June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Role of HIF-1α in TGF-β2 Induced Epithelial to Mesenchymal Transition in Lens Epithelial Cells.
Author Affiliations & Notes
  • Rooban B Nahomi
    Ophthalmology, University of Colorado Denver, Aurora, Colorado, United States
  • Ram H Nagaraj
    Ophthalmology, University of Colorado Denver, Aurora, Colorado, United States
  • Footnotes
    Commercial Relationships   Rooban Nahomi, None; Ram Nagaraj, None
  • Footnotes
    Support  EY022061, EY023286 and a challenge grant from RPB
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3788. doi:
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    • Get Citation

      Rooban B Nahomi, Ram H Nagaraj; Role of HIF-1α in TGF-β2 Induced Epithelial to Mesenchymal Transition in Lens Epithelial Cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3788.

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

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Abstract

Purpose : Purpose: Transforming growth factor (TGF-β2) mediated pathways are integral to the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) during secondary cataract formation. Previous studies have suggested that hypoxia inducible factor-1α (HIF-1α) could play a role in EMT. In this study we investigated the role of HIF-1α in TGF-β2-induced EMT of human LECs.

Methods : Methods: Human lens epithelial cells (FHL124) were cultured at 21% or 1% oxygen and treated with TGF-β2 (10 ng/ml) for 48 h to induce the EMT response. The HIF-1α and VEGF-A levels were measured by western blotting and ELISA, respectively. To determine the direct effect of HIF-1α on the EMT of LECs, FHL124 cells were treated with either a prolyl hydroxylase inhibitor, dimethyloxaloylglycine (DMOG, 0 to 10 μM or 500 μM) or it was overexpressed by transfecting with a HIF-1α plasmid. To further study the role of HIF-1α in TGF-β2 induced EMT, we used 200 nM of KC7F2, a HIF-1α translation inhibitor. To check the direct effect of VEGF-A on EMT, cells were cultured with 0 to 100 ng/ml of recombinant human VEGF-A. Expression of various EMT markers was analyzed by western blotting.

Results : Results: TGF-β2 treatment resulted in the upregulation of EMT markers (αSMA, αV and β1 -Integrin), which was largely independent of the oxygen levels. TGF-β2 treatment increased the HIF-1α levels in cells, along with the VEGF-A levels in the conditioned media. HIF-1α upregulation either by DMOG treatment or forced overexpression (without TGF-β2 treatment) failed to induce the EMT response. Direct VEGF-A treatment also showed no EMT response. However, the TGF-β2 mediated EMT response was significantly reduced in cells treated with KC7F2, and also in cells treated with high levels of DMOG (500 μM).

Conclusions : Conclusion: Our results suggest that the mild upregulation of HIF-1α might be necessary for the TGF-β2-mediated EMT response in lens epithelial cells, but that is independent of VEGF-A.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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