Effects Of Melatonin On Proliferation,Apoptosis And Signal Transduction Pathway Of Ox-LDL Induced In Endothelial Progenitor Cells

Effects of Melatonin on Proliferation,Apoptosis and Signal Transduction Pathway of Ox-LDL Induced in Endothelial Progenitor CellsBACKGROUNDStudies have identified a cell population termed endothelial progenitor cells(EPCs)that have the capacity to circulate,proliferate,and differentiate into mature endothelial cells,but which have not yet acquired characteristic mature endothelial markers and have not yet formed a lumen.EPCs were initially described by Asahara et al.The majority of circulating EPCs reside in the bone marrow in close association with hematopoietic stem cells and the bone marrow stroma that provides an optimal microenvironment.A lot of studies indicate that CD34,VEGFR-2,and AC133 is currently the best selective marker for identifying EPC and that circulating CD34~+,VEGFR-2~+,and AC133~+ cells constitute a phenotypically and functionally distinct population of circulating EC that may play a role in postnatal vasculogenesis.Laboratory evidence has suggested that EPCs participate in postnatal neovascularization and reendothelialization.Derived from bone marrow sources and peripheral mononuclear cells,circulating EPCs have been shown to possess several characteristics that may facilitate the use of these cells as novel markers of endothelial dysfunction as well as of ongoing tissue repair and/or regeneration.Characteristics such as their pluriopotency,ability to regenerate damaged endothelium,and the ability to "home" to damaged or ischemic tissue and contribute to neovascularization have elevated interest in these cells with novel prognostic and therapeutic goals in mind. Moreover,EPCs have recently been shown to affect the progression of coronary artery diseases(CAD).EPCs represent a promising biomarker of endothelial dysfunction,one of the earliest stages of atherogenesis.In a study of patients with risk factors for CAD,reduced levels of EPCs correlated with higher degrees of endothelial dysfunction. Conversely,augmenting EPCs volume through mechanisms,including transplantation,facilitates neovascularization and re-endothelialization and attenuates myocardial ischemia,supporting a role for EPCs in maintaining the homeostasis of the endothelial wall.Assessing for EPC levels may be more useful,given their correlation with established risk factors for CAD,their correlation with the presence of atherosclerosis, and with their prognostic ability in patients with established CAD.Compromise of endothelial integrity is felt to be fundamental to the initiation and progression of CAD.Irrespective of the underlying contributor,endothelial damage and dysfunction remain integral to atherogenesis and the development of CAD.Oxidized low density lipoprotein(Ox-LDL)is believed to play a key role in the development of atherosclerosis.Ox-LDL leads to endothelial activation,dysfunction and injury.Furthermore,ox-LDL have recently been reported to prevent EPCs differentiate and promote EPCs aging. However,the mechanisms mediating the effects of ox-LDL on EPCs remain to be determined.Subsequently,the present study is to investigate whether ox-LDL can decrease EPCs numbers,prevent EPCs proliferation,migration and adhesion in vitro,to investigate the possibl mechanisms.In addition,the present study is also to investigate the role of melatonin.PartⅠEffect of melatonin on the oxidized low-density lipoprotein--induced endothelial progenitor cells proliferation,apoptosis and functionOBJECTIVE:To explore the effect of Melatonin(MT)on the oxidized low-density lipoprotein(ox-LDL)-induced proliferation, apoptosis and function in endothelial progenitor cells(EPCs)from human umbilical cord blood in vitro.METHODS:Total mononuclear cells were isolated from human umbilical cord blood in vitro by Ficoll density gradient centrifugation, then the cells were plated on fibronectin-coated culture dishes.Grouping of cells after 7 days:control group contained normal cells;ox-LDL groups,the attached cells were incubated with different(5,10,20mg/ L)concentrations of ox-LDL for(6,12,24,48)hours;MT groups,the attached cells were incubated with different(0.5,1.0,2.0mmol/L) concentrations of MT respectively for(6,12,24,48)hours before incubated with 10mg/L ox-LDL.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fuorescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of CD34,VEGFR-2 and CD133 with flow cytometry.MTT assay was used to detect the effect of MT and ox-LDL on the multiplication ability of EPCs.EPCs adhesion assay was performed by replating it on fibronectin-coated dishes,and the adherent cells were then counted.EPCs migration were assayed by modified Boyden chamber assay.Flow cytometry was used to detect the apoptosis.RESULTS:After exposured to ox-LDL,the proliferation,adhesion,migration and amount of EPCs in ox-LDL group was lower than that in control group in dose-dependent manner and time-dependent manner(P＜0.01),and the apoptosis rate was higher in dose-dependent manner(P＜0.01);Addition of MT at different concentrations respectively before ox-LDL incubation yielded higher proliferation and lower apoptosis rate(P＜0.01);and function of EPCs in MT group was improved.CONCLUSION:Ox-LDL can inhibit the amount,adhesion, migration and proliferation of EPCs and promote the apoptosis of the cells in a concentration-dependent manner and time-dependent manner. MT can inhibit these effects of ox-LDL. PartⅡStudy of Signal Transduction Pathway of Ox-LDL Inducing endothelial progenitor cells apoptosisOBJECTIVE:To understand the signal transduction pathway of ox-LDL inducing apoptosis in endothelial progenitor cells(EPCs)from human umbilical cord blood in vitro.METHODS:Total mononuclear cells were isolated from human umbilical cord blood in vitro by Ficoll density gradient centrifugation, then the cells were plated on fibronectin-coated culture dishes.Grouping of cells after 7 days:control group contained normal cells;ox-LDL groups,the attached cells were incubated with different(5,10,20mg / L)concentrations of ox-LDL for 24 hours;MT + ox-LDL groups,the attached cells were incubated with different(0.5,1.0,2.0mmol/L) concentrations of MT respectively for 24 hours before incubated with 10mg / L ox-LDL.Wortmannin groups,the attached cells were incubated with Wortmannin(100μmol/L)for 24 hours before incubated with 10mg/L ox-LDL.MT + Wor + ox-LDL groups,the attached cells were incubated with MT(1.0mmol/L)for 24 hours before incubated with 10mg / L ox-LDL and 100μmol / L Wortmannin.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fuorescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of CD34,VEGFR-2 and CD133 with flow cytometry.MTT assay was used to detect the effect of MT and ox-LDL on the multiplication ability of EPC.Flow cytometry was used to detect the apoptosis.The expressions of bcl-2 mRNA and protein were detected respectively by RT-PCR and immunohistochemistry technology.The expressions of p-Akt protein were detected by western blot.RESULTS:After exposured to ox-LDL,the proliferation of EPCs in ox-LDL group was lower,and the apoptosis rate was higher than that in control group in dose-dependent manners(P＜0.01);Addition of MT at different concentrations respectively before ox-LDL incubation yielded higher proliferation and lower apoptosis rate(P＜0.01);Expression of bcl-2 mRNA,protein and p-Akt protein of EPCs in MT group was higher than that in ox-LDL group(P＜0.01).CONCLUSION:Ox-LDL can inhibit the proliferation of EPC and promote the apoptosis of the cells by down-regulating the p-Akt and bcl-2 expression.The PI3K/Akt signaling pathway mediates the ox-LDL-induced apoptosis.MT can inhibite these effects of ox-LDL.PartⅢEffect of melatonin on the proliferation and apoptosis in endothelial progenitor cells from patients with coronary artery disease in vitroOBJECTIVE:To explore the effect of melatonin(MT)on the proliferation and apoptosis in endothelial progenitor cells(EPCs)from patients with coronary artery disease in vitro.METHODS:Total mononuclear cells were isolated from patients with coronary artery disease in vitro by Ficoll density gradient centrifugation,then the cells were plated on fibronectin-coated culture dishes.Grouping of cells after 7 days:control group contained untreated cells;MT groups,the attached cells were incubated with different(0.5,1.0,2.0mmol/L)concentrations of MT for(6,12,24,48) hours.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fuorescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of CD34,VEGFR-2 and CD133 with flow cytometry.MTT assay was used to detect the effect of MT on the multiplication ability of EPCs.EPCs adhesion assay was performed by replating it on fibronectin-coated dishes,and the adherent cells were then counted.EPCs migration were assayed by modified Boyden chamber assay.Flow cytometry was used to detect the apoptosis.The expressions of bcl-2 mRNA and protein were detected respectively by RT-PCR and western blot.RESULTS:After exposured to MT,the proliferation,adhesion and migration of EPCs in MT group was higher,and the apoptosis rate was lower than that in control group in dose-dependent manner time-dependent manner(P＜0.01);Expression of bcl-2 mRNA and protein of EPC in MT group was higher than that in control group(P＜0.01).CONCLUSION:MY can promote the proliferation,adhesion and migration of EPCs and inhibit the apoptosis of the cells in a concentration-dependent manner and time-dependent manner.MT inhibits the apoptosis of EPCs by up-regulating the bcl-2 expression.