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Rapamycin is an immunosuppressive agent that induces apoptosis and inhibits vascular smooth muscle cell proliferation and migration. It is added to drug eluting stents to prevent restenosis, but it also impairs reendothelialization after percutaneous coronary intervention. Nicorandil is a hybrid agent with adenosine triphosphated (ATP)-sensitive K+ (KATP) channel opener and nitrate properties. It has been found to prevent rapamycin-induced production of reactive oxygen species (ROS), and decrease rapamycin-induced apoptosis in endothelial cells (ECs). We build the hypothesis that nicorandil may promote reendothelialization impaired by rapamycin by scavenging xanthine oxidase (XO)-mediated ROS formation and up-regulating eNOS expression.
In vivo carotid balloon injury model and in vitro microvascular cardiac endothelial cells (CMECs) were used to indentify this hypothesis. Balloon injury model was established in carotid arteries of SD rats. Rats were randomized to control group (n=10), balloon injury group (n=10), rapamycin group (n=20), and rapamycin-nicorandil co-treatment group (n=10). Reendothelialization was analyzed by Evans blue staining andimmunohistochemical staining with PECAM-1 (CD31). ROS generated in carotid arteries were analyzed by DHE staining of frozen arterial sections. Intimal hyperplasia was measured by HE staining of carotid sections. To investigate the effects of rapamycin and nicorandil on CMECs, cells were randomized to control group, rapamycin group, rapamycin-nicorandil co-treatment group. XO siRNA and Akt siRNA were transfected in CMECs. Intracellular ROS measurement was conducted by DHE staining and the fluorescence was detected by flow cytometry. Cell apoptosis was analyzed by Annexin V-FITC/PI kit and Caspase 3 activity assay kit.Cell migration and proliferation were analyzed by wound healing assay and BrdU incorporation, respectively.
Nicorandil increased reendothelialization impaired by rapamycin. 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 by XO siRNA 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.
The beneficial effect of nicorandil is mediated by inhibiting XO and up-regulating Akt pathway in ECs. Nicorandil combined with rapamycin in effect rescue the deficiencies of rapamycin alone in arterial healing after angioplasty.