Nicorandil Inhibits Carotid Intimal Hyperplasia And Promotes Re-endothelialization After Balloon Catheter Injury In Rats

Aims:We hypothesize that nicorandil promotes reendothelialization and inhibits intimal hyperplasia after balloon injury. A carotid balloon catheter injury model, diabetic rat model and in vitro primary cultured vascular smooth muscle cells (VSMCs) and cardiac microvascular endothelial cells (CMECs) were used to investigate this hypothesis.Methods:Diabetic rat model was established by introperitoneal injection of STZ. After balloon injury procedure in carotid arteries, rats were randomized to sham group, balloon injury group, nicorandil group, nicorandil-5-hydroxydecanoate (5-HD) co-treatment group. Perivascular delivery of εPKC siRNA was conducted to determine the role of sPKC pathway in intimal hyperplasia. In hyperglycemia environment, primary cultures of VSMCs were treated with nicorandil or 5-HD. To investigate the roles of εPKC in VSMCs, εPKC is knocked down by transient transfection of siRNA. Balloon injury model was established in carotid arteries of normal SD rats. Rats were randomized to sham group (n=10), balloon injury group (n=10), rapamycin group (n=20), and rapamycin-nicorandil co-treatment group (n=10). To measure the reendothelialization area, Evans blue and immunohistochemical staining with CD31 were used. ROS levels in frozen sections were measured by DHE staining. CMECs were isolated from neonatal rat hearts by enzyme digestion method. To investigate the effects of rapamycin and nicorandil on CMECs, cells were randomized to control group, rapamycin group, rapamycin-nicorandil co-treatment group. To investigate the roles of xanthine oxidase (XO) and Akt in CMECs, XO or Akt are knocked down by transient transfection of siRNA. Cell apoptosis was analyzed by Annexin V-FITC/PI kit and Caspase 3 activity assay kit. Cell migration was investigated by wound healing assay. Cell proliferation was assessed by BrdU cell proliferation assay kit and MTT assay. Intracellular ROS measurement was conducted by DHE staining in CMECs. eNOS mRNA expression was measured by qRT-PCR. XO、Akt、p-Akt、εPKC protein levels in carotid arteries and cells were measured by western blot.Results:Intimal hyperplasia significantly increased 14 days after balloon injury in diabetic rats. Nicorandil inhibited intima development, reduced inflammation and prevented cell proliferation in balloon-injured arteries. The protective effects of nicorandil were reversed by 5-HD.εPKC was activated in balloon-injured arteries. Nicorandil inhibited εPKC activation by opening mitoKATP channel. Perivascular delivery of εPKC siRNA inhibited intimal hyperplasia, inflammation and cell proliferation. High glucose-induced VSMCs proliferation and migration were inhibited by nicorandil.εPKC activation induced by high glucose was also inhibited by nicorandil and that is partially reversed by 5-HD.εPKC knockdown prevented VSMCs proliferation and migration. Nicorandil increased reendothelialization impaired by rapamycin, and it decreased ROS production and XO activation induced by rapamycin. In addition, eNOS expression was increased by nicorandil. In vitro, rapamycin-impeded CMECs migration, proliferation and rapamycin-induced ROS production were reversed by nicorandil. Knockdown of XO partially inhibited rapamycin-induced ROS production and cell apoptosis in CMECs, and it promoted CMECs migration and proliferation impeded by rapamycin. Knockdown of Akt by Akt siRNA partially prevents eNOS up regulation promoted by nicorandil.Conclusions:1. Nicorandil inhibits intimal hyperplasia, cell proliferation and inflammatory cell infiltration in balloon-injured arteries in diabetic rats. Nicorandil also prevents VSMCs proliferation and migration induced by high glucose.2. The inhibitive effect of nicorandil on intimal hyperplasia is conducted via opening mitoKATP channel and inhibiting εPKC activation.3. Nicorandil promotes reendothelialization after balloon injury. It combines with rapamycin in effect rescue the deficiencies of rapamycin alone in arterial healing after angioplasty.4. In the balloon-injured arteries and CMECs, rapamycin induces XO activation and thus improves production of ROS and promotes reendothelialization.5. Nicorandil inhibits XO activation in carotid arteries and decreases ROS generated by XO. Nicorandil promotes expression of Akt/eNOS inhibited by rapamycin. It decreases oxidative stress and cell apoptosis, and promotes proliferation and migration in CMECs.