The Basic Research Of The Impact Of Hyperinsulinemia On Endothelial Progenitor Cells And The Relationship Between Insulin Resistance And The Extent Of Coronary Artery Disease

Atherosclerosis and its clinic consequence,such as coronary atherosclerotic disease, have become the leading health problem worldwidely.Accumulating evidence indicates the origin of atherosclerosis(AS) as the imbalance between endothelial impairment caused by multiple risk factors and its endogenous repair processes.It has been recognized for many years that proliferation of mature endothelial cells adjacent to sites of damage was the only means of endothelial regeneration.However,Bone marrow-derived 'endothelial progenitor cells'(EPCs) which have the potential to proliferate and differentiate into mature endothelial cells have been identified as significant contributors to endogenous vascular repair. Researches have discovered that various pro-atherosclerosis risk factors such as hypercholesteremia,hypertension,hypercysteinemia,hyperglycemia can lead to decrease in the number and function of EPCs.This indicates that EPCs have an important role in the occurrence and progression of AS.Diseases featured by insulin resistance(IR) such as diabetes mellitus,obesity,impaired glucose tolerance and dyslipidemia are all important components of cardiovascular dysfunction.They lead to hypertension,coronary artery disease and coronary atherosclerosis by endothelial dysfunction.IR can cause endothelial dysfunction by various mechanisms.The number and function of EPCs in patients with diabetes mellitus(DM),metabolic syndrome or healthy volunteers with IR are found to be decreased,from which that IR can inhibit EPCs is speculated.However,nothing about the direct effect of IR on EPCs is known.Firstly,high insulin concentration was used in vitro to explore its impact on eNOS expression of cultured EPCs isolated from cord blood. Secondly,in order to find the influence of IR on EPCs,the difference of the number and function of EPCs from patients with stable angina pectoris(SAP) with and without IR was studied.Finally,the relationship between IR and the extent of coronary artery disease in patients with SAP but without DM was investigated to make clear the role of IR in the progression of AS.This study is aimed to explore the change of EPCs under IR and its role in AS in the aspect of cellular level and clinic epidemiology.It is made up of three parts:PartⅠ:The impact of hyperinsulinemia on eNOS expression of endothelial progenitor cells in vitro Objective Hyperinsulinemia is a kind of compensatory or post-treatment phenomena,it has been reported as an independent risk factor of atherosclerosis(AS) by many studies.However,the exact mechanism by which hyperinsulinemia leads to AS is unclear.Bone marrow-derived endothelial progenitor cells(EPCs) is a group of cells with the potential to proliferate and differentiate into mature endothelial cells and participate in the repair process of injured vascular.This study is aimed to investigate the impact of hyperinsulinemia on eNOS expression of human EPCs in vitro in order to find the mechanism by which hyperinsulinemia causes AS.Methods:Mononuclear cells(10~6-10~7 in total) were isolated by Ficoll density-gradient centrifugation from cord blood(80-90ml/unit).After seven days culture with VEGF,EPCs formed typical colony,which was confirmed by Dil-acLDL(Dil-acetylated low-density lipoprotein)as EPCs in differentiation.Each culture well(10~4-10~5/well) was treated with insulin at concentrations of 0,1nM,10nM,100nM,1000nM for 24 hours respectively. RNA was then extracted by Trizol and eNOS mRNA expression was assessed by RT-PCR.Results eNOS expression began to increase from 1nM,peaked at 10nM,and began to decrease at 100nM,which indicates that physical concentration of insulin(1nM) as well as 10 times of it promote eNOS expression while concentrations more than that(＞10nM) inhibit its expression.Conclusion To some extent,higher level concentration of insulin than physical concentration has promotive effect on EPCs function.However,if the concentration is too high,insulin will have inhibitory effect on EPCs.This indicates that with the severity of insulin resistance increases,the dysfunction of EPCs caused by hyperinsulinemia may have adverse effect on the development of atherosclerosis.PartⅡ:The number and function of circulating endothelial progenitor cells in patients with stable angina pectoris accompanied with insulin resistanceObjective:Diseases featured by insulin resistance such as diabetes mellitus, obesity,impaired glucose tolerance and dyslipidemia are all important components of cardiovascular dysfunction.They lead to coronary atherosclerosis and coronary artery disease by endothelial dysfunction.Recent research regards the origin of atherosclerosis (AS) as an imbalance between endothelial damage and repair process.Endothelial progenitor cells(EPCs) which derive from bone marrow can regenerate endothelium by differentiation into mature endothelial cells.Patients with stable angina pectoris(SAP) but without DM was chose in this study to explore the relationship between insulin resistance and the number and function of EPCs.Methods:27 patients with clinical diagnosis of SAP was chose,of which 19 are males and mean age is 58±7 years old.Biochemial indexes such as fasting serum insulin concentration, blood lipid levels,blood glucose were measured.The extent of insulin resistance was evaluated by McAuley Index and the whole subjects were divided into groups according to McAuley Index(McAuley index＜5.8 as insulin resistant group including 13 subjects and McAuley index＞5.8 as non-insulin resistant group including 14 subjects).Coronary score was used to judge the extent of AS.100μl peripheral blood was obtained to do flow cytometry analysis(EPCs were defined by the surface markers of CD133+VEGFR-2+) and 12ml was used to isolate mononuclear cells by Ficoll density-gradient centrifugation for culture,colony forming-units(CFUs) were counted after 7 days.Results:Insulin resistant group had higher coronary score(2.34±2.73 vs.0.68±0.82, p＜0.05 ) and lower HDL-C level((1.08±0.17mmol vs.1.30±0.32mmol/L,p＜0.05).The number and colony-forming units of EPCs are less in IR group than in non-IR group(0.035±0.046%vs.0.116±0.112%,54.9±28.9 vs.75.2±20.7,respectively,p＜0.05).Conclusion:In patients of SAP with insulin resisitance,coronary artery disease are severer and the number and function of EPCs are decreased.The dysfunction of EPCs may be one of the mechanisms by which insulin resistance leads to AS.PartⅢ:The relationship between insulin resistance and the extent of coronary artery disease in patients with stable angina pectorisObjective:Recent researches find out that insulin resistance leads to endothelial dysfunction which promotes the progression of atherosclerosis(AS) and it is an independent risk factor of coronary artery disease.Patients with clinical diagnosis of stable angina pectoris(SAP) accompanied with insulin resistance but without diabetes were chose to explore the relationship between insulin resistance and coronary artery disease. Methods:A total of 272 patients with SAP to do coronary angiography were enrolled into the study,of which 199 were males and mean age was 59±8 years old.Biochemical indexes such as fasting blood insulin concentration,blood lipids and blood glucose were measured.The extent of insulin resistance and coronary artery disease were judged by McAuley index(McAuley index＜5.8 as insulin resistant,McAuley index≥5.8 as non-insulin resistant) and coronary score separately.Results:85 patients(31.25%) were found insulin resistant,76(27.94%) without coronary atherosclerosis,78(28.68%) with coronary atherosclerosis but with stenosis＜50%, 118(43.3%) with stenosis≥50%.McAuley index had significant correlation with coronary score(R=-0.153,p＜0.05 ),while fasting insulin concentration had no statistically significant correlation with coronary score(p＞0.05).Groups were divided according to coronary score(＜1.89,1.9-4.83,≥4.84,3 groups in all) and McAuley index was lower in group with high coronary score(p＜0.05).Fasting insulin concentration showed no statistical significance among each group.Conclusion:The extent of insulin resistance has positive correlation with the extent of coronary artery disease,while fasting insulin concentration has no statistically significant correlation.