Effects And Mechanisms Of Apelin On Cardiac Collateral Circulation In Ischemic Myocardial Rats

IntroductionAcute myocardial infarction is one of most important disease threatening human life and health. It is the chief reasons of sudden death and heart failure. It is necessary to reperfute myocardium in order to save dying myocardium. There are many kinds of reperfusions, including nterventional therapy, coronary artery bybass graft, stem cell transplantation and so on. Among of these ways the collateral circulation formation is very important for preventing myocardial infarction and protecting heart functons. Apelin is a micromolecule polypeptide which has several effects on cardiovascular system. It was separated from bovine stomach by Tatemoto in 1998 and it is the endogenic ligand of angiotensionⅡprotein J (APJ). The Apelin/APJ could activate many intracellar signals and produce several physical functions. Apelin is expressed in cardiovascular system extensively. The Apelin/APJ system can reinforce myocardial contractile force, relax blood vessels, relieve cardiac loads, promote blood vessels growth and regulate body fluid equilibrium etc. Among theses effects the character of Apelin to promote blood vessels growth is more and more attracting interests to hakeems.Apelin signal has important effects on formation of blood vessels and hearts. And Apelin was found to express in retinal vessels endothelium extensively and Apelin can be a angiogenesis factor for retina endotheliocytes. Apelin expression is aμgmented under mionectic circumstances just like other angiogenesis factors. The vascularization would be dyspoiesis if Apelin expression was blocked with antisense technique. Apelin had effects of promoting karyokinesis, differentiation and inhibiting apoptosis. Apelin and its receptor, agtrllb, controlled the genesis of zebra-fish heart. Apelin and its receptor would effect heart development whether their expression was deficient or excessive. Moreover, Apelin would open the angiogenic switch and activate tumor neovascularization. Apelin is increased in one-third of human tumors. The character of Apelin enhancing neovascularization has been confirmed in mice neovascularization.Apelin has the character.of promoting endotheliocytes karyokinesis and angiogenesis, which means it could have the potent character of curing diseases of related angiogenesis. The APJ receptor agonist would treat ischemic diseases through angiogenesis while the APJ receptor blocker would prevent tumors growth and retinopathy. However, it is not reported that as a powerful factor of promoting endotheliocytes proliferation and angiogenesis whether or not Apelin could promote the angiogenesis of ischemic myocardium and lessen the injury or necrosis induced by ischemia.The aims of the study were to explore:if Apelin could take part in and protect myocardial ischemia injury; if Apelin given exogenously could promote proliferation, migration and angiogenesis of endotheliocytes; if Apelin given exogenously could enchance angiogenesis of ischemic hearts and on which pathway Apelin promote angiogenesis in order to find an effective way to save ischemic and dying myocardium.Objective1. To observe if Apelin could protect ischimic heart by setting up a rat myocardium ischemic model;2. To confirm if Apelin given exogenously could promote heart collateral circulation and reduce the injury of myocardil ischemia and myocardial infarction;3. To study the effects of Apelin on the proliferation, migration and tubeformation of HUVECs; To explore the possible mechanisms of Apelin promoting angiopoiesis and if these mechanisms have relations to Apelin and mTOR.Methods1. To detect the protcetion effects of Apelin on ischimic rat.(1) To ligate the male SD rats left anterior descending (LAD) coronary artery and set up a myocardial ischimia model. Apelin was given(intraperitoneal injection, 1μg/kg.d×2) to examine heart rate (HR), left ventricular end-diastolic pressure(LVEDP), left ventricular end-systolic pressure (LVESP) and maximal left ventricle developed pressure (LV±dp/dtmax) with a cardiac catheterization in order to evalue the effects of Apelin on heart function. (2) Apelin was given (intraperitoneal injection, lμg/kg.d×2) to detect the content of LDH by ligating the male SD rats'LAD and a ischemic model.2. To examine the effects of Apelin on the collateral circulation of ischemic rat hearts.(1) To ligate the male SD rats'left anterior descending (LAD) coronary artery and set up a myocardial ischimia model. Apelin was given (intraperitoneal injection, 1μg/kg.d×7) to observe the.effects of Apelin on collateral circulation in ischemic rats.(2) To detect the content of CD31 in the myocardium of ischemic rats with enzyme linked immunosorbent assay (ELISA).(3) To dye with Azo-Blue and TTC and to calculate the myocardial infarction aera with a image analysis system (IAS).(4) To observe the angiogenesis ischemic of myocardium dyed with CD31 immunohistochemistry under a microscope.(5) To observe the injury of ischemic myocardium dyed with HE under a microscope.(6) To oberve the protein expression of VEGF and CD31with western blot.3. To examine the effects of Apelin on the proliferation, migration and angiopoiesis of HUVECs.(1) To observe the effect of Apelin on the proliferation of HUVECs by methyl thiazolyl tetrazolium (MTT).(2) To explore the effect of Apelin on the migration of HUVECs with Transwell.(3) To study the effect of Apelin on the angiopoiesis of HUVECs with tubeformation.Results1. The effects of Apelin on heart function of myocardial ischemia in rats The group of Apelin (1μg/kg.d×2, intraperitoneal injection) enchanced LV±dp/dtmax,LVESP and reduced LVEDP significantly compared with other groups at ischemia 48h (P<0.05,0.01).2. The effects of Apelin on myocardial enzymes of myocardial ischemia in rats The Apelin group (1μg/kg.d×2, intraperitoneal injection) was lower LDH significantly compared with other groups at ischemia 48h showed by automatic biochemistry machine (P<0.01).3. Apelin had effects on CD31 content of myocardial ischemia in rats ELISA of rat CD31 showed that Apelin group (1μg/kg.d×7, intraperitoneal injection) was higher compared with other groups (P<0.05).4. The effects of Apelin on myocardial anfarction of myocardial ischemia in rats The results of infarction aera dyed with Azo-Blue and TTC and calculated with IAS discovered that Apelin group (1μg/kg.d×7, intraperitoneal injection) was significantly lower compared with other groups (P<0.05).5. The effects of Apelin on MVD of myocardial ischemia in rats The CD31immunohistochemistry dye showed that the MVD of Apelin group (1μg/kg.d×7, intraperitoneal injection) was significantly higher compared with other groups under microscope (P<0.01).6. The effects of Apelin on myocardial construction of myocardial ischemia in rats The HE dye showed that the scar of Apelin group (1μg/kg.d×7, intraperitoneal injection) were significantly much less compared with other groups under microscope.7. The effects of Apelin on VEGF and CD31 expression of myocardial ischemia in rats The expression of VEGF and CD31 were higher significantly compared with other groups (P<0.01).8. The effects of Apelin on the proliferation of HUVECs The proliferation of Apelin on HUVECs with MTT showed that Apelin group (Apelin lumol/L) could promote proliferation of HUVECs significantly compared with other groups (P<0.01).9. The effects of Apelin on the migration of HUVECs The migration of Apelin on HUVECs with transwell showed that.Apelin group(Apelin 1μg/mL) could promote migration of HUVECs compared with other groups (P<0.01).10. The effects of Apelin on the angiopoiesis of HUVECs The angiopoiesis of Apelin on HUVECs with tubeformation showed that.Apelin group(Apelin 60uM) could promote angiopoiesis of HUVECs compared with other groups (P<0.05).11. Although Apelin had effects above mentioned the effects, however, the effects of Apelin on myocardial protection and cardiac function in ischemic rats disappered when Apelin was adminstered with U-73122 simultaneously. And the effects of Apelin on collateral circulation in ischemic rats and proliferation, migration and tubeformation in HUVECs were disappeared when Apelin was adminstered with rapamycin simultaneously.Conclusions1. Apelin could protect cardiac function and injury in ischemic rat hearts.2. Apelin protected cardiac function and injury in ischemic rat hearts by acting PLC signal pathway.3. Apelin could promote myocardium to expression CD31, stimulate angiogenesis, reduce myocardial infarction area and enchance collateral circulation in ischemic myocardium.4. Apelin could facilitate prolifeation, migration and tubeformation in HUVECs.5. Apelin could promote collateral circulation in ischemic hearts and tubeformation in HUVECs by exciting mTOR receptors and could be blocked by the antagon of mTOR-rapamycin.