Antagonism Effect Of Amlodipine On Oxidative Low Density Lipoprotein Injuried Rat Marrow Endothelial Progenitor Cells

OBJECTIVE:To investigate the protective effect of amlodipine on oxidative low density lipoprotein treated rat marrow endothelial progenitor cell(sEPCs) and related mechanisms.METHODS:EPCs was isolated from rat marrow, and was divided into three groups,①control, added culture medium( contend 200μmol/L DMSO);②oxLDL group(50μg/ml oxLDL);③Amlodipine group(50μg/ml ox-LDL+0.5μmol/L amlodipine). Adhesion method to isolation EPCs, Immunofluorescence, flow cytometry, endocytosis ac-LDL and binding UEA-1 were associated to identify EPCs, and use MTT method for proliferation, transwell for migration, gelatin adhesion for EPCsadhesion, photshop7.0 for measuring vascular like tube information length, number and size of sigle cell clone form units(CFUs) were identified under microscope, RT-PCR and western blotting were used to measure mRNA and protein of eNOS, cells immunohistochemistry was used to measure eNOS expression within EPCs, and DCFH-DA dyeing for reactive oxygen species (ROS), Griess Reagent for nitric oxide (NO).RESULTS:①Almost 70 percent of CD133+/VEGFR-2+ double positive EPCs can be isolated from rat marrow by adhesion method;②CFUs was found at 3th days, endothelial cells like cobblestone cells was found at 14th days,③Antagonism effect of amlodipine on proliferation of EPCs was found at the concentration 0.5μmol/L;④The migration capacity of EPCs was reduced almost 3 times by 50μg/ml oxLDL, and this inhibitory effect can be recovered by 0.5μmol/L amlodipine;⑤The clone form capacity of EPCs was reduced by 50μg/ml oxLDL obviously(7.56±0.85 vs 2.3±0.45,P<0.01, n=5), and it was partly restored by administrating 0.5μmol/L amlodipine(5.6±1.54 vs 2.3±0.45,P<0.01,n=5);⑥The adhesion capacity of EPCs was remarkably reduced by 50μg/ml oxLDL(75.02±14.13cells/field vs 29.04±8.21 cells/field,P<0.01, n=5) , and it was partly restored by administrating 0.5μmol/L amlodipine (64.12±15.11cells/field vs 29.04±8.21cells/field,P<0.01, n=5);⑦The vascular like tube information was reduced by 50μg/ml oxLDL remarkably (15.13mm±1.62mm/field vs 1.52mm±0.71mm /field, P<0.01,n=5), and it was partly recovered by administrating 0.5μmol/L amlodipine(11.01mm±2.14mm/field vs 1.52mm±0.71mm /field, P<0.01,n=5).Researchs in mechanism:①Both in mRNA and protein of eNOS was reduced by 50μg/ml oxLDL, also the nitric oxide level was reduced(24.35±4.62 vs 11.44±1.15), and it can be restored remarkably by administrating 0.5μmol/L amlodipine;②The ROS level was increased by 50μg/ml oxLDL in EPCs(fluorescence intensity 1.00±0.25vs 2.53±0.32,P<0.01,n=3), and it was decreased by administrating 0.5μmol/L amlodipine(fluorescence intensity 1.56±0.18 vs 2.53±0.32,P<0.01,n=3).CONCLUSIONS:Injury effect of oxLDL can be antagonismed by amlodipine, This effect of amlodipine are related to up-regulate expression eNOS and reduce ROS.