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K. M. Ford, B. A. Bryan, P. A. D'Amore; Regulation of VEGF Expression During Retinal Pigment Epithelial Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):860.
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Despite a lack of active angiogenesis, VEGF is expressed in nearly all adult tissues. Recent evidence suggests that VEGF may serve as a survival factor for both vascular and non-vascular tissues. The mechanisms controlling constitutive VEGF expression in adult tissue are largely unknown; however, hypoxia, the best studied regulator of VEGF, is unlikely to be involved. Therefore, we sought to elucidate the molecular basis of VEGF regulation in adult tissues using differentiation models of mesenchymal (skeletal muscle) and epithelial (retinal pigment epithelium, RPE) cells.
VEGF expression was analyzed using semi-quantitative PCR in proliferative and terminally differentiated C2C12 (myogenic progenitor) cells and in ARPE-19, a line of human RPE cells. The possible coordinate control of VEGF regulation during skeletal muscle differentiation was investigated in C2C12 cells by overexpressing a dominant negative form of the myogenic transcription factor (MyoD). In ARPE-19 cells, VEGF regulation was analyzed by transient transfection with luciferase reporter constructs containing varying lengths of the 9 kb VEGF promoter.
During skeletal muscle differentiation, upregulation of VEGF isoform expression coincided with cell differentiation; stable overexpression of a dominant negative MyoD suppressed VEGF expression. In ARPE-19 cells, the 9 kb VEGF promoter exhibited a 2000-fold increase in luciferase activity over the promoterless construct and a 10-fold induction over the 5 kb promoter. Sequence analysis of the region of the VEGF promoter between -5 kb and -9 kb revealed putative binding sites for Pax6, Otx2, and MITF, which are key transcription factors that are essential for RPE specification.
The results in skeletal muscle indicate that VEGF is coordinately regulated with differentiation. Preliminary findings suggest that this paradigm may extend to the RPE. Elucidation of the mechanisms that regulate physiologic VEGF expression may permit the development of therapies that can control aberrant VEGF expression, as in the case of AMD, while sparing normal VEGF expression associated with vascular and neural cell survival.
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