CREG Genes Inhibits High Glucose-and High Palmitate-Induced Apoptosis In Human Umbilical Vein Endothelial Cells

Background and Object:Macrovascular complications(e.g.,coronary artery disease,peripheral vascular disease,and stroke)are the major causes of mortality in patients with type 2 diabetes mellitus(T2DM),accounting for more than 50%of diabetes-related deaths.Diabetic macrovascular complications usually feature accelerated atherosclerosis,which is anatomically similar to atherosclerosis in nondiabetic individuals but is more extensive and occurs much earlier.A growing body of evidence suggests that endothelial dysfunction and apoptosis are the earliest and the most critical events in the onset and progression of atherosclerosis in diabetic patients.During the progression of DM,endothelial cells(ECs)exposed to hyperglycaemia and hyperlipidaemia undergo apoptotic processes,leading to the detachment of endothelial cells from the intima.This intial denudation consequently triggers pro-atherosclerotic responses,resulting in the development of atherosclerosis and macrovascular complications in those with DM.Although a causal relationship between endothelial apoptosis and atherosclerosis is well established,the underlying mechanisms remain incompletely understood.Therefore,there is an increasing demand for investigations into new therapeutic targets directed at reducing ECs apoptosis and atherosclerosis in diabetic patients.Cellular repressor of E1A-stimulated genes(CREG)is a novel glycoprotein that is highly expressed in blood vessels under physiological conditions.It has been well established that CREG plays essential roles in maintaining homeostasis in vascular smooth muscle cells by regulating their proliferation,migration and phenotypic switches.Notably,results from our lab have also demonstrated that CREG is critically involved in the differentiation and functional maintenance of ECs.CREG has been found to be abundant in both the arterial endothelium and in primary human umbilical vein endothelial cells(HUVECs),implying its potential importance.We have also confirmed that CREG promotes the proliferation and migration of HUVECs through the activation of different signalling pathways.Moreover,CREG is essential for in vivo and in vitro angiogenesis.In a porcine coronary injury model,the implantation of a CREG protein eluting stent resulted in the acceleration of the re-endothelialization of the coronary artery.In ECs inflammation model induced by tumour necrosis factor alpha,over-expression of CREG inhibited the inflammatory responses and the hyper-permeability of ECs.In addition,CREG inhibits endothelial apoptosis via the VEGF/PI3K/Akt pathway.Meanwhile CREG has been reported to be involved in the apoptosis of vascular smooth muscle cells and bone marrow mesenchymal stem cells.Considering the above role of CREG in ECs and in apoptosis,we aimed to determine whether CREG aetiologically participates in ECs apoptosis under conditions associated with DM.Method:1.In the present study,the expression of hCREG and the index of apoptosis were assessed in atherosclerotic arteries from diabetic amputees and in control arteries from DM-free amputees by HE staining,immunofluorescence staining,TUNEL staining and Immunoblotting.Then we detected hCREG expression and endothelial cells apoptosis in vitro model.Primary human umbilical vein endothelial cells(HUVECs)were isolated and cultured in a high glucose/high palmitate medium(25 mmol/L D-glucose,0.4 mmol/L palmitate).The over-expression or knock-down of CREG was performed in HUVECs to determine the role of CREG in HUVECs apoptosis by Annexin V-FITC/PI dual-colour flow cytometry,TUNEL staining and western blotting.2.To elucidate the upstream transcriptional regulation of hCREG expression,a promoter-binding transcription-factor profiling array assay,a chromatin immunoprecipitation(ChIP)assay and mutations of the binding site were performed to determine the transcriptional factors regulated the expression of hCREG.3.To explicitly determine the role of GATA1 in relation to diabetic complications,human diabetic arteries were obtained and analysed by immunoblotting or real-time PCR,respectively.To determine the roles of GATA1 in hCREG expression and endothelial apoptosis,full-length human GATA1 cDNA(1245 bp)was amplified via PCR from whole HUVECs cDNA and inserted into the pcDNA3.1(+)expression vector via EcoRI/XhoI double digestion.The plasmid pcDNA3.1(+)-GATA1 was transfected into HUVECs and selected using 500 μg/ml G418,and cells transfected with an empty pcDNA3.1(+)plasmid were used as a vehicle control.Then,cells were incubated with 25 mM D-glucose and 0.4 mM palmitate for 24 h to induce apoptosis.The expression of GATA1,hCREG and the rate of apoptosis were assessed by Annexin V-FITC/PI dual-colour flow cytometry,TUNEL staining,and Real-time PCR and immunoblotting.Results:Our results demonstrate that over-expression of hCREG inhibits high glucose/high palmitate-induced apoptosis in HUVECs and that hCREG is upregulated expression by GATA1 in transcriptional level.It implicated that hCREG might be a potential therapeutic target for management of vascular complications in patients with DM.The major results are presented below:1.Expression of hCREG is reduced at atherosclerotic-prone sites associated with increased endothelial apoptosis in human diabetic arteries and in primary HUVECs.To explore the relationship between hCREG and endothelial apoptosis under diabetic conditions,we detected hCREG expression and endothelial apoptosis in atherosclerotic arteries from diabetic amputees and in control arteries from DM-free amputees.HE staining showed that the arteries used in this study from diabetic patients presented with significant atherosclerotic plaques,while arteries from patients without diabetes showed a relatively normal intimal structure.In addition,immunofluorescence staining showed that hCREG expression was significantly reduced in the endothelium of diabetic arteries compared with the expression in control arteries.TUNEL staining further showed that apoptosis occurred more frequently in the endothelium of diabetic arteries than in the control arteries.Immunoblotting also demonstrated significantly decreased hCREG expression and increased apoptosis in diabetic arteries.These data suggest that hCREG might be involved in endothelial apoptosis under diabetic conditions.High glucose/high palmitate-induced reduction of hCREG expression caused apoptosis in primary HUVECs.To elucidate the role of hCREG in ECs apoptosis,an in vitro model of HUVECs treated with high glucose(25 mM D-glucose)and a range of palmitate concentrations(0.2,0.3 and 0.4 mM)was established to mimic the pathological stimuli present in individuals with DM.Annexin V-FITC/PI dual-colour flow cytometry showed that compared with the normoglycemic control(5.5 mM D-glucose,19.5 mM L-glucose and 0.4 mM BSA),high glucose(25 mM D-glucose)alone increased HUVEC apoptosis and the combination of high glucose with different concentrations of palmitate(0.2,0.3 and 0.4 mM)further increased cell apoptosis in a dose-dependent.ECs apoptosis was also confirmed by detecting DNA fragments via TUNEL staining.Moreover,immunoblotting and RT-PCR analysis showed that the expression of hCREG was reduced,while Cleaved caspase-3 expression was gradually increased after treatment with high glucose and with high glucose plus palmitate at various concentrations.Because high glucose with high palmitate(0.4 mM)produced the most significant pro-apoptotic effect and hCREG reduction,it was employed in the following experiments.The above in vitro data accurately reflect the changes in hCREG expression and apoptosis observed in vivo from diabetic arteries,justifying further investigation into the causal effect of hCREG in the process of endothelial apoptosis under diabetic conditions.To determine whether a causal relationship exists between hCREG and HUVECs apoptosis,we established over-expressing CREG(CREG-OE)and CREG knock-down cells(CREG-KD),and HUVECs expressing scrambled shRNA sequences were used as a normal control(CREG-NR).Then,the ratio of cellular apoptosis and the expression of hCREG in three groups of HUVECs treated with high glucose(25 mM-glucose)and high palmitate(0.4 mM)for 24 h were assessed.Annexin V-FITC/PI flow cytometry and TUNEL staining showed that the apoptotic cells was dramatically increased in the CREG-KD cells and was significantly decreased in CREG-OE cells relative to the expression in CREG-NR HUVECs.Western blotting demonstrated that the expression of Cleaved caspase-3 was remarkably decreased in CREG-OE cells and was increased in CREG-KD cells.These data provide a direct evidence that high glucose/high palmitate-induced endothelial apoptosis is caused by a reduction of hCREG,which can be rescued by the over-expression of hCREG.Thus,hCREG might be a therapeutic target for altering endothelial apoptosis and atherosclerosis in patients with DM,and exploring factors which can directly regulate hCREG expression is of great importance.2.GATA1 directly binds to the core promoter of hCREG and regulates its activation.To investigate the upstream factors regulating hCREG,we first generated luciferase constructs to determine the core promoter of the hCREG gene.The hCREG 586 bp(-508/+78)fragment exhibited the highest promoter activity,which was defined as 100%,and this segment was identified to be the core promoter region.In addition,the 2003 bp(-1925/+78)fragment,945 bp(-867/+78)fragment and 478 bp(-400/+78)fragment produced similar results and displayed relatively high promoter activities(70.7%,62.6%and 77.2%in HUVECs and 67.1%,64.5%and 75.7%in 293T cells,respectively),while the 358 bp(-280/+78)fragment had extremely low activity(16.5%in HUVECs and 15%in 293T cells),indicating that the fragment at-508/-281 may play a pivotal role in regulating hCREG transcription.To characterize the transcription factors(TFs)that bind to the hCREG core promoter region(-508/+78)and regulate the activation of the hCREG gene,48 TFs in HUVECs were assessed using a competitive promoter-binding TF profiling array.The results demonstrate the possible presence of binding sites for Brn-3,C\EBP,EGR,ETS,GATA,GR/PR,HIF,IRF,NF-1,NF-KB and Pax-5 within the hCREG core promoter.Bioinformatic predictions showed there were 2,1 and 2 consensus binding sequences for C\EBP β,GATA1,and ETS-1,respectively.ChIP assay results further demonstrate that GATA1 and ETS1 bound to the hCREG core promoter(-508/-281)directly.To confirm this finding,reporter vectors of a mutant hCREG promoter with deletions of the 2 consensus GATA1 binding sequences[-467/-462(GATA1-MUT1)、-297/-292(GATA1-MUT2)]and ETS1 binding sequence(-392/-389 ETS1-MUT)were constructed and transiently transfected into HUVECs to assess luciferase activity.Compared with the activity of the core promoter of hCREG(-508/+78),which was defined as 100%,the transcriptional activity of GATA1-MUT1 and ETS1-MUT were almost no change,while that of GATA1-MUT2 was dramatically reduced.These data suggest that GATA1 can directly bind to a cis-acting DNA element in the hCREG promoter region,which is essential for the activation of hCREG transcription.3.Overexpression of GATA1 activates hCREG expression and inhibits HUVEC apoptosis induced by high glucose/high palmitate stimulationThe expression of GATA1 was shown to be markedly decreased in diabetic arteries relative to the expression in arteries from non-diabetic subjects as assessed using immunoblotting or real-time PCR,respectively,which indicates that GATA1 might be involved in the development of atherosclerosis in individuals with diabetes.Annexin V-FITC/PI flow cytometry showed that the percentage of apoptotic cells was reduced by 47%in GATA1 over-expressing HUVECs(pc-GATA1)relative to the expression in the vehicle control[pcDNA3.1(+)].TUNEL staining also showed consistent results,indicating that apoptosis was decreased by 21%in GATA1 over-expressing HUVECs relative to expression in the control group.We further detected the expression of GATA1 and hCREG at both the transcription and protein level.Real-time PCR analysis showed that GATA1 and hCREG expression increased by approximately 2-fold in GATA1 over-expressing HUVECs relative to expression in the control group.Immunoblotting showed that GATA1 and hCREG expression increased approximately 4-fold relative to expression in the control group.These data demonstrate that the over-expression of GATA1 activates hCREG expression,leading to the protection of HUVECs against high glucose/high palmitate-induced apoptosis.Conclusion:In conclusion,this study indicates that:①The expression of CREG was obviously decreased and endothelial apoptosis was increased in the atherosclerotic vasculature of diabetic patients when compared with the findings in non-diabetic control vessels.②Under high concentrations of glucose(25 mM D-glucose)and different concentrations of palmitate(0.2,0.3 or 0.4 mM),which simulate the pathological stimuli of poorly controlled T2DM in vivo,apoptosis increased and CREG expression decreased in HUVECs in a concentration dependent manner in vitro.③Elevating the expression of CREG antagonized HUVECs apoptosis,whereas suppressing CREG expression futher increased HUVECs apoptosis induced by high glucose/high palmitate concentrations.④The hCREG 586 bp(-508/+78)fragment exhibited the highest promoter activity and this segment was identified to be the core promoter region.GATA1 can directly bind to a cis-acting DNA element in the hCREG promoter region(-297/-292).⑤GATA1 activates CREG expression,leading to the protection of HUVECs from the apoptosis induced by high glucose/high palmitate stimulation.CREG might be a potential therapeutic target for the management of macrovascular complications in diabetic patients.