Anti-Vascular Endothelial Growth Factor Treatment Induces Blood Flow Recovery Through Vascular Remodeling In High-Fat Diet Induced Diabetic Mice

Objective:To explore the mechanism of anti-vascular endothelial growth factor induces blood flow recovery in a mouse hind limb ischemia model.Methods:1.Feeding diabetic mice: high fat,sugar feeding about C57/BL6 mice about 14 weeks,tail vein blood glucose value was measured.2.Established model of hind limb ischemia:After anesthesia,Ligation of the left lower limb femoral artery,then cut the femoral artery between two ligations.After rewarming,laser Doppler scanner to scan blood flow in the lower limbs in mice,recording its perfusion recovery.Measurements were performed pre-and post-artery ligation,additionally on postoperative days3,7,11 and 14.The left-to-right ratio(occluded-to-nonoccluded leg)was calculated for each animal.Mice were divided into four groups:control,bevacizumab in diabetic mice,control,and bevacizumab in C57/BL6 mice,Each group of 18 mice.Bevacizumab was intraperitoneally administered on the days of 1,3,7and11.Saline as control.In some cases,by using small animal in vivo imaging system,vascular leakage fluorescent dye was used to monitor vascular leakage in mice.3.Gastrocnemius muscles were harvested for Real time PCR,Western blotting,ELISA,immumofluorescence analysis.PDGF-BB was performed by RT-PCR.Both VEGF-A and PDGF-BB level wereanalyzed by Western blotting,and ELISA.Both anti-PECAM-1 and anti-NG-2 were observed by immumofluorescence.4.By using ELISA,in vitro human umbilical vein endothelial cells were used to analyze the PDGF-BB level by the treatment of bevacizumab.ip.5.Statistical analysis:Data are presented as the mean ± SEM and were analyzed by ANOVA and by unpaired two-tailed Student's t test.P values of <0.05 were regarded as statistically significant.Results:1.Bevacizumab promotted blood flow perfusion recovery in hind limb ischemia mouse model: In either diabetic or C57/BL6 mice,blood flow perfusion recovery treated by bevacizumab was significantly increased compared with vehicle group.2.We found abundant expression of VEGF in ischemic gastrocnemius muscles from diabetic mice in vivo,and bevacizumab potently decreased VEGF protein concentrations in ischemic muscles.Bevacizumab can improve pericytes recruitment through the induction of PDGF-BB.To compare the effects of VEGF blockage on the magnitude and kinetics of expression of PDGF-BB and VEGF,we determined the kinetics of protein expression by ELISA using samples from3,7,and14 days after surgery.The induction of VEGF protein by ischemia peaked at 7 days,and treatment of Bevacizumab can significantly inhibited the production of VEGF by3,7 and 14 days,respectively.Furthermore,the blockage of VEGF exhibited a specific up-regulation of PDGF-BB by 7 and 14 days.No significant induction ofPDGF-BB was observed by 3 days after surgery.3.Real-time PCR results showed that the PDGF-BB expression in ischemic diabetic mice in bevacizumab group higher than ischemic diabetic mice in the control group.4.Small animal in vivo imaging results showed that: the degree of vascular leakage in diabetic mice in bevacizumab group was significantly lower than the diabetic mice in control group.5.Human umbilical vein endothelial cell culture in vitro results showed that bevacizumab for human umbilical vein endothelial cells in the expression of PDGF-BB had no significant effect.6.Capillary density was determined using anti-PECAM-1antibody.Time-course analysis of capillary density showed the increase in capillaries observed in the ischemic hind limb at 3 days postoperatively versus that measured in the normal limb.A statistically significant increase in capillary density was noted by day 7,and increased further at days 14.In contrast,no significant alteration in capillary density was observed between days 0 and 14 in the non-ischemic hind limb.Conclusions:1.Bevacizumab promotes blood perfusion recovery in diabetic hind limb ischemia mice.2.Bevacizumab increases the level of gene expression of PDGF-BB.3.Bevacizumab promotes vessel maturation by blocking vascular leakage.4.Bevacizumab increases pericyte coverage,and promotes new vessel maturation.