Timing und Target der temporären Genexpression von VEGF165 in einem ischämischen Lappenmodell der Ratte

Introduction: In previous experiments to induce angiogenesis, we could show that fibroblasts produce VEGF165 temporarily after adenoviral transfection. The angiogenic effects of VEGF165 were also only temporary during in vivo long-term experiments without ischemia. This study was performed to investigate if timing and target of cell-based therapy could lead to different results for the induction of therapeutic blood vessels in an ischemic flap model. Methods: Isogenic rat fibroblasts were transfected adenovirally and implanted in a McFarlane flap model (2x8 cm) of the rat at different tissue locations (flap tissue (A) and flap plus the adjacent wound margin (B)). We established 24 animal groups with each 10 animals and 5 Mio. implanted cells in location A and 10 Mio. cells in B. Setting I: Implantation of VEGF165 modified cells at the time of flap operation in A (group VEGF165 A), simultaneous implantation in B (group VEGF165 B), implantation of GFP (green fluorescent protein) modified cells in A and B (group GFP B and GFP B), Implantation of non modified cells in A (group NMFB A) and B (group NMFB B), injection of control medium in A and B (groups DMEM A and DMEM B); Setting II: The same procedure one week before flap operation; Setting III: The same procedure two weeks before flap operation. 7 days after onset of ischemia the animals were sacrificed and the flaps were analysed for clinical outcome by planimetric measurements of the vital flap parts. Further, a microangiographical, immunhistological and proteoanalytical examination of the target tissue was performed. Results: Only group VEGF165 B in Setting II (implantation of VEGF165 modified cells into the flap plus adjacent wound margin 1 week before flap operation) showed significantly more vital flap tissue. In all other groups the results were not significantly different. This was demonstrated by planimetrical measurements of vital flap parts and by quantitative counting of the blood vessels in the target tissue using microangiography. Proteoanalysis showed that there were high amounts of VEGF165 in the groups of setting I and II only. Conclusion: In our model, temporary expression of VEGF165 induces therapeutically relevant angiogenesis only if applicated one week before the beginning of ischemia. It is important to include not only the ischemic area for induction of angiogenesis but also the surrounding area. In contrast the angiogenic effects are not effective in the target area and its surrounding tissue, if therapeutic gene expression is started during onset of ischemia or two weeks before ischemia in our model.