Low Frequency Pulsed Electromagnetic Fields Antagonize CRP-induced Inhibition Of Rat Bone Marrow-derived Endothelial Progenitor Cells Proliferation, Apoptosis And Nitrite Oxide Secretion

Background and AimsNeovascularization is a vital compensatory response in chronic ischemia. Myocardial ischemia provides a potent stimulus to angiogenesis and the sub- sequent development of collateral vasculature that maintains and/or revitalizes cardiac tissue. The mobilization and differentiation of bone marrow–derived endothelial progenitor cells (EPC) has recently been shown to be important in this process of adult neovascularization. The number and migratory activity of circulating EPC has also been shown to inversely correlate with risk factors for coronary artery disease. As one of the risk factors of coronary artery disease, its blood density elevates obviously.It had been proved that CRP could increase apoptosis,inhibit proliferation of EPC,attenuate the expression of EPC-specific marker,impair EPC-mediated angiogenesis.With the modern technology unceasing development, people more and more exposed in low frequency electromagnetic field. Its biology effect also more and more receives the people to pay attention. The low frequency pulsing electromagnetic field may promote the person umbilical vein endothelial cell and the cow aorta endothelial cell's multiplication, promotes angiogenesis; Stimulation the secretion of endothelial cell FGF, TGF-β- the main promotionfactors of arteries form.The present study intends to explore the effects of low frequency pulsed electromagnetic fields on EPC proliferation, apoptosis and nitric oxide(NO) secretion under CRP on marrow-derived EPC. The results may help to find the new way to treat ischemic heart disease. MethodsPartⅠEPC 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 DiI-ac-LDL and FITC-UEA-I to identify EPC.PartⅡThe effects of CRP on EPC.1. Collect EPCs at 4th day of culture, then plated in the absence or presence of CRP (5 to 20μg/ml).The treatment was removed after 72 hours, and EPCs were grown until day 7, with culture media changes every 48 hours.2. Test the proliferative ability of EPC by MTT assay. 3. Measure the secretion of NO by modified Griess reaction method.The effects of low frequency pulsed electromagnetic fields on EPC cultured with CRP(12μg/ml). 1. Collect EPCs at 4th day of culture, then plated in the absence or presence of LFPEMFs(0.2-1.4 mT,2 or 8 h), CRP (12μg/ml).The treatment was removed after 72 hours, and EPCs were grown until day 7, with culture media changes every 48 hours.2. Test the proliferative ability of EPC by MTT assay. 3. Measure the secretion of NO by modified Griess reaction method.4. Apoptosis of EPC was measured by a double staining flow cytometry assay using annexin V-FITC and propidium iodide (PI) co-staining.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 DiI-ac-LDL and FITC- UEA-I, indicating their endothelial lineage and progenitor property, i.e., they are EPCs.2. Compared with control group,incubated with low concentration(4,8μg/ml) CRP for 72 h didn't show obvious promotive effect in proliferation and NO secretion of EPC.Incubated with high concentration(≥12μg/ml) CRP resulted in a marked dose-dependent decrease in EPC proliferation and NO secretion.It was statistically significant at concentrations of CRP≥12μg/ml.3. Treatment with LFPEMFs resulted in increase in EPC proliferation and NO secretion,reduction in EPC apoptosis compared with CRP(12μg/ml) group, but only at 0.6mT with 8h and 1.0mT with 2h has statistical significance. There was not marked effect compared with control group. At 1.4mT 8h has statistical reduction in EPC proliferation and increase in EPC apoptosis comp- areed with CRP(12μg/ml) group,no marked effect on NO secretion.Conclusion1.EPC could be isolated by density gradient centrifugation method and adherence seceening method.2. CRP inhibit EPC proliferation and NO secretion ex vivo in a dose-dependent manner.3.LFPEMFs could attenuate the multiplication inhibition,apoptosis promotion and secretion of EPC induced by CRP.4.High strength electromagnetic field may aggravate the EPC multiplica- tion inhibition and apoptosis promotion,indicates the LFPEMFs have the bidi- rectional function to EPC, its biological effect was related with the intensity and the response time.