Abstract
Abstract: :
Purpose: Hypoxia inducible factor–1 (HIF–1) is a heterodimeric transcription factor capable of transactivating a variety of hypoxia–responsive genes. Small interfering RNA (siRNA) represents a novel approach to post–transcriptional inhibition of target gene expression. In human RPE cell cultures, a gain–of–function model was developed to test whether HIF–1 directly upregulates VEGF, independent of hypoxia. A knockdown model was used to test the ability of siRNA against HIF–1 to block hypoxic induction of both HIF–1 and VEGF. Methods: HIF–1 gain–of–function RPE cells were generated by transfection with a plasmid encoding a mutant form of HIF–1, with the oxygen–dependent domain excised. Unlike native HIF–1, which is rapidly degraded under normoxic conditions, this mutant HIF–1 protein accumulates irrespective of oxygen tension. HIF–1 knockdown RPE cells were generated by transfection with a siRNA targeting HIF–1 transcripts. Hypoxia was induced by exposing cells to 0.3% oxygen tension for 24 hours. HIF–1 and VEGF protein levels were determined by Western blotting and ELISA, and mRNA levels were measured by Northern blotting and quantitative RT–PCR. Results: Under hypoxic conditions both HIF–1 and VEGF protein levels were significantly elevated, as was VEGF mRNA as measured by quantitative RT–PCR. In cells transfected with the constitutively active mutant HIF–1, levels of HIF–1 mRNA and protein were increased. Furthermore, VEGF was upregulated by the mutant HIF–1 even in the absence of hypoxia. In HIF–1 siRNA–treated cells, Northern and Western blotting demonstrated a reduction of HIF–1 mRNA and protein in excess of 90%. Concurrent abrogation of the hypoxic induction of VEGF by HIF–1 knockdown was evident on ELISA and qRT–PCR. Conclusions: RPE cells in response to hypoxia express HIF–1, which in turn is sufficient to upregulate VEGF independent of hypoxia. SiRNA targeting HIF–1 transcripts was effective in reducing both HIF–1 levels and the hypoxic induction of VEGF. These results suggest that HIF–1 plays a pivotal role in ocular angiogenesis, and RNA interference targeting HIF–1 represents a potential therapeutic approach to neovascular eye diseases.
Keywords: retinal neovascularization • growth factors/growth factor receptors • retinal culture