Pulsed Electromagnetic Field Improves Angiogenesis In Response To Ischemia

Objective:Ischemic diseases have become a threat to human health and life quality since its high incidence of morbidity,disability and mortality.Therapeutic post-ischemic angiogenesis can improve symptoms and prognosis of patents by promoting regional ischemic perfusion,as an important means of treating ischemic diseases.It is reported that Pulsed electromagnetic fields(Pulsed electromagnetic field,PEMF)could repair damaged tissue and stimulate angiogenesis,via a variable electromagnetic field inducing voltage and current.This study aimed at clarifying the effect of PEMF on human umbilical vein endothelial cells(HUVECs)and hindlimb ischemia of mice model,and detecting the underlying molecular biological mechanism,providing new ideas for the clinical treatment to ischemic disease.Method:1.To explore the effect of PEMF on angiogenesis in HUVECs: HUVECs were simulated by PEMF(30 ± 3 Hz,5 mT)for 1-4 cycles(8 minutes / cycle)respectively,we detected the cell viability using CCK-8,evaluated apopotosis via Hoechst 33258 staining,tested tube formation ability by Matrigel experiment,observed cell migrate function by scratches and Transwell experiments,determined the expression of VEGF and Sirt1,phosphorylation of Akt and eNOS by Western Blot.2.To evaluate the effect of PEMF on the PI3k/Akt/eNOS signal pathway: After pretreated with eNOS inhibitors L-NAME eNOS or PI3 K inhibitor LY294002,HUVECs were exposed to PEMF for 4 cycles,then we tested the angigenesis function of HUVECs,the expression of VEGF and the phosphorylation levels of Akt,eNOS.3.To study the effect of PEMF on angiogenesis in the hind limb ischemia of mice model: Hindlimb ischemia was induced by unilateral femoral artery resection using 6-8 week-old male C57BL6 mice.Then,mice were exposed to PEMF therapy(4 cycles,8min/cycle,30 ± 3 Hz,5 mT)every day until day 14.Infrared thermal imaging analysis was used to evaluate skin temperature of hind limb on postoperative day 0 and 14,the score of hind limb ischemia was assessed according to grading standard and capillary density of ischemic tissue was observed using CD31 Immunofluorescence staining,protein expression of VEGF and Sirt1,the phosphorylation levels of Akt and eNOS were analyzed by Western Blot.Results:1.The pro-angiogenesis effect of PEMF on HUVECs: The ability of proliferation,migration and tube formation enhanced by PEMF treatment compared with control group,and the VEGF,Sirt1 protein levels,phosphorylation of eNOS and Akt also increased significantly,moreover,we found a time-dependent effect of PEMF on HUVECs.2.The angiogenesis function of PEMF on HUVECs may be associated with activation of the PI3 K / Akt / eNOS signaling pathways: The promotion effects of PEMF on tube formation and cell migration were significantly inhibited by pretreatment with L-NAME,and phosphorylation of eNOS also significantly decreased;the promotion effects of PEMF on tube formation and cell migration were significantly inhibited by pretreatment with LY294002 intervention,as well as the expression of VEGF and phosphorylation of eNOS.3.PEMF can promote angiogenesis in mice model of hind limb ischemia: The ischemia score of hind limb lowered,skin temperature of the left hind limb approached to normal,CD31 immunofluorescence staining showed increased capillary density in the ischemic area,and the expression of VEGF and Sirt1,the phosphorylation of eNOS and Akt in the muscle were significantly increased after PEMF treatment.Conclusion:PEMF promote angiogenesis in HUVECs and ischemic hind limb of mice model,with increased expression of VEGF,Sirt1 and phosphorylation of eNOS and Akt.It is suggested that the effect of PEMF may relate to activating PI3K-Akt-eNOS or Sirt1-eNOS signaling pathway.