OBJECTIVE: The importance of leukocyte subsets in angiogenesis is emerging. The aim of this project is to investigate if vascular repair during muscle ischemia can be promoted by strategic recruitment of specific leukocytes to the afflicted site as well as induce local leukocyte polarization towards a proangiogenic phenotype. METHODS AND RESULTS: Plasmids encoding the chemokines CCL2 and CXCL12 were constructed together with a GFP and luciferase reporter system and injected intramuscularly in wild type or CX3CR1-GFP mice. Muscle ischemia was induced immediate before plasmid delivery by excision of the femoral artery, where after restoration of functional blood flow was assessed by vascular responsiveness to heat challenge at day 1, 2, 3 and 7 post-insult in anesthetized mice using Laser Doppler flowmetry. The role of macrophages was studied by clodronate liposome-depletion two days prior to induction of ischemia. Leukocyte subsets in the injured muscle, perfused vessels and total vascular densities were quantified using confocal microscopy of ischemic tissues at 3 and 7 days post-insult. Plasmid gene expression correlated to luminescent signal and was demonstrated to peak at day 3, but could be detected in the muscle for a period extending 4 weeks. Blood flow response to heat challenge was improved in ischemic limbs over expressing CXCL12 at 2, 3 and 7 days post induction of ischemia, and higher density of perfused capillaries was detected at days 3 and 7. Further, increased number of macrophages (F4/80+) and polarization towards the proangiogenic M2 phenotype (F4/80+/MRC1+) were observed in the ischemic muscles expressing CXCL12, while functional blood flow was not improved by CXCL12 over expression in mice depleted of macrophages. Over expression of CCL2 did not increase vascular repair as demonstrated by vascular density and blood flow recordings, and the M2 macrophage population was not amplified at the site of ischemia. CONCLUSIONS: Treatment of ischemic hind limbs with plasmid encoded CXCL12 partially restores a functional blood perfusion in response to heat by increasing total capillary density and perfusion, which is completely dependent on the expansion and polarization of the macrophage population into the M2 phenotype.