title: Angiogenic gene-modified fibroblasts for induction of localized angiogenesis.
abstract: BACKGROUND
  Induction of localized angiogenesis is of particular interest in the field of plastic surgery. Topical application of recombined vascular endothelial growth factor (VEGF) shows only little effect on graft healing. This study describes the establishment of an ex vivo engineered VEGF slow release system based on autogenous fibroblasts.
  
  
  METHODS
  Primary fibroblasts were subjected to nucleofection and analyzed for transgene expression 1, 2, 3, 4, 7, and 9 d post-nucleofection using FACS. VEGF transgene expression was measured in cell culture supernatants using ELISA. Transgenic protein functionality was examined in the HUVEC proliferation assay. The effect of VEGF transgene expression on the neovascularization of a collagen membrane was investigated in a rat model.
  
  
  RESULTS
  Primary fibroblasts were nucleofected with an efficiency of 63.331% and transgene expression showed persistence up to day 9 post-nucleofection. VEGF was expressed in the cell culture up to day 14 with an expression peak at day 3. The cytokine was functionally active. VEGF transgene was released in vivo at day 7 and enhanced neovascularization of a collagen membrane.
  
  
  CONCLUSIONS
  Nucleofection is the ideal nonviral tool to engineer transgenic primary fibroblasts for transplantation purpose in an ex vivo gene therapeutic approach. Such ex vivo gene therapy is safe and efficient. Its suitability should be further evaluated in more complex flap models, for example the transfer of a free myocutaneous gracilis flap in the pre-irradiated neck region.
authors.affiliation: Department of Oral and Cranio- Maxillofacial Surgery/Plastic Surgery, University of Jena, Germany. cornelia.mueller1@med.uni-jena.de
authors.forename: Cornelia K
authors.initials: CK
authors.lastname: Mueller
authors.forename: Michael Wolf
authors.initials: MW
authors.lastname: Thorwarth
authors.forename: Stefan
authors.initials: S
authors.lastname: Schultze-Mosgau
mesh_terms: D000818:Animals; D021461:Animals, Outbred Strains; D002196:Capillaries / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D013518:Surgery, Plastic; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
chemical_list: D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D049452:Green Fluorescent Proteins; D003094:Collagen