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Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells

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Abstract

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).

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Acknowledgments

We thank Dr. Harrington of the Microarray Core for technical assistance in the DNA microarray assays.

Disclosure

Dr. Shearer is a paid consultant for Senju Pharmaceutical Co., Ltd., a company that may have a commercial interest in the results of this research and technology. Drs. Azuma, T. Nakajima, and E. Nakajima are employees of Senju Pharmaceutical Co., Ltd. These potential conflicts of interest were reviewed, and management plans approved by the OHSU Conflict of Interest in Research Committee were implemented.

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Authors and Affiliations

  1. Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan

    Takeshi Nakajima, Emi Nakajima & Mitsuyoshi Azuma

  2. Department of Integrative Biosciences, Oregon Health & Science University, Portland, OR, USA

    Thomas R. Shearer & Mitsuyoshi Azuma

  3. Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., OHSU West Campus, 20000 NW Walker Rd., Suite JM508, Beaverton, OR, 97006, USA

    Mitsuyoshi Azuma

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  1. Takeshi Nakajima
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  2. Emi Nakajima
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  4. Mitsuyoshi Azuma
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Correspondence to Mitsuyoshi Azuma.

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Nakajima, T., Nakajima, E., Shearer, T.R. et al. Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells. Mol Cell Biochem 383, 113–122 (2013). https://doi.org/10.1007/s11010-013-1760-1

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  • DOI: https://doi.org/10.1007/s11010-013-1760-1

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