted the previous night for ⱖ12 hours. They were kept in the supine position in a quiet, dark, air-conditioned room (constant temperature 22°C to 25°C) throughout the study. Thirty minutes after maintaining the supine position, basal FBF was measured. Then, FBF responses to acetylcholine (Daiichi Pharmaceutical Corporation, Tokyo, Japan), an endothelium-dependent vasodilator; SNP (Maruishi Pharmaceutical Co., Ltd., Osaka, Japan), an endothelium-independent vasodilator; and the co-infusion of acetylcholine and edaravone (Mitsubishi-Tokyo Pharmaceuticals, Inc., Osaka, Japan) were measured. Acetylcholine was administered at doses of 3.75, 7.5, and 15 g/min for 5 minutes, and SNP was administered at doses of 0.75, 1.5, and 3.0 g/min for 5 minutes. FBF was measured during the last 2 minutes of the infusion. These studies were carried out in a randomized fashion. Each study proceeded after FBF had returned to baseline values. In the preliminary study, FBF returned to baseline values ⱕ30 minutes after the infusion of acetylcholine or SNP. Thus, the end of the infusion of acetylcholine or SNP was followed by a 30-minute recovery period. To examine the effect of edaravone on the release of NO, we measured FBF during the infusion of acetylcholine and edaravone (100 g/min for 5 minutes) in the presence of the NO synthase inhibitor L-NMMA (Sigma Chemical Corporation, St. Louis, Missouri) in all subjects. The responses of the forearm vasculature to acetylcholine and edaravone after the infusion of L-NMMA were evaluated. Routine chemical methods were used to determine serum concentrations of total cholesterol, triglycerides, high-density lipoprotein cholesterol, insulin, glucose, creatinine, and electrolytes. Serum concentrations of low-density lipoprotein cholesterol were determined using the methods of Friedewald et al.7 The plasma con-
TABLE 1 Baseline Clinical Characteristics of Smokers and Nonsmokers Smokers (n ⫽ 10)
Variable Body mass index (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/min) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Serum glucose (mmol/dL) Serum insulin (pmol/L) Serum creatinine (umol/L) Plasma norepinephrine (ng/mL) Plasma angiotensin II (pg/mL) Urinary 8-OHdG (ng/mg of creatinine) FBF (ml/min/100 ml tissue)
22.2 117.8 61.8 59.7 4.16 1.08 1.31 2.36 5.3 40.0 88.4 0.20 7.0 20.9 5.1
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.5 3.0 1.1 7.6 0.86 0.52 0.20 0.82 0.9 22.4 8.8 0.10 2.1 2.6* 0.6
All results are presented as means ⫾ SDs. *p ⬍0.05 for comparison with nonsmokers. HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein.
FIGURE 1. Effects of acetylcholine on FBF in smokers (filled circles) and nonsmokers (open circles). Results are presented as means ⴞ SDs. The p value refers to a comparison of time course curves by analysis of variance for repeated measures.
myocardial injury after ischemia and reperfusion in rat hearts.2 Edaravone prevents the hydroxyradical-induced injury of cultured bovine aortic endothelial cells.3 In addition, edaravone stimulates the conversion of arachidonic acid to prostacyclin and inactivates reactive oxygen species (ROS), resulting in the protection of endothelial cells.4 To determine whether edaravone improves endothelial function in smokers, we evaluated the endothelium-dependent vasodilation induced by acetylcholine and the endothelium-independent vasodilation induced by sodium nitroprusside (SNP) before and after the administration of edaravone with and without the administration of the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). •••
The subjects were 10 healthy young male smokers (mean age 23 ⫾ 2 years) and 10 healthy age-matched young male nonsmokers (mean age 26 ⫾ 3 years). All of
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FIGURE 2. Effects of SN on FBF in smokers (filled circles) and nonsmokers (open circles). Results are presented as means ⴞ SDs. The p value refers to a comparison of time course curves by analysis of variance for repeated measures.
FIGURE 4. Effects of the co-infusion of acetylcholine with edaravone on FBF before (open circles) and after (filled circles) L-NMMA in smokers (A) and nonsmokers (B). Results are presented as means ⴞ SD. The p value refers to a comparison of time course curves by analysis of variance for repeated measurements.
FIGURE 3. Comparison of the responses of FBF to the co-infusion of acetylcholine with edaravone (filled circles) and to the infusion of acetylcholine alone (open circles) in smokers (A) and nonsmokers (B). Results are presented as means ⴞ SDs. The p value refers to a comparison of time course curves by analysis of variance for repeated measures.
1072 THE AMERICAN JOURNAL OF CARDIOLOGY姞
VOL. 94
centration of angiotensin II was assayed by radioimmunoassay. The plasma concentrations of norepinephrine were measured by high-performance liquid chromatography. The urinary concentrations of 8-hydroxy-2=-deoxyguanosin (8-OHdG) were assayed by enzyme-linked immunosorbent assay using 8-OHdG kits (Nihon Yushi, Tokyo, Japan). The results are presented as means ⫾ SDs. Values of p ⬍0.05 were considered to indicate statistical significance. The Mann-Whitney U statistic test was used to evaluate differences between current smokers and nonsmokers with regard to parameters at baseline. Comparisons between the 2 groups with respect to changes in parameters were performed with adjusted means by analysis of covariance, with baseline data used as the covariates. Comparisons of dose-response curves of parameters during the infusion of drug were analyzed by analysis of variance for repeated measures with Bonferroni’s correction. The baseline clinical characteristics of the 10 smokers and 10 nonsmokers are listed in Table 1. All parameters, including insulin, plasma angiotensin II, norepinephrine, and lipid profiles and systemic and forearm OCTOBER 15, 2004
hemodynamics, were similar in smokers and nonsmokers. The urinary excretion of 8-OHdG was greater in smokers than in nonsmokers. The intra-arterial infusion of acetylcholine increased FBF in a dose-dependent manner in smokers and nonsmokers. The vasodilator response to acetylcholine was significantly less in smokers than in nonsmokers (maximal FBF 29.2 ⫾ 2.6 vs 15.7 ⫾ 2.0 ml/min/100 ml tissue; p ⬍0.01, Figure 1). No significant change was observed in arterial blood pressure or heart rate with the intra-arterial infusion of acetylcholine. The intra-arterial infusion of SNP significantly increased FBF in a dose-dependent manner in smokers and nonsmokers. There was no significant difference between FBF responses to SNP in the 2 groups (Figure 2). No significant change was observed in arterial blood pressure or heart rate with the intra-arterial infusion of SNP. The FBF response to acetylcholine was significantly augmented by the co-infusion of edaravone in smokers (Figure 3). At the maximal dose of acetylcholine (15 g/min), FBF increased from 15.7 ⫾ 2.0 to 22.6 ⫾ 3.4 ml/min/100 ml tissue during the coinfusion of edaravone (p ⬍0.05). In contrast, FBF responses to acetylcholine in nonsmokers were not altered by co-infusion of acetylcholine and edaravone (Figure 3). At the maximal dose of acetylcholine (15 g/min), FBF before and during the co-infusion of edaravone was similar: 29.2 ⫾ 2.6 and 27.6 ⫾ 4.8 ml/min/100 ml tissue, respectively (p ⫽ NS). No significant change was observed in arterial blood pressure or heart rate with the intra-arterial infusion of acetylcholine and edaravone. The intra-arterial infusion of L-NMMA significantly decreased basal FBF in smokers and nonsmokers (5.6 ⫾ 0.7 to 3.4 ⫾ 0.4 and 6.9 ⫾ 0.8 to 4.5 ⫾ 0.7 ml/min/100 ml tissue, respectively, p ⬍0.01). The intra-arterial infusion of acetylcholine and edaravone after L-NMMA significantly decreased FBF from 22.6 ⫾ 3.4 to 13.4 ⫾ 2.1 ml/min/100 ml tissue (p ⬍0.01) in smokers (Figure 4) and from 27.6 ⫾ 4.9 to 11.7 ⫾ 2.5 ml/min/100 ml tissue (p ⬍0.01) in nonsmokers (Figure 4). No significant change was observed in arterial blood pressure or heart rate with the intraarterial infusion of acetylcholine and edaravone after L-NMMA. •••
The present findings demonstrate that endothelium-dependent vasodilation in forearm arteries was impaired in smokers compared with that in nonsmokers, that the urinary excretion of 8-OHdG was significantly increased in smokers compared with that in nonsmokers, and that edaravone augmented acetylcholine-induced vasodilation in smokers but not in nonsmokers. The forearm vascular response to acetylcholine, but not to SNP, was impaired in smokers. Endothelium-dependent vasodilation of the brachial artery is selectively impaired in smokers. Our results are consistent with the results of previous studies
showing that smoking is associated with endothelial dysfunction. The most interesting findings in the present study were that the radical scavenger edaravone restored impaired endothelium-dependent vasodilation in smokers to the same level as that in nonsmokers and that the enhancement of FBF response to acetylcholine by edaravone was completely abolished by LNMMA. These findings suggest that edaravone improves endothelial-dependent vasodilation in smokers through a decrease in ROS. It is well known that a balance between ambient levels of superoxide and NO release plays a critical role in the maintenance of normal endothelial function.8,9 ROS, including hydroxy radicals, directly scavenge NO and produce toxic peroxynitrite.10 Therefore, the improvement in endothelium-dependent vasodilation by edaravone may be due to an inhibition of ROS-induced NO degradation rather than increased NO production. The urinary levels of 8-OHdG, a principal stable maker of hydroxyl radical damage to DNA, were increased in smokers compared with that in nonsmokers. These findings suggest that oxidative stress may be at least be partially involved in impaired endothelium-dependent, NO-mediated vasodilation in smokers. Recently, we have shown that 1 mechanism by which endothelium-dependent vasodilation is impaired is an increase in oxidative stress in patients with renovascular hypertension, who are ideal models of excess angiotensin II and an angiotensin II–related increase in oxidative stress.6 It is well known that cigarette smoke contains a large number of oxidants. However, the precise mechanism responsible for increased oxidative stress in smokers remains unknown. Acknowledgment: We thank Izumi Yamashita for her secretarial assistance.
1. Abe K, Yuki S, Kogure K. Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988;19:480 – 485. 2. Minhaz U, Tanaka M, Tsukamoto H, Watanabe K, Koide S, Shohtsu A, Nakazawa H. Effect of MCI-186 on postischemic reperfusion injury in isolated rat heart. Free Radical Res 1996;24:361–367. 3. Watanabe T, Morita I, Nishi H, Murota S. Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro. Prostaglandins Leukot Essent Fatty Acids 1988;33:81– 87. 4. Murota S, Morita I, Suda N. The control of vascular endothelial cell injury. Ann N Y Acad Sci 1990;598:182–187. 5. Higashi Y, Sasaki S, Kurisu S, Yoshimizu A, Sasaki N, Matsuura H, Kajiyama G, Oshima T. Regular aerobic exercise augments endothelium-dependent vascular relaxation in normotensive as well as hypertensive subjects: role of endothelium-derived nitric oxide. Circulation 1999;100:1194 –1202. 6. Higashi Y, Sasaki S, Nakagawa K, Matsuura H, Oshima T, Chayama K. Endothelial function and oxidative stress in renovascular hypertension. N Engl J Med 2002;346:1954 –1962. 7. Friedewald WT, Levy RI, Fredrickson DS. Estimation of low density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem 1972;18:499 –502. 8. Nakae D, Kobayashi Y, Akai H, Andoh N, Satoh H, Ohashi K, Tsutsumi M, Konishi Y. Involvement of 8-hydroxyguanine formation in the initiation of rat liver carcinogenesis by low dose levels of N-nitrosodiethylamine. Cancer Res 1997;57:1281–1287. 9. Fraga C, Shigenaga M, Park J, Degan P, Ames BN. Oxidative damage to DNA during aging: 8-hydroxy-2=-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci U S A 1990;87:4533– 4537. 10. Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 2000;18:655– 673.
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1. Glagov S, Weisenberg E, Zarins CK, Stankunavicius R, Kolettis GJ. Com-
pensatory enlargement of human atherosclerotic coronary arteries. N Engl J Med 1987;316:1371–1375. 2. McPherson DD, Sirna SJ, Hiratzka LF, Thorpe L, Armstrong ML, Marcus ML, Kerber RE. Coronary arterial remodeling studied by high-frequency epicardial echocardiography: an early compensatory mechanism in patients with obstructive coronary atherosclerosis. J Am Coll Cardiol 1991;17:79 – 86. 3. Clarkson TB, Prichard RW, Morgan TM, Petrick GS, Klein KP. Remodeling of coronary arteries in human and nonhuman primates. JAMA 1994;271:289 –294. 4. Mintz GS, Kent KM, Pichard AD, Satler LF, Popma JJ, Leon MB. Contribution of inadequate arterial remodeling to the development of focal coronary artery stenoses. Circulation 1997;95:1791–1798. 5. Hirose M, Kobayashi Y, Mintz GS, Moussa I, Mehran R, Lansky AJ, Dangas G, Kreps EM, Collins MB, Stone GW, et al. Correlation of coronary arterial remodeling determined by intravascular ultrasound with angiographic diameter reduction of 20% to 60%. Am J Cardiol 2003;92:141–145. 6. Kornowski R. Impact of smoking on coronary atherosclerosis and remodeling as determined by intravascular ultrasonic imaging. Am J Cardiol 1999;83:443– 445. 7. Kornowski R, Mintz GS, Lansky AJ, Hong MK, Kent KM, Pichard AD, Satler LF, Popma JJ, Bucher TA, Leon MB. Paradoxic decreases in atherosclerotic plaque mass in insulin-treated diabetic patients. Am J Cardiol 1998;81:1298 –1304. 8. Sabate M, Kay IP, de Feyter PJ, van Domburg RT, Deshpande NV, Ligthart JM, Gijzel AL, Wardeh AJ, Boersma E, Serruys PW. Remodeling of atheroscle-
rotic coronary arteries varies in relation to location and composition of plaque. Am J Cardiol 1999;84:135–140. 9. von Birgelen C, Mintz GS, Sieling C, Bose D, Eggebrecht H, Baumgart D, Neumann T, Herrmann J, Haude M, Erbel R. Relation between plaque composition and vascular remodeling in coronary lesions with different degrees of lumen narrowing as assessed with three-dimensional intravascular ultrasound in patients with stable angina pectoris. Am J Cardiol 2003;91: 1103–1107. 10. Iyisoy A, Schoenhagen P, Balghith M, Tsutsui H, Ziada K, Kapadia S, Nissen S, Tuzcu M. Remodeling pattern within diseased coronary segments as evidenced by intravascular ultrasound. Am J Cardiol 2002;90:636 – 638. 11. Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation 2002; 105:297–303. 12. Schmermund A, Denktas AE, Rumberger JA, Christian TF, Sheedy PF II, Bailey KR, Schwartz RS. Independent and incremental value of coronary artery calcium for predicting the extent of angiographic coronary artery disease: comparison with cardiac risk factors and radionuclide perfusion imaging. J Am Coll Cardiol 1999;34:777–786. 13. Di Mario C, The SH, Madretsma S, van Suylen RJ, Wilson RA, Bom N, Serruys PW, Gussenhoven EJ, Roelandt JR. Detection and characterization of vascular lesions by intravascular ultrasound: an in vitro study correlated with histology. J Am Soc Echocardiogr 1992;5:135–146. 14. Lafont A, Guzman LA, Whitlow PL, Goormastic M, Cornhill JF, Chisolm GM. Restenosis after experimental angioplasty. Intimal, medial, and adventitial changes associated with constrictive remodeling. Circ Res 1995;76:996 – 1002. 15. Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation 2002; 105:297–303. 16. Dortimer AC, Shenoy PN, Shiroff RA, Leaman DM, Babb JD, Liedtke AJ, Zelis R. Diffuse coronary artery disease in diabetic patients: fact or fiction? Circulation 1978;57:133–136. 17. Ledru F, Ducimetiere P, Battaglia S, Courbon D, Beverelli F, Guize L, Guermonprez JL, Diebold B. New diagnostic criteria for diabetes and coronary artery disease: insights from an angiographic study. J Am Coll Cardiol 2001;37: 1543–1550. 18. Johnstone MT, Creager SJ, Scales KM, Cusco JA, Lee BK, Creager MA. Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. Circulation 1993;88:2510 –2516. 19. Davi G, Catalano I, Averna M, Notarbartolo A, Strano A, Ciabattoni G, Patrono C. Thromboxane biosynthesis and platelet function in type II diabetes mellitus. N Engl J Med 1990;322:1769 –1774.
Effect of Edaravone, a Novel Free Radical Scavenger, on Endothelium-Dependent Vasodilation in Smokers Daisuke Jitsuiki, MD, Yukihito Higashi, MD, PhD, Chikara Goto, PhD, Masashi Kimura, MD, Kensuke Noma, MD, Keiko Hara, MD, Keigo Nakagawa, MD, PhD, Tetsuya Oshima, MD, PhD, Kazuaki Chayama, MD, Masao Yoshizumi, MD, PhD The forearm blood flow (FBF) responses to acetylcholine and to sodium nitroprusside were evaluated before and after administration of edaravone in 10 smokers and 10 nonsmokers. FBF response to acetylcholine was lower in smokers than in nonsmokers. The vasodilatory effects of sodium nitroprusside were From the Departments of Cardiovascular Physiology and Medicine, Medicine and Molecular Science, Developmental Biology, and Clinical Laboratory Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. This study was supported in part by grants-in-aid 1559075100 and KOSEI151201 for scientific research from the Ministry of Education, Science and Culture of Japan, Tokyo, Japan. Dr. Higashi’s address is: Department of Cardiovascular Physiology and Medicine, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail: [email protected]. Manuscript received April 5, 2004; revised manuscript received and accepted June 28, 2004.
1070
©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 October 15, 2004
PhD,
and
similar in both groups. Co-infusion of edaravone increased the FBF response to acetylcholine in smokers, but did not affect the FBF response to acetylcholine in nonsmokers. The administration of NG-monomethylL-arginine abolished edaravone-induced augmentation of the FBF response to acetylcholine in smokers. The antioxidative agent edaravone increases nitric oxide mediated vasodilation through a decrease in oxidative stress. 䊚2004 by Excerpta Medica, Inc. (Am J Cardiol 2004;94:1070 –1073)
daravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a strong novel free radical scavenger, is used in E patients with acute brain infarctions. Edaravone prevents brain edema after ischemia and reperfusion injury.1 Moreover, edaravone has preventive effects on 0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.06.072
the subjects were recruited from healthy volunteers. The study protocol Nonsmokers was approved by the Ethics Commit(n ⫽ 10) tee of the Hiroshima University Faculty of Medicine. Informed consent for 21.5 ⫾ 2.2 118.9 ⫾ 1.3 participation in the study was obtained 62.7 ⫾ 2.2 from all subjects. The definition of 61.0 ⫾ 4.9 smokers was those who fulfilled the 4.03 ⫾ 0.42 prespecified entry criteria: a regular 0.95 ⫾ 0.40 1.26 ⫾ 0.30 smoking history of ⬎5 pack-years. 2.33 ⫾ 0.39 The degree of smoking was measured 4.7 ⫾ 0.4 in pack-years. One pack-year was 43.6 ⫾ 9.8 equivalent to 20 cigarettes smoked per 88.4 ⫾ 17.6 day for 1 year. Twenty-five cigarettes 0.22 ⫾ 0.13 5.4 ⫾ 2.5 smoked per day for 1 year would equal 11.2 ⫾ 1.0 1.25 pack-years. All of the smokers 5.6 ⫾ 0.6 (9.5 ⫾ 2.6 pack-years) had a smoking history of ⬎5 years and abstained from smoking for ⱖ3 hours before the forearm blood flow (FBF) measurements. We defined nonsmokers as those who had never smoked. FBF was measured with a mercury-filled Silastic strain-gauge plethysmograph (EC-5R, D. E. Hokanson, Inc., Bellevue, Washington) as previously described.5,6 The study began at 8:30 A.M. Subjects fasted the previous night for ⱖ12 hours. They were kept in the supine position in a quiet, dark, air-conditioned room (constant temperature 22°C to 25°C) throughout the study. Thirty minutes after maintaining the supine position, basal FBF was measured. Then, FBF responses to acetylcholine (Daiichi Pharmaceutical Corporation, Tokyo, Japan), an endothelium-dependent vasodilator; SNP (Maruishi Pharmaceutical Co., Ltd., Osaka, Japan), an endothelium-independent vasodilator; and the co-infusion of acetylcholine and edaravone (Mitsubishi-Tokyo Pharmaceuticals, Inc., Osaka, Japan) were measured. Acetylcholine was administered at doses of 3.75, 7.5, and 15 g/min for 5 minutes, and SNP was administered at doses of 0.75, 1.5, and 3.0 g/min for 5 minutes. FBF was measured during the last 2 minutes of the infusion. These studies were carried out in a randomized fashion. Each study proceeded after FBF had returned to baseline values. In the preliminary study, FBF returned to baseline values ⱕ30 minutes after the infusion of acetylcholine or SNP. Thus, the end of the infusion of acetylcholine or SNP was followed by a 30-minute recovery period. To examine the effect of edaravone on the release of NO, we measured FBF during the infusion of acetylcholine and edaravone (100 g/min for 5 minutes) in the presence of the NO synthase inhibitor L-NMMA (Sigma Chemical Corporation, St. Louis, Missouri) in all subjects. The responses of the forearm vasculature to acetylcholine and edaravone after the infusion of L-NMMA were evaluated. Routine chemical methods were used to determine serum concentrations of total cholesterol, triglycerides, high-density lipoprotein cholesterol, insulin, glucose, creatinine, and electrolytes. Serum concentrations of low-density lipoprotein cholesterol were determined using the methods of Friedewald et al.7 The plasma con-
TABLE 1 Baseline Clinical Characteristics of Smokers and Nonsmokers Smokers (n ⫽ 10)
Variable Body mass index (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/min) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Serum glucose (mmol/dL) Serum insulin (pmol/L) Serum creatinine (umol/L) Plasma norepinephrine (ng/mL) Plasma angiotensin II (pg/mL) Urinary 8-OHdG (ng/mg of creatinine) FBF (ml/min/100 ml tissue)
22.2 117.8 61.8 59.7 4.16 1.08 1.31 2.36 5.3 40.0 88.4 0.20 7.0 20.9 5.1
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.5 3.0 1.1 7.6 0.86 0.52 0.20 0.82 0.9 22.4 8.8 0.10 2.1 2.6* 0.6
All results are presented as means ⫾ SDs. *p ⬍0.05 for comparison with nonsmokers. HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein.
FIGURE 1. Effects of acetylcholine on FBF in smokers (filled circles) and nonsmokers (open circles). Results are presented as means ⴞ SDs. The p value refers to a comparison of time course curves by analysis of variance for repeated measures.
myocardial injury after ischemia and reperfusion in rat hearts.2 Edaravone prevents the hydroxyradical-induced injury of cultured bovine aortic endothelial cells.3 In addition, edaravone stimulates the conversion of arachidonic acid to prostacyclin and inactivates reactive oxygen species (ROS), resulting in the protection of endothelial cells.4 To determine whether edaravone improves endothelial function in smokers, we evaluated the endothelium-dependent vasodilation induced by acetylcholine and the endothelium-independent vasodilation induced by sodium nitroprusside (SNP) before and after the administration of edaravone with and without the administration of the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). •••
The subjects were 10 healthy young male smokers (mean age 23 ⫾ 2 years) and 10 healthy age-matched young male nonsmokers (mean age 26 ⫾ 3 years). All of
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FIGURE 2. Effects of SN on FBF in smokers (filled circles) and nonsmokers (open circles). Results are presented as means ⴞ SDs. The p value refers to a comparison of time course curves by analysis of variance for repeated measures.
FIGURE 4. Effects of the co-infusion of acetylcholine with edaravone on FBF before (open circles) and after (filled circles) L-NMMA in smokers (A) and nonsmokers (B). Results are presented as means ⴞ SD. The p value refers to a comparison of time course curves by analysis of variance for repeated measurements.
FIGURE 3. Comparison of the responses of FBF to the co-infusion of acetylcholine with edaravone (filled circles) and to the infusion of acetylcholine alone (open circles) in smokers (A) and nonsmokers (B). Results are presented as means ⴞ SDs. The p value refers to a comparison of time course curves by analysis of variance for repeated measures.
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centration of angiotensin II was assayed by radioimmunoassay. The plasma concentrations of norepinephrine were measured by high-performance liquid chromatography. The urinary concentrations of 8-hydroxy-2=-deoxyguanosin (8-OHdG) were assayed by enzyme-linked immunosorbent assay using 8-OHdG kits (Nihon Yushi, Tokyo, Japan). The results are presented as means ⫾ SDs. Values of p ⬍0.05 were considered to indicate statistical significance. The Mann-Whitney U statistic test was used to evaluate differences between current smokers and nonsmokers with regard to parameters at baseline. Comparisons between the 2 groups with respect to changes in parameters were performed with adjusted means by analysis of covariance, with baseline data used as the covariates. Comparisons of dose-response curves of parameters during the infusion of drug were analyzed by analysis of variance for repeated measures with Bonferroni’s correction. The baseline clinical characteristics of the 10 smokers and 10 nonsmokers are listed in Table 1. All parameters, including insulin, plasma angiotensin II, norepinephrine, and lipid profiles and systemic and forearm OCTOBER 15, 2004
hemodynamics, were similar in smokers and nonsmokers. The urinary excretion of 8-OHdG was greater in smokers than in nonsmokers. The intra-arterial infusion of acetylcholine increased FBF in a dose-dependent manner in smokers and nonsmokers. The vasodilator response to acetylcholine was significantly less in smokers than in nonsmokers (maximal FBF 29.2 ⫾ 2.6 vs 15.7 ⫾ 2.0 ml/min/100 ml tissue; p ⬍0.01, Figure 1). No significant change was observed in arterial blood pressure or heart rate with the intra-arterial infusion of acetylcholine. The intra-arterial infusion of SNP significantly increased FBF in a dose-dependent manner in smokers and nonsmokers. There was no significant difference between FBF responses to SNP in the 2 groups (Figure 2). No significant change was observed in arterial blood pressure or heart rate with the intra-arterial infusion of SNP. The FBF response to acetylcholine was significantly augmented by the co-infusion of edaravone in smokers (Figure 3). At the maximal dose of acetylcholine (15 g/min), FBF increased from 15.7 ⫾ 2.0 to 22.6 ⫾ 3.4 ml/min/100 ml tissue during the coinfusion of edaravone (p ⬍0.05). In contrast, FBF responses to acetylcholine in nonsmokers were not altered by co-infusion of acetylcholine and edaravone (Figure 3). At the maximal dose of acetylcholine (15 g/min), FBF before and during the co-infusion of edaravone was similar: 29.2 ⫾ 2.6 and 27.6 ⫾ 4.8 ml/min/100 ml tissue, respectively (p ⫽ NS). No significant change was observed in arterial blood pressure or heart rate with the intra-arterial infusion of acetylcholine and edaravone. The intra-arterial infusion of L-NMMA significantly decreased basal FBF in smokers and nonsmokers (5.6 ⫾ 0.7 to 3.4 ⫾ 0.4 and 6.9 ⫾ 0.8 to 4.5 ⫾ 0.7 ml/min/100 ml tissue, respectively, p ⬍0.01). The intra-arterial infusion of acetylcholine and edaravone after L-NMMA significantly decreased FBF from 22.6 ⫾ 3.4 to 13.4 ⫾ 2.1 ml/min/100 ml tissue (p ⬍0.01) in smokers (Figure 4) and from 27.6 ⫾ 4.9 to 11.7 ⫾ 2.5 ml/min/100 ml tissue (p ⬍0.01) in nonsmokers (Figure 4). No significant change was observed in arterial blood pressure or heart rate with the intraarterial infusion of acetylcholine and edaravone after L-NMMA. •••
The present findings demonstrate that endothelium-dependent vasodilation in forearm arteries was impaired in smokers compared with that in nonsmokers, that the urinary excretion of 8-OHdG was significantly increased in smokers compared with that in nonsmokers, and that edaravone augmented acetylcholine-induced vasodilation in smokers but not in nonsmokers. The forearm vascular response to acetylcholine, but not to SNP, was impaired in smokers. Endothelium-dependent vasodilation of the brachial artery is selectively impaired in smokers. Our results are consistent with the results of previous studies
showing that smoking is associated with endothelial dysfunction. The most interesting findings in the present study were that the radical scavenger edaravone restored impaired endothelium-dependent vasodilation in smokers to the same level as that in nonsmokers and that the enhancement of FBF response to acetylcholine by edaravone was completely abolished by LNMMA. These findings suggest that edaravone improves endothelial-dependent vasodilation in smokers through a decrease in ROS. It is well known that a balance between ambient levels of superoxide and NO release plays a critical role in the maintenance of normal endothelial function.8,9 ROS, including hydroxy radicals, directly scavenge NO and produce toxic peroxynitrite.10 Therefore, the improvement in endothelium-dependent vasodilation by edaravone may be due to an inhibition of ROS-induced NO degradation rather than increased NO production. The urinary levels of 8-OHdG, a principal stable maker of hydroxyl radical damage to DNA, were increased in smokers compared with that in nonsmokers. These findings suggest that oxidative stress may be at least be partially involved in impaired endothelium-dependent, NO-mediated vasodilation in smokers. Recently, we have shown that 1 mechanism by which endothelium-dependent vasodilation is impaired is an increase in oxidative stress in patients with renovascular hypertension, who are ideal models of excess angiotensin II and an angiotensin II–related increase in oxidative stress.6 It is well known that cigarette smoke contains a large number of oxidants. However, the precise mechanism responsible for increased oxidative stress in smokers remains unknown. Acknowledgment: We thank Izumi Yamashita for her secretarial assistance.
1. Abe K, Yuki S, Kogure K. Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988;19:480 – 485. 2. Minhaz U, Tanaka M, Tsukamoto H, Watanabe K, Koide S, Shohtsu A, Nakazawa H. Effect of MCI-186 on postischemic reperfusion injury in isolated rat heart. Free Radical Res 1996;24:361–367. 3. Watanabe T, Morita I, Nishi H, Murota S. Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro. Prostaglandins Leukot Essent Fatty Acids 1988;33:81– 87. 4. Murota S, Morita I, Suda N. The control of vascular endothelial cell injury. Ann N Y Acad Sci 1990;598:182–187. 5. Higashi Y, Sasaki S, Kurisu S, Yoshimizu A, Sasaki N, Matsuura H, Kajiyama G, Oshima T. Regular aerobic exercise augments endothelium-dependent vascular relaxation in normotensive as well as hypertensive subjects: role of endothelium-derived nitric oxide. Circulation 1999;100:1194 –1202. 6. Higashi Y, Sasaki S, Nakagawa K, Matsuura H, Oshima T, Chayama K. Endothelial function and oxidative stress in renovascular hypertension. N Engl J Med 2002;346:1954 –1962. 7. Friedewald WT, Levy RI, Fredrickson DS. Estimation of low density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem 1972;18:499 –502. 8. Nakae D, Kobayashi Y, Akai H, Andoh N, Satoh H, Ohashi K, Tsutsumi M, Konishi Y. Involvement of 8-hydroxyguanine formation in the initiation of rat liver carcinogenesis by low dose levels of N-nitrosodiethylamine. Cancer Res 1997;57:1281–1287. 9. Fraga C, Shigenaga M, Park J, Degan P, Ames BN. Oxidative damage to DNA during aging: 8-hydroxy-2=-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci U S A 1990;87:4533– 4537. 10. Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 2000;18:655– 673.
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