Aim The mechanism of basic fibroblast growth factor(bFGF)in mediating increase of intracellular free magnesium ([Mg2+]i) in human umbilical vein endothelial cells (HUVECs), and the relationship between Mg2+and angiogenesis were investigated in this study.Methods The change of[Mg2+]i in HUVECs were quantitatively detected in intracellular cation measurement system via loaded with the fluorescent magnesium indicator mag-fura-2. Endothelial cells were primarily acquired by infusion of collagen enzyme solutioninto the lumens of human umbilical veins and cultured in M199 with 0.2 fetal bovine serum. The role of bFGF in angiogenesis was observed in presence of 0,1 mmol?L-1 or 2 mmol?L-1 of extracellular Mg2+.Results bFGF dose-dependently increased [Mg2+]i, and there was not any significant difference among the groups of 0,1 mmol?L-1and 2 mmol?L-1 of extracellular Mg2+;similar results were obtained in groups done with Na+ and Ca2+. Pretreatment with bFGF receptor-2 (KDR) inhibitor (SU1498) blocked the increase of [Mg2+]i induced by bFGF.Unlike in the group of 0 mmol?L-1extracellular Mg2+,the apparent angiogeneses were observed in the groups of 1 mmol?L-1 and 2 mmol?L-1 extracellular Mg2+ in the presence of bFGF.bFGF-induced angiogenesis was significantly blocked with SU1498 in the presence of 1 mmol?L-1 extracelluar Mg2+.Conclusions These results suggest that the increase of [Mg2+]i by bFGF come from intracellular Mg2+ pools mediated by KDR-dependent signaling pathways,thereby resulting in the bFGF-induced angiogenesis.

Objective To investigate the therapeutic effects of erythropoietin sustained-release gelatin hydrogel microspheres (EPO-GHM) on a murine model of hindlimb ischemia and related mechanisms.Methods Fifty two ten weeks old male C57BL/6J mice were assigned to 5 groups:shamoperated group (the right femoral artery suture was passed through the right femoral artery but not tied,n =8);saline group (right femoral artery ligation and intramuscular injection of saline at a dose of 4 ml/kg into the right hind limb,n =12);EPO group(right femoral artery ligation and intramuscular injection of EPO at a dose of 5 000 IU/kg into the right hind limb,n =12),empty GHM group (right femoral artery ligation and intramuscular injection of empty GHM at a dose of 4 ml/kg into the right hind limb,n =8);EPO-GHM group(right femoral artery ligation and intramuscular injection of EPO-GHM at a dose of 5 000 IU/kg into the right hind limb,n =12).The blood flow ratio of ischemic limb (right)/nonischemic limb (left) was measured using a laser Doppler perfusion imager.After 8 weeks,immunohistochemical analysis were used to evaluate the vessel density (vessel density of CD31 positive),arteriole density(vessel density of α-smooth muscle actin(α-SMA) positive) and muscle area(HHF35 positive area).The proliferating index of vessels was evaluated by double immunofluorescent labeling to evaluate effect of EPO-GHM on angiogenesis of ischemia limb.Western blot was used to evaluate the protein expression of EPO receptor,protein kinase B (AKT),p-AKT,endothelial nitric oxide synthase (eNOS),p-eNOS and matrix metalloproteinase 2(MMP-2).Results (1) Eight weeks later,the blood flow ratio of ischemic limb/nonischemic limb was significantly higher in the EPO-GHM group compared with other groups(0.810 ±0.080,0.563 ±0.051,0.570 ±0.056and 0.561 ± 0.052 respectively,all P ＜ 0.05).(2) CD31 antibody positive and α-SAM antibody positive densities were higher in the EPO-GHM group compared with other groups(P ＜ 0.01 or 0.05).(3)HHF35 positive area in saline group,EPO group,empty GHM group and EPO-GHM group were smaller than that of sham-operated group(all P ＜0.05).HHF35 positive area in saline group,EPO group,empty GHM group and EPO-GHM group were similar(all P ＞ 0.05).(4) The proliferating index of vessels was higher in the EPO-GHM group compared with other groups (P ＜ 0.01 or 0.05).(5) Compared with other groups,the protein levels of EPO receptor,AKT,p-AKT,p-eNOS and MMP-2 were significantly higher in EPO-GHM group(P ＜ 0.01 or 0.05) and level of eNOS was similar among five groups (P ＞ 0.05).Conclusion Results from present study suggest EPO-GHM could improve blood perfusion of ischemia limb in mice through increasing capillary and arteriolar densities and these beneficial effects are possibly mediated by EPOR up-regulation and AKT/p-eNOS/MMP-2 signaling pathway activation.