title: Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications.
abstract: BACKGROUND
  Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo.
  
  
  METHODS
  Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo.
  
  
  RESULTS
  Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days.
  
  
  CONCLUSIONS
  FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.
authors.affiliation: INSERM Unit 460, Cardiovascular Remodeling, CHU Xavier Bichat, Paris, France.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Elmadbouh
authors.forename: Patrick
authors.initials: P
authors.lastname: Rossignol
authors.forename: Olivier
authors.initials: O
authors.lastname: Meilhac
authors.forename: Roger
authors.initials: R
authors.lastname: Vranckx
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
authors.forename: Bruno
authors.initials: B
authors.lastname: Pouzet
authors.forename: Patrick
authors.initials: P
authors.lastname: Midoux
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Michel
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000378:metabolism; D002412:Cations; D002470:Cell Survival; D002478:Cells, Cultured; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D011917:Rats, Inbred Lew; D013997:Time Factors; D014162:Transfection; D015032:Zinc / Q000737:chemistry; D017967:Zinc Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
chemical_list: D002412:Cations; D002712:Chlorides; D017967:Zinc Compounds; D011107:Polylysine; C016837:zinc chloride; D011094:Polyethyleneimine; D000469:Alkaline Phosphatase; D015032:Zinc