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A more recent version of this article appeared on February 1, 2010
 (Investigative Ophthalmology and Visual Science. )
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.09-3622
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Microarray Analysis Identifies Changes in Inflammatory Gene Expression in Response to Amyloid-Beta Stimulation of Cultured Human Retinal Pigment Epithelial Cells

Khaliq Kurji,1 Jing Cui,2 Tony Lin,3 David Harriman,4 Shiv Prasad,5 Ljuba Kojic,6 and Joanne Matsubara7

1Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, Canada 2Ophthalmology, Eye Care Centre, vancouver, Canada 3Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, Canada 4Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, Canada 5Centre for Biologics Research, Health Canada, Ottawa, Canada 6Brain Research Centre, University of British Columbia, Vancouver, Canada 7Ophthalmology, University of British Columbia, Vancouver, Canada

Joanne Matsubara, Email: jms{at}interchange.ubc.ca

Abstract

Purpose: Age related macular degeneration (AMD) is a common cause of irreversible vision loss in the elderly. We hypothesize that in vitro stimulation of RPE cells with Aβ1-40, a constituent of drusen, will promote changes in gene expression and cellular pathways associated with the pathogenesis of AMD, including oxidative stress, inflammation, and angiogenesis.

Methods: Confluent human RPE cells were stimulated with Aβ1-40, or the reverse peptide Aβ40-1, and genome wide changes in gene expression were studied with Agilent Oligo microarrays. Selected genes were verified by qRT-PCR and ELISA. Pathway analysis with Gene Set Enrichment Analysis (GSEA) and Ingenuity revealed top functional pathways in RPE after Aβ1-40 stimulation.

Results: RPE cells stimulated with Aβ1-40 (0.3 µM) for 24 hrs resulted in 63 upregulated and 22 downregulated previously known genes. The upregulated genes were predominantly in inflammatory and immune response categories, but other categories were also represented, including apoptosis, cell signalling, cell proliferation and signal transduction. Categories of downregulated genes included immune response, transporters, metabolic functions and transcription factors. ELISA confirmed that secreted levels of IL-8 were 2 fold higher than controls. GSEA and Ingenuity analysis confirmed that the top affected pathways in RPE cells following with Aβ1-40 stimulation were inflammation and immune response related. Surprisingly, few angiogenic pathways were activated at the dosages and exposure times studied here.

Conclusions: Aβ1-40 promotes RPE gene expression changes in pathways associated with immune response, inflammation, cytokine and interferon signalling pathways. Results may relate to in vivo mechanisms associated with the pathogenesis of AMD.

Key Words: drusen • age-related macular degeneration • cell culture • gene expression • complement factor I • RPE






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