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T. Nakajima, E. Nakajima, T. R. Shearer, M. Azuma; Hypoxia-Inducible Factor (HIF)-2 siRNA Augments Inhibition of VEGF Expression by HIF-1 siRNA in Hypoxic, Cultured RPE Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1188.
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© ARVO (1962-2015); The Authors (2016-present)
Hypoxia inducible factors (HIF-1 and HIF-2) are fragile proteins under normal physiological conditions. In response to hypoxia, HIF-1 and HIF-2 become stable, form a hetero-dimer with HIF-1β or HIF-2β, and induce transcription of angiogenic factors such as VEGF. Disruption of regulated HIF activity causes induction of pathologic angiogenesis observed in diabetic retinopathy and age-related macular disease. Although it has well known that HIF-1 plays an important role in VEGF induction in hypoxic retinal pigment epithelial (RPE) cells, the involvement of the other isoform, HIF-2, remains unclear. Thus, the purpose of present study is to clarify the contribution of HIF-2 to the induction of angiogenic genes, including VEGF, in cultured RPE cells.
Human RPE cells (ARPE-19) were cultured under hypoxic conditions. Small interference RNAs (siRNA) were used to elucidate the involvement of HIFs. Expression of mRNAs for HIF-1, HIF-2, and VEGF was measured by quantitative PCR. HIF-1 and HIF-2 proteins were analyzed by immunoblotting. VEGF protein secreted from RPE cells into the medium was measured using ELISA. Other angiogenic-regulated genes were analyzed in RPE by PCR arrays.
HIF-1 and HIF-2 proteins increased in hypoxic RPE cells. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. Inhibition of expression of mRNAs for HIF-1 and HIF-2 by siRNAs decreased HIF proteins to normal baseline levels. The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1 siRNA, but not by HIF-2 siRNA. However, the addition of HIF-2 siRNA with HIF-1 siRNA did greatly augment the inhibitory effect of HIF-1 siRNA against VEGF induction in hypoxic RPE cells. Reduction of VEGF protein in medium was well correlated with VEGF mRNA levels. PCR arrays revealed that not only VEGF, but also other angiogenic-regulated genes were regulated by both HIF-1 and HIF-2.
HIF-1 appeared to be the major isoform of HIF regulating VEGF expression in RPE cells cultured under hypoxia. However, augmentation of VEGF inhibition by HIF-2 siRNA suggested an important role of HIF-2 in the regulation of VEGF expression. Thus, inhibition of both HIF-1 and HIF-2 during VEGF production may be a possible therapy against retinal angiogenesis in conditions such as diabetic retinopathy and age-related macular disease.
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