Protective Mechanisms Of Grape Seed Proanthocyanidin B2 On Advanced Glycation End Products Induced Endothelial Cell Apoptosis

Part One Protection effects of grape seed proanthocyanidin B2 on endothelial damage caused by AGEsBackgroundWith the development of social economy, the improvement of people living and population aging, the incidence of diabetes mellitus has increased in the worldwide. Diabetes mellitus have been a globe public health issue next to cardiovascular and cerebrovascular disease and tumor that may seriously harm human healthy. All forms of diabetes are characterized by chronic hyperglycaemia and the development of diabetes-specific vascular complications. Vascular endothelial cells are the first barrier of human body vessel wall. Under normal circumstances, the vascular endothelium influences vessel tone and modulates structure, vessel permeability, adhesion and migration of inflammatory cells, and hemostasis. The endothelium is an early target in diabetes, and dysfunction of vascular endothelial cells plays a critical role in the diabetic vascular disease process.Grape seed proanthocyanidin extracts (GSPE) derived from grape seeds, have been reported to possess a variety of potent properties including anti-oxidant, anti-nonenzymatic glycation and cardioprotective effect. Dimeric procyanidin B2 is one of the main components of GSPE, composed of two molecules of the flavan-3-ol (-)-epicatechin linked by a 4b→8 bonds. Several studies have shown that procyanidin B2 exerts a more potent anti-tumor-promoting effect greater than other dimers, such as procyanidins B1, B4, and B5. In our present study, we examined the effects of AGEs on the human umbilical vein endothelial cells (HUVEC) to evaluate the efficacy of HUVEC protection by grape seed procyanidin B2 (GSPB2). To delineate the underlying molecular mechanism, we investigated the lactadherin and phosphorylation of glycogen synthase kinase 3β(GSK3β) signaling pathway using quantitative RT-PCR, Western blot.Objective1. To study the effects of GSPB2 on the nonenzymatic glycation damage of vascular endothelial cells.2. To study the effects of GSPB2 on the lactadherin and phosphorylation of glycogen synthase kinase 3β(GSK3β) signaling pathway.MethodsHUVEC were incubated in complete medium RPMI 1640 containing 10% FBS in Costar flasks. GSPB2 (0.78,1.56,3.12.6.25,12.50,25.00,50.00μmol/L) were added for incubation for 48 hours. To examine the effects of the GSPB2 on HUVEC proliferation, cells were harvested by trypsinization. Cell survival was estimated with MTT and CCK-8 colorimetric assay.2.5μmol/L,5.0μmol/L and 10.0μmol/L of GSPB2 were added for preincubation for 1 hour. After preincubation, the HUVEC were stimulated separately with 200μg/ml of AGEs or unmodified BSA for 48 hours. Cell survival was estimated with CCK-8 colorimetric assay. Apoptosis of cells was analyzed using annexin V-FITC apoptosis detection kit. The production of intracellular reactive oxygen species (ROS) was estimated fluorometrically using 2',7'-dichlorofluorescein diacetate. We determined the lactadherin, cleaved caspase 3, caspase 3, and phosphorylation of GSK-3P in HUVEC by quantitative real-time PCR or western blotting analysis.Results1. Effects of GSPB2 on HUVEC viability The cell viability was increased when HUVEC were exposed to GSPB2 for 48h at the concentration (<12.50μmol/L). GSPB2 increased the cell viability significantly at 6.25μmol/L. Meanwhile, GSPB2 caused approximately 25-35% inhibition of cell viability at 25.00μmol/L or high concentrations.2. Effects of GSPB2 on viability in HUVEC stimulated by AGEsBoth unmodified BSA and DMSO did not affect cell viability. Figure 2 showed the results of comparisons of cell viability findings every group with CCK-8 assay. The cell viability of AGEs was significantly lower approximately 30%. The pretreatment of HUVEC with different concentrations of GSPB2 significantly improved the AGEs-stimulated cell viability in a dose dependent manner.3. Effects of GSPB2 on anti-apoptosis in HUVEC stimulated by AGEsBoth unmodified BSA and DMSO did not affect cell apoptosis. It was found that AGEs-stimulated HUVEC significantly increased the percentage of apoptotic cells (P＜0.05). Moreover, pretreatment of HUVEC with different concentrations of GSPB2 significantly improved the AGEs-stimulated cell apoptosis in a dose dependent manner (P<0.05).4. Effects of GSPB2 on intracellular ROS formation in HUVEC stimulated by AGEsThe levels of ROS were low in the unstimulated HUVEC. AGEs-stimulated HUVEC significantly increased the ROS formation, whereas pretreatment of GSPE in a dose-dependent manner apparently prevented AGEs-induced ROS generation (P<0.05).5. Effects of GSPB2 on cleaved caspase-3 and lactadherin in HUVEC stimulated by AGEsStimulation of HUVEC with AGEs (200μg/mL) resulted in a significant increase in the cleaved caspase-3 and lactadherin by western blotting. The pretreatment with GSPB2 significantly improved the AGEs-stimulated the cleaved caspase-3 and lactadherin for 48h (P＜0.05). By quantitative real-time PCR assay showed that lactadherin mRNA expression in HUVEC exposed to AGEs were significantly upregulated within 48h (P＜0.05). Moreover, pretreatment of HUVEC with different concentrations of GSPB2 significantly inhibited the expression of lactadherin mRNA of HUVEC stimulated by AGEs (P＜0.05). 6. Effects of GSPB2 on the levels of phospho-GSK3βin HUVEC stimulated by AGEsStimulation of HUVEC with AGEs (200μg/mL) resulted in a significant decrease in the levels of phospho-GSK3β, whereas pretreatment of HUVEC with different concentrations of GSPB2 significantly attenuated AGEs-stimulated the decreased levels of phospho-GSK3βunder AGEs stimulation for 48h (P<0.05)Conclusion1. AGEs induced HUVEC apoptosis and upregulated the expression of caspase-3 activation and lactadherin. Treatment of HEVECs with GSPB2 significantly inhibited the cell apoptosis and the expression of caspase-3 activation and lactadherin induced by AGEs.2. The regulation of lactadherin and phospho-GSK3βby GSPB2 contributes to the improvement of endothelial dysfunction resulting from the damage from AGEs.Part Two Investigate the mechanism of grape seed proanthocyanidin B2 on advanced glycation end products induced endothelial cell apoptosis based on gene transfectionBackgroundNonenzymatic protein glycation by glucose is a complex cascade of reactions yielding a heterogeneous class of compounds, collectively termed advanced glycation end products (AGEs). The Schiff base can undergo an intramolecular rearrangement to form the Amadori products. Among the many metabolic abnormalities of diabetes mellitus, AGEs are one showing in epidemiological studies the most consistent and significant correlation with diabetic vascular complications. AGEs lead endothelial dysfunction and apoptosis, and endothelial dysfunction play a critical role in the pathophysiology of vascular complications in diabetes mellitus. AGE-modified adducts on long-lived proteins in extracellular matrix alter basement membrane structure by trapping plasma macromolecules and by increasing vessel wall rigidity through formation of cross-links. Meanwhile, interaction of AGEs with receptor for advanced glycation end product results in triggering a range of cellular responses, including transcription factor activation and changes in gene expression. However, the pathophysilogical mechanism of AGEs-related vascular endothelium and clinical complications remains largely elusive.In the first part, the pretreatment with GSPB2 significantly improved the AGEs-stimulated the expression of lactadherin. Our previous proteomic studies showed that the expression of lactadherin was significantly increased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts (GSPE) significantly inhibited the expression of lactadherin in diabetic rats. Lactadherin is a secreted glycoprotein of milk-fat globule that shares structural domain homology with Del-1. Mouse lactadherin is also known as milk-fat globule-EGF factor 8 (MFG-E8). Lactadherin is expressed in the mammary epithelium, breast cancer, pancreas, aortic endothelial cells and smooth muscle cells, and macrophages and dendritic cells, etc. In our present study, we focused on the molecular mechanisms of human umbilical vein endothelial cells (HUVEC) apoptosis induced by AGEs, particularly 1) the role and molecular mechanism of the lactadherin (overexpression and siRNA) in the AGEs-induced endothelial cells apoptosis,2) the efficacy of HUVEC protection by grape seed procyanidin B2 (GSPB2).MethodsHUVEC were cultured in six-well plates overnight and transfected with siRNA against lactadherin using Lipofectamine2000. Moreover, HUVEC were transduced with lentiviral vectors of lactadherin overexpression. HUVEC transfected lactadherin genes and siRNA were incubated for 48 h in the presence or absence of GSPB2 (10μmol/L). Meanwhile, HUVEC transfected lactadherin siRNA were stimulated with 200μg/mL of AGEs. Cell survival was estimated with MTT and CCK-8 colorimetric assay. The apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. To evaluate changes in morphology, HUVEC transfected lactadherin genes were visualized by transmission electron microscope (TEM). We determined the lactadherin, cleaved caspase 3, caspase 3, phosphorylation of GSK-3P and Bax/Bcl-2 in HUVEC by quantitative real-time PCR or western blotting analysis.Results1. Transduction efficiency with lactadherin siRNA and overexpression plasmidsWe transduced the HUVEC with siRNA or lentivirus vector. Transduction conditions were optimized by using different MOI and the transduction efficiency was assessed by fluorescence microscopy, real-time PCR and western blotting. HUVEC carrying negative control siRNA (NC), HUVEC carrying siRNA against lactadherin (LsiRNA) and HUVEC carrying GFP (LV-C), HUVEC carrying both GFP and lactadherin genes (LV) were harvested. The transduction efficiency was about 95% or higher at day 5 or longer. Transgene expression was confirmed by fluorescent microscopic imaging for lactadherin protein expression. The mRNA and protein expression of lactadherin in LsiRNA group decreased to more than 60% the level of the NC group at 48 h after transfection. Lactadherin mRNA and protein expression reached its highest level at day 5 after virus removal.2. Effects of lactadherin on viability in HUVEC stimulated by AGEsThere were no differences in the cell viability between LsiRNA group and NC group. Stimulation of NC group with AGEs (200μg/mL) resulted in a significant decrease in the cell viability, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the decrease of cell viability compared with NC group under AGEs stimulation for 48h (P＜0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated cell viability for 48h (P<0.05). Both HUVEC transfected GFP and negative control did not affect cell viability. Moreover, the overexpression of lactadherin significantly decreased the cell viability. The cell viability was increased when LV group was exposed to GSPB2 (10μmol/L) for 48h (P<0.05).3. Effects of lactadherin on apoptosis in HUVEC stimulated by AGEsStimulation of NC group with AGEs (200μg/mL) resulted in a significant increase in the cell apoptosis, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the cell apoptosis compared with NC group under AGEs stimulation for 48h (P<0.05). The treatment of NC group with GSPB2 (10μmol/L) also significantly improved the AGEs-stimulated cell apoptosis for 48h (P＜0.05). HUVEC overexpression lactadherin was susceptible to cell apoptosis, while GSPB2 (10μmol/L) significantly attenuated the cell apoptosis for 48 h.4. The morphological changes of HUVEC overexpression lactadherinThe cell apoptosis significantly increased in the HUVEC overexpression lactadherin, while GSPB2 (10μmol/L) significantly attenuated the cell apoptosis for 48 h.5. Effects of lactadherin on cleaved caspase-3 and Bax/Bcl-2 ratioThe uncleaved caspase-3 levels were unchanged in LV-C group, LV group, LsiRNA group and NC group. Stimulation of NC group with AGEs (200μg/mL) resulted in a significant increase in the cleaved caspase-3 and Bax/Bcl-2 ratio, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the cleaved caspase-3 and Bax/Bcl-2 ratio compared with NC group under AGEs stimulation for 48h (P＜0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated the cleaved caspase-3 and Bax/Bcl-2 ratio for 48h (P<0.05). The cleaved caspase-3 and Bax/Bcl-2 ratio significantly increased in LV group comparing to LV-C group, while GSPB2 (10μmol/L) significantly inhibited the cleaved caspase-3 and Bax/Bcl-2 ratio in HUVEC overexpression lactadherin for 48 h (P<0.05).6. Effect of lactadherin on the levels of phospho-GSK3βStimulation of NC group with AGEs (200μg/mL) resulted in a significant decrease in the levels of phospho-GSK3β, whereas siRNA against lactadherin significantly attenuated AGEs-stimulated the decreased levels of phospho-GSK3βcompared with NC group under AGEs stimulation for 48h (P<0.05). The treatment of NC group with GSPB2 (10μmol/L) significantly improved the AGEs-stimulated the decreased levels of phospho-GSK3P for 48h (P＜0.05). The levels of phospho-GSK3βsignificantly decreased in LV group comparing to LV-C group, while GSPB2 (10μmol/L) significantly improved the phospho-GSK3βin HUVEC overexpression lactadherin for 48 h (P＜0.05).Conclusion1. Up-regulation of lactadherin plays a critical role in endothelial cells apoptosis induced by AGEs. 2. Mitochondria apoptosis pathway played a pivotal role in the cell apoptosis by lactadherin.3. GSPB2 might have benefits in the early stage of diabetic endothelial dysfunction by inhibition of lactadherin.