Effect Of AGEs On The Microvascular Endothelial-cells Angiogenesis By SDF-1/CXCR4 Axis Sinnaling Pathway

BackgroundDiabetes mellitus is one of the popular chronic diseases which badly hurt human's health. In fact, from the point of view of cardiovascular medicine, it may be appropriate to say, diabetes is a cardiovascular disease. Research have shown that metabolic disorder triggered diabetic cardiomyopathy, leading to the myocardial cell fibrosis, apoptosis, and causes arrhythmia, chronic cardiac insufficiency. It is the main reason cause of death in diabetes patients. The main pathological manifestation in diabetes-induced ischemic cardiovascular disease are the dysfunction in myocardial angiogenesis, including reduced coronary artery collateral vessel formation and low capillary density.Diabetic cardiomyopathy were highly correlated with hyperglycemia-induced metabolic, of which, advanced glycation end products (AGEs) played an important role in, was considered the main pathogenic substances. AGEs are modifications of proteins or lipids that become nonenzymatically glycated and oxidized after contact with aldose sugars, Early glycation and oxidation processes result in the formation of Schiff bases and Amadori products, Further glycation of proteins and lipids causes molecular rearrangements that lead to the generation of AGEs. AGEs through the promotion of retinal endothelial cells, glomerular endothelial cells, umbilical vein endothelial cells from the secretion of VEGF and affect the intracellular signal transduction in vascular complications of diabetes.Previous studies pointed out that, AGEs can affect the secretion of VEGF in retinal endothelial cells and unbilical vein endothelial cells, and affect intracellular signal transduction in vascular complications of diabetes. Studies have shown that AGEs promote the development and progression of microvascular disease.Stromal cell-derived factor-1α(SDF-1α)is a member of CXC chemokine family, which was originally isolated form murine bone marrow stromal cells. CXCR4, 7-transmembrane-spanning G protein-coupled receptor, is the only known receptor for SDF-1αand the specific antagonist is AMD3100. Recent studies have shown that SDF-1α/CXCR4 interaction also plays an important role in the regulation of a variety of cellular functions such as cell migration, proliferation, surivival and angiogenesis.Currently, the mechanisms of diabetes cardiomyopathy remains unclear. In our study, CMECs were cultured with AGEs at different concentrations. Check out the expression of SDF-1/CXCR4 whether has changed and observe the effects of the AGEs on proliferation, migration, angiogenesis and lumen formation of cardiac microvascular endothelial cells.MethodsPart I: CMECs were isolated from adult male Sprague-Dawley rat left ventricle. The identification CMECs is according to the origin of cells, characteristic morphology, uptaking of Dil-Ac-LDL and expression of factorⅧ.Use nonenzymatic reaction to BSA and glucose at constant temperature of 37℃, incubated for 100 days with dark condition. Cultured BSA( bovine serum albumin) with same condition as a control. After the success of incubation, using fluorescence spectrophotometer and calculate the protein content of AGEs indentified.Part II CMECs were cultured with AGEs at different concentrations (100mg/L,200mg/L and 400mg/L) for 48h. Randomly divided into groups and each experimental group were added with 0.1mg/L AMD3100. The proliferation of CMECs were assessed by MTT colorimetry, the migration of CMECs were detected by transwell, capillary lumen formation test was applied to detect the angiogenesis.Part III CMECs were cultured in vitro. Add AGEs with the concentration of 200mg/L and experimental group add AMD3100 with concentration of 0.1mg/L. Then maintained in a regular incubator for 24h,48h,72h and 96h respectively. The proliferation of CMECs were assessed by MTT colorimetry, the apoptosis of CMECs were detected by Hocheset staining, capillary lumen formation test was applied to detect the angiogenesis. CXCR4 were analyzed by Western blotting.Results1. CMEC were isolated and cultured in vitro successfully,the cells displayed a uniform cobblestone morphology and reached confluence after 7～10 days.2 AGEs were successfully incubate and the average fluorescence for AGEs were 93.6kU/g and 2.5kU/g for BSA.3 Comparing with the control group, CMECs in the AGEs concentration of 100mg/L,200mg/L and 400mg/L after 48h incubation, enhance the proliferation(P<0.05), migration(P<0.05) and lumen formation(P<0.05) with concentration dependent.Block the pathway of SDF-1/CXCR4 can suppression the proliferation, migration and lumen formation.4 Comparing with the control group,AGEs can enhance the apoptosis of CMECs. In the early state of AGEs can enhance the proliferation and lumen formation, But with the longer duration of action, late AGEs can accelerated the collapse of tube-like structure(P<0.05).Conclusions1. In the early state, AGEs can enhanced proliferation,migration and tuble-like structure formation with concentration dependent by SDF-1/CXCR4 pathway.2. In the late state, AGEs surppress the proliferation and tube-like structure formation and increased cell apoptosis by inhibiting the expression of CXCR4.