Department of Internal Medicine, Sakura Medical Center, School of Medicine, Toho University, Chiba, Japan

Original Article 621 Effects of Olmesartan, an Angiotensin Receptor Blocker, and Amlodipine, a Calcium Channel Blocker, on Cardio-Ankle Vascular Index (CAVI) in Type 2 Diabetic Patients with Hypertension Yoh Miyashita, Atsuhito Saiki, Kei Endo, Noriko Ban, Takashi Yamaguchi, Hidetoshi Kawana, Daiji Nagayama, Masahiro Ohira, Tomokazu Oyama, and Kohji Shirai Department of Internal Medicine, Sakura Medical Center, School of Medicine, Toho University, Chiba, Japan Aim: Recently, a novel device for measuring the cardio-ankle vascular index (CAVI) as an arterial stiffness parameter has been developed. In this study, we evaluated the effect of angiotensin receptor blocker (ARB) and calcium channel (Ca) blocker on CAVI in type 2 diabetic patients with hypertension. Methods: Seventy type 2 diabetes mellitus patients with hypertension were enrolled and randomly divided into two groups. One group was administered olmesartan medoxomil 10 mg/day for 12 months (), and the other group was administered amlodipine besilate 5 mg/day for 12 months (). Results: In the, a significant decrease in CAVI was observed after 12 months; however, no significant change in CAVI was observed in the although changes in blood pressure were almost the same. By simple regression analyses, CAVI changes correlated positively with 8-OHdG changes. Conclusions: Olmesartan, an ARB, improved arterial stiffness more than amlodipine, and this effect might be due to not only the blood pressure-lowering effect but also to reducing the potential of oxidative stress recognized in olmesartan. J Atheroscler Thromb, 2009; 16:621-626. Key words; Angiotensin receptor blocker, Calcium channel blocker, Oxidative stress, 8-hydroxy-2 -deoxyguanosine, Cardio-ankle vascular index Introduction In diabetic patients, hypertension is a frequent complication and accelerates cardiovascular diseases 1). Angiotensin receptor blockers (ARBs) and calcium channel blockers are frequently used as the first choice antihypertensive in diabetes mellitus 2). The main purpose of blood pressure-lowering therapy is to protect the progression of atherosclerosis, so evaluation of vascular function is needed during blood pressure-lower- Address for correspondence: Yoh Miyashita, Department of Internal Medicine, Sakura Medical Center, School of Medicine, Toho University, 564-1 Shimoshizu, Sakura-City, Chiba, 285-0841, Japan E-mail: mumon@sf6.so-net.ne.jp Received: September 9, 2008 Accepted for publication: April 2, 2009 ing therapy. For the evaluation of arteriosclerosis or vascular function, pulse wave velocity (PWV) and stiffness parameter, evaluated by changes in the diameter of the vessel wall, are used 3-6). The problem of PWV in clinical use is that PWV itself essentially depends on blood pressure and age. Although Hasegawa et al. established the aortic PWV method, which is independent of blood pressure 5), this method had several drawbacks, such as difficulty in finding the notch of the pulse wave, need for technical skill, and low reproducibility. Stiffness parameter is based on the change in vascular diameter corresponding to arterial pressure variance 4, 7), and the value does not depend on blood pressure 8) ; however, there are also various problems: reflects the local property of a segment of the artery, and the method requires special ultrasonic equipment.

622 Recently, a novel arterial stiffness parameter, called the cardio-ankle vascular index (CAVI), has been developed, which essentially reflects the stiffness of the aorta, femoral artery and tibial artery 9). CAVI is independent of blood pressure, and has adequate reproducibility for clinical use 9). Furthermore, no special technique is required to measure CAVI. Several reports have demonstrated the usefulness of CAVI to detect atherosclerotic diseases 9-12). In the present study, we evaluated the effect of ARB and calcium channel blocker on CAVI in type 2 diabetic patients with hypertension. Subjects and Methods Subjects A randomized, open study was performed. Seventy type 2 diabetes mellitus patients with hypertension, who attended Sakura Medical Center of Toho University as outpatients, were enrolled. Patients were excluded if they had received insulin therapy or had diabetic retinopathy, nephropathy and previous cardiovascular and cerebrovascular diseases. All patients had taken no antihypertensive drugs before this study. The enrolled subjects were randomly divided into two groups. One group was administered olmesartan 10 mg/day for 12 months (, n 35), and the other group was administered amlodipine 5 mg/day for 12 months (, n 35). These drugs were taken in the morning after 12 hours of fasting. During this study, all patients maintained the same diet and exercise therapies, and did not change medications. All subjects received nutrition education from a dietitian every month. This study was approved by the institutional review board. The purpose of this study was explained to the subjects, and consent was obtained for participation in the study and also for release of the study data. Measurement of Body Weight and Blood Pressure Body weight (BW) and blood pressure (BP) were measured in the morning after 12 hours of fasting. Before measuring BW and BP, the subjects took only ARB or calcium channel blocker. Blood pressure was measured at least twice in a sitting position. Assay of HbA1c and Serum Lipids Blood samples were collected in the morning after 12 hours of fasting and taking only ARB or calcium channel blocker. Serum was separated within 1 hour, and samples were used to measure the following chemical parameters. Glycosylated hemoglobin (HbA1c), including stable and unstable fractions, was measured by high pressure liquid chromatography using the Hi-Auto A1c kit (Kyoto Daiichi Kagaku, Kyoto, Japan). Data of the stable type were used in the present analysis. Total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were measured with an automatic analyzer (Hitachi 7150 available from Hitachi Tokyo, Japan). High-density lipoprotein cholesterol (HDL-C) was measured by the selective inhibition method (Daiichi Pure Chemicals, Tokyo, Japan) 13). Urinary 8-OHdG Analysis Urine samples were centrifuged at 800 g for 10 min and the supernatant was used to determine 8-hydroxy-2 -deoxyguanosine (8-OHdG) with a competitive enzyme-linked immunosorbent assay (8-Hydroxydeoxyguanosine Check; Japan Institute for the Control of Aging, Shizuoka, Japan). The monoclonal antibody has been characterized previously and the antibody found to be specific for 8-OHdG 14). The results are expressed as a ratio to the creatinine content (per mg Cr) measured in the same urine sample. Measurement of CAVI CAVI was measured using a VaSera CAVI instrument (Fukuda Denshi Co. Ltd., Tokyo, Japan) by the methods described previously 9). CAVI was measured in the morning after 12 hours of fasting and taking only ARB or calcium channel blocker. Briefly, cuffs were applied to bilateral upper arms and ankles, with the subject lying supine and the head held in the midline position. Examinations were performed after resting for 10 minutes. To detect brachial and ankle pulse waves with cuffs, a low cuff pressure from 30 to 50 mmhg was used to ensure the minimal effect of cuff pressure on hemodynamics. Blood pressure was measured thereafter. CAVI was calculated by the following formula: CAVI a{(2 / P) ln(ps/pd)pwv 2 } b where Ps is systolic blood pressure, Pd is diastolic blood pressure, PWV is pulse wave velocity, P is Ps Pd, is blood density, and a and b are constants. Scale conversion was performed to compare CAVI with PWV (Hasegawa s method). The VaSera was equipped with both measurement and calculation systems, and automatically calculated the CAVI. The average coefficient of variation of CAVI was less than 5%, which was small enough for clinical usage and indicated that CAVI has good reproducibility 9). Statistical Analysis All data are expressed as the mean S.D. Stat-

623 Table 1. Backgrounds of the two groups n (male/female) age BW (kg) BMI (kg/m 2 ) BP (mmhg) systolic diastolic HbA1c (%) TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) 35 (19/16) 62.7 8.2 61.3 6.9 24.6 2.9 152 17 91 9 7.1 1.2 210 24 181 67 55.2 8.2 131 21 35 (21/14) 64.3 7.4 64.0 7.7 23.9 3.1 158 17 91 11 7.7 1.5 206 31 183 83 50.2 9.4 115 28 BW: body weight, HbA1c: glycosylated hemoglobin, BP: blood pressure, TC: total cholesterol, TG: triglyceride, HDL-C: high density lipoprotein-cholesterol, LDL-C: high density lipoprotein-cholesterol Data; mean S.D. Blood pressure (mmhg) 180 160 140 120 100 80 60 0 3 6 9 12 (months) Systolic BP Diastolic BP Fig. 1. Changes in BP during administration of ARB or calcium channel blocker. Open circle, ; closed circle,. There was no significant difference in blood pressure at any point between ARB and calcium channel blocker. Data are presented as the mean S.D. p 0.05 vs 0 month by paired t-test. View ver. 4.51 (Abacus Concepts, Inc.) for Macintosh was used for statistical processing. Comparison between groups was performed using Student s t-test or the paired t-test. The relationship between changes in CAVI and 8-OHdG was analyzed using simple regression analysis. In all comparisons, p 0.05 was considered significant. Results Profile Comparison between ARB and Ca Blocker Group The clinical profile of the subjects is shown in Table 1. The two groups did not differ significantly in any baseline parameters. BP Changes in ARB and Ca Blocker Group BP changes during this study are shown in Fig. 1. In both groups, BP fell from 3 months of ARB or calcium channel blocker administration, and significant decreases in BP were observed from baseline to 6 and 12 months, respectively (Fig. 1); however, no significant differences in BP changes between groups were observed. Changes in BW, HbA1c and Serum Lipid Levels After 12 Months of ARB or Calcium Channel Blocker Administration Slight decreases of BW, HbA1c, TC, TG and LDL-C were recognized in the two groups, but were not significant (Table 2). Table 2. Changes in BW, HbA1c and serum lipid levels after 12 months of ARB or calcium channel blocker administration BW (kg) HbA1c (%) TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) 0.5 4.3 0.6 1.1 13 21 32 47 1.0 6.2 7 19 0.7 5.2 0.5 1.4 22 16 40 53 3.2 5.1 11 21 BW: body weight, HbA1c: glycosylated hemoglobin, TC: total cholesterol, TG: triglyceride, HDL-C: high density lipoprotein-cholesterol, LDL-C: high density lipoprotein-cholesterol Data: mean S.D. 8-OHdG Levels Before and After Administration of ARB or Calcium Channel Blocker The levels of 8-OHdG before and after this study are shown in Fig. 2. In the, a significant decrease in 8-OHdG was observed after 12 months (from 9.59 3.9 to 7.4 2.9 ng/mg Cr). However, no significant change in 8-OHdG was observed in the Ca blocker group (from 8.4 4.0 to 8.2 4.0 ng/mg Cr). Changes of CAVI in ARB and Ca Blocker Group CAVI changes are shown in Fig. 3. In the ARB group, a significant decrease in CAVI was observed after 12 months (from 9.40 0.72 to 9.00 0.81); however, no significant change in CAVI was observed in the (from 9.70 0.78 to 9.60

624 20 4 2 8-OHdG (ng/mg Cr) 15 10 5 8-OHdG (ng/mg Cr) 0-2 -4-6 -8 r=0.478 p<0.05-10 0 before 12 months before 12 months Fig. 2. 8-OHdG levels before and after administration of ARB or calcium channel blocker. Open bar, ; closed bar,. Data are presented as the mean S.D. p 0.05 vs before by paired t-test. CAVI 11.0 10.5 10.0 9.5 9.0 8.5 p<0.05 0.79). No significant difference was observed between ARB and s in baseline CAVI. Correlation between Changes in CAVI and 8- OHdG To clarify the correlation between CAVI changes NS before 12 months before 12 months Fig. 3. Changes in CAVI before and after administration of ARB or calcium channel blocker. Open bar, ; closed bar,. Data are presented as the mean S.D. p 0.05 vs before by paired t-test. -1.0-0.8-0.6-0.4-0.2 0 0.2 0.4 0.6 0.8 1.0 CAVI Fig. 4. Correlation between changes in CAVI and 8-OHdG by simple regression analyses. and oxidative stress, simple regression analyses were performed (Fig. 4). CAVI changes correlated positively with 8-OHdG changes. Discussion Recently, a novel arterial stiffness parameter called CAVI has been developed as a noninvasive and easy technique for the diagnosis of atherosclerosis 9-12). Shirai et al. have reported that CAVI is independent of blood pressure and has adequate reproducibility for clinical use 9). In this study, we have shown a difference in the effects of ARB or calcium channel blocker on improvement of CAVI in type 2 diabetes patients with hypertension in spite of the same changes in BP in the two groups. CAVI is also known to correlate positively with age with different standard criteria by gender 9). In this study, the age and gender ratio were matched in the two groups so the results were considered to be not influenced by these factors. Recent reports indicate that ARB has a favorable effect on glucose and lipid metabolism and protective effects on various organs in addition to the blood pressure-lowering effect 15, 16). Among them, reducted oxidative stress is considered an important anti-atherosclerotic effect in ARB, because enhanced oxidative stress may be closely related to the pathogenesis of atherosclerosis 17-19). 8-OHdG is a product of oxidative DNA damage following specific enzymatic cleavage after 8-hydroxylation of the guanosine base. Urinary 8-OHdG is a biomarker of total in vivo systemic oxidative stress 20, 21). Urinary 8-OHdG has been shown

625 to be higher in diabetic patients than in non-diabetic subjects 21, 22). A recent report indicates that candesartan, an ARB, decreases intima-media thickness by decreasing 8-OHdG 23). In the present study, a significant decrease in 8-OHdG was observed in but not in. This result indicates that ARB is superior to calcium channel blocker in reducing the potential for oxidative stress. Furthermore, CAVI was significantly decreased in but not in although the BP changes were the same in the two groups. These results suggest that olmesaltan improved an arterial stiffness parameter called CAVI by decreasing 8-OHdG. As for the mechanisms of CAVI reduction, there are at least two possible explanations. The first is that vascular organic lesions are regressed. Recent reports indicate that CAVI could detect vascular organic lesions 9-12), which regressed by reducing oxidative stress. For example, Ono et al. reported that carotid intimamedia thickness was decrease by reducing 8-OHdG from 10.98 to 7.61 ng/mg Cr 23). In the present study, the decrease of 8-OHdG was almost identical to Ono s report. Thus, the reduction of CAVI may be reflected by the regression of vascular organic lesions. Another possibility is that endothelial function is improved. Oxidative stress is reported to be the cause of endothelial dysfunction 24), and high levels of 8-OHdG are reported to contribute to atherogenetic processes by vascular endothelial dysfunction 25, 26). So, the improvement of CAVI recognized in our study might be due to not only the regression of organic lesions but also the improvement of endothelial dysfunction. Endothelial function is known to be affected by many factors, especially by dietary conditions so to clarify it is necessary the effects of these factors on CAVI in the future. 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