Effects Of Glucose And Insulin On Proliferation And Function Of Rat Bone Marrow-derived Endothelial Progenitor Cells

Background and AimsEndothelial progenitor cells (EPCs) are bone marrow-derived progenitor cells that express surface markers like AC133 and flk-1(vascular endothelial growth factor receptor), and differentiate into mature endothelial cells on the vascular wall, thus display important role in repair of endothelial injuries and revascularization of infarcted areas. However, many risk factors of cardiovascular diseases, like hyperlipidemia and hyperglycemia, can reduce the number of circulating EPCs and impair its functions. Researches indicate that decreased circulating EPCs number predicts higher cardiovascular event risks. Many researches have been carried on to fully understand the modulation mechanism of EPC proliferation and function. However, the effect of insulin on EPC has not been clarified.The present study intends to explore the effects of insulin on EPC proliferation, senescence,adhesion and nitric oxide(NO) secretion under both normal and high glucose conditions, and the effects of glucose on marrow-derived EPC. The results may help to understand the cardiovascular effects of insulin and glucose from a new aspect. MethodsPartⅠ: EPC culture and surface marker identification. 1. Harvest rat bone marrow, collect mononuclear cells through density gradient centrifugation, and culture them in Medium 199 supplemented with vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF). 2. Immunofluorescent co-stain with flk-1 and AC133 to identify EPC.PartⅡ: The effects of insulin and glucose on EPC. 1. Collect EPCs at 5th day of culture, then add different doses of glucose and insulin to the medium and incubate for 2 or 7 days. 2. Test the proliferative ability of EPC by MTT assay. 3. Detect the senescent level of EPC by senescence associatedβ-Gal staining. 4. Measure the adhesion ability of EPC by adhesion assay. 5. Measure the secretion of NO by modified Griess reaction method after 24 h of incubation.Statistical analysis was performed with SPSS version 13.0. One-way analysis of variation and post hoc t (LSD-t) test were employed.Results1. The cells obtained showed double positive for AC133 and flk-1, indicating their endothelial lineage and progenitor property, i.e., they are EPCs.2. Compared with control group, insulin markedly promoted EPC proliferation and adhesion after 7 d of incubation. Low concentration insulin(0.1, 1 nmol/L) can lower the EPC senescent rate after 7 d of incubation, while insulin group didn't differ from control group in senescent rate. Low concentration insulin (0.1, 1 nmol/L) promoted NO production of EPC, while higher concentration insulin didn't show obvious promotive effect.3. High concentration glucose(20 mmol/L) promoted EPC proliferation after 2 d of incubation. But after 7 d, high glucose(40 mmol/L) inhibited EPC proliferation and adhesion, accelerated EPC senescence. High glucose inhibited NO secretion after 24 h of incubation. Osmotic control (5 mmol/L glucose and 35 mmol/L mannitol) was comparable with 5 mmol/L glucose group in proliferation, senescence and NO secretion.4. Compared with 40 mmol/L glucose group, insulin incubation under high glucose condition for 7 d markedly promoted EPC proliferation, which peaked at 1 nmol/L. 0.1 nmol/L and 1 nmol/L insulin decreased EPC senescent rate, while 10 nmol/L insulin didn't. Insulin enhanced NO production under high glucose conditions after 24 h, which peaked at 10 nmol/L. However, insulin didn't have promotive effects on EPC adhesion under high glucose conditions.Conclusion1. High glucose can promote EPC proliferation ex vivo in a short period.2. High glucose can inhibit EPC proliferation and adhesion, quicken EPC senescence, and inhibit NO production ex vivo after a longer period.3. High glucose affects EPC through an osmosis-independent way.4. Insulin can promote EPC proliferation and NO secretion, slow its senescence under both normal and high glucose conditions. In other words, insulin can alleviate high glucose's damage to EPCs.5. Low level insulin can promote EPC adhesion under normal glucose conditions but not high glucose conditions.6. EPC's sensitivity to insulin stimulation in NO production decreases in high glucose conditions.7. Overhigh concentration of insulin (10 nmol/L,100 nmol/L) has none or only minor promotive effect in EPC proliferation and NO secretion.