April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Exogenous regulation of the HIF pathway in RPE cells
Author Affiliations & Notes
  • Helder Andre
    Clinical Neurosciences, St Erik Eye Hospital, Stockholm, Sweden
  • Malena Ekstrom
    Clinical Neurosciences, St Erik Eye Hospital, Stockholm, Sweden
  • Anna Takei
    Clinical Neurosciences, St Erik Eye Hospital, Stockholm, Sweden
  • Yu Ma
    Clinical Neurosciences, St Erik Eye Hospital, Stockholm, Sweden
  • Anders P Kvanta
    Clinical Neurosciences, St Erik Eye Hospital, Stockholm, Sweden
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3440. doi:
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      Helder Andre, Malena Ekstrom, Anna Takei, Yu Ma, Anders P Kvanta; Exogenous regulation of the HIF pathway in RPE cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3440.

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

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Abstract

Purpose: In this study we investigate the effects of a series of hypoxia-inducible factors (HIF)-regulating molecules (HRM) on the hypoxia pathway in retinal pigment epithelial (RPE) cells, critically involved in neovascular age-related macular degeneration (nAMD) pathogenesis.

Methods: ARPE-19 were maintained at normoxia (21% O2), hypoxia (1% O2), or in hypoxia-mimicking agents (CoCl2). HUVEC were kept at normoxia. ARPE-19 cells were transfected with plasmids encoding FLAG-tagged HRM (PHD1, PHD2, PHD3, VHL, FIH-1), as well as HIF-1α and HIF-2α, as needed. Effects of HMR on ARPE-19 were analyzed by a luciferase reporter assay (DLR), Western blot, and soluble vascular endothelial factor (VEGF)-capture assay. Effects on endothelial cells (EC) were studied by exposing HUVEC cells to ARPE-19 pre-conditioned media after transfection with particular HRM.

Results: ARPE-19 cells displayed endogenous expression of HIF-1α, but not -2α, at the protein level. Moreover, transfection of all plasmids encoding FLAG-tagged HRM was normalized for comparable levels of protein expression. The DLR denoted a marked negative regulation of HIF-1α activity in ARPE-19 in the presence of all exogenous PHDs. Analysis of HIF-1α protein showed a very considerable decrease, particularly in the presence of PHD2. Additionally, PHD2 also showed the most dramatic reduction of HIF-1α protein life-time. Following, ARPE-19 were transfected with PHD2-encoding plasmid, and the level of secreted VEGF was analyzed by immunocapturing assay using bevacizumab (anti-VEGF). The immunoprecipitated level of VEGF from the media of ARPE-19 cells transfected with PHD2 was considerably lower than that of an empty control. Subsequently, HUVEC were exposed to the media from these experiments, and EC proliferation was decreased in pre-conditioned media from ARPE-19 transfected with PHD2.

Conclusions: In this study we have compared the effects of canonical HMR on the hypoxia-mediated response in ARPE-19 cells. Our data indicates that PHD2 seems to be the most potent negative-regulator of the HIF pathway. Furthermore, the negative effects of PHD2 were clearly associated with decrease in secreted VEGF. This decrease in VEGF level displayed further effects in reducing EC proliferation. These results may have implications for the clinical treatment of patients with nAMD, particularly regarding the use of gene therapy to negatively regulate the neoangiogenesis present in these patients.

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium • 548 hypoxia  
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