Microarray analysis identifies changes in inflammatory gene expression in response to amyloid-beta stimulation of cultured human retinal pigment epithelial cells.
OBJECTIVE
Age-related macular degeneration (AMD) is a common cause of irreversible vision loss in the elderly. The hypothesis was that in vitro stimulation of RPE cells with Abeta(1-40), a constituent of drusen, promotes 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 Abeta(1-40), or the reverse peptide Abeta(40-1), and genome wide changes in gene expression were studied with gene 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 Abeta(1-40) stimulation.


RESULTS
RPE cells stimulated with Abeta(1-40) (0.3 microM) for 24 hours 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 signaling, 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 two times higher than control levels. GSEA and ingenuity analysis confirmed that the top affected pathways in RPE cells after Abeta(1-40) stimulation were inflammation and immune response related. Surprisingly, few angiogenic pathways were activated at the doses and exposure times studied.


CONCLUSIONS
Abeta(1-40) promotes RPE gene expression changes in pathways associated with immune response, inflammation, and cytokine and interferon signaling pathways. Results may relate to in vivo mechanisms associated with the pathogenesis of AMD.
Department of Ophthalmology and Visual Sciences, University of British Columbia, British Columbia, Canada.
Khaliq H
KH
Kurji
Jing Z
JZ
Cui
Tony
T
Lin
David
D
Harriman
Shiv S
SS
Prasad
Ljuba
L
Kojic
Joanne A
JA
Matsubara
D016229:Amyloid beta-Peptides / Q000494:pharmacology*; D043925:Angiogenesis Inducing Agents / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D018625:Microscopy, Atomic Force; D020411:Oligonucleotide Array Sequence Analysis; D010446:Peptide Fragments / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016229:Amyloid beta-Peptides; D043925:Angiogenesis Inducing Agents; D016207:Cytokines; D005136:Eye Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; C078592:amyloid beta-protein (1-40)