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Ann M. Chan, Shawn A. Morales, David G. Telander, Madhuri Wadehra, Lynn K. Gordon; Epithelial Membrane Protein 2 (EMP2) Modulates Hypoxia-Inducible Factor 1α (Hif-1α and VEGF Expression by ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4778. doi: https://doi.org/.
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The "wet" form of age-related macular degeneration (AMD) is associated with aberrant angiogenesis, largely through VEGF production by retinal pigmented epithelial (RPE) cells. Epithelial membrane protein 2 (EMP2), a member of GAS3/PMP22 tetraspan protein family, is highly expressed in the RPE, and has been shown to control VEGF expression in the retinal pigment epithelial cell line, ARPE-19. The goal of this study is to determine the relationship between EMP2 and VEGF expression under normoxic and hypoxic conditions and to define whether the observed changes in VEGF are functionally significant.
ARPE-19 cells were engineered to have reduced or overexpressed levels of EMP2. Cells were grown under normoxic conditions or in 0.5% oxygen (hypoxia). The panel of ARPE-19 cells (over-expressing, knock-down, and control) were evaluated for Hif-1α, VEGF, E2F1, and EMP2 expression via Western Blot. In response to cultured media from ARPE-19 EMP2 modified cells, HUVEC migration was assayed using Boyden chambers. HUVEC vessel formation was evaluated using light microscopy.
Under hypoxic conditions, overexpression of EMP2 in ARPE-19 cells is associated with a significant increase in both Hif-1α and VEGF expression (P< 0.0001 for each). Condordantly, under hypoxic conditions, reduction of EMP2 expression using specific shRNA lentiviral vectors reduced Hif-1α and VEGF expression. Studies of HUVEC cell migration, vessel tube formation, and vessel tube length all demonstrated that the observed changes in VEGF were functionally significant.
Increased EMP2 expression in ARPE-19 cells is associated with a robust and physiologically significant increase in Hif-1α and VEGF expression under hypoxic conditions. These changes have functional consequences as EMP2 expression promoted capillary-like vessel formation. Concordantly, knockdown of EMP2 leads to decreased, but not absent, Hif-1α and VEGF. The relationship between EMP2 and VEGF may lead to a new therapeutic target for treatment of neovascular AMD, through control of EMP2 expression by the retinal pigment epithelium, and potentially other diseases involving pathologic angiogenesis.
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