| BackgroundThe prevalence of diabetes mellitus is rising year by year, and cardiovascular complications are the leading causes of morbidity and mortality. Endothelial cell apoptosis has been postulated to be an initial trigger of the cardiovascular dysfunction and even cardiovascular disease. Many metabolic disorders in diabetic patients are the risk factors of endothelial micro-environmental imbalances, endothelial dysfunction, and even apoptosis. Endothelial cells apoptosis not only weaken the vessels defense functions such as anti-inflammatory, but also destroy the balance of anticoagulation fibrinolysis system, induce thrombosis, promote the formation of the atheromatous plaque, which can lead to a series of diabetic vascular complications, such as Atherosclerosis (AS). Therefore, the inhibition of endothelial cells apoptosis and the protection of endothelial cells have a meaningful, positive effect on control diabetic disease onset and progression.Advanced glycation end products (AGEs), formed by non-enzymatic glycation reactions between sugars and macromolecules, deposit on blood vessel wall and induce endothelial cells apoptosis, are crucially involved in the diabetic vascular complications. The large accumulation of AGEs accumulate with aging and abundantly increase while hyperglycemia. AGEs in serum and organs in diabetic patients were significantly higher than health person. Moreover, the severity degree of diabetic complications correlates with AGEs levels.AGEs play a central role in the pathogenesis of cardiovascular complications in diabetes. Now, oxidative stress evoked by AGEs is considered to be the key cause of endothelium injury. A large number of AGEs which formed in chronic hyperglycemia accumulate on the vessel wall and specially the lesions of AS. AGEs and RAGE interaction evokes oxidative stress generation, with elevated production of reactive oxygen species (ROS), activates NF-κB and elicits inflammation and thrombosis, thus inducing endothelial cells injury and apoptosis. A large number of AGEs in body is difficult to be removed by own metabolic or drugs, so how to protect diabetic vascular injury induced by AGEs has gradually attract more and more scholars. Recently study has identified that glucagon-like peptide-1(GLP-1) can protect endothelial cells against AGEs-induced oxidative damage via down-regulating the expression of RAGE. Further, GLP-1also counteracts the AGEs induced-detrimental effects such as redox imbalance and insulin secretion dysfunction.GLP-1is a polypeptide secreted by the intestinal L cell. GLP-1is best known as a safe and effective insulinotropic hormone and has been proposed as prospective approach to clinical treatment of type2diabetes mellitus (T2DM). Besides the well-known actions that in pancreatic cells, GLP-1stimulate survival and proliferation, and increased insulin secretion, GLP-1also plays an important role in diabetic cardiovascular complications. GLP-1inhibits adhesion of monocytes to aortic endothelial cells, in order to alleviate inflammatory injury and inhibit AS progression. GLP-1can reduce TNF-alpha-induced inflammatory, in order to protect endothelial cells against hypercoagulation and inflammatory. Therefore, the long-acting GLP-1analogue, liraglutide, has been suggested to be used for improve the endothelial cell dysfunction associated with premature atherosclerosis identified in type2diabetic patients. Moreover, GLP-1receptor agonist exendin-4promotes endothelial repair quickly due to stimulating proliferation of vascular endothelial cells.The mechanisms of GLP-1protecting endothelial cells remain unknown, and maybe involved in oxidative stress. GLP-1decreases the level of ROS induced by AGEs in endothelial cells in a dose dependent manner. GLP-1can also counteract the oxidative effects induced by H2O2by up-regulating the expression of anti-oxidative gene. Exendin-4may promote NO activity due to reduce the oxidative stress induced by hyperhomocysteinemia (HHcy).In addition to the above antioxidant effects, PI3K/Akt and its downstream signaling may contribute to the protective effects of GLP-1on endothelial cells. Exendin-4stimulates proliferation of endothelial cells through PKA-PI3K/Akt activation pathways. The activated PI3K/Akt can interact with its substrate Bad, induce the change of apoptotic related gene Bcl-2/Bax ratio and mitochondrial permeability, leading to cytochrome c release, and subsequently the activation of caspases-9,-3and cell apoptosis. Zhou YJ et al suggested that AGEs can increase the expression of the pro-apoptotic protein Bax, activated caspase-9and-3, and subsequently induced the apoptosis of HUVECs. In different cell types, including pancreatic P-cells, cardiomyocytes, and cholangiocytes, GLP-1could exert anti-apoptotic effects by regulating the activity of Bcl-2family and caspase. Then, in endothelial cells, is the anti-apoptotic effects of GLP-1against AGEs involved in mitochondrion-cytochrome c-caspase protease pathway?Investigation of the effects and mechanisms of GLP-1on blood vessels which extra-pancreatic tissues represents promising targets of therapeutics in struggle against T2DM.especially against vascular complications associated with T2DM. In this study, we established the apoptotic model by treating human umbilical vein endothelial cells (HUVECs) with AGE-BSA, in order to explore the effects and related mechanisms of GLP-1on endothelial cells induced by AGEs, and highlight the importance of antioxidant injury in prevention and treatment of diabetic vascular complications, and try to provide the broad clinical value of GLP-1with experimental basis. ObjectivesIn this project we aim to on the base of apoptotic model induced by AGEs, to observe the effects of GLP-1on apoptosis of endothelial cells induced by AGEs. and investigate the effects of GLP-1on apoptotic related molecules and PISKAkt pathway, also to discuss the effects and Mechanisms of GLP-1on apoptosis of endothelial cells induced by AGEs.ContentThe whole project includes three parts:Part I The effects of GLP-1on apoptosis of endothelial cells induced by AGEsObjectivesTo establish the apoptotic model by treating HUVECs with AGE-BSA, and analyze the effects of GLP-1on apoptosis of endothelial cells induced by AGEs.Methods1. Preparation of AGE-BSA in vitroBriefly, BSA was dissolved in phosphate-buffered saline (PBS;pH=7.4) with D-glucose, and incubated for12weeks in the dark. AGE-BSA was identified using a fluorescence spectrophotometer.2. Isolation, culture and identification of cellsHUVECs were isolated from healthy umbilical cord and digested by type I collagenase. HUVECs were cultured in M199medium and at37℃in a humidified atmosphere of5%CO2. These cells were identified for von Willebrand factor antigen by immunofluorescence.3. Cell viability was assessed by the colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-dihenyltetrazolium bromide (MTT) assayGroups:AGE-BSA treated the cells at concentrations of100,200,400p.g/mL for48h;. AGE-BSA alone (final concentration200μg/mL) incubated with cells for12,24,36,48h respectively; The protective effect of GLP-1on AGEs-induced apoptosis was tested by co-incubation with different concentrations GLP-1(0,10,50,100,150nmol/L)for48h.The cells are planted in96-well plates. After each experimental treatment, add MTT solution to each hole, and add150μL DMSO to each hole. Absorption was measured at570nm with a microplate reader. Meanwhile, Then, set up blank holes which only contain culture medium and without cells, regulate to zero based on blank cells while comparing the colors. The absorbance measured by MTT assay reflects cells injury.4. Morphology of cells apoptosis was observed by Wright’s-Giemsa staining, Acridine Orange and Hoechst33258staining.5. Flow cytometry assay apoptosisCells were divided into four groups:control group,200μg/mLAGE-BSA group,200μg/mLAGEs+100nmol/LGLP-1group,100nmol/LGLP-1group. After incubation for48h, cells were collected, centrifuged and drop the supernatant, washed twice with PBS. The cells were resuspended in PBS and stained with Annexin V and PI for15min at room temperature. Flow cytometric analyses were performed on a FACSCalibur flow cytometer, and the early apoptotic rate were analyzed with the Cell Quest analysis program.Statistical AnalysisAll analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. We used Spearman correlation analysis for correlation analysis. Apoptotic rate was determined by the factorial design ANOVA. Statistical significance was defined as two-sided p<0.05.Results1.After treatment with100,200,400μg/mL AGEs for48h, the OD value were0.29±0.03,0.13±0.03,0.07±0.02, respectively, and significantly lower than control group (vs. control, P=0.000). A significant negative correlation exists between the OD value of HUVECs and the AGEs concentrations (100,200,400μg/mL)(r=-0.931, P=0.000). 2. Comparison OD value of AGEs incubated for different time showed that a significant negative correlation exists between the OD value of HUVECs and time (12h,24h,36h,48h)(r=-0.956, P=0.000).3. The OD value of10,50,100,150nmol/L GLP-1were0.38±0.03,0.42±0.03,0.50±0.02,0.51±0.04.The results of HUVECs treated with different concentrations GLP-1alone showed that the OD value of control was significantly higher than GLP-1groups (vs.50,100,150nmol/LGLP-1group, P=0.000). However, there was no statistical difference between control and10nmol/LGLP-1group (P=0.907). Spearman correlation analysis showed that a significant positive correlation exists between the OD value and GLP-1concentrations (10,50,100,150nmol/L)(r=0.820, P=0.001).4. The results of GLP-1inhibiting cytotoxicity induced by AGEs showed that the OD value of AGEs is significantly different with control ones (P=0.000). the OD value of AGEs+GLP-1(10,50,100,150nmol/L) groups were0.11±0.02,0.14±0.01,0.22±0.03,0.23±0.02respectively, and compared with AGEs group, results showed that the OD value of in AGEs+GLP-1(50,100,150nmol/L) groups were significantly higher than AGEs group(AGEs vs.50nmol/LGLP-1+AGEs, P=0.036; AGEs vs.100,150nmol/LGLP-1+AGEs, P=0.000).However, there was no statistical difference between AGEs+10nmol/L GLP-1and AGEs group(P=0.932). The OD value of control was higher than the otheer five groups (P=0.000). Spearman correlation analysis showed that there was a significant positive correlation exists between the OD value and GLP-1concentrations in whichl0,50,100,150nmol/L GLP-1+AGEs groups (r=0.885, P=0.000).5. The apoptotic cells in AGEs groups showed topic morphological changes were observed using Wrihgt’s-Gimesa, Acridine Orange and Hoechst33258staining. Apoptotic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.6. The results obtained from Hoechst33258staining showed that the apoptotic rate in AGEs group is (22.07±2.20)%, which significantly higher than control (P=0.000), and higher than AGEs+GLP-1group (16.33±1.23)%(P=0.001).There was no statistical difference between GLP-1group and control.7. The results obtained from FCM showed that the apoptotic rate in AGEs group is (20.56±1.16)%, which significantly higher than that of control (7.07±0.65)%(P=0.000), and higher than AGEs+GLP-1group (13.28±1.19)%(P=0.001). Apoptotic rate in GLP-1group was (4.92±0.39)%, which significantly lower than that of control (P=0.020).ConclusionApoptosis was induced by AGEs in a dose, time-dependent manner. Certain concentration range (50-150nmol/L) GLP-1increase endothelial cell viability in a dose-dependent manner. We found for the first time that (50-100nmol/L) GLP-1can restore the cell viability inhibited by AGEs, and attenuate the apoptosis induced by AGEs in a dose dependent fashion.Part II The molecular mechanisms of the anti-apoptotic action of GLP-1Section I The effects of GLP-1on oxidative stress and important caspases induced by AGEs in endothelial cellsObjectivesWe analyzed the level of oxidative stress by confocal laser, and tested the activities of caspase-9,-3using enzyme-linked immunosorbent assay (ELISA), in order to analyze the mechanisms of the anti-apoptotic action of GLP-1.Methods1. The level of reactive oxygen species (ROS) was quantified by2’,7’-dichlorofluorescein diacetate assay.2. The activities of caspase-9and-3were detected by ELISA.Statistical AnalysisAll analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. In this part, all results were determined by the factorial design ANOVA. Statistical significance was defined as two-sided p<0.05.Results1. GLP-1inhibits the ROS generation induced by AGEsCompared with control group, DCF fluorescence in cells exposed to AGEs was strikingly increased (P=0.000). DCF fluorescence of AGEs+GLP-1group was36.70±1.17, and significantly higher than that of AGEs (P=0.000), but still significantly higher than control group (P=0.000).2. Caspase-9,-3activities of AGEs group were significantly higher than that of control group (P=0.000,0.000) and AGEs+GLP-1group (P=0.036,0.006), respectively. GLP-1could attenuate the activities of caspase-9,-3induced by AGEs. Factorial variance analysis showed that there was interaction effect between AGEs and GLP-1(F=68.549, P=0.000; F=42.132, P=0.000).Conclusion Exposure to AGEs for48h caused a significant increase in ROS generation and activities of caspase-9and-3, but co-incubation with GLP-1attenuate these AGEs-induced effects in HUVECs. Therefore, we speculated that oxidative stress and caspase-9,-3may contribute to the anti-apoptotic actions of GLP-1against AGEs.Section Ⅱ The effects of GLP-1on PI3K/Akt pathway induced by AGEs in endothelial cellsObjectivesTo analyze the effects of GLP-1on PI3K/Akt pathway and the downstream Bcl-2, Bax, cyto-c level in AGEs-treated endothelial apoptosis.Methods1. Cell viability was assessed by MTT assay. Use the same method with that of Chapter Ⅰ.2. Flow cytometry assay apoptosis. Use the same method with that of Chapter Ⅰ.3. Western-blot assay p-Akt (ser473), Bcl-2, Bax and cyto-c Cells with various treatments were collected and lysed in protein lysis buffer. The supernatant contained the cell extracts and the protein concentration was measured using the BCA protein assay. Equal amounts of protein (20μg) from each sample were separated on12%SDS-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in5%non-fat milk for2h and incubated with either anti-p-Akt (ser473)(1:2,000), anti-Bcl-2(1:1,000), anti-Bax (1:2,000), anti-cytochrome c (1:2,000), or anti-β-actin (1:2,000) antibody. Secondary specific horseradish-peroxidase-linked antibodies (1:5,000) were added for1h, and immune complexes were detected by ECL chemiluminescence, the band intensity was measured and quantified with ImageJ software.Statistical AnalysisAll analyses were carried out with SPSS13.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. We used Spearman correlation analysis for correlation analysis. Apoptotic rate was determined by the factorial design ANOVA. Statistical significance was defined as two-sided p<0.05.Results1. The effects of pretreatment with PI3K inhibitor LY294002on the protective action of GLP-1against AGEsThe OD value of AGEs group is0.11±0.03, which was significantly higher than that of control group (P=0.000). The OD value of cells that100nmol/L GLP-1and AGEs co-incubated was0.22±0.03, and was significantly higher than that of AGEs treated cells (P=0.001). The effects of AGEs+GLP-1on OD value was markedly inhibited by pretreatment with LY294002(P=0.030).2. The effects of pretreatment with LY294002on the anti-apoptotic action of GLP-1against AGEsCells in groups were treated with indicated treatment for10h. Apoptotic rate in AGEs group was (20.89±2.67)%, which was significantly higher than that of control group (P=0.000), and also higher than that of AGEs+GLP-1group (P=0.002). Apoptotic rate in LY294002pretreatment group was (16.69±2.06)%, which was significantly higher than that of AGEs+GLP-1group (P=0.045). 3. The effects of LY294002on the endothelial p-Akt levels induced by AGEsThe levels of p-Akt of endothelial cells after treatment with200μg/mL AGEs for48h was significantly lower than that of control(P=0.039).In the presence of100nmol/L GLP-1and AGEs, Akt was significantly activated, resulting in a1.3-fold increase of p-Akt in AGEs-treated HUVECs (P=0.003). The effects of GLP-1on Akt activity was completely abolished by pretreatment with LY294002(vs. AGEs+GLP-1, P=0.030). The expression of p-Akt in100nmol/LGLP-1-treated cells was significantly higher than control ones (P=0.000). Factorial variance analysis showed that there was interaction effect between AGEs and GLP-1(F=52.244, P=0.000).4. The effects of different concentrations of GLP-1on the p-Akt levels for indicated time induced by AGEsHUVECs were incubated with different concentrations of (0,10,50,100nmol/L) GLP-1for48h, and10nmol/L GLP-1did not significantly influence p-Akt levels (vs. control, P=0.947). However,50,100nmol/L GLP-1both were significantly increased the p-Akt levels (vs. control, P=0.022,0.000).The results of comparison the p-Akt levels in different concentrations of (10,50,100nmol/L) GLP-1showed that a significant positive correlation exists between the p-Akt levels in HUVECs and concentrations of GLP-1(r=0.945, P=0.000).5. The effects of pretreatment with LY294002on the expression of Bcl-2, Bax, cyto-c in HUVECsAGEs significantly increased the expression of Bax (P=0.000vs. control), but didnot significantly influence Bcl-2levels (P=0.958vs. control), and markedly decreased the Bcl-2/Bax ratio (P=0.000vs. control). Cells were treated with AGEs and GLP-1for48h, and the Bcl-2levels was significantly higher than that of AGEs (P=0.000), but Bax levels was significantly decreased (P=0.000Vs. AGEs), so the ratio of Bcl-2/Bax was increased significantly (P=0.000Vs. AGEs). Pretreatment with LY294002partially attenuated the effects of GLP-1and restored the AGEs-induced Bax down-regulation (P=0.004vs. AGEs+GLP-1), but was not significantly influence the expression of Bcl-2(P=0.139vs. AGEs+GLP-1), so decreased the ratio of Bcl-2/Bax significantly (P=0.000vs. AGEs+GLP-1). AGEs significantly increased the release of cyto-c (P=0.000vs. control). Cells were treated with AGEs and GLP-1for48h,and the release of cyto-c significantly lower than that of AGEs-treated cells(P=0.000). Pretreatment with LY294002for1h partially attenuated the effects of GLP-1(P=0.000vs. AGEs+GLP-1).ConclusionThis study revealed for the first time that GLP-1prevent cell apoptosis of primary human endothelial cells expose to AGEs in a PI3K/Akt pathway-dependent manner. We also have provided the first evidence that GLP-1can regulate the expression of Bcl-2, Bax. cyto-c induced by AGEs, and these effects were involved in PI3K/Akt pathway. |
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