Abstract
Purpose: :
In choroidal neovascularization (CNV), newly formed blood vessels enter the subretinal space, where leakage and bleeding lead to retinal detachment and photoreceptor death. Factors such as vascular endothelial growth factor (VEGF) have been shown to be important in CNV. Their expression is related to hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of a constitutive β- and an oxygen-dependent -subunit. Hypoxia can increase HIF-1 expression in retinal pigment epithelial (RPE) cells, suggesting an important role of HIF-1 in ocular angiogenesis. Yet, how HIF-1 is related to RPE cell death and subsequent photoreceptor atrophy is unclear. We aim to investigate the association of HIF-1 with expression of angiogenic factors and cell death in RPE cells under reduced oxygen tension using an in vitro hypoxia model.
Methods: :
Small interfering RNA (siRNA) were transfected into ARPE-19 cells to down-regulate HIF-1 expression. Cells were then subjected to hypoxia in an anoxic chamber (95% N2, 5% CO2) for various time periods. HIF-1 expression was analyzed by Western blot and VEGF secretion was measured by ELISA. LDH assay was performed to assess for cytotoxicity. Protein expression profile will be examined by 2-dimensional gel electrophoresis and mass spectrometry.
Results: :
The level of HIF-1 expression and VEGF secretion from un-transfected ARPE-19 cells increased with prolonged exposure to hypoxia and peaked at 24 hour. Interestingly, cell death remained low under hypoxia, even after 24 hours when HIF-1 expression was the highest. On the other hand, siRNA treatment significantly down-regulated the expression of HIF-1 at all time points despite exposure to hypoxia. Furthermore, VEGF secretion from the HIF-1-diminished RPE cells under hypoxia were reduced. Proteomic analysis results will be presented later.
Conclusions: :
Treatment with siRNA down-regulated the expression of HIF-1 and reduced the secretion of VEGF in APRE-19 cells under hypoxia. Differential protein expressions in RPE cells upon hypoxia after HIF-1 down-regulation will be discussed.
Keywords: hypoxia • RNAi • retinal pigment epithelium