Induction of an aging mRNA retinal pigment epithelial cell phenotype by matrix-containing advanced glycation end products in vitro.
OBJECTIVE
To determine an extensive mRNA phenotype of the established RPE cell line ARPE-19 when grown on a matrix modified by advanced glycation end products (AGEs).


METHODS
Growth Factor Reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) was nonenzymatically glycated with glycolaldehyde. ARPE-19 cells were seeded on both AGE-Matrigel and Matrigel and grown to confluence, and serum was withdrawn for 3 days. RNA was extracted, and microarray analysis was performed to characterize the genes, which are altered by a matrix modified by AGEs. Gene expression changes were confirmed by RT-PCR/Southern and Northern blot analysis. Apoptosis was measured by annexin V/propidium iodide labeling.


RESULTS
Clusters of genes with altered expression were found related to cell differentiation, growth factors that regulate the RPE cell and basement membrane, and apoptosis. RT-PCR/Southern and Northern blot analysis confirmed the expression patterns of selected genes, and flow cytometry showed increased annexin V/propidium iodide-labeled cells when grown on AGE-Matrigel.


CONCLUSIONS
Microarray analysis identified clusters of genes that could promote an aging RPE phenotype in vitro induced by a matrix modified with AGEs.
Honda
S
S
Department of Ophthalmology, University of California, Davis, USA.
Farboud
B
B
Hjelmeland
L M
LM
Handa
J T
JT
D000375:Aging / Q000378:metabolism*; D017304:Annexin A5 / Q000378:metabolism; D001485:Basement Membrane / Q000502:physiology; D015152:Blotting, Northern; D015139:Blotting, Southern; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006133:Growth Substances / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017304:Annexin A5; D017931:DNA Primers; D005136:Eye Proteins; D017127:Glycation End Products, Advanced; D006133:Growth Substances; D012333:RNA, Messenger