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Altered Gene Expression of Angiogenic Factors Induced by Calcium-Mediated Dissociation of Retinal Pigment Epithelial Cells

From the Department of Ophthalmology and Visual Sciences and Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

PURPOSE. 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 VEGF₁₂₁ and VEGF₁₆₅ was upregulated on calcium-induced dissociation of RPE cells, that of VEGF₁₄₅ and VEGF₁₈₉ was unchanged. VEGF₂₀₆ 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.

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