High Concentration Of Glucose Induced Apoptosis Of Endothelial Progenitor Cells By Endoplasmic Reticulum Stress Pathway And The Role In The Diabetic Coronary Vasculopathy

BackgroundThe endothelial progenitors cells(EPCs)were reported in 1997 to differentiate in vitro into endothelial cell(EC)and seemed to be incorporated at sites of active angiogenesisin various animal models of ischaemia,which led to the new concept that vasculogenesis and angiogenesis may occur simultaneously in postnatal.These cells are derived from the bone marrow and can circulate in the blood.The majority of circulating EPCs reside in the bone marrow where exist an optimal microenviroment. EPCs are capable of differentiating into endothelial cells and homing to sites of ischaemia and play a important role in vascular repair and reendothelialisation of injured blood vessels and collateral vessels formation.Moreover,diabetic macro -angiopathy develop as the result of an atherogenesis process initiated by endothelial damage and dysfunction.And recent insights show that the injured endothelial monolayer is regenerated by circulating EPCs,which accelerate reendothelialization and limits atherosclerotic lesion formation.There is a balance between endothelial cell damage and endothelial cell regeneration.Apoptotic and necrotic endothelial cells are regenerated by circulating EPCs.However,the system becomes imbalanced in conditions of enhanced endothelial cell damage and exhausted EPCs-mediated endothelial cell repair.The resulting the disequilibrium between damage and regeneration,results in the atherogenesis progression.So the EPCs would play a vital role in the diabetic macroangiopathy.It has been reported that the number of circulating EPCs which origin from bone marrow were impaired in patients with diabetes mellitus(DM).Moreover,recent studies have revealed that ER stress mediiated apoptosis inβcells could be a cause of DM.Based on these results,this present study hypothesized that the EPCs might be impaired in bone marrow in diabetes mellitus and that ER stress mediated apoptosis in bone marrow derived EPCs (BM-derived EPCs)might be a cause of diabetic macroangiopathy.The objectives of this study are as follow:Objectives1.To establish a DM animal model.2.To investigate the methods of isolating,culturing and identifying BM-derived EPCs3.To examine the occurrence of apoptosis of BM-derived EPCs in DM rat and control group to determine whether or not the EPCs is impaired in bone marrow in DM.4.To assess the mRNA express of GRP78 and CASPASE-12 of BM-derived EPCs in diabetic or non-diabetic rats to investigate whether or not the ER stress mediated apoptosis in bone marrow derived EPCs.Methods1.Establishment of DM animal modelAll protocols were approved by the Shandong University experimental animal center. Twenty-seven male age-matched Wistar rats(Experimental Animal Center of Shandong University,Ji'nan,China)were randomly divided into 2 groups:control group(n=12)and DM group(n=15).Diabetes was induced by a single intraperitoneal injection of streptozotocin(STZ,65 mg/kg body wt and dissolved in 0.1 mol/l citrate buffer,pH 4.2)in DM group.The rats in control group received citrate buffer alone. One week after injection of STZ,fasting plasma glucose levels was measured,and rats with plasma glucose at least two times higher than 16.7mmol/L were used.DM group rats were fed high fat and glucose diet and the rats in control group were fed standard rat diet.2.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and Characterization2.1 EPCs culture BM cells were obtained by flushing the tibias and femurs of rat.BM mononuclear cells were isolated by density centrifugation over rat lymphocyte separating medium(LTS-1083)at 2000 rpm for 30 min.Then mononuclear cells were isolated and plated culture flasks in M199 medium(Gibco,USA)supplemented with 20% fatal calf serum,100μg/mL streptomycin,100U/ml penicillin.After 24 h in culture, the nonadherent cells were harvested and resuspended,then 1×10~6 cells were plated on 24-well culture dishes or T25 flasks pre-coated with 0.1%gelatin(Sigma,USA) and maintained in M199 medium supplemented with 20%fatal calf serum,100μg/mL streptomycin,100U/ml penicillin,40 ng/mL vascular endothelial growth factor (VEGF,Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA), 10 ng/mL epidermal growth factor(EGF,Peprotech,USA),and 10U/mL heparin. After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS,new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.2.2 Characterization of EPCsFluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FIYC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,St.Louis,USA)and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo -carbocyanine(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular ProbeCo,Eugene,USA).To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%parafor -maldehyde for 10 minutes.After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..3.EPCs apoptosis detection by flow cytometer.The BM-derived EPCs apoptosis of the rats in DM and control groups was determined by flow cytometric detection according to the manufacturer's instructions of the annexin V-FITC apoptosis detection kit(Jingmei,China).Briefly,EPCs were washed once with PBS and resuspended in binding buffer were stained with annexin Ⅴ(0.5μg/ml)and propidium iodide(0.6μg/ml)for 5 min at room temperature.After staining,cells were immediately analyzed using a FACScan flow cytometer(Becton Dickinson,CA)with simultaneous monitoring of green fluorescence(530 nm,30 nm band-pass filter)for annexin V-FITC and red fluorescence(long-pass emission filter that transmits light＞650 nm)associated with propidium iodide.10,000 events were collected and analyzed to determine the percentage of annexin-ⅤFITC and PI-positive cells.The cells were analyzed using Cell Quest analysis software (Becton-Dickinson,CA).4.The mRNA expression of GRP78 and CASPASE-12 by RT-PCRTotal RNA from DM and control rat BM-defived EPCs was isolated using the TRIzol Reagent(Invitrogen)as described by the manufacturer.For RT-PCR,lug total RNA and oligo(dT)(Promega)was reverse-transcribed into cDNA with 1 unit/ml M-MLV reverse transcriptase(Promega)at 42℃for 1b.Primers sequences for rat grp78, caspase-12 andβ-actin are available with Taq Polymerase(Takara)for PCR.Results1.General features of the experimental ratsAt the end of the experiment,11 diabetic rats induced by STZ and 12 control rats survived.Glucose levels were significantly elevated in diabetic rats compared with control rats after STZ injection.Other symptoms,such as lower body weights, polyuria and polyphagia,which are normally associated with diabetic state were also observed in the diabetic rats.2.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and characterizationThe secondarily attached cells stretched after 3 days.The cells proliferated faster after 7-10 days' incubation when cord-like structure was observed.After 2 weeks' induction, most of the cells exhibited multangular morphology.More than 80%attached cells took up DiI-ac-LDL,bound FITC-UEA-1(double positive fluorescence).3.EPCs apoptosis detection by flow cytometer.BM-derived EPCs significantly increased apoptosis in DM rat compared with control group(20.3±4.64%vs.11.2±2.85,P＜0.05).4.The mRNA expression of GRP78 and CASPASE-12 by RT-PCRThe mRNA expression of GRP78 and CASPASE-12 significantly increased in DM rat when compared with control group(GRP78:0.64±0.07%vs.0.43±0.05%,P＜0.05; CASPASE-12:0.56±0.09%vs.0.36±0.04,P＜0.05).Conclusions1.BM-derived EPCs significantly increased apoptosis in DM rat.2.The mRNA expression of GRP78 and CASPASE-12 significantly increased in DM rat which suggest that ER stress mediated apoptosis in bone marrow derived EPCs might be one of causes of diabetic macroangiopathy. BackgroundEndoplasmic reticulum(ER)stress is a conditions interfering with endoplasmic reticulum function will perturb the folding of newly synthesized membrane or secreted proteins.The accumulation of unfolded proteins in ER causes cells ER stress response,which reduces the amount and aggregation of unfolded proteins in ER.In case the response fails to solve ER stress,the apoptosis pathway will be activated.ER stress can lead to apoptosis by various pathways,among them caspase-12 has been proposed as a specific mediator of ER-stress-induced apoptosis.It has been suggested that ER stress might induce cell apoptosis via caspase-12 independently.The involvement of apoptosis via ER stress is a relatively new pathway other than the death receptor or mitochondrial death pathways.It has been shown that type 2 diabetic patients have reduced pancreatic isletsβcell mass and increasedβcell apoptosis rates.Recent in vitro studies suggest that elevated glucose levels induced apoptosis in culturedβcells possibly through ER stress.And we observed the occurrence of apoptosis of EPCs elevated in DM rat bone marrow as well as ER stress was actived in vivo study.Moreover,the endothelial progenitor cells (EPCs)play a important role in vascular repair and reendothelialisation of injured blood vessels,which means the EPCs play a key factor in diabetic vascular disease. Based on these results,we hypothesized that elevated glucose levels impair the bone marrow-derived EPCs(BM-derived EPCs)survival and induce the cells apoptosis. Then whether the ER stress-mediated apoptotic pathway is a component of high concentration of glucose induced the cells apoptois is unknown.In order to test this hypothesis,we cultured rat BM-derived EPCs under different concentration of glucose to investigate that the effect of high concentration of glucose on EPCs and a possible role of ER stress in the apoptosis.Objectives1.To assess the effect of apoptosis indued by high concentration of glucose on rat BM-derived EPCs.2.To assess whether the ER stress was actived in rat BM-derived EPCs by high concentration of glucose.3.To assess whether the ER stress-mediated apoptotic pathway is a component of high concentration of glucose induced rat BM-derived EPCs apoptois.Methods1.rat BM-derived EPCs culture and characterization1.1 EPCs cultureAdult male Wistar age-matched(6 weeks)rats(Experimental Animal Center of Shandong University,Ji'nan,China)weighing 250-300 g were used.All protocols were approved by the shandong university experimental animal center.BM cells were obtained by flushing the tibias and femurs of rat.BM mononuclear cells were isolated by density centrifugation over LTS-1083 at 2000 rpm for 30 min.Then mononuclear cells were isolated and plated culture flasks in M199 medium(Gibco,USA) supplemented with 20%fatal calf serum,100μg/mL streptomycin,100U/ml penicillin.After 24 h in culture,the nonadherent cells were harvested and resuspended, then 1×10~6 cells were plated on 24-well culture dishes or T25 flasks pre-coated with 0.1%gelatin(Sigma,USA)and maintained in M199 medium supplemented with 20% fatal calf serum,100μg/mL streptomycin,100U/ml penicillin,40 ng/mL vascular endothelial growth factor(VEGF,Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA),10 ng/mL epidermal growth factor(EGF,Peprotech, USA),and 10U/mL heparin.After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS,new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.1.2 Characterization of EPCsFluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FITC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,St.Louis,USA)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo -carbocyanine(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular Probe,Eugene,USA).To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%parafor -maldehyde for 10 minutes.After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..1.3 Passage of EPCsThe cells should be subcultured when they reach 80-85%confluence.Aspirate the medium and wash the cells with PBS,then add trypsin-EDTA.Incubate 37℃for a few minutes meanwhile should be monitored using an inverted phase-contrast microscope.Once the cells have begun to detach inactivate the trypsin with medium and centrifuge at 1000 rpm for 8 min.Aspirate the supernatants and resuspend cells with medium then split the cells at 1:3 ratio to expand and be ready to use.2.EPCs apoptosis detection by flow cytometer.2.1 Flow cytometer was performed to characterize the occurrence of apoptosis of various concentration of glucose(control,5mmol/L,12.Smmol/L,25mmol/L and 25mmol/L manitol)treatment for 24 h on EPCs.2.2 Flow cytometer was performed to characterize the occurrence of apoptosis of pre-treating or no pretreating the EPCs with Z-ATAD-FMK(caspase-12 specific inhibitor)in the presence or absence of high glucose(25mmol/L).3.Western blot analysis3.1 The protein expression of the effect of various concentration of glucose(control, 5mmol/L,12.Smmol/L and 25mmol/L)treatment for 24h on EPCs were investigated by western blot analysis for the GRP78,CASPASE-12,CASPASE-3,respectively.3.2 The protein expression of the effect of pre-treating or no pretreating the EPCs with Z-ATAD-FMK in the presence or absence of high concentration of glucose(25mmol/L) by western blot analysis for the CASPASE-12,CASPASE-3,respectively. Results1.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and characterizationThe BM mononuclear cells were isolated and the secondarily attached cells stretched after 3 days.The cells proliferated faster after 7-10 days resulted in a spindle-shaped, EC-like morphology.After 2 weeks' induction,most of the cells exhibited multangular morphology.The cord-like structures and cobblestone appearance was observed.Fluorescent chemical detection of EPCs was performed on second attached cells after 7 days in culture.Uptake of ac-LDL was detected red-stained cells,binding UEA-1 was detected green-stained cells and dual-stained cells for both UEA-1 and ac-LDL were judged to be EPCs.More than 80%attached cells were double positive fluorescence.2.The effect of high concentration of glucose on BM-derived EPCs apoptosis FAC were performed to characterize the effect of high concentration of glucose treatment on EPCs.Treatment with high concentration of glucose(Smmol/L, 12.5mmol/L and 25mmol/L)for 24h significantly increased apoptosis in cultured rat BM-EPCs compared with control group in a dose-dependent manner(7.34±0.44%vs 6.18±0.33%,P＜0.05;11.59±1.22%vs 6.18±0.33%,P＜0.05;36.82±2.16%vs. 6.18±0.33%,P＜0.05;n=3).Treatment of the cells with 25mmol/L mannitol,used as an osmotic control,had no significant effect on apoptosis compared with control group in rat BM-EPCs(5.95±0.81%vs.6.18±0.33%,P＜0.05;n=3).3.The ER stress by high concentration of glucoseTreatment with various concentration of glucose(5mmol/L,12.5mmol/L and 25mmol/L)for 24h significantly increased the protein expression of the GRP78 in EPCs compared with control in a dose-dependent manner(127.33±9.71 vs 39±6.56, P＜0.05;152.33±7.09 vs 39±6.56,P＜0.05;183.33±4.51 vs 39±6.56,P＜0.05;n=3).4.Protein expression of high concentration of glucose on EPCs for CASPASE-12 and CASPASE-3Treatment of BM-EPCs with various concentration of glucose(Smmol/L,12.5mmol/L and 25mmol/L)for 24h markedly increased protein expression of cleaved-caspase-12 and cleaved-caspase-3 in a dose-dependent manner(cleaved-caspase-12:34.67±9.07, 71.33±4.16,125.33±11.68;cleaved-caspase-3:64.67±7.37,131.67±9.02,165.33±5.69)compared with control group(cleaved-caspase-12:0,P＜0.01;cleaved-caspase-3: 0,P＜0.01;n=3).5.The effect of caspase-12 specific inhibitor(Z-ATAD-FMK)on apoptosis induced by high concentration of glucose in rat BM-derived EPCsPretreatment with Z-ATAD-FMK significantly decreased apoptosis in high glucose (25mmol/L)cultured rat BM-EPCs compared with those of no pretreating group(17.91±0.94%vs.36.82±2.16%,P＜0.05;n=3).In contrast,pretreatment or no with Z-ATAD-FMK had no significant effect on apoptosis of rat BM-EPCs in normal glucose(5.62±0.92%vs.6.18±0.33%,P＞0.05;n =3).6.The effect of caspase-12 specific inhibitor(Z-ATAD-FMK)on protein expression of caspase-12 and caspase-3 induced by high concentration of glucose in rat BM-derived EPCsPretreatment with Z-ATAD-FMK significantly decreased protein expression of cleaved-caspase-12 and cleaved-caspase-3 compared with those of no pretreatment (cleaved-caspase-12:0 vs.58±7.21,P＜0.01;cleaved-caspase-3.155±10.58 vs. 185.33±12.01,P＜0.05;n=3)in group with high concentration of glucose.In contrast,pretreatment or no with Z-ATAD-FMK had no significant effect on protein expression of cleaved-caspase-12 and cleaved-caspase-3(both had no expression of cleaved-caspase-12 and cleaved-caspase-3;n=3).Conclusions1.High concentration of glucose may induce the rat BM-derived EPCs apoptosis in a dose-dependent manner.2.ER stress in rat BM-derived EPCs was actived by high concentration of glucose.3.ER stress pathway mediated by caspase-12 may play a important role in rat BM-derived EPCs apoptosis induced by high concentration of glucose. BackgroundThe prevalence of type 2 diabetes is increasing year by year.As an independent risk factor for cardiovascular disease,type 2 diabetes has been implicated as a coronary heart disease equivalent and it is an important component of the complex of common cardiovascular risk factors.Cardiovascular complications are the leading causes of morbidity and mortality in patients with diabetes mellitus(DM).Among them the acute coronary syndroms(ACS)is the extremely severe outcome.Recent studies have shown that the key pathobiological processes are responsible for transforming stable atherosclerotic plaques into active rupture-prone plaques in ACS.Moreover,the processes may be enhanced in diabetes.So it is important for clinician to detect the vulnerable plaque and seek the method to stabilize the plaques.As a method to diagnose the coronary disese,the coronary angiography(CAG)has several inherent limitations because it is unable to take account of arterial remodeling.Incontrast with angiography,Intravascular ultrasound(IVUS)is unique in its ability to assess both the intraluminal plaque and the surrounding arterial wall morphology.IVUS provides cross-sectional morphometric detail and quantifies atherosclerotic plaque area and plaque burden.Endothelial progenitor cells(EPCs)are capable of differentiating into endothelial cells and homing to sites of ischaemia and play a important role in vascular repair and reendothelialisation of injured blood vessels and collateral vessels formation. Moreover,the endothelial dysfunction and damage are common starting points for diabetic macroangiopathy.And recent insights show that the injured endothelial monolayer is regenerated by circulating EPCs,which accelerate reendothelialization and limits atherosclerotic lesion formation.So the EPCs would play a vital role in the diabetic macroangiopathy.Then it would be of significance to assess the correlation between EPCs levels and extent of lesion in diabetic coronary disease.There was analysis of the CAG show that the lesion is more extensive and severe,the ability to form collateral circulation is descent in patients with DM.And it was reported that the number of circulating EPCs has been impaired in patients with diabetes mellitus and been increased in patients with ACS.However,few data are available on the relationship between the number of circulating EPCs and the instability of plaque as well as the extension of lesion assessed by intravascular ultrasound in diabetic patients with ACS.The objectives of this study are as follow:objectives1.To investigate the coronary culprit plaque in diabetic patients with ACS by IVUS.2.To investigate the number of circulating EPCs in diabetic patients with ACS.3.To investigate the potential relation between the number of circulating EPCs and plaque burden and severity of coronary culprit lesions using IVUS in diabetic patients with ACS.Methods1.SubjectsFrom July 2007 to January 2008,40 patients with diagnosis of ACS with de novo culprit lesions underwent preintervention IVUS were enrolled in this study.Among the subjects,there were 24 patients with unstable angina,16 patients with acute myocardial infarction.Acute myocardial infarction was defined as continuous chest pain at rest with abnormal levels of cardiac enzymes(creatininekinase-MB or troponin T).The culprit lesion in ACS was identified by the combination of left ventricular wall motion abnormalities,electrocardiographic findings,angiographic -lesion morphology.The lesion with the worst diameter stenosis was selected as the target lesion for IVUS analysis.According to the American Diabetes Association 2005 criteria for insulin-independent diabetes mellitus,the patients were classified to diabetic group and non-diabetic group:diabetic group(n=20),nondiabetic group (n=20).The medical history,including the pre-existing coronary riskfactors such as hypertension,hypercholesterolemia,and smoking,was recorded for each patient.2.Examinational Methods2.1 Coronary angiography:Coronary angiography was performed according to standard angiographic techniques.Angiographic scoring was done by interventional cardiologists who were blinded to the study protocol.Angiographic diagnosed criteria fulfilling the Coronary Artery Surgery Study:The left main coronary arterywas defined as a lumen diameter decrease of at least 50%,the anterior descending,left circumflex,and right coronary arteries were at least 70%,respectively.2.2 intravascular ultrasoundAfter completion of the diagnostic coronary angiography,the culprit lesion was localized according to the angiographic signs of vessel morphology and electrocardiogrampatterns.Before the IVUS procedure,200μg intracoronary nitroglycerine was administered to prevent vasospasm,and a 2.9F IVUS imaging catheter(Eagle Eye;Volcano Corporation,USA)was advanced distally to the culprit lesion and pulled back at a pull-back speed of 0.5 mm/sec.In parallel,the images were recorded on videotapes for subsequent off-line analysis.IVUS images were analyzed independently by 2 experienced observers.Conventional gray-scale quantitative IVUS analyses were performed according to criteria of the clinical expert consensus document on IVUS to include external elastic membrane(EEM),luminal, and plaque areas.The external elastic membrane cross-sectional area(defined as the area encompassed by the media-adventitia border)were measured in diastolic frames. The plaque cross-sectional area,defined as the intimamedia area,was calculated from the difference between the external elastic membrane and lumen cross-sectional areas. The plaque burden on each cross section was calculated as 100×(external elastic membrane cross-sectional area-lumen cross-sectional area)/external elastic membrane cross-sectional area).2.3 EPCs Culture,Characterization and Count2.3.1 EPCs Culture Assay After placement of an arterial sheath,3 to 5 ml of blood was wasted and then 20 ml of whole blood obtained for cell culture.The heparinized specimen was diluted with phosphate buffered saline(PBS),and the specimen was centrifuged across a 1.077 density gradient over human lymphocyte separating medium(LTS-1077)at 2000 rpm for 30 min.Then mononuclear cells were isolated from the diluted peripheral blood. Immediately after isolation,1×10~6 mononuclear cells were plated on 24-well culture dishes pre-coated with 0.1%gelatin(Sigma,USA)and maintained in M199 medium (Gibco,USA)supplemented with 20%fatal calf serum,100μg/mL streptomycin, 100U/ml penicillin,40 ng/mL human vascular endothelial growth factor(VEGF, Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA),10 ng/mL epidermal growth factor(EGF,Peprotech,USA),and 10U/mL heparin.After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS, new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.2.3.2 Characterization of EPCsFluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FITC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,USA)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyani -ne(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular Probe,USA). To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%paraformaldehyde for 10 minutes. After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..2.3.3 Count of EPCsDual-stained positive cells were counted per well.Two independent investigators who was blinded to the clinical profile of the patients evaluated the number of EPCs per well by counting 3 randomly selected high-power fields.Results The culprit lesion was localized in the left anterior escending,left circumflex,or right coronary artery in 22(55%),8(20%)and 10(25%)patients,respectively.IVUS dataIVUS findings of culprit lesions,comparing diabetic patients and without diabetes. The minimum luminal area site lumen,external elastic membrane,and plaque plus media cross-sectional areas were 3.3±1.6,14.3±3.0 and 11.1±2.7 mm~2 respectively, whereas the plaque burden was 76.5%±11.7%in diabetes group.Another group,the minimum luminal area site lumen,external elastic membrane,and plaque plus media cross-sectional areas were 4.9±2.2,13.8±3.4 and 9.0±2.6 mm~2 respectively,and the plaque burden was 66.2%±13.2%.DM group had greater pique burden compared with the nondiabetes.EPCs NumberWhen patients were classified according to DM or nondiabete group,the significantly lower levels of EPCs were found in the patients with DM,compared with EPCs levels in the patients without DM(30.4±6.99 vs.51.7±11.1 EPCs/×200 field,P＜0.05).Correlation between EPCs number and plaque burden determined with IVUSThe lower levels of circulating EPCs were measured in association with lesion with greater plaque burden.In addition,the plaque burden of the lesion was associated with significantly decreased levels of EPCs(r=-0.427,P＜0.05)in diabetes group.Conclusions1.Coronary culprit plaque in ACS patients with DM showed a higher extent of coronary stenosis,a greater plaque burden and a smaller lumen cross-sectional area.2.The number of circulating EPCs was significantly lower in patients with DM compared to those without DM.3.The number of EPCs was negatively associated with the plaque burden.Decreased number of circulating EPCs may reflect the severity of coronary culprit lesion in diabetic patients with ACS.4.Given the important role of EPCs in process of atherogenesis,a decrease in the number of EPCs may contribute to the accelerated atherogenesis and impaired endothelial regeneration in diabetic patients with ACS.