title: Efficient transduction and seeding of human endothelial cells onto metallic stents using bicistronic pseudo-typed retroviral vectors encoding vascular endothelial growth factor.
abstract: BACKGROUND
  Stents seeded with genetically modified endothelial cells (EC) may provide an attractive therapeutic modality for treating vascular diseases by combining the mechanical properties of the metallic stent with the biologic activity of native or genetically engineered ECs. The clinical feasibility of implanting seeded stents depends on the ability to achieve adequate stent coverage within a clinically applicable time frame. We tested the hypothesis that this goal could be achieved by seeding stents with human ECs overexpressing vascular endothelial growth factor (VEGF) and by using an efficient gene transfer system.
  
  
  RESULTS
  Efficiency of gene transfer to human ECs using an amphotropic retroviral vector and a gibbon ape leukemia virus (GALV) pseudo-typed retroviral vector was examined and compared. For assessment of transduction rates, LacZ-encoding vectors were used and beta-galactosidase activity was determined 48 h after gene transfer. The transduction rate of primary human ECs using the amphotropic retroviral vector encoding the LacZ gene was low (2.9+/-2% of cells). Under the same conditions, the GALV pseudo-typed vector encoding LacZ transduced 94+/-2% of cells (P<.001). To test the effect of VEGF gene transfer on stent coverage, we transduced ECs using a bicistronic GALV pseudo-typed retroviral vector encoding either GFP alone or both VEGF and GFP. Since all transduced cells expressed GFP, stent coverage by ECs could be assessed by fluorescent inverted microscopy, which demonstrated that stent coverage by ECs overexpressing VEGF was more rapid and effective than coverage by ECs overexpressing GFP. Progressively increasing quantities of VEGF protein were detected in the conditioned medium of stents seeded with endothelia cells expressing VEGF 2, 3, and 5 days after seeding.
  
  
  CONCLUSIONS
  High-rate gene transfer to human primary ECs was observed 48 h after transduction with GALV pseudo-typed retroviral vectors, eliminating the need for the time-consuming process of cell selection. Seeding with ECs overexpressing VEGF improved stent coverage and was associated with continuing secretion of the protein. The findings provide support for the feasibility of implanting genetically engineered biologically active cellular-coated stents.
authors.affiliation: Department of Cardiovascular Medicine, Lady Davis Carmel Medical Center, Bruce Rappaport School of Medicine, Technion-IIT, Haifa 34362, Israel.
authors.forename: Belly
authors.initials: B
authors.lastname: Koren
authors.forename: Anat
authors.initials: A
authors.lastname: Weisz
authors.forename: Lukas
authors.initials: L
authors.lastname: Fischer
authors.forename: Zoya
authors.initials: Z
authors.lastname: Gluzman
authors.forename: Meir
authors.initials: M
authors.lastname: Preis
authors.forename: Naomi
authors.initials: N
authors.lastname: Avramovitch
authors.forename: Tzafra
authors.initials: T
authors.lastname: Cohen
authors.forename: Francois-Loic
authors.initials: FL
authors.lastname: Cosset
authors.forename: Basil S
authors.initials: BS
authors.lastname: Lewis
authors.forename: Moshe Y
authors.initials: MY
authors.lastname: Flugelman
mesh_terms: D001807:Blood Vessel Prosthesis; D015153:Blotting, Western; D042783:Endothelial Cells / Q000378:metabolism / Q000821:virology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000601:surgery; D004797:Enzyme-Linked Immunosorbent Assay; D004867:Equipment Design; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use*; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D008670:Metals; D012501:Saphenous Vein / Q000166:cytology / Q000378:metabolism / Q000601:surgery; D015607:Stents; D014161:Transduction, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D008670:Metals; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D001616:beta-Galactosidase