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J. Cai, W. Jiang, A. Ahmed, M.B. Grant, S. Li Calzi, A. Afzal, M.E. Boulton; Placenta Growth Factor–Mediated Upregulation of VE–Cadherin Expression in Human Retinal Endothelial Cells Involves the Transcription Factor Sp1 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5332.
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We have previously reported that placenta growth factor (PlGF) upregulates VE–cadherin expression in microvascular endothelial cells. The aim of this study was to investigate promoter regulation of VE–cadherin expression.
Human retinal endothelial cells (HREC) were treated with 100ng/ml of either VEGF, PlGF or the VEGF:PlGF heterodimer for 20min or 24 hours. Expression of VE–cadherin at both the mRNA and protein level was determined using a combination of QRT–PCR and Western blotting. The electrophoretic mobility shift assay (EMSA) and antibody supershift assay were used to analyse the role of Sp1 and Sp3 in PlGF–mediated VE–cadherin gene transcription.
QRT–PCR data showed an increase in VE–cadherin mRNA expression by 20 min in cultures exposed to VEGF and VEGF:PlGF heterodimers while mRNA levels were reduced in PlGF treated cells. However, by 24 hours, PlGF significantly increased mRNA levels of VE–cadherin by 8 fold above those of VEGF or VEGF: PlGF. By contrast, Western blotting showed that overall expression of VE–cadherin did not change at 20 minutes after addition of either VEGF or PlGF. However by 24 hours, PlGF increased VE–cadherin protein expression by 10 fold. EMSA showed that the binding of endothelial cell proteins to a GT–rich sequence (–49/–39) of VE–cadherin promoter was increased dramatically when the cells were exposed to VEGF, VEGF: PlGF at 20 minutes and PlGF at 24 hours. Antibody supershift assay with antibodies against Sp1 and Sp3 revealed that interaction proteins were composed of Sp1 and Sp3. PlGF increased Sp1 binding and decreased Sp3 binding at 24 hours treatment.
Prolonged treatment of PlGF reduces interendothelial permeability by upregulating VE–cadherin expression in HREC, in part, via binding of the transcription factor Sp1 to the VE–cadherin promoter region.
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