Comparison of human retinal pigment epithelium gene expression in macula and periphery highlights potential topographic differences in Bruch's membrane.
OBJECTIVE
To describe gene expression differences between healthy, young human retinal pigment epithelium (RPE) cells from the macular area and RPE cells from two locations in the retinal periphery.


METHODS
RPE cells from six human donor eyes, ages 17-36, without histopathological abnormalities, were dissected by laser and isolated from cryosections. Total RNA was isolated, amplified, and hybridized to a custom made oligonucleotide array containing 22,000 genes. Bioinformatic analysis was carried out using the computer programs Rosetta Resolver and the webtools EASE/David and GoStat. Confirmatory real time PCR (RT-PCR) and immunohistochemistry were performed according to standard protocols.


RESULTS
Microarray and statistical analysis yielded 438 genes that were differentially expressed between macular RPE, and at least one out of two peripheral RPE locations. Out of these genes, 33 that showed fold changes of four, or higher, were selected for RT-PCR confirmation. For 17 genes (51%), a significant differential expression was found, while 11 additional genes (33%) showed a similar trend. Immuno-staining of one target (WFDC1) confirmed its differential expression on the protein level. Functional annotation and overrepresentation analysis independently defined extracellular matrix (ECM) genes as a statistically overrepresented class of genes in our RPE dataset. In total, 33 ECM genes were differentially expressed between macular and peripheral RPE regions. A subset of proteins corresponding to these genes is known to be present in Bruch's membrane.


CONCLUSIONS
Our data showed that consistent topographical gene expression differences in the human RPE constitute around 1-5% of the RPE transcriptome. These changes may underlie topographical differences in RPE physiology, and pathology and may reflect local differences in the molecular composition and turnover of Bruch's membrane.
van Soest
Simone S
SS
Department of Clinical and Molecular Ophthalmogenetics, The Netherlands Institute for Neuroscience, an institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), Meibergdreef, Amsterdam, The Netherlands.
de Wit
Gerard M J
GM
Essing
Anke H W
AH
ten Brink
Jacoline B
JB
Kamphuis
Willem
W
de Jong
Paulus T V M
PT
Bergen
Arthur A B
AA
D000293:Adolescent; D000328:Adult; D016570:Bruch Membrane / Q000378:metabolism*; D003199:Computer Systems; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D008266:Macula Lutea / Q000378:metabolism*; D046228:Microarray Analysis; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012160:Retina / Q000378:metabolism*; D014018:Tissue Distribution