Aim To investigate the effect of combined gene transfer of angiopoietin-1(Ang-1) gene and vascular endothelial growth factor(VEGF) gene,with different proportions,on angiogenesis,arteriogenesis and neovascular structure and function in a rat model of hindlimb ischemia.Methods Expression plasmid,pcD2(control plasmid),pcD2/Ang-1,pcD2/VEGF,or pcD2/Ang-1+pcD2/VEGF at different proportions,was injected intramuscularly into ischemic hindlimb rat model,followed by electroporated.VEGF and Ang-1 expressions were evaluated by RT-PCR.Angiogenesis and arteriogenesis were assessed by capillary density,arteriole density and angiography.Vascular permeability was evaluated by Evans blue uptake assays.Results Collateral vessel development was assessed at 14 days after treatment.Vessel numbers increased slightly in the rat transfected with pcD2/Ang-1 or pcD2/VEGF compared with that in the rats transfected with pcD2 alone(p<0.05).In the rats transfected with pcD2/Ang-1+pcD2/VEGF,with the increase of pcD2/Ang-1 during the pcD2/VEGF at 100 μg,the number of artery increased and was 1.9,2.4 and 2.45 times higher than that of control rats after gene delivery.With the increase of pcD2/VEGF during the pcD2/Ang-1 at 100 μg,the number of artery increased and was 2.9,2.07 and 2.39 times higher than that of control.Evans blue amount in skeletal muscle increased companied with the dosages of pcD2/VEGF.Combination with pcD2/Ang-1 decreased the vessel leakage.Evans blue uptake in group of pcD2/VEGF 100 μg+pcD2/Ang-1 200 μg was the closest to that of the normal group(p>0.05).Conclusions The current study demonstrated that co-expression of Ang-1 and VEGF genes in the ischemic muscle effectively develop leakage-resistant vessels in rat model.At a ratio of pcD2/VEGF to pcD2/Ang-1 by 1:2 is the optimal dosage.Therefore,this approach may provide a more appropriate therapeutic strategy in ischemic vascular diseases.