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W.J. Lukiw, P.K. Mukherjee, J.–G. Cui, N.G. Bazan; Mechanism of Hypoxia and Inflammation–Induced Pathoangiogenic Gene Expression in Retinal Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5920.
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© ARVO (1962-2015); The Authors (2016-present)
The induction of retinal–choroidal neovascularization (CNV), the pathogenic growth of new blood vessels from the retinal–choriodal capillaries via the proliferation of retinal vascular endothelial cells, is a major trigger for the retinal–vascular effects of age–related macular degeneration and diabetic retinopathy. There are no effective treatments for these complex, multifactorial disorders, and their molecular–genetic basis is not well understood. Here we have investigated early genetic events associated with cytokine– or hypoxia–induced onset of inflammation and patho–angiogenesis in a retinal cell model.
Retinal choridal endothelial cells (RF/6A) were stressed using the cytokine interleukin–1ß (IL–1ß) or hypoxia (5% O2). Total cellular proteins and RNA were isolated and total genome gene expression patterns were examined using HG U133 plus 2.0 DNA arrays (Affymetrix). Protein abundance was determined using Western immunoassay. Transcription factors (TFs) were quantified using gel–shift assay, and the extent of vessel tube formation (an index of CNV) was monitored using quantitative phase contrast microscopy. Promoter DNA sequence analysis was performed using DNAsis MAX algorithms (Hitachi Genetic Systems).
Tube formation in cytokine– or hypoxia–stressed RF/6A cell cultures was found to be preceded by the significant, coordinated up–regulation of a small family of pro–inflammatory genes that included beta–amyloid precursor protein (ßAPP), cyclooxygenase–2 (COX–2), endothelin–1 (ET–1), interleukin–1ß (IL–1ß) and vascular endothelial growth factor (VEGF). In turn, these changes in gene expression were preceded by increases in DNA–binding of a family of TFs that include hypoxia inducible factor–1 (HIF–1), the nuclear factor NF–ΚB and promoter specificity protein–1 (SP1).
Cytokine–induced inflammatory signaling or hypoxia may be early triggers for retinal pathoangiogenesis, and these events may be regulated upstream by a family of TFs that include HIF–1, NF–ΚB and SP1. DNA recognition features for these oxygen– and stress–sensitive DNA binding proteins are enriched in the immediate promoters of several potentially pathogenic genes that include ßAPP, COX–2, ET–1, IL–1ß and VEGF. Hence, the translation products of this gene family either directly through their expressed proteins, or indirectly through their catalytic activities, may coordinately up–regulate pro–inflammatory signaling pathways that direct endothelial cell fate towards tube–formation and CNV.
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