Treatment of ischemic limbs by transplantation of G-CSF stimulated bone marrow cells in diabetic rabbits

Abstract

To evaluate the efficacy of autologous transplantation of granulocyte colony-stimulating factor (G-CSF) stimulated bone marrow mononuclear cells in the treatment of diabetic lower limb ischemic disease in a rabbit model. The diabetic model was produced by intravenous injection of 5% alloxan into New Zealand white rabbits. The lower limb ischemia model was created by femoral artery ligation in diabetic rabbits (n=50) 14 days after alloxan injection. Animals were then randomized into five groups (10 rabbits per group): group 1, transplanted with G-CSF immobilized peripheral blood mononuclear cells (PBMNC); group 2, transplanted with G-CSF stimulated bone marrow mononuclear cells (BMMNC); group 3, animals were transplanted with non-stimulated BMMNCs; group 4, G-CSF group injected with G-CSF alone without transplantation; and group 5, PBS group treated with PBS alone. Necrosis of foot or toes and blood flow recovery in ischemic limbs was assessed. Expression of von Willebrand factor (vWF) and vascular endothelial cell growth factor (VEGF) were measured in ischemic muscles by immunohistochemistry analysis. Blood flow in ischemic limbs was significantly improved in the G-BMMNC group (ratio of blood flows: 0.82+/-0.06) in comparison with the G-PBMNC (0.61+/-0.09) and BMMNC (0.62+/-0.08) groups (P < 0.001). In the G-BMMNC group, the capillary density, a measure of vWF expression, was significantly higher than in either the G-PBMNC or BMMNC groups (47.9+/-2.51 vs. 36.8+/-4.16 and 39.6+/-2.72, respectively, P < 0.05). The expression of VEGF in G-BMMNC animals was significantly increased in comparison with the G-PBMNC and BMMNC groups (16.93+/-0.70 vs. 11.83+/-0.98 and 12.32+/-0.96, respectively, P < 0.05). A combination of G-CSF stimulation and autologous transplantation of bone marrow stem cells synergistically improved neovascularization and angiogenesis in ischemic limb tissues in diabetic rabbits.