title: Altered gene expression of angiogenic factors induced by calcium-mediated dissociation of retinal pigment epithelial cells.
abstract: OBJECTIVE
  To examine the effect of loss of cell-cell contacts on the gene expression of vascular endothelial growth factor (VEGF) and other factors in primary culture of human retinal pigment epithelial (RPE) cells with real-time reverse transcription-PCR.
  
  
  METHODS
  The dissociation of postconfluent RPE cells was induced by calcium chelation, low-calcium medium, anti-E-cadherin, and anti-N-cadherin antibodies. Total RNA was isolated from the cultured RPE cells and reverse transcribed to cDNA. VEGF was quantified by real-time PCR with a fluorescence detector. VEGF isoforms were differentially measured by specific exon-spanning primers. Besides VEGF, the gene expression levels of some other growth factors were also examined in calcium-mediated dissociation.
  
  
  RESULTS
  Disruption of cell-cell contacts of RPE cells was induced by calcium chelation and low-calcium medium, but not by anti-E-cadherin and anti-N-cadherin antibodies. Calcium-mediated dissociation of RPE cells significantly increased the gene expression levels of VEGF. The mRNA levels of VEGF increased by 6.3-fold on treatment with EGTA and by 4.7-fold in the low-calcium medium at 6 hours. Splice variants of VEGF showed the differential pattern of gene expression. Whereas the expression of VEGF(121) and VEGF(165) was upregulated on calcium-induced dissociation of RPE cells, that of VEGF(145) and VEGF(189) was unchanged. VEGF(206) was not detected. On calcium-induced dissociation, bFGF, IL-6, matrix metalloproteinase (MMP)-1, and placental growth factor (PlGF) were upregulated, whereas acidic (a)FGF and pigment epithelium-derived factor (PEDF) were both downregulated.
  
  
  CONCLUSIONS
  The results show that loss of intercellular contacts promotes increased gene expression of VEGF and other angiogenic factors in human RPE cells.
authors.affiliation: Department of Ophthalmology and Visual Sciences and Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Xue-Feng
authors.initials: XF
authors.lastname: Wang
authors.forename: Jing Z
authors.initials: JZ
authors.lastname: Cui
authors.forename: Shiv S
authors.initials: SS
authors.lastname: Prasad
authors.forename: Joanne A
authors.initials: JA
authors.lastname: Matsubara
mesh_terms: D000199:Actins / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D002450:Cell Communication / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D000199:Actins; D015820:Cadherins; D002614:Chelating Agents; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D002118:Calcium