The Effects And Possible Mechanisms Of Advanced Glycation End-products On The Tube Formation And Collapse Of Cardiac Microvascular Endothelial Cells

Background and ObjectiveVarious hyperglycemia-induced metabolic have been reported to contribute to the characteristic histopathological changes of diabetic icroangiopathy .Among them, advanced glycation end products (AGEs), have been strongly implicated in the pathogenesis of diabetic vascular complications. AGEs are a complex group of compounds formed via a non-enzymatic reaction between reducing sugars and amine residues on proteins, lipids or nucleic acids. AGEs have been shown to promote the angiogenesis of umbilical cord vein endothelial cells, human dermal microvascular cells and retinal endothelial cells through autocrine VEGF in vitro.Diabetic cardiomyopathy has been recognized as a microvascular disease that may lead to heart failure, accounting for significant morbidity and mortality in the diabetic population. By serial clinicopathological investigation of diabetic rats, Yoon has documented a progressive decrease of myocardial VEGF expr- ession and reduced capillary density in the myocardial microcirculation. These findings suggest that diabetic rats present a dysfunction of angiogenesis.Angiogenesis is a multistep process including degradation of the extracellular matrix, migration and proliferation of vascular endothelial cells, and capillary formation.β-catenin, a key factor of wnt signaling , recently is found to be a critical mediator during development of angiogenesis. When Wnt signaling is activated,β-catenin accumulates and translocates into nucleus and represses several important target genes and enhances the expression of VEGF. So the translocation of theβ-catenin from cystol to the nuclei is considered an index of the activation inβ-catenin.Apoptosis is a negative mediator of angiogenesis. The caspase family plays an important role in apoptosis. Previous study showed that the apoptosis of microvascular endothelial cell increased in diabetic heart.Because of the heterogeneity in different types of cells and organs and the fact that the capillary density in diabetic rat decreased, we therefore hypothesized that AGEs could have different effects on the angiogenesis of the cardiac microvascular endothelial cells (CMECs). To test this hypothesis, we investigated the effect of AGE-BSA on the Tube-like structure (TLS) formation and collapse of the cultured rat CMECs in different time and possible mechanisms.MethodsPart one: the isolation, culture and identification of CMECs: 1.The CMECs were isolated enzymatically from left ventricle of rats. 2. The CMECs were identified by origin, characteristic morphology, and expression of factorⅧand uptaking of Dil-AC-LDL. Part two: Preparation and identification of AGE proteins: 1. bovine serum albumin (BSA) was incubated under sterile conditions with D-glucose in sodium phosphate buffer (PBS, pH 7.4) at 37℃in dark for 12 weeks. BSA incubated without glucose under the same conditions was used as the negative control in all experiments. AGE-BSA content was determined spectrofluorometrically.Part three: The effect of AGEs on TLS formation of CMECs and possible mechanisms: the primary passage cells after cultivated for 7 days were administrated in the study. After incubating CMECs with AGE-BSA for 1 day, we performed the following experiments: 1. the TLS formation assay was used to evaluated the TLS. 2. MTT assay was performed to test the proliferation. 3. Westen-blot was conducted to investigate the expression of VEGF and totalβ-catenin protein. 4. Confocal microscopy was performed to detect the translocation ofβ-catenin. 5. ELISA was used to test the secretive VEGF of CMECs.Part four: The effect of AGEs on TLS collapse of CMECs and possible mechanisms: the primary passage cells after cultivated for 7 days were administrated in the study. 1. After treating CMECs with AGE-BSA for 3 days, we used the TLS formation assay to test the collapse of the TLS. 2. After incubating CMECs with AGE-BSA for 1days and 3 days, we conducted flow cytometry to detect the apoptosis of CMECs. 3. After incubating CMECs with AGE-BSA for 1days and 3 days, we conducted ELISA to detect the relative activity of casepase-3 of CMECs.Statistical analysis was performed with SPSS version 11.0. One-way analysis of variation and LSD-t test were employed.Results Part one: CMECs were isolated enzymatically from rat left ventricle successfully. After 1～2 days the cells can form TLS. After 7～9 days, the cells reached confluence and displayed a uniform''cobblestone''morphology.90.68 percent of the cells were factorⅧpositive. 93.82 percent of the cells could intake Dil-AC-LDL.Part two: The average fluorescence for AGE and control were 218.32 units per mg protein and 0.65 units per mg protein, respectively.Part three:After 1 day of incubation, compared with BSA group and control group, AGE-BSA induced a greater degree of TLS formation, as well as proliferation in a time-dependent manner (P<0.05); the AGE-BSA not only enhanced the totalβ-catenin and VEGF expression but also increased the translocation ofβ-catenin from cytocl to nuclei in CMECs (P<0.05); The AGE-BSA also increased the VEGF autocrine in supernatant of CMECs (P<0.05).Part three: after 3 days of incubation, compared with BSA group and control group, AGE-BSA sharply accelerated the collapse of the TLS (P<0.05); AGE-BSA increased the rate of apoptosis and the activity of caspase-3 dramatically (P<0.05), However, after just 1 day of incubation, compared with BSA group and control group, AGE-BSA had no effects on the rate of apoptosis and the activity of caspase-3.Conclusions1. AGE-BSA increases the TLS formation of CMECs possibly associated withβ-catenin pathway at early stage.2. AGE-BSA accelerates the TLS collapse of CMECs possibly involved in caspase-3 pathway at advaced stage.