Therapeutic effects and related mechanisms of erythropoietin sustained-release gelatin hydrogel microspheres on a murine model of hindlimb ischemia

Abstract

To investigate the therapeutic effects of erythropoietin sustained-release gelatin hydrogel microspheres (EPO-GHM) on a murine model of hindlimb ischemia and related mechanisms. Fifty two ten weeks old male C57BL/6J mice were assigned to 5 groups: sham-operated 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). (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±0.056 and 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). 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.