title: Overexpression of human HMW FGF-2 but not LMW FGF-2 reduces the cytotoxic effect of lentiviral gene transfer in human corneal endothelial cells.
abstract: OBJECTIVE
  Recently, insertion of immuno-modulatory or anti-apoptotic genes into corneal endothelial cells (HCECs) came into focus. Basic FGF-2 occurs in one secreted (low molecular weight, LMW, 18 kD) and four nuclear (high molecular weight, HMW, 22-34 kD) isoforms. HMW isoforms are known differentiation and survival factors, while LMW FGF-2 is a known mitogen. The effect of FGF-2 overexpression of each of the five known isoforms on HCEC cell survival after lentiviral gene transfer in different culture media was investigated.
  
  
  METHODS
  Cells were transduced with lentiviral vectors encoding for each of the five FGF-2 isoforms. Transduction efficiency and expression of individual FGF-2 isoforms was assessed by marker gene transfer and western blotting. Primary HCECs were cultured and transduced in four different media previously described for HCEC cultivation or corneal organ cultivation. Cytotoxic effect of virus infection and a possible rescue effect of FGF-2 overexpression were determined by resazurin conversion assay.
  
  
  RESULTS
  Transduction with FGF-2 encoding lentiviral vectors resulted in overexpression of the respective isoform in all tested cell populations. Western blotting after total cell lysis proved nuclear localization of transgenic HMW isoforms. Overexpression of HMW FGF-2-especially 34 kD FGF-2-reduced lentiviral cytotoxicity, while overexpression of LMW FGF-2 aggravated viral cytotoxicity.
  
  
  CONCLUSIONS
  Cytotoxicity of lentiviral gene transfer in corneal endothelial cells may be reduced by using bicistronic vectors that encode for the target gene and the 34-kD isoform of human FGF-2. Such cotransduction of a survival factor may increase cell survival after gene transfer, thereby improving gene therapeutic approaches.
authors.affiliation: Institute of Anatomy, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstrasse 74, Dresden, Germany. monika.valtink@tu-dresden.de
authors.forename: Monika
authors.initials: M
authors.lastname: Valtink
authors.forename: Lilla
authors.initials: L
authors.lastname: Knels
authors.forename: Nicole
authors.initials: N
authors.lastname: Stanke
authors.forename: Katrin
authors.initials: K
authors.lastname: Engelmann
authors.forename: Richard H W
authors.initials: RH
authors.lastname: Funk
authors.forename: Dirk
authors.initials: D
authors.lastname: Lindemann
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000235:genetics*; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000821:virology; D015828:Eye Infections, Viral / Q000235:genetics / Q000517:prevention & control* / Q000821:virology; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007634:Keratitis / Q000235:genetics / Q000517:prevention & control* / Q000821:virology; D016086:Lentivirus / Q000235:genetics*; D008875:Middle Aged; D008970:Molecular Weight; D014161:Transduction, Genetic; D055815:Young Adult
chemical_list: D016222:Fibroblast Growth Factor 2; D004247:DNA