EPCs and ERS

Homocysteine-Induced Caspase-3Activationby Endoplasmic Reticulum Stress in Endothelial Progenitor Cells from Patients with Coronary Heart Disease and Healthy DonorsLi L I,1Bang-chuan H U,2Shi-jin G ONG,1and Jing Y AN1;y1Intensive Care Unit and Zhejiang Provincial Key Laborary of Geriatrics,Zhejiang Hospital,12Lingyin Road,Hangzhou,China2Intensive Care Unit,Zhejiang Provincial People’s Hospital,158Shangtang Road,Hangzhou,ChinaReceived January28,2011;Accepted April15,2011;Online Publication,July7,2011[doi:10.1271/bbb.110074]Previous studies have suggested an association of hyperhomocysteinemia-induced vascular pathology with enhanced apoptotic potential of endothelial pro-genitor cells in patients with coronary heart disease. Our results indicate that500 mol/L homocysteine induced endothelial progenitor cell apoptosis and acti-vation of caspase-3,both of which were abolished by 100 mol/L and200 mol/L salubrinal,an agent that prevents endoplasmic reticulum stress-induced apopto-sis.The addition of500 mol/L homocysteine caused a release of Ca2þfrom intracellular stores,and enhanced phosphor-eukaryotic initiation factor2 phosphoryla-tion at Ser51and the expression of a glucose-regulated protein of78kDa and a C/EBP homologous protein independently of extracellular Ca2þ.These effects of homocysteine on endothelial progenitor cells were significantly greater in patients with coronary heart disease than in healthy donors.Thesefindings suggest that homocysteine induces endoplasmic reticulum stress-mediated activation of caspase-3in endothelial progenitor cells,an event that is enhanced in patients with coronary heart disease.Furthermore,enhanced endoplasmic reticulum stress-mediated activation of caspase-3in endothelial progenitor cells might be involved in hyperhomocysteinemia-associated vascular pathology.Key words:homocysteine;endothelial progenitor cells;apoptosis;endoplasmic reticulum stressIt is generally recognized that hyperhomocysteinemia (HHcy)is an important risk factor for coronary artery disease(CAD),1)but the pathogenesis of its vascular action remains to be elucidated.Accumulating evidence has demonstrated that endothelial dysfunction plays a major role in HHcy-related vascular pathology.2)Endo-thelial dysfunction is a loss of balance between injury and repair capacity.Recent studies suggest an important role of endothelial progenitor cells(EPCs)in endothelial repair after injury.3)CAD Patients have decreased numbers of EPCs with impaired functional capacity.4) In vitro,homocysteine(Hcy)dose-and time-depend-ently decreases EPC numbers and impairs cell prolifer-ation,migration,and vasculogenesis capacity.5)Im-paired EPC numbers and functional capacity have been identified in a murine model with HHcy.6)One possible explanation for the reduced capacity of EPCs is increased apoptosis of premature progenitor cells.Recently,Bao found that Hcy induces apoptosis of EPCs in vitro.7) Taken together,thesefindings suggest an association of Hcy with the enhanced apoptotic potential of EPCs in CAD patients.The endoplasmic reticulum(ER)has a high sensitivity to alteration in Ca2þhomeostasis,and a low luminal Ca2þconcentration([Ca2þ]ER)leads to oxidative stress and the accumulation of misfolded or unfolded proteins, resulting in ER dysfunction and stress.8)ER stress is triggered by an accumulation of misfolded proteins in the ER that bind to an ER chaperone glucose-regulated protein of78kDa(Grp78).This causes the unfolded protein response(UPR),resulting in the dissociation of Grp78from the three major ER stress sensors:protein kinase RNA-like ER kinase(PERK),activating tran-scription factor-6(ATF6),and inositol-requiring pro-tein-1(IRE-1).9)PERK-dependent phosphorylation of eukaryotic initiation factor2 (eIF2 )results in trans-lational attenuation,reducing the load of new protein synthesis on the ER.These responses re-establish homeostasis and normal ER function.If ER stress is severe or prolonged,apoptotic cell death is induced.8) Several apoptosis pathways are known to be involved, the central role being played by the proapoptotic transcription factor C/EBP homologous protein (CHOP).8)Transcriptional induction of CHOP depends mostly on activation of PERK/eIF2 .ER stress has been found to be involved in endothelial dysfunction and related vascular pathologies.Upregulation of Grp78 and other ER stress-related genes was detected in a murine model of myocardial infarction.9)Both cytopro-tective and apoptotic components of ER stress signaling are upregulated in heart failure.High levels of apoptosis and of the expression of Grp78and CHOP were found withinfibrous caps of atheroma and ruptured plaques of both autoptic human coronary specimens and atherec-tomy specimens from patients with unstable angina pectoris.10)A previous report provided evidence of the relevance of ER stress to macrophage apoptosis and enlargement of the necrotic core in advanced athero-y To whom correspondence should be addressed.Tel:+86-571-87987373ext5083;Fax:+86-571-87980175;E-mail:yanjing2011@yahoo.-Biosci.Biotechnol.Biochem.,75(7),1300–1305,2011sclerotic plaques.11)Although there are no reports regarding ER stress in EPCs,other resources of adult stem and progenitor cells,such as mesenchymal stem cells,have been reported to be induced to apoptosis via the ER pathway.12)Hence,we hypothesized that there is ER stress-induced apoptosis in EPCs.Hcy is a well-known inducer of ER stress.Apolipo-protein E-deficient mice fed a HHcy diet exhibit increased expression of molecular markers of ER stress in both early and late atherosclerotic lesions.13)In a rat model of HHcy,increased expression of ER stress-associated proteins,including Grp78,CHOP,and cas-pase-12,was found in myocardial tissue.14)In vitro,Hcy induces ER stress and cell apoptosis in cardiomyo-cytes,14)smooth muscle cells,15)and endothelial cells.16) Although these studies indicate that Hcy is closely associated with ER stress-mediated cell apoptosis,it is yet not clear whether ER stress contributes to HHcy-related vascular pathology,or how this might occur. Hence,we investigated the possible role of Hcy in ER stress-mediated apoptosis in EPCs from CAD patients and healthy donors in an attempt to elucidate the mechanisms underlying HHcy-related vascular pathol-ogy associated with CAD.Materials and MethodsMaterials.EGM-2endothelial medium and EGM-2-MV-Single-Quots were purchased from Clonetics(San Diego,CA).Fura-2AM, fura-FF AM,and pluronic acid were from Molecular Probes(Leiden, Netherlands).Hcy,fibronectin(FN),thapsigargin(TG),ionomycin, 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid(BATPA), and anti-glyceraldehyde-3-phosphate dehydrogenase(GAPDH)anti-body were from Sigma-Aldrich(St.Louis,MO).Salubrinal was from Tocris(Bristol,UK).Anti-Grp78and-phospho-eIF2 (Ser51)anti-bodies were from Cell Signaling Technology(Danvers,MA).Anti-CHOP antibody and horseradish peroxidase-conjugated secondary IgG antibody were from Santa Cruz Biotechnology(Santa Cruz,CA). Other reagents are indicated below.Subjects.Patients with CAD were recruited from the Cardiovascular Disease Clinic and Ward at Zhejiang Hospital(Zhejiang,China).A diagnosis of CAD was reached by coronary angiography.Patients with malignant disease,peripheral vascular disease,proliferative retinop-athy,or recent(<2months)acute coronary syndrome,inflammation, bleeding,or blood transfusion were excluded,since these conditions and procedures might have affected the number or function of EPCs. None of the patients had been treated with statins or erythropoietin in the preceding2months.The control subjects were age-and sex-matched healthy volunteers.This study was approved by the Research Ethics Review Board of Zhejiang Hospital and complied with the Code of Ethics of the World Medical Association(Helsinki Declaration of 1964,as revised in2002).Informed consent was obtained in writing from all subjects.Isolation,incubation,and identification of EPCs.EPCs were prepared as described previously.5)Briefly,mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation and cultured on human FN-coated dishes in EGM-2endothelial medium supplemented with EGM-2-MV-SingleQuots and20%FBS.Cellular apoptosis detected byflow cytometer.EPCs were seeded and pre-cultured at37 C for24h.After treatment with various drugs for various durations,EPCs were collected and treated according to the protocol in the Annexin V-FITC Apoptosis Detection Kit (Merck,Germany).The percentages of apoptotic cells were determined with a FACSCaliburflow cytometer with CellQuest software(BD Pharmingen,San Diego,CA).Measurement of caspase-3activity.The activity of caspase-3was evaluated using a colorimetric activity assay kit(Chemicon Interna-tional Molecular,Temecula,CA)following the manufacturer’s instructions.Briefly,the cells were lysed for30min on an ice bath. The lysed cells were centrifuged at12;000Âg for10min.After the protein concentration was determined,100m g of the protein was incubated with50m L of a reaction buffer and5m L of the caspase-3 substrate(Ac-DEVD-pNA)on a96-well plate.The activity of caspase-3was measured using a spectrophotometer at405nm,and the results were expressed as fold increase over mean value of the control.Measurement of the intracellular free calcium concentration ([Ca2þ]c).To monitor[Ca2þ]c,cells were incubated at37 C with 2m mol/L of fura-2AM for45min.Fluorescence was recorded from 1mL aliquots of magnetically stirred cell suspensions(108cells/mL) at37 C using a spectrophotometer(Zeiss,Jena,Germany)with ex 340and380nm and em505nm.Changes in[Ca2þ]c were monitored using the fura-2340/380fluorescence ratio and calibrated.Ca2þrelease was estimated using the integral of the rise in[Ca2þ]c for 2.5min after the addition of agents.Measurement of the free calcium concentration in the intracellular stores([Ca2þ]ER).Endothelial progenitor cells were incubated with 5m mol/L of fura-FF AM for1h in the presence of0.025%pluronic acid,and were loaded with10m mol/L dimethyl BATPA for30min. EPCs were then collected by centrifugation at1;000Âg for10min and resuspended in HEPES-buffered saline.Fluorescence was recorded from1mL aliquots of magnetically stirred cell suspensions (108cells/mL)at37 C using a spectrophotometer(Zeiss)with ex 340nm and em505nm.Changes in[Ca2þ]ER were expressed as F n/F0,considering F0asfluorescence of fura-FF in the absence of free Ca2þ.Immunoblotting.Following treatment,whole-cell lysates were prepared as described previously.17)In some experiments,immuno-blotting for CHOP protein was additionally performed on nuclear extracts prepared using a Nuclear Extraction Kit following the manufacturer’s protocol(Millipore,Billerica,MA).The protein concentration was determined by the Lowry method.Ten m L of protein was loaded in each lane and subjected to SDS–PAGE(8%), and then transferred to a polyvinylidene difluoride(PVDF)membrane. The blots were probed with antibodies against phospho-eIF2 ,Grp78, CHOP,or GAPDH.Horseradish peroxidase-conjugated secondary antibodies were used in conjunction with an ECL chemiluminescence detection system(Roche,Mannheim,Germany)and quantified by scanning densitometry.Statistical analysis.All data are presented as meanÆSEM. Statistical evaluation of the data was performed by Student’s t test for paired or unpaired observations and by analysis of variance (ANOVA).Scheffe’s test for multiple comparisons was used to identify differences among groups.Values were considered to be significantly different at p<0:05.ResultsComparison of the numbers of EPCs from CAD patients and healthy donorsApproximately5Â106mononuclear cells from every CAD patient and healthy control were seeded.The number of EPCs was significantly higher(0:064Æ0:004)in the CAD patients than in the healthy controls (0:032Æ0:004;p<0:01,n¼12in each group) (Fig.1).Comparison of apoptosis of EPCs induced by Hcy from CAD patients and healthy donorsThe apoptosis of EPCs induced by Hcy was examined in the presence of various concentrations of Hcy(0,50, 100,and500m mol/L)over24h and in the presence of 500m mol/L Hcy for0,12h,24h,and48h.As shown inEnhanced Caspase-3Activation in EPCs from CAD Patients1301Fig.2,after 24h Hcy induced a detectable increase in the percentage of apoptotic EPCs with 100m mol/L to 3:8Æ0:8%and 5:1Æ0:9%in healthy donors and CAD patients respectively,with a maximal effect at 500m mol/L (7:4Æ1:2%vs.8:2Æ0:8%,p <0:05),andalso after 48h of treatment (13:7Æ1:3%vs.15:9Æ0:9%,p <0:05)in healthy donors and CAD patients.Hcy induced ER-dependent caspase-3activation in EPCs from CAD patients and healthy donorsCaspase-3activation of EPCs induced by Hcy was examined in the presence of various concentrations of Hcy (0,50,100,and 500m mol/L)for 24h and in the presence of 500m mol/L Hcy for 0,12h,24h,and 48h.Hcy induced caspase-3activity in a dose-and time-dependent manner in healthy donors and CAD patients,with a maximal effect at 500m mol/L (3:8Æ0:5-and 4:7Æ0:4-fold increases vs.control untreated cells;p <0:05,n ¼6in each group)and after 48h of treatment (4:8Æ0:5-and 5:7Æ0:4-fold increases vs.untreated cells;p <0:05,n ¼6in each group)(Fig.3A and B).To determine whether the caspase-3activation in-duced by Hcy is a result of endoplasmic reticulum stress,we used salubrinal,a cell-permeant,selectiveinhibitorparison of Numbers of Endothelial Progenitor Cells (EPCs)from Patients with Coronary Artery Disease (CAD)and Healthy Donors.Mononuclear cells from CAD patients and healthy controls (5Â106per subject)were seeded.Values are mean ÆSEM .ÃÃp <0:01as compared with healthy donors (n ¼12).ABparison of Apoptotic Rates of EPCs from Patients with CAD and Healthy Donors in the Presence of Various Concentrations ofHomocysteine (Hcy)(0,50,100,and 500m mol/L)for Different Durations (0,12h,24h,and 48h).Values are mean ÆSEM .Ãp <0:05as compared with healthy donors (n ¼6).A BC DFig.3.Hcy-Induced Caspase-3Activation Is Endoplastic Reticulum (ER)-Dependent in EPCs.A,EPCs from CAD patients and healthy donors were incubated with various concentrations of Hcy (0,50,100,and 500m mol/L)for 24h and caspase-3activation was evaluated using a colorimetric activity assay kit.B,EPCs from CAD patients and healthy donors were incubated with 500m mol/L Hcy for different durations (0,12h,24h,and 48h).C and D,Pretreatment for 30min with various concentrations of salubrinal (10,100,200m mol/L)or vehicle with 500m mol/L Hcy for a further 24h.Caspase-3activation and the apoptosis rate of EPCs were measured.Values are mean ÆSEM .Ãp <0:05as compared with healthy donors;y p <0:05as compared with 500m mol/L Hcy-treated cells (n ¼6).1302L.L I et al.of eIF2 dephosphorylation that protects cells from endoplasmic reticulum stress.18)EPCs from healthy donors and CAD donors were stimulated with Hcy (500m mol/L)after pretreatment with various concen-trations of salubrinal(10,100,and200m mol/L)for 30min.The Hcy-evoked increase in caspase-3activity was inhibited by pretreatment with salubrinal in both CAD patients and healthy donors,with a maximum effect at200m mol/L,with1:3Æ0:5-and1:6Æ0:4-fold increases vs.control(untreated cells)in healthy donors and CAD patients respectively(p<0:05,n¼6in each group)(Fig.3C).Figure3D shows that salubrinal inhibited Hcy-induced apoptosis of EPCs from healthy donors and CAD patients.Hcy mobilized Ca2þfrom intracellular poolsThe intracellular free calcium concentration and [Ca2þ]ER were measured in the presence of50,100, and500m mol/L Hcy.In a Ca2þ-free medium,treat-ment of fura-2-loaded EPCs with Hcy induced a dose-dependent increase in cytosolic Ca2þdue to Ca2þrelease from intracellular Ca2þcompartments.Cyto-solic Ca2þconcentrations,both at rest and after stimulation with Hcy,were significantly greater in CAD patients than in healthy donors(Fig.4A–D).To investigate further the ability of Hcy to release Ca2þfrom intracellular stores,EPCs were loaded with fura-FF,a common tool to measure compartmentalized Ca2þ.In addition,cells were loaded with dimethyl BAPTA and suspended in a Ca2þ-free medium (100m mol/L EGTA added)to avoid interference with cytosolic and with extracellular Ca2þrespectively.As shown in Fig.4E,Hcy(0,50,100,and500m mol/L) decreased[Ca2þ]ER due to Ca2þrelease in a dose-dependent manner.The Ca2þremaining in the stores after10min of treatment with Hcy was estimated by the addition of TG(1m mol/L),an inhibitor of sarcoendoplasmic Ca2þATPase,in combination with ionomycin(50nmol/L),to deplete the intracellular Ca2þstores fully(Fig.4F).ACEBDFFig.4.Hcy Mobilized Ca2þfrom Intracellular Pools in EPCs.Fura-2-loaded EPCs from CAD patients and healthy donors were suspended in a Ca2þ-free medium and then treated with Hcy(50,100, 500m mol/L).A–C,Traces are representative of four independent experiments.D,Values are meansÆSEM.Ãp<0:05as compared with healthy donors.EPCs from healthy donors were loaded with fura-FF and dimethyl1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid and suspended in a Ca2þ-free medium.Cells were stimulated with Hcy(10,100,500m mol/L)and changes in[Ca2þ]ER were monitored.E, Traces are representative of four independent experiments.F,Ca2þremaining in the stores after10min of treatment with Hcy was estimated by adding a combination of thapsigargin and ionomycin to deplete the intracellular Ca2þstores fully.Values are meanÆSEM.Ãp<0:05as compared with untreated cells.Enhanced Caspase-3Activation in EPCs from CAD Patients1303Hcy induced UPR activation in EPCs from CAD patients and healthy donorsSince Hcy induces Ca2þefflux from intracellular stores,which can induce ER stress,UPR activation was detected by immunoblotting using specific anti-phos-pho-eIF2 and anti-Grp78antibodies.As shown in Fig.5A and B,in a Ca2þ-free medium,Hcy(500 m mol/L)induced time-dependent phosphorylation of eIF2 and expression of Grp78.These were signifi-cantly greater in the EPCs from the CAD patients than from the healthy donors.The maximal effect of Hcy was obtained after48h of treatment in CAD patients and healthy donors.In a medium containing1mmol/L of Ca2þ,similar results were obtained for the phos-phorylation of eIF2 and the expression of Grp78(data not shown).Since prolonged or severe ER stress is linked to the triggering of programmed cell death,the effect of Hcy on CHOP,a major ER stress-inducible pro-apoptotic transcription factor that operates as a downstream component of the ER-stress pathway,was investigated. We found that CHOP expression increased after treatment with Hcy(500m mol/L)for various times (0,12,24,and48h).Maximum CHOP expression was obtained at48h of exposure to Hcy(Fig.5C).In a medium containing1mmol/L of Ca2þ,similar results were obtained for the expression of CHOP(data not shown).DiscussionIn this study,we confirmed the observations of Bao7) and Alam,19)who reported that Hcy induces apoptosis of peripheral EPCs in vitro.We report for thefirst time that the Hcy-induced apoptotic potential of EPCs from patients with CAD was higher than that from healthy donors.Furthermore,Hcy-induced EPC apop-tosis was operational via ER stress-mediated caspase-3 activation.Patients with CAD show decreased numbers of EPCs with impaired functions,which can be due to several mechanisms such as calcium signaling and oxidative stress.The imbalance between oxygen radical genera-tion and antioxidant power plays an important role in the pathogenesis of Hcy-related vascular pathology,and oxidative stress has been found to be involved in the development of vascular and blood cell apoptosis, including EPCs.7)We report for thefirst time that Hcy induced ER stress in EPCs,independently of extracel-lular Ca2þlevels,by promoting Ca2þefflux from intracellular stores.The mechanism underlying Ca2þrelease from intracellular stores by Hcy was not further investigated,but it is probably attributable to the oxidant actions of Hcy.Oxygen radicals have been reported to induce Ca2þrelease in various cell types,including endothelial cells,20)smooth muscle cells,21)and cardio-myocytes.22)ABCFig.5.Hcy Induced Unfolded Protein Response(UPR)Activation in EPCs.EPCs from healthy donors and CAD patients were treated in a Ca2þ-free medium with500m mol/L of Hcy for various durations,and eIF2 phosphorylation and Grp78and CHOP expression were determined by immunoblotting.GAPDH was assayed to verify equal loading of cell lysates.The left panels show representative immunoblots,indicating that Hcy induced eIF2 phosphorylation and Grp78and CHOP expression in a time-dependent manner.The three right panels show quantification of the bands by densitometry.Results are normalized to the levels of GAPDH and are shown as percentages of untreated control cells from four independent experiments.Ãp<0:05as compared with healthy donors.1304L.L I et al.In our study,Hcy enhanced phosphorylation of eIF2 at Ser,and increased the expression of Grp78and CHOP in a time-dependent manner.Other ER stress activators, such as TG can increase Grp78and CHOP,confirming the ability of Hcy to induce ER stress in EPCs.ER stress has been suggested to lead to the activation of certain caspases,including caspase-3,caspase-4,and caspase-9.23)We found that Hcy caused time-and dose-depend-ent activation of caspase-3.The activation of caspase-3 is probably attributable to ER stress,since salubrinal,a cell-permeant inhibitor of eIF2 phosphatase at Ser, prevented the caspase-3activation induced by Hcy. However,another mechanism might be involved in Hcy-induced caspase-3activation.Alam et al.reported that Hcy induces apoptosis in EPCs through the mitochon-drial pathway.19)We found that high concentrations of Hcy induced greater survival/rescue molecules,phosphorylated eIF2 and Grp78expression,and apoptotic molecules, and enhanced CHOP and activated caspase-3expression in peripheral EPCs from CAD patients.Thesefindings might be explained by a loss of efficiency of the UPR due to UPR dysfunction induced by Hcy.UPR dysfunc-tion has been found in a number of disorders,including non-alcoholic fatty liver disease,24)renal disorders,25) and diabetes mellitus.Additional evidence suggests that ER stress and UPR dysfunction play important roles in the pathogenesis of atherosclerosis,contributing to endothelium dysfunction.26)Here we report for thefirst time that there was UPR dysfunction in CAD.Our finding that Hcy-induced EPC actions,including Ca2þmobilization,eIF2 phosphorylation,Grp78and CHOP expression,and caspase-3activation,were significantly greater in patients with CAD than in healthy donors, suggests that Hcy is more efficient at inducing EPC effects in CAD patients than in healthy donors.Taken together with the reported higher Hcy levels in CAD patients,thesefindings suggest that plasma Hcy plays an important role in the pathogenesis of HHcy-related vascular pathology.Our results indicate that ER stress, and subsequently eIF2 phosphorylation and Grp78and CHOP expression,were similar in EPCs from healthy donors and CAD patients at rest.This might be because, despite thefinding that resting[Ca2þ]c in EPCs from CAD patients was enhanced as compared with healthy controls,the amount of Ca2þaccumulated in the stores was comparable in the EPCs from CAD patients and healthy donors.Thus,the ER stress level at rest was comparable in the EPCs from CAD patients and healthy donors.In summary,our results indicate for thefirst time that the Hcy-induced apoptotic potential of EPCs from CAD patients is greater than that from healthy donors. Furthermore,Hcy-induced EPCs apoptosis is opera-tional via ER stress-induced caspase-3activation.The EPCs from CAD patients showed a higher sensitivity to Hcy than those from healthy donors.Thesefindings provide evidence of a new mechanism underlying Hcy-induced apoptosis that might be involved in the patho-genesis of HHcy-related vascular pathology.AcknowledgmentsThis study was partly supported by the Science and Technology Department of Zhejiang Province,China (2006C13018),the Health Bureau of Zhejiang Provence (2009A017,2010KYB017),and the Zhejiang Province Natural Science Foundation(Z2100237,Y2110801). 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