Liraglutide Protects The Cardiac Microvascular Endothelial Cells Against Hypoxia/Reoxygenation Injury

Aim:The microvasculature located at the terminal end of circulation determines to some extent the myocardial perfusion level and the coronary reserve. Although percutaneous coronary interventional (PCI) therapy for acute myocardial infarction (AMI) could open the blocked blood vessels, it induced ischemia/reperfusion(I/R) injury in cardiac cells due to the excessive production of reactive oxygen species (ROS), which perturbed the intracellular redox balance and led to oxidative stress that contributed to cell dysfunction and programmed cell death. Based on previous research, under oxidative stress conditions, xanthine oxidase(XO) was the main sources of ROS production in CMEC. Therefore, the protection of CMEC against excessive ROS production and subsequent apoptosis under I/R injury is vital for improving the clinical outcome of patients with acute PCI. Glucagon-likepeptide-1 (GLP-1) has been found that hold the antioxidant effects. This study is aimed at exploring the effects of Liraglutide, GLP-1 analogue, on CMEC and the mechanisms by which Liraglutide reduce the CMEC oxidative stress apoptosis under hypoxia/reoxygenation (H/R) model.Methods:In vitro cultured CMEC of SD rats were purified by differential adhesion method and identified immunocytochemically using CD31 antibody and factor ?. GLP-1R and CD31 was assessed by co-location immunohistochemistry. MTT assay was performed to assess the proliferation of the first-generation cells exposed to different concentrations (0-100 nM) of Liraglutide. BrdU fluorescent labeling and scratch assay were performed to observe the proliferation and migration of CMEC following Liraglutide treatment. H/R model was used to induce cellular apoptosis, and the apoptotic rate was assessed by low cytometry. Fura-3 AM was applied to observed the concentration of Ca2+. DCFHDA was used to evaluate the ROS contents. Western blots and immunohistochemistry were used to assess the expression of XO and caspase3. Pathways inhibitors and siRNA technology were used to illustrate the role of PI3K/Akt-Survivin in Liraglutide-mediated anti-apoptotic functions.Results:1. Separation of CMEC from SD rats in vitro, it presented a typical "aura" cell growth after 72 h. Immunocytochemical staining of CD31 and VIII factors demonstrated a proportion of double positive cells isolated exceeding 95% which means high purity of CMEC in vitro.2. Co-immunocytochemical staining of CD31 and GLP-1 receptor promoted that GLP-1R was expressed on the surface of CMEC. Liraglutide exposure concentration-dependently promoted the proliferation of CMEC with the optimal concentration of 100 nmol/L.3. BrdU and scratch assay showed that 100 nmol/L Liraglutide concentration-dependently promoted the proliferation and migration of CMEC.4. H/R model was established by 4 h hypoxia and 4 h reoxygenation, which increased the contents of ROS that was responsible for cellular apoptosis. However, pretreatment with Liraglutide for 12 h could reversed such changes.5. H/R model induced the higher expression of XO, and consequently generated excessive ROS which was responsible for cellular apoptosis. Knockdown of XO by siRNA technique or pretreatment with Liraglutide could significantly reduce cytoplasm ROS level and the expression of apoptosis proteins meanwhile.6. H/R model leaded to cell cytoplastic calcium overload, inducing the increased XO expression and consequently excessive ROS and apoptosis protein production. Using BAPTA or pretreatment with Liraglutide could significantly reduce the cytoplasm calcium level and XO-ROS induced oxidative apoptosis.7. Excessive cytoplastic XO-ROS generation, induced by H/R model, finally decreased the mitochondrial membrane potential, and induced the leakage of cytochrome c (cyt-c) into cytoplasm, and finally activated mitochondrial apoptotic pathways. Knockdown of XO or pretreatment with Liraglutide could partly reverse the loss of mitochondrial membrane potential, cyt-c leakage and thus promoted cell survival.8. H/R model down-regulated SERCA2a and up-regulated IP3R transcription that induced the imbalance function of endoplasmic reticulum channel, which leaded to Ca2+overload and subsequently increased XO expression and ROS contents. Inhibition of IP3R, chelation of Ca2+, knockdown of XO significantly reduced ROS contents and caspase3 expression. Lirglautide pretreatment for 12 h could reverse SERCA2a transcription and downregulation IP3R transcription which finally suppressed the overload of Ca2+and consequent XO activation, ROS outburst and cellular apoptosis.9. Liraglutide pretreatment for 12 h could activate higher expression of p-Akt and survivin protein. After treatment with LY294002, the blocker of PI3K/Akt pathway, the expression of survivin was decreased.10. Inhibition of GLP-1R, knockdown of survivin or blocking PI3K/Akt could depress the regulated effects of Liraglutide on transcription of SERCA2a and IP3R, which induced the imbalance of cytoplastic calcium in downstream, excessive ROS generation, and increased apoptosis protein expression.11. Besides, Liraglutide could increase the SOD, GSH and GPx to maintain the normal ROS contents.Conclusions:1. Liraglutide promotes the proliferation and migration of CMEC in vitro via PI3K/Akt and MAPK/ERK signaling pathways.2. H/R induced excessive ROS via SR-Ca2+-XO to induce mitochondrial apoptotic pathways, which was reversed by Liraglutide.3. Liraglutide, on one hand, could enhance SOD, GSH and GPx to conteract excessive ROS, and on the other hand, it activated GLP-1R/PI3K/Akt/survivin to sustain SERCA2a expression and activity to reduce SR-Ca2+-XO-mediated oxidative stress apoptosis.