Synergistic effect of vascular endothelial growth factor and granulocyte colony-stimulating factor double gene therapy in mouse limb ischemia.

BACKGROUND: Vascular endothelial growth factor (VEGF) has mostly been tested to treat ischemic diseases, although the outcomes obtained are not satisfactory. Our hypothesis is that the local transient expression of VEGF and stem cell mobilizer granulocyte colony-stimulating factor (G-CSF) genes in ischemic limbs can complement their activities and be more efficient for limb recovery. METHODS: Limb ischemia was surgically induced in mice and 50 microg of VEGF and/or G-CSF genes were locally transferred by electroporation. After 3-4 weeks, evidence of necrosis by visual inspection, capillary density, muscle mass, muscle force and hematopoietic cell mobilization were evaluated. RESULTS: After 4 weeks, 70% and 90% of the animals of the ischemic group (IG) and VEGF-treated group (VG), respectively, presented limb necrosis, in contrast to only 10% observed in the group of mice treated with both VEGF and G-CSF genes (VGG). Recovery of muscle mass and muscle force was higher than 60% in the VGG compared to the non-ischemic group. The mobilization of Sca1+ cells and neutrophils was also higher in the VGG, which may explain the lower level of necrosis observed in this group (22%, in contrast to 70% in the IG). Capillary density and degree of fibrosis were determined in weeks 3 and 4, and also showed a clear benefit as a result of the use of the G-CSF and VEGF genes together. CONCLUSIONS: Gene therapy using VEGF and G-CSF demonstrated a synergistic effect promoting vessel and tissue repair in mouse hind limb ischemia.

Granulocyte-macrophage colony-stimulating factor gene based therapy for acute limb ischemia in a mouse model.

BACKGROUND: Granulocyte-colony-stimulating factor (GM-CSF) is a pleiotropic factor for hematopoiesis that stimulates myeloblasts, monoblasts and mobilization of bone marrow stem cells. Therefore, the GM-CSF gene is a potential candidate for vessel formation and tissue remodeling in the treatment of ischemic diseases. METHODS: A new mouse limb ischemia was established by surgery and gene transfer was performed by injection of 100 microg of a plasmid carrying GM-CSF. Muscle force and weight, histology, capillary density, circulating stem cells and monocytes were determined after 3-4 weeks. RESULTS: More than 60% of nontreated ischemic animals showed gangrene below the heel after 4 weeks, whereas the GM-CSF gene-treated animals showed only darkening of nails or toes. These animals demonstrated a full recovery of the affected muscles in terms of weight, force and muscle fiber structure, but the muscles of nontreated ischemic animals lost approximately 50% weight, 86% force and their regular structure. When the GM-CSF gene was injected into the contralateral limb, only partial loss was observed, demonstrating a distant effect of GM-CSF. The capillary density in the GM-CSF-treated group was 52% higher in relation to the nontreated group. Blood analysis by flow cytometry showed that the GM-CSF-treated group had 10-20% higher levels of circulating monocytes and Sca-1(+). CONCLUSIONS: We conclude that the direct administration of GM-CSF gene in limb ischemia had a strong therapeutic effect because it promoted the recovery of muscle mass, force and structure by mobilizing therapeutic cells and augmenting the number of vessels.