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

Molecular and Cellular Biochemistry volume 383, pages 113–122 (2013)Cite this article

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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References

  1. Weidemann A, Johnson RS (2008) Biology of HIF-1alpha. Cell Death Differ 15:621–627

    PubMed  Article  CAS  Google Scholar 

  2. Bracken CP, Whitelaw ML, Peet DJ (2003) The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses. Cell Mol Life Sci 60:1376–1393

    PubMed  Article  CAS  Google Scholar 

  3. Bressler NM, Bressler SB, Fine SL (1988) Age-related macular degeneration. Surv Ophthalmol 32:375–413

    PubMed  Article  CAS  Google Scholar 

  4. McLeod DS, Grebe R, Bhutto I, Merges C, Baba T, Lutty GA (2009) Relationship between RPE and choriocapillaris in age-related macular degeneration. Invest Ophthalmol Vis Sci 50:4982–4991

    PubMed  Article  Google Scholar 

  5. Martin G, Schlunck G, Hansen LL, Agostini HT (2004) Differential expression of angioregulatory factors in normal and CNV-derived human retinal pigment epithelium. Graefes Arch Clin Exp Ophthalmol 242:321–326

    PubMed  Article  CAS  Google Scholar 

  6. Zhang P, Wang Y, Hui Y, Hu D, Wang H, Zhou J, Du H (2007) Inhibition of VEGF expression by targeting HIF-1 alpha with small interference RNA in human RPE cells. Ophthalmologica 221:411–417

    PubMed  Article  CAS  Google Scholar 

  7. Forooghian F, Razavi R, Timms L (2007) Hypoxia-inducible factor expression in human RPE cells. Br J Ophthalmol 91:1406–1410

    PubMed  Article  Google Scholar 

  8. Mowat FM, Luhmann UF, Smith AJ, Lange C, Duran Y, Harten S, Shukla D, Maxwell PH, Ali RR, Bainbridge JW (2010) HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia. PLoS One 5:e11103

    PubMed  Article  Google Scholar 

  9. Wang V, Davis DA, Haque M, Huang LE, Yarchoan R (2005) Differential gene up-regulation by hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha in HEK293T cells. Cancer Res 65:3299–3306

    PubMed  CAS  Google Scholar 

  10. Akeno N, Czyzyk-Krzeska MF, Gross TS, Clemens TL (2001) Hypoxia induces vascular endothelial growth factor gene transcription in human osteoblast-like cells through the hypoxia-inducible factor-2alpha. Endocrinology 142:959–962

    PubMed  Article  CAS  Google Scholar 

  11. Sheridan CM, Pate S, Hiscott P, Wong D, Pattwell DM, Kent D (2009) Expression of hypoxia-inducible factor-1alpha and -2alpha in human choroidal neovascular membranes. Graefes Arch Clin Exp Ophthalmol 247:1361–1367

    PubMed  Article  CAS  Google Scholar 

  12. Koh JY, Choi DW (1987) Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods 20:83–90

    PubMed  Article  CAS  Google Scholar 

  13. Donovan D, Brown NJ, Bishop ET, Lewis CE (2001) Comparison of three in vitro human angiogenesis assays with capillaries formed in vivo. Angiogenesis 4:113–121

    PubMed  Article  CAS  Google Scholar 

  14. Bishop ET, Bell GT, Bloor S, Broom IJ, Hendry NF, Wheatley DN (1999) An in vitro model of angiogenesis: basic features. Angiogenesis 3:335–344

    PubMed  Article  CAS  Google Scholar 

  15. Mahoney DJ, Carey K, Fu MH, Snow R, Cameron-Smith D, Parise G, Tarnopolsky MA (2004) Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise. Physiol Genomics 18:226–231

    PubMed  Article  CAS  Google Scholar 

  16. Ryan HE, Lo J, Johnson RS (1998) HIF-1 is required for solid tumor formation and embryonic vascularization. EMBO J 17:3005–3015

    PubMed  Article  CAS  Google Scholar 

  17. Peng J, Zhang L, Drysdale L, Fong GH (2000) The transcription factor EPAS-1/hypoxia-inducible factor 2alpha plays an important role in vascular remodeling. Proc Natl Acad Sci USA 97:8386–8391

    PubMed  Article  CAS  Google Scholar 

  18. Xiao Q, Zeng S, Ling S, Lv M (2006) Up-regulation of HIF-1alpha and VEGF expression by elevated glucose concentration and hypoxia in cultured human retinal pigment epithelial cells. J Huazhong Univ Sci Technolog Med Sci 26:463–465

    PubMed  Article  CAS  Google Scholar 

  19. Arjamaa O, Nikinmaa M, Salminen A, Kaarniranta K (2009) Regulatory role of HIF-1alpha in the pathogenesis of age-related macular degeneration (AMD). Ageing Res Rev 8:349–358

    PubMed  Article  CAS  Google Scholar 

  20. Carroll VA, Ashcroft M (2006) Role of hypoxia-inducible factor (HIF)-1alpha versus HIF-2alpha in the regulation of HIF target genes in response to hypoxia, insulin-like growth factor-I, or loss of von Hippel-Lindau function: implications for targeting the HIF pathway. Cancer Res 66:6264–6270

    PubMed  Article  CAS  Google Scholar 

  21. Schulz K, Milke L, Rubsamen D, Menrad H, Schmid T, Brune B (2012) HIF-1alpha protein is upregulated in HIF-2alpha depleted cells via enhanced translation. FEBS Lett 586:1652–1657

    PubMed  Article  CAS  Google Scholar 

  22. Le Jan S, Amy C, Cazes A, Monnot C, Lamande N, Favier J, Philippe J, Sibony M, Gasc JM, Corvol P, Germain S (2003) Angiopoietin-like 4 is a proangiogenic factor produced during ischemia and in conventional renal cell carcinoma. Am J Pathol 162:1521–1528

    PubMed  Article  Google Scholar 

  23. Cazes A, Galaup A, Chomel C, Bignon M, Brechot N, Le Jan S, Weber H, Corvol P, Muller L, Germain S, Monnot C (2006) Extracellular matrix-bound angiopoietin-like 4 inhibits endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton. Circ Res 99:1207–1215

    PubMed  Article  CAS  Google Scholar 

  24. Ito Y, Oike Y, Yasunaga K, Hamada K, Miyata K, Matsumoto S, Sugano S, Tanihara H, Masuho Y, Suda T (2003) Inhibition of angiogenesis and vascular leakiness by angiopoietin-related protein 4. Cancer Res 63:6651–6657

    PubMed  CAS  Google Scholar 

  25. Gealekman O, Burkart A, Chouinard M, Nicoloro SM, Straubhaar J, Corvera S (2008) Enhanced angiogenesis in obesity and in response to PPARgamma activators through adipocyte VEGF and ANGPTL4 production. Am J Physiol Endocrinol Metab 295:E1056–E1064

    PubMed  Article  CAS  Google Scholar 

  26. Korfhagen TR, Le Cras TD, Davidson CR, Schmidt SM, Ikegami M, Whitsett JA, Hardie WD (2009) Rapamycin prevents transforming growth factor-alpha-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 41:562–572

    PubMed  Article  CAS  Google Scholar 

  27. Leker RR, Toth ZE, Shahar T, Cassiani-Ingoni R, Szalayova I, Key S, Bratincsak A, Mezey E (2009) Transforming growth factor alpha induces angiogenesis and neurogenesis following stroke. Neuroscience 163:233–243

    PubMed  Article  CAS  Google Scholar 

  28. Cui JZ, Kimura H, Spee C, Thumann G, Hinton DR, Ryan SJ (2000) Natural history of choroidal neovascularization induced by vascular endothelial growth factor in the primate. Graefes Arch Clin Exp Ophthalmol 238:326–333

    PubMed  Article  CAS  Google Scholar 

  29. Krzystolik MG, Afshari MA, Adamis AP, Gaudreault J, Gragoudas ES, Michaud NA, Li W, Connolly E, O’Neill CA, Miller JW (2002) Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol 120:338–346

    PubMed  Article  CAS  Google Scholar 

  30. Wells JA, Murthy R, Chibber R, Nunn A, Molinatti PA, Kohner EM, Gregor ZJ (1996) Levels of vascular endothelial growth factor are elevated in the vitreous of patients with subretinal neovascularisation. Br J Ophthalmol 80:363–366

    PubMed  Article  CAS  Google Scholar 

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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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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

Authors
  1. Takeshi Nakajima
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  2. Emi Nakajima
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  3. Thomas R. Shearer
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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

Keywords

  • Hypoxia-inducible factor-1α and -2α
  • Retinal pigment epithelial cells
  • Hypoxia
  • Vascular endothelial growth factor
  • Age-related macular degeneration