The Study Of Advanced Glycation End Products Regulating Endothelial Progenitor Cells' Function Via Akt/eNoS Sign Pathway

BACKGROUND AND OBJECTIVE:EPCs,known as angioblast or endothelial stem cells,was a precursor vascular endothelial cells,which could differentiate into mature endothelial cells.In 1997,Asahara et al,by the first,separated and confirmed that there existed and could be divided EPCs from human peripheral blood cycle after birthed.EPCs played an important role in the angiogenesis and the restoration of endothelial injury.Studies also showed that EPCs played an important role in the occurrence and development of coronary heart disease recently.The decreased number of EPCs in the peripheral blood result to reduce the capacity of repaireing vascular endothelial dysfunction and increase the incidence of cardiovascular disease.Diabetes mellitus have the same risk with coronary heart disease.In patients with diabetes,Such as atherosclerosis(AS),chronic vascular complications had become a major cause of death and disability.Research showed that the number of EPCs in the peripheral blood of patients with diabetes was less than that of normal man.Study found that EPCs proliferation,adhesion,and other functional was impaired in high glucose conditions in vitro.Study also showed that the advanced glycation end products(AGEs) played an role in the development process of diabetes induced to atherosclerosis.Therefore AGEs on the impact of EPCs had the same significance in vitro study.Our previous study had proved that AGEs effect the functions of EPCs via the p-38 sign pathway.Now,we want to explore whether AGEs regulate the function of EPCs via PI3K/Akt/eNOS pathway also.According to the theory of traditional Chinese medicine(TCM) Tong-xing Luo(TXL) capsule possess the capability to benefit gas,promote blood flow,activate meridians to stop pain,improve the circulation of arteries and veins.TXL capsule can significantly improve the function endothelium in patients with coronary heart disease,stabilize the plaque and prevent the development of atherosclerosis.We also explore the role of Tongxinluo mechanism further.【Methods】一,Culture and identification of human EPCs from peripheral bloodEPCs were cultured according to the previously described techniques.The mononuclear cells(MNCs) were isolated from peripheral blood of healthy human volunteers with Ficoll density gradient centrifugation.Cells were plated on culture dishes coated with human fibronectin(Chemicon) and maintained in Medium 199 supplemented with 10 percent fetal bovine serum,VEGF(10ng/ml),penicillin(100ug/ml) and streptomycin(100μg/ml).After 4d of culture,nonadherent cells were removed by washing with PBS,new medium was applied,and the culture was maintained through 7d.EPCs were characterized as adherent cells double positive for Di-LDL uptaking and FITC-lectin binding by laser confocal microscope,and further demonstrated by expressing CD34,CD133 and VEGFR-2 using flow cytometry.二,Effects of AGEs on the expression of p-Akt,p- eNOS protein of EPCsAt the 7th day,EPCs were synchronized for 24 hours.At the 8th day,EPCs were divised with different concentrations of AGEs and pre-intervention factors(anti-RAGE antibodies,rosiglitazone,Tongxinluo).Akt,p-Akt,p-eNOS protein Expression were test through Western-blot test.Using image-pro plus6.0 software for quantitative analysis,each group were from the gray value p-Akt/Akt,p-eNOS /β-actin compared to a control group compared to the value of the denominator percentage.Study of different concentrations and pre-intervention on the p-Akt,p-eNOS expression of EPCs.三,Effects of AGEs on the function of EPCsAt the 7th day,EPCs were synchronized for 24 hours.Divised with the blank control, AGEs,anti-RAGE antibody,rosiglitazone,Tongxinluo,SNP,L-NAME,LY294002 group,we detected migration with small rooms Transwell,CCK-8 kit to proliferation,FCM to apoptosis,NO kit to NO generation,VCAM-1 ELISA kit to VCAM-1 expression of EPCs.【Results】一,Characteristics of human EPCsAfter 7 days culture the MNCs showed as spindles-shaped,endothelial cells-like morphology.EPCs were characterized as adherent cells with double positive for Di-LDL-uptake and lectin binding with laser scanning confocal microscope.Further the cells were demonstrated by expressing CD34~+(0.823±0.072) and CD133~+(0.855±0.050) using flow cytometry,VEGF-R2~+(0.826±0.053). 二,Effects of AGEs on the expression of p-Akt,p- eNOS protein of EPCsWith the increasing concentration of AGEs,Akt expression of each group had no significant difference,but p-Akt,p-eNOS expression reduced,100μg / ml AGEs group p-Akt/Akt ratio than the control group dropped 34.5%,p-eNOS /β-actin dropped 56.3%, 200μg / ml AGEs group p-Akt/Akt ratio than the control group dropped 66.7%,p-eNOS /β-actin decreased 84.3%(P <0.05),200μg / ml AGEs group to join anti-RAGE antibody, rosiglitazone,Tongxinluo pre-intervention,p-Akt/Akt ratio respectively over 200μg / ml AGEs group increased by 56.2%,53.8%,30.7%(P<0.05);p-eNOS /β-actin ratio respectively over 200μg / ml AGEs group increased by 55.1%,46.8%,25.6%(P<0.05).三,Effects of AGEs on the function of EPCs200μg/ml AGEs group could significantly promote EPCs apoptosis and increase the expression of VCAM-1,AGEs reduce the capacity of EPCs migration,adhesion, proliferation,NO production.L-NAME and LY294002 can produce the same effect.SNP, anti-RAGE antibodies,rosiglitazone and Tongxinluo can reverse this effect partially.【Conclusion】一,EPCs could be isolated and cultured from human peripheral blood through the method of Ficoll density gradient centrifugation.二,AGE-HSA promoted EPCs apoptosis and increase the expression of VCAM-1, reduced the capacity of EPCs migration,adhesion,proliferation,NO production.三,AGEs might impact EPCs functions via Akt / eNOS signaling pathway四,SNP,anti-RAGE antibodies,rosiglitazone,TXL could inhibit the effect of AGEs partially...