June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Epithelial Membrane Protein 2 (EMP2) Modulates VEGF During Hypoxia in ARPE-19 Cells
Author Affiliations & Notes
  • Ann Chan
    Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, CA
  • Shawn Morales
    Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, CA
  • Yu Qin
    Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, CA
  • Madhuri Wadehra
    Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA
  • Lynn Gordon
    Jules Stein Eye Institute, University of California, Los Angeles, Los Angeles, CA
  • Footnotes
    Commercial Relationships Ann Chan, None; Shawn Morales, None; Yu Qin, None; Madhuri Wadehra, None; Lynn Gordon, Paganini (I), Paganini (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 6104. doi:
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    • Get Citation

      Ann Chan, Shawn Morales, Yu Qin, Madhuri Wadehra, Lynn Gordon; Epithelial Membrane Protein 2 (EMP2) Modulates VEGF During Hypoxia in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6104.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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 hypoxic conditions.

Methods: ARPE-19 cells were engineered to have reduced (EMP2 shRNA) or increased (EMP2 OE) expression levels of EMP2. Cells were exposed to 0.5% oxygen conditions (hypoxia) for 4 hours. The panel of ARPE-19 cells (over-expressing, knock-down, and control) were evaluated for Hif-1α, Hif-2, VEGF, E2F1, Glut1, and EMP2 expression via Western Blot. Cultured media collected from ARPE-19 cells were used to detect secreted VEGF via ELISA. In response to cultured media from EMP2 modified cells, HUVEC migration was assayed using Boyden chambers. HUVEC vessel formation in Geltrex (reduced growth factor basement membrane matrix) was evaluated using fluorescent microscopy.

Results: Under hypoxic conditions, increased expression of EMP2 in ARPE-19 cells is associated with a significant increase in both Hif-1α and VEGF expression (P<0.0001 for each) by Western blot. Concordantly, under hypoxic conditions, reduction of EMP2 expression using specific shRNA lentiviral vectors reduced Hif-1α and VEGF expression. As compared to supernatant from ARPE-19 controls, supernatant from EMP2 OE cells caused an increase in HUVEC cell migration and vessel tube formation, demonstrating that the observed increase in VEGF protein was functionally significant.

Conclusions: 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 expression of Hif-1α and VEGF under conditions of hypoxia. The relationship between EMP2 and VEGF under hypoxic conditions is a new observation. This association, if confirmed in vivo, may lead to new approaches to control VEGF indirectly through modulating EMP2 expression.

Keywords: 688 retina • 548 hypoxia • 412 age-related macular degeneration  
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