rss
Br J Ophthalmol 2007;91:1406-1410 doi:10.1136/bjo.2007.123125
  • Extended report
    • Laboratory science - Extended reports

Hypoxia-inducible factor expression in human RPE cells

  1. Farzin Forooghian1,4,
  2. Rozita Razavi2,4,
  3. Lee Timms3,4
  1. 1
    Department of Ophthalmology and Vision Sciences and Institute of Medical Science, University of Toronto, Toronto, ON, Canada
  2. 2
    Department of Immunology, University of Toronto, Toronto, ON, Canada
  3. 3
    The Centre for Applied Genomics, Toronto, ON, Canada
  4. 4
    Hospital for Sick Children, Toronto, ON, Canada
  1. Dr Farzin Forooghian, Room 10128, Elm Wing, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada, M5G 1X8; farzin.forooghian{at}utoronto.ca
  • Received 3 May 2007
  • Revised 20 May 2007
  • Accepted 6 June 2007
  • Published Online First 13 June 2007

Abstract

Background: Hypoxia-inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.

Methods: ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme-linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real-time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.

Results: HIF-1α was readily produced by ARPE-19 cells under hypoxia, but HIF-2α and HIF-3α could not be detected even after HIF-1α silencing. HIF-1α protein levels showed an increasing trend for the first 24 h while HIF-1α mRNA levels fluctuated during this time. After 36 h HIF-1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF-1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.

Conclusions: HIF-1α appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1α levels are likely self-regulated by a feedback loop that involves both transcriptional and post-translational mechanisms. VEGF production by human RPE cells is regulated by HIF-1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.

Footnotes

  • Competing interest: None declared.

This Article

  1. Abstract
  2. Full text
  3. PDF
  4. All versions of this article:
    1. bjo.2007.123125v1
    2. 91/10/1406 most recent

Responses

  1. Submit a response
  2. No responses published

Register for free content

The full back archive is now available for all BMJ Journals. Institutional subscribers may access the entire archive as part of their subscription. Personal subscribers will also have access to all content when logged in. Non-subscribers who register have free access to all articles published before 2006 right back to volume 1 issue 1. Register here to access the free archive of all BMJ Journals.

Don't forget to sign up for content alerts so you keep up to date with all the articles as they are published.