Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats

Cardiovascular Diabetology Nicorandil prevents endothelial dysfunction due to antioxidative effects via normalisation of NADPH oxidase and nitric oxide synthase in streptozotocin diabetic rats Ken-ichi Serizawa 0 Kenji Yogo 0 Ken Aizawa 0 Yoshihito Tashiro 0 Nobuhiko Ishizuka 0 0 Product Research Department, Chugai Pharmaceutical Co., Ltd. , Gotemba, Shizuoka 412-8513 Japan Background: Nicorandil, an anti-angina agent, reportedly improves outcomes even in angina patients with diabetes. However, the precise mechanism underlying the beneficial effect of nicorandil on diabetic patients has not been examined. We investigated the protective effect of nicorandil on endothelial function in diabetic rats because endothelial dysfunction is a major risk factor for cardiovascular disease in diabetes. Methods: Male Sprague-Dawley rats (6 weeks old) were intraperitoneally injected with streptozotocin (STZ, 40 mg/ kg, once a day for 3 days) to induce diabetes. Nicorandil (15 mg/kg/day) and tempol (20 mg/kg/day, superoxide dismutase mimetic) were administered in drinking water for one week, starting 3 weeks after STZ injection. Endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anaesthetised rats. Cultured human coronary artery endothelial cells (HCAECs) were treated with high glucose (35.6 mM, 24 h) and reactive oxygen species (ROS) production with or without L-NAME (300 M), apocynin (100 M) or nicorandil (100 M) was measured using fluorescent probes. Results: Endothelial function as evaluated by FMD was significantly reduced in diabetic as compared with normal rats (diabetes, 9.7 1.4%; normal, 19.5 1.7%; n = 6-7). There was a 2.4-fold increase in p47phox expression, a subunit of NADPH oxidase, and a 1.8-fold increase in total eNOS expression in diabetic rat femoral arteries. Nicorandil and tempol significantly improved FMD in diabetic rats (nicorandil, 17.7 2.6%; tempol, 13.3 1.4%; n = 6). Nicorandil significantly inhibited the increased expressions of p47phox and total eNOS in diabetic rat femoral arteries. Furthermore, nicorandil significantly inhibited the decreased expression of GTP cyclohydrolase I and the decreased dimer/monomer ratio of eNOS. ROS production in HCAECs was increased by high-glucose treatment, which was prevented by L-NAME and nicorandil suggesting that eNOS itself might serve as a superoxide source under high-glucose conditions and that nicorandil might prevent ROS production from eNOS. Conclusions: These results suggest that nicorandil improved diabetes-induced endothelial dysfunction through antioxidative effects by inhibiting NADPH oxidase and eNOS uncoupling. Endothelial dysfunction; Diabetes; Nicorandil; Reactive oxidative species; eNOS; NADPH oxidase - Background Diabetes mellitus is regarded as an independent major risk factor for the development of cardiovascular disease, since long-term survival and freedom from cardiac events were reduced in diabetic coronary angioplasty patients [1-3]. Endothelial dysfunction plays a central role in diabetic vascular diseases [4]. A common mechanism underlying this endothelial dysfunction could involve increased production of reactive oxygen species (ROS) in vascular tissue [5]. High glucose greatly increases endothelial superoxide production [6], leading to an eNOS uncoupling state, followed by reduction of NO production and increased ROS production [7-11] which act to quench NO. Reduced NO availability will lead to attenuation of its beneficial vascular effects such as vasodilation, regulation of vascular smooth muscle proliferation, and expression of cellular adhesion molecules involved in the initiation of atherosclerotic plaque formation [12]. Therefore, increased ROS production in diabetes has been speculated to reduce endothelial NO availability, leading to endothelial dysfunction [13,14]. Nicorandil, an anti-angina agent with ATP-sensitive potassium channel opening and nitrate-like activity, reportedly improves prognosis in patients with angina pectoris via preconditioning effects [15], and also exerted endothelial protective effects in both clinical settings and animal studies. Long-term administration of nicorandil significantly improved endothelial function in patients with ischemic heart disease or with cardiovascular risk factors, as evaluated by measurement of flow-mediated dilation (FMD) in forearm arteries [16,17]. In the swine heart, nicorandil reduced myocardial no-reflow after ischemia reperfusion by protecting endothelial function [18]. In human umbilical vein endothelial cells, nicorandil inhibited apoptosis induced by serum starvation by inhibiting ROS production [19]. Furthermore, nicorandil protected from diabetic through inhibition of the production of ROS stimulated by high glucose [20]. Therefore, we hypothesised that nicorandil can prevent diabetic endothelial dysfunction. In the present study, we investigated the protective effect of nicorandil on endothelial function in streptozotocin (STZ)-induced diabetic rats by measuring FMD in femoral arteries using a high-resolution ultrasound system under in vivo conditions in which blood flow, many humoral factors and nerve activity were maintained. The mechanism underlying the protective action of nicorandil was also investigated in relation to ROS production in the endothelium both in vivo and in vitro. and allowed free access to water under a constant light and dark cycle of 12 h. Diabetes was induced by intraperitoneal administration of STZ (40 mg/kg) once a day for 3 days. One week after STZ administration, glucose concentrations were measured. Diabetes was considered to have been induced when the glucose level was higher than 250 mg/dL. Nicorandil (15 mg/kg/day) and tempol (20 mg/kg/day) were administered in drinking water for one week, starting 3 weeks after STZ administration. All animal procedures were conducted in accordance with Chugai Pharmaceuticals ethical guidelines for animal care, and all experimental protocols were approved by the Animal Care Committee of the institution and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National institutes of Health. Measurement of FMD Four weeks after STZ administration, blood pressure (tail-cuff method, BP-98A Softron, Tokyo, Japan) and blood glucose were measured, and the rats were anaesthetised with thiobutabarbital with constant monitoring of rectal temperature. The animals were kept stable with a heated sheet and warming lamps directed at each rat. Femoral arterial diameter and Doppler flow were measured using a high-resolution ultrasound system (Vevo 770, VisualSonics, Toronto, Canada). The femoral artery was visualised with a 30- or 40-MHz transducer. After identification of the femoral artery by its characteristic flow pattern, the probe position was optimised to show clear vessel wall/lumen interfaces and fixed throughout the investigations. Experiments were started over a 15mi (...truncated)