title: Endogenic regulation of proliferation and zinc transporters by pigment epithelial cells nonvirally transfected with PEDF.
abstract: OBJECTIVE
  Genetic modification of cells before transplantation may allow the delivery of neuroprotective and other functional molecules to patients with neurodegenerative diseases. To avoid complications associated with virally transfected cells, we have explored the use of nonviral methods to insert genetic material into RPE cells.
  
  
  METHODS
  After transfection with plasmids encoding different pigment epithelium-derived factor (PEDF) fusion proteins, transfected cells were established and passaged up to 100 times. Gene expression of PEDF, ZnT3, ZIP2, CRALBP, CATD, and ZO-1 was determined by RT-PCR. Secretion dynamics were analyzed using ELISA and a spheroid-based assay was used to confirm the anti-angiogenic activity of the recombinant PEDF.
  
  
  RESULTS
  Transfection efficiency reached up to 98.7% with a plasmid encoding PEDF and enhanced green fluorescent protein (EGFP) separately and 87.2% with a plasmid encoding an EGFP-PEDF fusion. Immunoblotting revealed that transfected RPE cells express the appropriate PEDF or EGFP-PEDF. Expression of recombinant PEDF is stable, as shown by its secretion for the 2 years and the 100 passages the cells have been followed. PEDF expression was overexpressed and the transfected cells exhibited increased proliferation, up-regulation of ZnT3 and ZIP2, and inhibited sprouting in human umbilical vein endothelial cell spheroids.
  
  
  CONCLUSIONS
  Genetic in vitro modification of pigment epithelial cells using nonviral transfection protocols should improve the potential therapeutic treatment of neurodegenerative diseases by transplantation of genetically modified cells without the disadvantages of virally mediated transfection. Here we have shown that genetically modified RPE cells overexpress a functional human recombinant PEDF, as evidenced by the autogenic regulation of proliferation, up-regulation of two distinct zinc transporters, and in vitro inhibition of endothelial cell sprouting.
authors.affiliation: Interdisciplinary Center for Clinical Research Aachen, Rheinisch-Westfälische Technische Hochschule Aachen University, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Johnen
authors.forename: Olga
authors.initials: O
authors.lastname: Kazanskaya
authors.forename: Narendra
authors.initials: N
authors.lastname: Armogan
authors.forename: Christiane
authors.initials: C
authors.lastname: Stickelmann
authors.forename: Michael
authors.initials: M
authors.lastname: Stöcker
authors.forename: Peter
authors.initials: P
authors.lastname: Walter
authors.forename: Gabriele
authors.initials: G
authors.lastname: Thumann
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins; D027682:Cation Transport Proteins / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000235:genetics*; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D009414:Nerve Growth Factors / Q000235:genetics*; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics*; D014162:Transfection; D015032:Zinc / Q000378:metabolism*; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D005136:Eye Proteins; D049452:Green Fluorescent Proteins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D015843:Serpins; D015032:Zinc; D000099174:Pigment Epithelium-Derived Factor; C020932:11-cis-retinal-binding protein; C102806:SLC30A3 protein, human; C498424:SLC39A2 protein, human; C485184:enhanced green fluorescent protein