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In the United States, over 8.5 million people suffer from peripheral arterial disease (PAD). Previously we reported that Pellino-1(Peli1) gene therapy reduces ischemic damage in the myocardium and skin flaps in Flk-1 [Fetal Liver kinase receptor-1 (Flk-1)/ Vascular endothelial growth factor receptor-2/VEGFR2] heterozygous (Flk-1+/‐−) mice. The present study compares the angiogenic response and perfusion efficiency following hind limb ischemia (HLI) in, Flk-1+/− and, MAPKAPKINASE2 (MK2−/−) knockout (KO) mice to their control wild type (WT). We also demonstrated the use of Peli1 gene therapy to improve loss of function following HLI. Femoral artery ligation (HLI) was performed in both Flk-1+/− and MK2−/− mice along with their corresponding WT. Another set of Flk-1+/− and MK2−/− were injected with either Adeno-LacZ (Ad.LacZ) or Adeno-Peli1 (Ad.Peli1) after HLI. Hind limb perfusion was assessed by laser doppler imaging at specific time points. A standardized scoring scale is used to quantify the extent of ischemia. Histology analysis performed includes capillary density, fibrosis, pro-angiogenic and anti-apoptotic proteins. Flk-1+/− and MK2−/− had a slower recovery of perfusion efficiency in the ischemic limbs than controls. Both Flk-1+/− and MK2−/− KO mice showed decreased capillary density and capillary myocyte ratios with increased fibrosis than their corresponding wild types. Ad.Peli1 injected ischemic Flk-1+/− limb showed improved perfusion, increased capillary density, and pro-angiogenic molecules with reduced fibrosis compared to Ad.LacZ group. No significant improvement in perfusion was observed in MK2−/− ischemic limb after Ad. Peli1 injection. Deletion of Flk-1 and MK2 impairs neovascularization and perfusion following HLI. Treatment with Ad. Peli1 results in increased angiogenesis and improved perfusion in Flk-1+/− mice but fails to rectify perfusion in MK2 KO mice. Overall, Peli1 gene therapy is a promising candidate for the treatment of PAD. • Therapeutic vascularization in the hind-limb ischemia model with Pellino-1 gene therapy, an E3 ligase is promising. • Pellino-1 repairs and rectifies downstream abnormal VEGF signaling in ischemic tissues. • Pellino-1 is more effective and appropriate in inducing angiogenic response than VEGF-A. • VEGF-A increases vascular permeability and thrombosis, whereas Pellino-1 bypasses these effects. • The role of E3 Ligases in therapeutic angiogenesis is new. [ABSTRACT FROM AUTHOR]