| Background: The natural processes leading to collateral development are extremely complex, requiring multiple growth factors interacting in concert and in sequence and cell-cell interaction. Optimal neovascularization may, therefore, require multiple angiogenic factors. Because BM-MNC contains various kinds of primitive cells, which differentiate into endothelial cells and could secret several growth factors, it may provide optimal therapeutic neovascularization. We attempt to: (1)define in vitro whether BM-MNC secret vascular endothelial growth factor (VEGF^ basic fibroblast growth factor(bFGF) and interleukin-1 (3 (IL-1β); (2) evaluate the effect of BM-MNC on endothelial cell(EC) proliferation; (3) examine whether in vivo implantation of autologous BM-MNCs can give rise to functional EPCs and augmentation postnatal neovascularization in rat model of hind limb ischemia; (4)investigate efficiency and safety of implantation of autologous BM-MNCs.Methods: Rat BM-MNCs were isolated by centrifugation through a Histopaque density gradient. Part of BM-MNCs was cultured four weeks in vitro. Conditioned medium was assayed for VEGF, bFGF and IL-1β and was added to cultured human umbilicus vein endothelial cells(HUVEC) to assess the effect of BM-MNC on endothelial cell(EC) proliferation. A rat ischemic hind limb model was made by the ligation of the right femoral artery and its branches in imbred Wistar rats. BM-MNCs were injected into 7 points of the ischemic muscles (20ul/point). To determine the growth factors and cytokines contributing to angiogenesis, the levels of VEGF, bFGF and IL-1β were measured by ELISA.To assess angiogenesis, histologic evaluation was performed at 2 and 4 weeks postligation. Severity of ischemic insult was evaluated by determining the femoral arteriovenous oxgen difference (AVDO2) at 2 and 4 weeks postligation.Results: Vascular endothelial growth factor, basic fibroblasts growth factor and interleukin-1β levels in t IV mditioned medium increased than control culture medium which was not exposed to BM-MNCs. TheIVconditioned medium enhanced, in a dose-related manner, HUVEC proliferation. Levels of bFGF and IL-1B in the MNC groups were significantly elevated commpared with those in the sham groups and in the PBS groups .The histologic evaluation showed that the implanted BM-MNCs induced angiogenesis. The MNC groups had a higher capillary density than the sham(9.68±1.35 vs 3.32±0.95; 6.81±1.54 vs 3.28±1.34 P<0.01) and PBS groups(9.68±1.35 vs 5.29±1.15; 6.81±1.54 vs 3.76±1.55 /><0.01) at 2 and 4 weeks postligation.The capillary/muscle fiber ratios in the MNC groups were significantly higher than those in the sham groups(2.62±0.98 × 10-2 vs 1.11±0.38×10-2; 1.69±0.97×10-2 vsl.06± 0.61 × 10-2 P<0.01) and the PBS groups(2.62±0.98xlO'2 vsl.54±0.60 ×10-2; 1.69±0.97× 10-2 vs 1.22±0.54×10-2 P<0.01). AVDO2 in the MNC group significantly decreased when compared with that in the PBS groups (5.19±1.67 ml/dL vs 8.70±3.60 ml/dL and 4.72±1.71 ml/dL vs7.54±1.69 ml/dL ; P<0.05) at 2 and 4 weeks postligation.Conclusions:(l)BM-MNCs is a valuable source of multiple factors involved in angiogenesis.(2) BM-MNCs secret potent angiogenic cytokines such as VEGF , bFGF and IL-1B and induce proliferation of endothelial cell.(3) The implantation of autologous BM-MNCs induced neovascularization in a rat ischemic hind limb model.(4) The mechanism seemed to be related to the ability of the MNCs secrete potent angiogenic ligands and cytokines.(5) The implantation of autologous BM-MNCs angment collateral perfusion of ischemic hind limb.(6) Direct local implantation of autologous BM-MNCs represents a simple and novel and promising method of inducing therapeutic neovascularization for ischemic disease.
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