title: The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.
abstract: BACKGROUND
  Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation.
  
  
  OBJECTIVE
  To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis.
  
  
  RESULTS
  Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.
authors.affiliation: Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia. petercelec@hotmail.com
authors.forename: Peter
authors.initials: P
authors.lastname: Celec
authors.forename: Roman
authors.initials: R
authors.lastname: Gardlík
authors.forename: Roland
authors.initials: R
authors.lastname: Pálffy
authors.forename: Július
authors.initials: J
authors.lastname: Hodosy
authors.forename: Stanislav
authors.initials: S
authors.lastname: Stuchlík
authors.forename: Hana
authors.initials: H
authors.lastname: Drahovská
authors.forename: Martina
authors.initials: M
authors.lastname: Stuchlíková
authors.forename: Gabriel
authors.initials: G
authors.lastname: Minárik
authors.forename: Ján
authors.initials: J
authors.lastname: Lukács
authors.forename: Ingrid
authors.initials: I
authors.lastname: Jurkovicová
authors.forename: Ivan
authors.initials: I
authors.lastname: Hulín
authors.forename: Ján
authors.initials: J
authors.lastname: Turna
authors.forename: Ján
authors.initials: J
authors.lastname: Jakubovský
authors.forename: Martin
authors.initials: M
authors.lastname: Kopáni
authors.forename: Lubos
authors.initials: L
authors.lastname: Danisovic
authors.forename: Dávid
authors.initials: D
authors.lastname: Jandzík
authors.forename: Matús
authors.initials: M
authors.lastname: Kúdela
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Yonemitsu
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D043925:Angiogenesis Inducing Agents / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015316:Genetic Therapy; D005822:Genetic Vectors; D018919:Neovascularization, Physiologic / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D043925:Angiogenesis Inducing Agents; D020033:Protein Isoforms; D042461:Vascular Endothelial Growth Factor A