title: Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats.
abstract: BACKGROUND
  Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy.
  
  
  RESULTS
  Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed.
  
  
  CONCLUSIONS
  These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.
authors.affiliation: Department of Plastic Surgery and Hand Surgery, Faculty of Medicine, University Hospital Rechts der Isar, Technische Universität München, Munich, Germany. zhangziyang776@googlemail.com
authors.forename: Ziyang
authors.initials: Z
authors.lastname: Zhang
authors.forename: Alex
authors.initials: A
authors.lastname: Slobodianski
authors.forename: Wulf D
authors.initials: WD
authors.lastname: Ito
authors.forename: Astrid
authors.initials: A
authors.lastname: Arnold
authors.forename: Jessica
authors.initials: J
authors.lastname: Nehlsen
authors.forename: Shaoxiang
authors.initials: S
authors.lastname: Weng
authors.forename: Natalie
authors.initials: N
authors.lastname: Lund
authors.forename: Jihong
authors.initials: J
authors.lastname: Liu
authors.forename: José-Tomás
authors.initials: JT
authors.lastname: Egaña
authors.forename: Jörn A
authors.initials: JA
authors.lastname: Lohmeyer
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Müller
authors.forename: Hans-Günther
authors.initials: HG
authors.lastname: Machens
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003097:Collateral Circulation / Q000235:genetics / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000637:transplantation*; D006614:Hindlimb / Q000473:pathology*; D007511:Ischemia / Q000628:therapy*; D008297:Male; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2