Hepatocyte Growth Factor Protects Human Endothelial Cells Against Advanced Glycation End Products-induced Apoptosis And Its Mechanism

IntroductionCardiovascular complications are the leading cause of morbidity and mortality in patients with diabetes mellitus. Apoptosis of endothelial cells is associated with cardiovascular diseases. Injury of endothelial cells has been postulated to be an initial trigger of the progression of atherosclerosis. Endothelial cell apoptosis is thought to play an important role in the pathogenesis of atherosclerosis. Recent study has identified that AGEs can induced endothelial cell death through the induction of apoptosis. AGEs formation may contribute to the progression of atherosclerosis, particularly in diabetes. It has been well documented that AGEs progressively accumulate on the tissues and organs that develop chronic complications of diabetes mellitus, such as retinopathy, nephropathy, neuropathy and also macrovascular disease atherosclerosis. AGEs intervention reduce diabetes - accelerated atherosclerosis. Several growth factors have been shown to provide protection against programmed cell death. Hepatocyte growth factor ( HGF) can act as an anti - apoptotic factor for endothelial cells. Therefore , we examine whether HGF prevents AGEs - induced injury of endothelial cells.HGF, also known as scatter factor, is a multifunctional cytokine possessing a wide spectrum of biological activities. It regulates cell growth, cell motility, and morphogenesis of various cell types, including endothelial cells. Moreover, HGF is a potent angiogenic factor that can induce endothelial cell proliferation and migration, without induction of vascular smooth muscle cell division. Endo-thelial cell migration, proliferation, and apoptosis contribute to the pathogenesis of atherosclerosis. In addition, HGF can act as a survival factor for endothelial cells.The anti - apoptotic effects of HGF occur through several different pathways in other cell types. The different signals that converge on mitochondria to trigger or inhibit apoptosis and their downstream effects delineate several major pathways in physiological cell death. The effectors of apoptosis are now well known to be represented by a family of intracellular cysteine proteases known as caspas-es. A feature of apoptosis that impinges on caspases is altered mitochondrial function characterized by a reduction in the electrochemical gradient across the mitochondrial membrane and release of mitochondrial cytochrome c to cytoplasm, and it is inhibited by the presence of Bel -2 in these organelles. Bel -2 and Bax are homologous proteins that have opposing effects on cell life and death, with Bel - 2 serving to prolong cell survival and Bax acting as an accelerator of apoptosis. Bel -2 and Bax reciprocally control apoptosis by respectively inhibiting or stimulating mitochondrial cytochrome c release. Cytosolic cytochrome c and Apaf - 1 cooperatively activate initiator caspase - 9 that triggers a caspase cascade leading to apoptosis. Another regulator of apoptosis is the transcription factor, nuclear factor kB ( NF - kB ) , participates in many important processes in endothelial cells including apoptosis and regulation of inflammatory responses. It can play a role in promoting cell survival. Several mediators of atherosclerosis, including cytokines and growth factors, have been shown to activate NF - kB. Regulation of NF - kB activity by HGF may provide an additional anti -apoptotic mechanism in endothelial cells. However, the molecular mechanisms of the anti - apoptotic action of HGF on endothelial cells are not fully understood. We therefore use an in vitro model to further investigate the pathways of HGF against AGEs - induced apoptosis in the endothelium.Section A. Effect of Advanced Glycation End ProductsOn Expression of Hepatocyte GrowthFactor in Human Endothelial CellsObjectiveTo investigate the effects of advanced glycation end products ( AGEs) on protein and mRNA expression of hepatocyte growth factor ( HGF) in cultured human umbilical vein endothelial cells ( HUVECs).Methods1. Cell culture. Cells isolated from human umbilical cords were treated with AGEs in the presence or absence of HGF. Cells were cultured on gelatin -coated dishes and propagated in RPMI -1640 medium supplemented with 20% (v/v) heat -inactivated FBS, 50jxg/mL endothelial cell growth factor, 90/xg/ mL heparin, lOOIU/mL penicillin and 100|xg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 95% air-5% carbon dioxide.2. Preparation of AGE - HSA in vitro. 0. 5g HSA was dissolved in 10ml of 0.5mol/L sodium phosphate buffer (pH 7.4) with 3. Og D - glucose, 1000|xg penicillin and 500|xg gentamicin. The samples were filter - sterilized by using a 0. 22 - jxm MiUipore filter and incubated at 37°C for 120 days under sterile and dark conditions, dialyzed against phosphate -buffered saline (PBS, pH 7.4)in order to eliminate unconjugated glucose. While nonglycated HSA was prepared simultaneously as the same method but the solution lacked glucose.3. Cell treatment. HUVECs were treated with AGEs at different concentrations for 24h and at a concentration of 400mg/L for different time.4. The HGF mRNA was determined by reverse transcriptase - polymerase chain reaction ( RT - PCR) and HGF protein was analyzed by immunocytochem-istry.Results1. Immunocytochemical staining showed that after treated with AGEs at different concentrations ( 100mg/L,200mg/L,400mg/L) for 24h, integrated optical density (OD) of HGF in HUVECs were significantly higher than that of control group(P < 0.05).2. After treated with AGEs at a concentration of 400mg/L for 6 hN12 h and 24 h, the OD of HGF in HUVECs were strikingly higher than that of 0 h. After 48h, the OD of HGF was decreased.3. RT - PCR showed that treated with AGEs at different concentrations for 24h, expression of HGF mRNA were increased in HUVECs compared with control group(P < 0.05).4. The expression of HGF mRNA was elevated after treated with 400mg/L AGEs for 6 h and reached the peak at 24h. After 48 h, the expression of HGF mRNA was decreased.Conclusion1. Advanced glycation end products may induce the expression of HGF mRNA and protein in the early stage then caused a gradient decrease of HGF expression in HUVECs.2. The protective action of HGF against endothelial cell injury elevate with AGEs concentration in the early stage then decrease in HUVECs.Section B. Effects of Hepatocyte Growth Factor on Apoptosis of Endothelial Cells Induced by Advanced Glycosylation End ProductsObjectiveTo investigate inhibiting effect of hepatocyte growth factor (HGF)o thelial apoptosis induced by advanced glycosylation end products (AGEsMethods1. HUVECs were cultured in vitro2. Preparation of AGE - HSA in vitro.3. Cell treatment. AGE - HSA was added at concentrations of IOC and 400mg/L. HSA alone (final concentration 200mg/L) was used to tr< trol cells. Cell lysates were collected after 48 hours of AGE - HSA exp< investigate the presence of apoptosis. For the time course study of apop dose of 200mg/L was used, and apoptosis was examined after 12, 24, 2 48 hours of AGE - HSA exposure. The protective effect of HGF on AG1 duced apoptosis was tested by pretreatment with either 50 or lOOng/mL r pretreatment for 12, 24, and 48 hours was performed with HGF (lOOng Cell lysates were collected after 48 hours of AGE - HSA exposure.4. AGEs - induced cell growth inhibition was quantified by MMT a5. Morphology of cell apoptosis was observed by Wright's - Giem ning, Acridine Orange and Hoechst 33258 fluorescence staining.6. Cell apoptosis was detected by flow cytometry with PI staining a nexin - FITC/PI double staining.Results1. Cell viability with AGE - HSA treatment was significantly decreased compared with those with HSA treatment (control). This cytotoxic effect was time and concentration - dependent.2. HUVECs undergo apoptosis. Typical morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed using Acridine Orange and Hoechst 33258 staining under fluorescence microscope.3. Flow cytometric analysis with Annexin - V FITC/PI double staining revealed that the apoptotic rate was significantly elevated by AGE - HSA treatment compared with those with HSA treatment.4. The percentage of cells undergoing AGEs - mediated apoptosis was decreased by pretreatment with HGF.Conclusion1. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability and induced DNA fragmentation, leading to apoptosis.2. Apoptosis was induced by AGEs in a dose - and time - dependent fashion.3. Pretreatment with HGF protected against AGEs - induced cytotoxicity in the endothelial cells.Section C. The Molecular Mechanisms of the Anti - apoptotic Action of HGFObjectiveThe molecular mechanisms of the anti - apoptotic action of HGF on endo-thelial cells are not fully understood. We therefore use an in vitro model to further investigate the pathways of HGF against AGEs - induced apoptosis in the endothelium.Methods1. HUVECs were cultured in vitro2. Preparation of AGE - HSA in vitro.3. Cell treatment. AGE - HSA was added at concentrations of 100, 200, and 400mg/L. HSA alone (final concentration 200mg/L) was used to treat control cells. Cell lysates were collected after 48 hours of AGE - HSA exposure to investigate the presence of apoptosis. For the time course study of apoptosis, a dose of 200mg/L was used, and apoptosis was examined after 12, 24, 36, and 48 hours of AGE - HSA exposure. The protective effect of HGF on AGEs - induced apoptosis was tested by pretreatment with either 50 or lOOng/mL HGF. A pretreatment for 12, 24, and 48 hours was performed with HGF (lOOng/mL). Cell lysates were collected after 48 hours of AGE - HSA exposure.4. The expression of apoptosis — associated genes Bax^Bcl - 2 and NF - kB were determined by western blot analysis.5. The activities of caspase -3 and -9 were detected by enzyme -linked immunosorbent assay.Results1. AGEs markedly elevated Bax and decreased NF - kB , but not Bel - 2 expression.2. AGEs significantly inhibited cell growth through a pro -apoptotic action involving caspase -3 and -9 activations in HUVECs.3. HGF significantly promoted the expression of Bel - 2 and NF - kB , while decreased the activities of caspase - 3 and - 9 without affecting Bax level.