Cilostazol improves endothelial dysfunction by increasing endothelium-derived hyperpolarizing factor response in mesenteric arteries from Type 2 diabetic rats

European Journal of Pharmacology 599 (2008) 102–109

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Cardiovascular Pharmacology

Cilostazol improves endothelial dysfunction by increasing endothelium-derived hyperpolarizing factor response in mesenteric arteries from Type 2 diabetic rats Takayuki Matsumoto, Eri Noguchi, Keiko Ishida, Naoaki Nakayama, Tsuneo Kobayashi, Katsuo Kamata ⁎ Department of Physiology and Morphology, Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan

a r t i c l e

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Article history: Received 24 June 2008 Received in revised form 18 September 2008 Accepted 5 October 2008 Available online 10 October 2008 Keywords: Cilostazol Diabetes Endothelium-derived hyperpolarizing factor Endothelial dysfunction Mesenteric artery

a b s t r a c t Diabetes mellitus impairs endothelial function, an effect that can be considered a hallmark of the development of cardiovascular diseases in diabetics. Cilostazol, a selective phosphodiesterase 3 inhibitor, is currently used to treat patients with diabetic vascular complications. However, the effects of cilostazol on responses mediated by endothelium-derived relaxing [in particular, nitric oxide (NO) and hyperpolarizing factors (EDHF)] and contracting factors remain unclear. Here, we hypothesized that cilostazol could improve endothelial dysfunctions in mesenteric arteries isolated from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Using cilostazol-treated (100 mg/kg/day for 4 weeks) or -untreated OLETF and control (Long Evans Tokushima Otsuka) rats, we examined the acetylcholine-induced endothelium-dependent responses and the cell-permeant cyclic adenosine monophosphate (cAMP) analog-induced relaxations in the superior mesenteric artery. We also determined blood parameters in these animals. In OLETF rats, chronic treatment with cilostazol reduced the blood levels of triglyceride, non-esterified fatty acids, and leptin, and increased antioxidant capacity, but did not alter the blood glucose or insulin levels. In studies on mesenteric arteries from cilostazol-treated OLETF animals, the cilostazol treatment improved: (a) the acetylcholineinduced EDHF-mediated relaxation and (b) the cAMP-mediated relaxation. However, cilostazol did not alter the NO-mediated relaxation or the endothelium-derived contracting factor-mediated contraction. These results suggest that cilostazol improves endothelial functions in OLETF mesenteric arteries by increasing EDHF signaling, and that it normalizes some metabolic abnormalities in OLETF rats. On that basis, cilostazol may prove to be a potent drug for the clinical treatment of diabetic vasculopathy. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Cilostazol, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3, 4-dihydro2(1H)-quinolinone, increases the intracellular cyclic adenosine monophosphate (cAMP) level by inhibiting the activity of phosphodiesterase 3 (Lugnier, 2006). Cilostazol is currently used in the treatment of intermittent claudication in diabetic patients (Dawson et al., 1998) and for peripheral vascular occlusive diseases (Kwon et al., 2005). Cilostazol has also been shown to be effective in preventing both silent brain infarction in Japanese patients with type 2 diabetes (ShinodaTagawa et al., 2002) and atherosclerosis (Meadows and Bhatt, 2007), effects considered to result from its anti-platelet, vasodilator, and antiproliferative actions. Although there is an accumulating body of evidence to show that cilostazol has beneficial effects on several pathophysiological states, the effects of this drug on endothelium-dependent vasomotor responses in type 2 diabetes remain uncertain. Macro- and micro-vascular diseases are the principal contributors to the increased morbidity and mortality associated with both type 1 and type 2 diabetes [indeed, type 2 diabetic patients have a mortality ⁎ Corresponding author. Tel./fax: +81 3 5498 5856. E-mail address: [email protected] (K. Kamata). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.006

rate 3–4 times that of the general population (Haffner et al., 1998)]. Endothelial dysfunction may play a key role in the development of both macro- and micro-angiopathy both in diabetic patients and in animal models of diabetes (De Vriese et al., 2000; Matsumoto et al., 2006a; Pieper, 1998). Evidence is accumulating to indicate (a) that endothelium-dependent relaxation is impaired in several blood vessels in type 2 diabetes [both in animal models and in patients (De Vriese et al., 2000; Kobayashi et al., 2004; Van Gaal et al., 2006)] as well as in type 1 diabetes (De Vriese et al., 2000; Hattori et al., 1991; Kamata et al., 1989; Matsumoto et al., 2003, 2005a, 2007c; Mayhan and Patel, 1998), and (b) that a decreased production of endotheliumderived relaxing factors [in particular, nitric oxide (NO) and hyperpolarizing factors (EDHF)] and/or defects in endothelium-derived relaxing factors-signaling may underlie the impairment of endothelium-dependent relaxation seen in type 2 diabetic vessels (De Vriese et al., 2000; Ding and Triggle, 2005). Moreover, this impairment of vascular relaxation may be attributable not only to reduced amounts of endothelium-derived relaxing factor and/or EDHF, but also to increased amounts of endothelium-derived contracting factor, leading to diabetic vasculopathy (Cohen, 2005; De Vriese et al., 2000; Feletou and Vanhoutte, 2006; Matsumoto et al., 2006c). Although the balance between endothelium-derived relaxing factors and endothelium-

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derived contracting factors is known to play an important role in setting vascular tone in both physiological and pathophysiological states, little information is available as to whether (and to what extent) manipulation of such factors by drugs might be able to improve vasculopathy in type 2 diabetes. Otsuka Long-Evans Tokushima Fatty (OLETF) rats are characterized by an early increase in serum insulin, and also by late-onset hyperglycemia, mild obesity, and mild type 2 diabetes (Kawano et al., 1992), and there are several reports of abnormalities of vascular function in this diabetic model (Kagota et al., 2000; Matsumoto et al., 2006b,c, 2007b). Moreover, we recently demonstrated: (a) that endothelial dysfunction is present in the mesenteric arteries of aged OLETF rats, (b) that this may result from an imbalance between endothelium-derived factors (reduced endothelium-derived relaxing factors signaling and increased endothelium-derived contracting factors signaling), (c) that the mechanisms underlying this abnormality may involve increments in both cyclooxygenase-1 and -2 activities (Matsumoto et al., 2007a), and (d) that the impairment of EDHF-type relaxation seen in this diabetic rat may be attributable not only to a reduction in cAMP/ protein kinase A signaling, but also to reduced endothelial Ca2+activated potassium channel (KCa) activities (Matsumoto et al., 2006c). Although there is considerable evidence to show that cilostazol has beneficial effects on several pathophysiological states, no study has yet investigated the effects of chronic cilostazol treatment on the endothelial dysfunction present in type 2 diabetic mesenteric arteries. Therefore, in the present study we carried out just such an investigation, using mesenteric arteries isolated from OLETF rats. 2. Materials and methods 2.1. Reagents Apamin, phenylephrine, indomethacin, NG-nitro-L-arginine (L-NNA), N , O2-dibutyryl-adenosine-3′, 5′-cyclic monophosphate (db-cAMP), and TRAM34 [1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole] were all purchased from Sigma Chemical Co. (St. Louis, MO, USA), while acetylcholine chloride was purchased from Daiichi-Sankyo Pharmaceuticals (Tokyo, Japan). Drugs were dissolved in saline, except for TRAM34 (dissolved in dimethyl sulfoxide) and indomethacin. Indomethacin was dissolved first in a small amount of 0.1 M Na2CO3 solution, and then made up to the final volume with distilled water. 6

2.2. Animals and experimental design Five-week-old male rats [OLETF rats and Long-Evans Tokushima Otsuka (LETO) rats, a genetic control for OLETF] were supplied by the Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan). Food and water were given ad libitum in a controlled environment (room temperature 21–22 °C, room humidity 50 ± 5%) until the rats were 36–42 weeks old. Some OLETF and LETO rats were chronically given cilostazol (100 mg/kg/day, p.o.) for 4 weeks starting at 36–42 weeks old. Thus, we studied four groups: cilostazol-untreated LETO and OLETF groups and cilostazol-treated LETO and OLETF groups. This study was approved by the Hoshi University Animal Care and Use Committee, and all studies were conducted in accordance with “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health, and “Guide for the Care and Use of Laboratory Animals” adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University (which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology, Japan). 2.3. Measurement of blood glucose, cholesterol, triglyceride, insulin, leptin, antioxidant, and non-esterified fatty acid, and blood pressure Plasma parameters and systemic blood pressure were measured as described previously (Matsumoto et al., 2003, 2004, 2005a,b, 2007c).

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Briefly, plasma glucose, cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol, and serum non-esterified fatty acid levels were each determined by the use of a commercially available enzyme kit (Wako Chemical Company, Osaka, Japan). Plasma insulin was measured by enzyme-immunoassay (Shibayagi, Gunma, Japan). Plasma leptin was determined by enzyme-linked immunosorbent assay (Morinaga Institute of Biological Science, Yokohama, Japan). The plasma antioxidant level was determined by the use of a commercially available kit (Cayman Chemical, Ann Arbor, MI, USA). This Trolox equivalent antioxidant-capacity assay is based on the scavenging of the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical [ABTS(⁎)] converting it into a colorless product. After a given rat had been in a constant-temperature box at 37 °C for a few minutes, its blood-pressure was measured by the tail-cuff method using a blood pressure analyzer (BP-98A; Softron, Tokyo, Japan). 2.4. Measurement of isometric force Vascular isometric force was recorded as in our previous papers (Matsumoto et al., 2003, 2005a, 2006c, 2007a). At 40–46 weeks of age, rats were anesthetized with diethyl ether, then euthanized by decapitation. The superior mesenteric artery was rapidly removed and immersed in oxygenated, modified Krebs-Henseleit solution (KHS). This solution consisted of (in mM) 118.0 NaCl, 4.7 KCl, 25.0 NaHCO3, 1.8 CaCl2, 1.2 NaH2PO4, 1.2 MgSO4, and 11.0 dextrose. The artery was carefully cleaned of all fat and connective tissue, and ring segments 2 mm in length were suspended by a pair of stainless-steel pins in a well-oxygenated (95% O2- 5% CO2) bath containing 10 mL of KHS at 37 °C. The rings were stretched until an optimal resting tension of 1.0 g was loaded, and then allowed to equilibrate for at least 60 min. Force generation was monitored by means of an isometric transducer (model TB-611T; Nihon Kohden, Tokyo, Japan). For the relaxation studies, mesenteric rings were precontracted with an equally effective concentration of phenylephrine (100 nM–3 μM) (i.e., so that the tension developed in response to phenylephrine was similar among all groups). There was no significant difference in the response to phenylephrine among the LETO (n=32), OLETF (n =32), cilostazol-treated LETO (n=32), and cilostazol-treated OLETF (n=32) groups (1.65 ± 0.05 g, 1.68± 0.05 g, 1.63 ± 0.04 g, and 1.69 ± 0.05 g, respectively). When the phenylephrine-induced contraction had reached a plateau level, acetylcholine (1 nM–10 μM) was added in a cumulative manner. After the addition of sufficient aliquots of the agonist to produce the chosen concentration, a plateau response was allowed to develop before the addition of the next dose of the same agonist. To investigate the influences of the various factors that might constitute endotheliumderived relaxing factor in the present preparations, we examined acetylcholine-induced relaxation in the absence or presence of various inhibitors, as follows: 1) 10 μM indomethacin plus 10 μM TRAM34 (specific inhibitor of the intermediate-conductance KCa channel) plus 100 nM apamin (specific inhibitor of the small-conductance KCa channel) (to investigate NO-mediated relaxation), 2), 10 μM indomethacin plus 100 μM L-NNA (to investigate EDHF-type relaxation). Rings were incubated with the appropriate inhibitor(s) for 30 min before administration of phenylephrine. To investigate the cAMP-mediated relaxation, db-cAMP (a cell-permeable cAMP analog) (1 μM–100 μM) was added in a cumulative manner in the presence of 10 μM indomethacin plus 100 μM L-NNA. For the contraction studies, mesenteric rings were first contracted using 80 mM K+, these responses being taken as 100%. There was no significant difference in the response to 80 mM K+ among the LETO (n = 8), OLETF (n = 8), cilostazol-treated LETO (n = 8), and cilostazoltreated OLETF (n = 8) groups (1.54 ± 0.03 g, 1.57 ± 0.06 g, 1.60 ± 0.04 g, and 1.54 ± 0.03 g, respectively). To investigate the endotheliumderived contracting factor-mediated response, mesenteric rings were treated with 100 μM L-NNA for 30 min. After this incubation period, acetylcholine (10 nM–10 μM) was cumulatively applied.

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Table 1 Diabetic parameters in cilostazol-treated and -untreated LETO and OLETF rats LETO Body weight (g) Glucose (mg/dl) Insulin (ng/ml) Leptin (ng/ml) Systolic blood pressure (mmHg) Heart rate (beats/min)

550.5 ± 8.5 130.6 ± 7.3 2.4 ± 0.3 9.2 ± 1.2 118.1 ± 2.3 414.8 ± 10.4

(12) (12) (12) (9) (12) (12)

Cilostazol-treated LETO

OLETF

Cilostazol-treated OLETF

516.3 ± 12.9 (12)a 117.0 ± 5.3 (12) 2.8 ± 0.4 (12) 8.0 ± 1.0 (9) 119.0 ± 1.9 (12) 429.1 ± 9.2 (12)

703.3 ± 14.1 (12)c 235.1 ± 18.4 (12)c 5.8 ± 1.3 (12)a 24.0 ± 3.3 (9)c 142.5 ± 3.3 (12)c 424.4 ± 13.4 (12)

681.1 ± 20.0 (12)c 218.9 ±24.5 (12)b 4.9 ± 1.2 (12)a 13.7 ± 2.4 (9)d 137.1 ± 2.3 (12)c 410.8 ± 12.2 (12)

Values are means ± S.E.M. Number of determinations is shown within parentheses. LETO, Long-Evans Tokushima Otsuka; OLETF, Otsuka Long-Evans Tokushima fatty. aP b 0.05, b P b 0.01, cP b 0.001 vs. LETO. dP b 0.05 vs. OLETF.

2.5. Statistical analysis Data are expressed as means ± S.E.M. Each relaxation response is expressed as a percentage of the contraction induced by phenylephrine. Contractile responses are expressed as a percentage of the response to 80 mM KCl. When appropriate, statistical differences were assessed by Dunnett's test for multiple comparisons after a one-way analysis of variance (ANOVA), a probability level of P b 0.05 being regarded as significant. Statistical comparisons between concentration-response curves were made using a two-way ANOVA, with Bonferroni's correction for multiple comparisons being performed post hoc (P b 0.05 again being considered significant). 3. Results 3.1. Diabetic status As shown in Table 1, at the time of the experiment all OLETF rats (non-fasted) exhibited hyperglycemia, their blood glucose concentrations being significantly higher than those of the age-matched nondiabetic control LETO rats (also non-fasted). The body weight of the OLETF rats was greater than that of LETO rats. The plasma insulin and leptin levels were significantly higher in OLETF rats than in LETO rats. The systolic blood pressure of OLETF rats was higher than that of LETO rats, while heart rate was similar between the two groups. Treatment with cilostazol did not alter plasma glucose, insulin, systolic blood pressure, or heart rate in OLETF rats, but it significantly lowered plasma leptin level. In LETO rats, treatment with cilostazol significantly lowered body weight (versus non-treated LETO rats). 3.2. Blood lipid status To evaluate the effect of cilostazol on lipid metabolism, we measured the levels of plasma cholesterol, HDL, triglyceride, and serum non-esterified fatty acid in all groups (Table 2). The plasma triglyceride

Table 2 Blood lipid parameters in cilostazol-treated and -untreated LETO and OLETF rats LETO

Cilostazoltreated LETO

OLETF

Cilostazol-treated LETO

Cholesterol 121.8 ± 4.8 (12) 114.6 ± 3.4 (12) 133.8 ± 9.6 (12) 123.9 ± 11.1 (12) (mg/dl) HDL (mg/dl) 68.6 ± 7.6 (12) 74.1 ± 5.7 (12) 68.7 ± 9.5 (12) 73.9 ± 8.9 (12) Triglyceride 78.8 ± 4.6 (12) 59.1 ± 4.3 (12)b 290.0 ± 42.4 (12)c 139.2 ± 21.7 (12)a,d (mg/dl) 0.29 ± 0.02 (12) 0.31 ± 0.03 (10) 0.45 ± 0.04 (12)b 0.31 ± 0.02 (12)d Nonesterified fatty acid (mEq/l) Values are means ± S.E.M. Number of determinations is shown within parentheses. LETO, Long-Evans Tokushima Otsuka; OLETF, Otsuka Long-Evans Tokushima fatty; HDL, high-density lipoprotein. aP b 0.05, bP b 0.01, cP b 0.001 vs. LETO. dP b 0.01 vs. OLETF.

and serum non-esterified fatty acid levels were significantly higher in OLETF rats than in LETO rats, while total cholesterol and HDL levels were similar between the two groups (Table 2). Treatment with cilostazol did not alter plasma total cholesterol or HDL in OLETF rats, but it significantly lowered plasma triglyceride and serum nonesterified fatty acid (versus non-treated OLETF rats). In LETO rats, treatment with cilostazol significantly lowered triglyceride (versus non-treated LETO rats). 3.3. Effects of cilostazol treatment on endothelium-dependent relaxation We previously demonstrated that mesenteric arteries from aged OLETF rats exhibit endothelial dysfunction and that this results from an imbalance of endothelium-derived factors (reduced endotheliumderived relaxing factor signaling and increased endothelium-derived contracting factor signaling) (Matsumoto et al., 2007a). To evaluate the effect of chronic cilostazol treatment on endothelial function in mesenteric arteries from OLETF rats, the vasorelaxation response to acetylcholine (1 nM–10 μM) was examined in all four groups (Fig. 1 and Table 3). Acetylcholine induced a concentration-dependent relaxation (with the maximum response being at 100–300 nM, and responses then being progressively weaker up to 10 μM), and this relaxation was significantly weaker in rings from OLETF rats than in those from LETO rats (Fig. 1A). In LETO rats, cilostazol treatment caused no significant alteration in the acetylcholine-induced relaxation (vs. untreated LETO group) (Fig. 1B). In the OLETF group, however, cilostazol significantly increased the relaxation responses to lower concentrations of acetylcholine (i.e., 3 nM to 30 nM), but did not alter the tendency for the relaxation to weaken (or reverse) as the concentration of acetylcholine was increased (0.3–10 μM) (Fig. 1C). To investigate which endothelium-derived factors might be improved in mesenteric arteries from cilostazol-treated OLETF rats,

Table 3 Sensitivity (EC50) and magnitude (Rmax) values for the relaxations induced by acetylcholine (with or without inhibitors) in mesenteric rings from cilostazol-treated and -untreated LETO and OLETF rats LETO EC50 (nM) Control 16.2 ± 5.3 (8) Indo + Apa + TRAM34 17.9 ± 1.8 (8) L-NNA + Indo 46.2 ± 6.9 (8)

CilostazolOLETF treated LETO

Cilostazol-treated OLETF

13.7 ± 2.7 (8) 18.2 ± 3.3 (8) 31.2 ± 4.3 (6)

8.5 ± 1.3(8)c 13.4 ± 2.2 (8) 36.2 ± 6.5 (8)

Rmax (%) Control 96.5 ± 0.8 (8) 89.9 ± 4.0 (8) Indo + Apa + TRAM34 98.4 ± 0.6 (8) 98.1 ± 0.9 (8) L-NNA + Indo 70.2 ± 2.6 (8) 68.5 ± 2.5 (8)

32.1 ± 10.3 (8) 22.4 ± 5.4 (8) 47.4 ± 7.2 (8)

71.2 ± 10.0 (8)a 93.4 ± 1.0 (8)c 92.2 ± 1.0 (8)b 94.4 ± 1.0 (8) 23.8 ± 6.0 (8)b 60.7 ± 4.0(8)d

Values are means±S.E.M. Acetyicholine-induced relaxation in the absence (Control) or presence of various inhibitors, as follows: Indo, indomethacin (10−5 M); Apa, apamin (10− 7 M); TRAM34 (10−5 M); L-NNA, NG-nitro-L-arginine (10−4 M). LETO, Long-Evans Tokushima Otsuka; OLETF, Otsuka Long-Evans Tokushima fatty. Number of determinations is shown within parentheses. aPb 0.05, bPb 0.001 vs. corresponding LETO group. cPb 0.05, dPb 0.001 vs. corresponding OLETF group.

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performed a series of experiments in which acetylcholine was added cumulatively to rings precontracted by phenylephrine in the presence of 100 μM L-NNA plus 10 μM indomethacin (Fig. 3, Table 3). This EDHFtype relaxation was significantly weaker in rings from OLETF rats than in those from LETO rats (Fig. 3A). In OLETF rats (Fig. 3C), but not in LETO rats (Fig. 3B), cilostazol treatment significantly improved the acetylcholine-induced EDHF-type relaxation. 3.4. Effects of cilostazol treatment on endothelium-dependent contraction As described above (Fig. 1), at higher concentrations of acetylcholine (i.e., 0.3–10 μM) a reduced acetylcholine-induced relaxation was

Fig. 1. Acetylcholine-induced endothelium-dependent relaxations in mesenteric arteries obtained from Long-Evans Tokushima Otsuka (LETO) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats. A: Concentration-response curves for acetylcholine-induced relaxations of isolated rings of mesenteric arteries obtained from LETO and OLETF rats. B, C: effects of chronic treatment with cilostazol on such acetylcholine-induced relaxations in mesenteric arteries isolated from LETO (B) and OLETF (C) rats. Details are given under Materials and methods. Data are means ± S.E.M. Number of determinations is shown within parentheses. For comparison, the curves obtained for acetylcholine-induced relaxation in intact preparations (A) are shown again in (B) and (C). ⁎P b 0.05, ⁎⁎⁎P b 0.001 vs. LETO group. # P b 0.05, ##P b 0.01 vs. OLETF group.

we examined the acetylcholine-induced relaxation in the presence of various inhibitors (Figs. 2 and 3). To investigate the acetylcholineinduced NO-mediated relaxation, we added acetylcholine cumulatively to rings precontracted by phenylephrine in the combined presence of 10 μM indomethacin, 100 nM apamin, and 10 μM TRAM34 (Fig. 2, Table 3). Under these conditions the acetylcholine-induced NOmediated relaxation was significantly weaker in rings from OLETF rats than in those from LETO rats (Fig. 2A). Treatment with cilostazol did not alter this NO-mediated relaxation in either LETO (Fig. 2B) or OLETF (Fig. 2C) rats. To investigate the component of the acetylcholine-induced endothelium-dependent relaxation that is mediated by EDHF, we

Fig. 2. Acetylcholine-induced NO-mediated relaxations in mesenteric arteries obtained from LETO and OLETF rats. A: Concentration-response curves for acetylcholine-induced NO-mediated relaxations of isolated rings of mesenteric arteries (i.e., relaxations induced by acetylcholine in the presence of 10 μM indomethacin plus 100 nM apamin plus 10 μM TRAM34). B, C: effects of chronic treatment with cilostazol on such NOmediated relaxations in mesenteric arteries isolated from LETO (B) and OLETF (C) rats. Details are given under Materials and methods. Data are means ± S.E.M. Number of determinations is shown within parentheses. For comparison, the curves obtained for acetylcholine-induced NO-mediated relaxation (A) are shown again in (B) and (C). ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs. LETO group.

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those from LETO rats (Fig. 4A). Cilostazol had no significant effect on this acetylcholine-induced contraction in either the LETO group (Fig. 4B) or the OLETF group (Fig. 4C).

Fig. 3. Acetylcholine-induced EDHF-mediated relaxations in mesenteric arteries obtained from LETO and OLETF rats. A: Concentration-response curves for acetylcholine-induced EDHF-mediated relaxations of isolated rings of mesenteric arteries (i.e., in the presence of 10 μM indomethacin plus 100 μM L-NNA). B, C: effects of chronic treatment with cilostazol on such EDHF-mediated relaxations in mesenteric arteries isolated from LETO (B) and OLETF (C) rats. Details are given under Materials and methods. Data are means ± S.E.M. Number of determinations is shown within parentheses. For comparison, the curves obtained for acetylcholine-induced EDHFmediated relaxation (A) are shown again in (B) and (C).⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs. LETO group. ##P b 0.01, ###P b 0.001 vs. OLETF group.

observed, with the relaxation being more nearly abolished in rings from OLETF rats than in those from LETO rats. To investigate the effect of cilostazol on the contractile component of these acetylcholine-induced responses, we added acetylcholine (10 nM–10 μM) cumulatively to rings in the presence of L-NNA (100 μM), as in a previous report (Matsumoto et al., 2007a). As shown in Fig. 4A, under these conditions an acetylcholine-induced contraction was observed at higher acetylcholine concentrations (i.e., 0.3–10 μM) in rings from both groups. This response was significantly greater in mesenteric arteries from OLETF rats than in

Fig. 4. Endothelium-dependent contractions in mesenteric arteries obtained from LETO and OLETF rats. A: Concentration-response curves for acetylcholine-induced endotheliummediated contractions of isolated rings of mesenteric arteries (i.e., in the presence of 100 μM L-NNA). B, C: effects of chronic treatment with cilostazol on such acetylcholineinduced endothelium-dependent contractions in mesenteric arteries isolated from LETO (B) and OLETF (C) rats. Details are given under Materials and methods. Data are means±S.E.M. Number of determinations is shown within parentheses. For comparison, the curves obtained for acetylcholine-induced contraction in the presence of 100 μM L-NNA (A) are shown again in (B) and (C). ⁎Pb 0.05 vs. LETO group.

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3.5. Effects of cilostazol treatment on cAMP-mediated relaxation To investigate whether chronic cilostazol treatment would effectively inhibit phosphodiesterase 3 and modulate cAMP signaling in the mesenteric artery, we examined the relaxing effect of a cellpermeant cAMP analog in rings from all groups (in the presence of 100 μM L-NNA plus 10 μM indomethacin) (Fig. 5). The relaxation response induced by this cAMP analog (db-cAMP) was significantly weaker in the OLETF group than in the LETO group. This impaired relaxation was significantly enhanced by chronic cilostazol treatment of OLETF rats. 3.6. Evaluation of antioxidants It is known that antioxidants play an important role in the prevention of cardiovascular diseases (Aguirre et al., 1998). Since it takes into account the cumulative effect of all the antioxidants present in the plasma, measurement of overall antioxidant capacity may provide more relevant biological information than measurement of individual components (such as superoxide dismutase, catalase, glutathione peroxidase, and relevant macro- and/or small-molecules). We therefore measured the total antioxidant capacity in rat plasma

Fig. 6. Plasma antioxidant levels in cilostazol-treated and -untreated LETO and OLETF rats. Details are given under Materials and methods. Data are means ± S.E.M. Number of determinations is shown within parentheses. ⁎P b 0.05 vs. LETO group. #P b 0.05 cilostazol-treated OLETF vs. OLETF.

(Fig. 6). The plasma total antioxidant level was lower in OLETF rats than in LETO rats, and it was significantly higher in the cilostazoltreated OLETF group than in the untreated OLETF group. 4. Discussion

Fig. 5. cAMP derivative-induced relaxations in mesenteric arteries obtained from cilostazol-treated and -untreated LETO and OLETF rats. A: Concentration-response curves for the relaxations induced by db-cAMP in isolated rings of mesenteric arteries in the presence of 10 μM indomethacin plus 100 μM L-NNA. B: EC50 values for all groups. Details are given under Materials and methods. Data are means ± S.E.M. Number of determinations is shown within parentheses. ⁎P b 0.05 vs. LETO group. ##P b 0.01, ### P b 0.001 cilostazol-treated OLETF vs. OLETF.

In the present study, we detected two major effects of chronic cilostazol treatment of type 2 diabetic OLETF rats: modulation of certain blood parameters and a specific improvement in EDHF-type relaxation in isolated mesenteric arteries. Cilostazol has been shown to be effective in the prevention and treatment of diabetic complications (via improvement in the peripheral circulation) (Mohler, 2007; Shinoda-Tagawa et al., 2002). In some clinical studies, administration of cilostazol to diabetic patients was associated with decreased triglyceride levels and increased levels of HDL cholesterol (Elam et al., 1998; Nakamura et al., 2003) in the blood. Although cilostazol thus has beneficial effects on lipids, we know of no previous reports of any positive effects on endothelial dysfunction after the chronic administration of this drug to diabetic animals. In our OLETF rats, plasma triglyceride, leptin, and serum non-esterified fatty acid levels were all significantly increased, and these increased lipid and leptin levels were normalized by the chronic administration of cilostazol. Further, the endotheliumdependent relaxation of mesenteric artery rings seen in response to acetylcholine was significantly attenuated in OLETF rats, and this impaired endothelium-dependent relaxation was restored by chronic administration of cilostazol. A possible interpretation of these results is that the endothelial dysfunction present in OLETF rats is due to increases in blood triglyceride, non-esterified fatty acid, and/or leptin levels, and that the endothelium-dependent relaxation is improved by the chronic administration of cilostazol at least in part through a lowering of these parameters. In fact, evidence of endothelial dysfunction has been observed in several vessels in states in which there is an increment in the circulating lipid profile [including an elevation in triglyceride (Abe et al., 1998; Creager et al., 2003) or free fatty acids (Creager et al., 2003; Steinberg et al., 1997), or hyperleptinemia (Beltowski, 2006; Knudson et al., 2005; Piatti et al., 2003)]. A novel, intriguing, and potentially important finding made in this study was that a specific improvement in EDHF-type relaxation is present in mesenteric arteries from cilostazol-treated OLETF rats. This conclusion is supported by several pieces of evidence obtained by comparing the present OLETF group with the LETO (control) group. First, the acetylcholine-induced endothelium-dependent relaxation was impaired in the OLETF group, and the relaxation became weaker

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at higher concentrations of acetylcholine (Fig. 1A). Cilostazol significantly increased the relaxation responses to lower concentrations of acetylcholine without affecting the tendency for the relaxation to reverse at higher concentrations of acetylcholine (Fig. 1C). Second, under the treatment used to prevent NO-mediated relaxation (i.e., indomethacin plus apamin plus TRAM34) (Matsumoto et al., 2006d), the acetylcholine-induced relaxation was significantly reduced in the OLETF group (Fig. 2A). However, this NO-mediated relaxation was similar between the cilostazol-treated and -untreated OLETF rats (Fig. 2C). Third, the acetylcholine-induced EDHF-type relaxation observed in the presence of L-NNA plus indomethacin was greatly impaired in the OLETF group (Fig. 3A), and cilostazol significantly increased this relaxation (Fig. 3C). Fourth, the acetylcholine-induced endothelium-derived contracting factor-mediated contraction observed in the presence of L-NNA was significantly increased in the OLETF group (Fig. 4A), but cilostazol did not affect this contraction (Fig. 4C). An imbalance between endothelium-derived relaxing factors and endothelium-derived contracting factors is implicated in the endothelial dysfunction seen in OLETF mesenteric arteries [Figs. 1–4 and Ref. (Matsumoto et al., 2007a)], and the present data suggest that cilostazol improves endothelial function at least partly by improving EDHF-mediated responses in the type 2 diabetic mesenteric artery. A seminal study published very recently by Suzuki et al. (2008) demonstrated (a) that cilostazol leads to increased NO production and suppressions of cytokine-induced nuclear-κB activation and vascular cell adhesion molecule-1 expression via AMP-activated protein kinase activation in human umbilical vein endothelial cells, and (b) that administration of cilostazol to diabetic OLETF rats for 24 weeks (i.e., starting at 4 weeks old, with the experiments being performed at 28 weeks of age) restores endothelial function in the thoracic aorta (viz. improvements in acetylcholine-induced endotheliumdependent relaxation and biopterin metabolism). On the other hand, in the present study we set out to examine the therapeutic, not preventive, effects of cilostazol. Therefore, we administered 4-weeks' cilostazol treatment to OLETF rats at age 36–42 weeks (i.e., in an established diabetic model), and we found that such cilostazol-treated OLETF rats exhibited restored endothelial function in the mesenteric artery (viz. improved EDHF signaling). The differences between the two studies in the beneficial effects of cilostazol might be due to differences in the starting age and/or in the duration of the cilostazol treatment and/or in the artery studied. The mechanisms underlying the improvement we observed in EDHF-type relaxation following chronic treatment with cilostazol remain uncertain, but a modulation of the cAMP-signaling pathway may be involved. This speculation is supported by several lines of evidence. First, it has recently been reported that cAMP facilitates EDHF-type relaxation in conduit arteries by enhancing electrotonic conduction via gap junctions (Griffith, 2004; Griffith et al., 2002). Second, our laboratory (Matsumoto et al., 2005a) found a few years ago that cilostazol improves the EDHF response through increases in cAMP- and protein kinase A-signaling in the streptozotocin-induced type 1 diabetic mesenteric artery (in which cAMP signaling is impaired) (Matsumoto et al., 2003, 2004, 2005b). Third, our previous study (Matsumoto et al., 2006c) of rat mesenteric arteries found that: (a) the acetylcholine-induced EDHF-type relaxation was significantly reduced both by a protein kinase A inhibitor and by a gap-junction inhibitor in the control LETO group, but not in the OLETF group; (b) the phosphodiesterase 3 inhibitor-induced relaxation was significantly weaker in the OLETF group; (c) the relaxation induced by a cAMP analog was significantly weaker in the OLETF group, and (d) both cAMP analog-stimulated and -unstimulated protein kinase A activity levels were decreased in the OLETF group. Indeed, in the present study (Fig. 5), the cAMP analog-induced relaxation was significantly reduced in the OLETF (vs. LETO) group; however, this impaired relaxation showed significant recovery in mesenteric arteries obtained from cilostazol-treated OLETF rats. From these results and the published

evidence, we suggest that cilostazol improves EDHF-type relaxations in OLETF rats via an increase in cAMP signaling. Another possibility as to the mechanism underlying the improvement of EDHF-type relaxation following chronic treatment with cilostazol is that there may be reductions in blood triglycerides and oxidative stress. Although the role of triglycerides in the regulation of vasomotion remains poorly characterized, hypertriglycemic patients exhibit diminished vasodilation of skin vessels, an impairment than can be reversed by a lowering of their triglyceride levels (Tur et al., 1994). Furthermore, cutaneous acetylcholine-induced vasodilation shows a negative correlation with triglyceride levels in humans (Algotsson, 1996). In addition, recent evidence suggests that radical scavengers improve the impaired EDHF-mediated responses seen in several disease models (Feletou and Vanhoutte, 2004; Ozkan and Uma, 2005). Since previous reports and the present study suggest that cilostazol decreases triglyceride levels [(Elam et al., 1998; Nakamura et al., 2003), Table 2] and reduces oxidative stress [(Lee et al., 2005), Fig. 6], the correction of EDHF-signaling in OLETF rats by cilostazol may be mediated by its antilipemic and/or antioxidative actions. As yet, it remains unclear whether there is a direct relationship between the effects of cilostazol on EDHF responses and its effects on these cellular pathways. Further investigations are required on this point. Previous studies have suggested that impairments of EDHFmediated responses are present in disease states, indicating the potential for therapeutic interventions (Feletou and Vanhoutte, 2004; Matsumoto et al., 2006a). For example, chronic treatment with an angiotensin converting enzyme inhibitor, with an angiotensinreceptor antagonist, or with a diuretic normalizes EDHF-mediated responses in spontaneously hypertensive rats (Feletou and Vanhoutte, 2004). In addition, dietary supplements and exercise have beneficial effects on EDHF responses (Feletou and Vanhoutte, 2004). However, the mechanisms underlying these drug-induced and adjutantinduced improvements in EDHF responses remain poorly understood. EDHF-mediated responses are clearly affected in a variety of pathological conditions, and use of the above therapeutic or adjutant interventions to restore EDHF-mediated responses might be beneficial for the patient(s). Especially interesting is the possibility that an enhancement of EDHF-mediated responses might contribute to improvements in diabetic microvascular complications such as retinopathy, nephropathy, and neuropathy (because EDHF plays important roles in the microvasculature). Cilostazol is already used clinically in several diseases, such as peripheral artery disease (Dawson et al., 1998; Kwon et al., 2005; Mohler, 2007; ShinodaTagawa et al., 2002), and the present study provides evidence of its potential to be a therapeutic drug for the improvement of EDHFmediated responses in diabetic states. In conclusion, our study suggests that cilostazol improves endothelial functions in OLETF mesenteric arteries by increasing EDHF signaling, and also that it normalizes some metabolic abnormalities in such type 2 diabetic animals. We believe that our findings should stimulate further interest in cilostazol as a potential therapeutic drug for use against diabetes-associated vasculopathy. Acknowledgments We thank Otsuka Pharmaceutical for providing LETO and OLETF rats. This study was supported in part by the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the Open Research Center Project. References Abe, H., Yamada, N., Kamata, K., Kuwaki, T., Shimada, M., Osuga, J., Shionoiri, F., Yahagi, N., Kadowaki, T., Tamemoto, H., et al., 1998. Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1. J. Clin. Invest. 101, 1784–1788.

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