Comparison of human RPE gene expression in macula and peripheryhighlights potential topographic differences in Bruch's membrane

[摘要] Purpose: To describe gene expression differences between healthy,young human retinal pigment epithelium (RPE) cells from the macular areaand RPE cells from two locations in the retinal periphery.Methods: RPE cells from six human donor eyes, ages 17-36, withouthistopathological abnormalities, were dissected by laser and isolatedfrom cryosections. Total RNA was isolated, amplified, and hybridized toa custom made oligonucleotide array containing 22,000 genes.Bioinformatic analysis was carried out using the computer programsRosetta Resolver and the webtools EASE/David and GoStat. Confirmatoryreal time PCR (RT-PCR) and immunohistochemistry were performed accordingto standard protocols.Results: Microarray and statistical analysis yielded 438 genes thatwere differentially expressed between macular RPE, and at least one outof two peripheral RPE locations. Out of these genes, 33 that showed foldchanges of four, or higher, were selected for RT-PCR confirmation. For17 genes (51%), a significant differential expression was found, while11 additional genes (33%) showed a similar trend. Immuno-staining of onetarget (WFDC1) confirmed its differential expression on the proteinlevel. Functional annotation and overrepresentation analysisindependently defined extracellular matrix (ECM) genes as astatistically overrepresented class of genes in our RPE dataset. Intotal, 33 ECM genes were differentially expressed between macular andperipheral RPE regions. A subset of proteins corresponding to thesegenes is known to be present in Bruch's membrane.Conclusions: Our data showed that consistent topographical geneexpression differences in the human RPE constitute around 1-5% of theRPE transcriptome. These changes may underlie topographical differencesin RPE physiology, and pathology and may reflect local differences inthe molecular composition and turnover of Bruch's membrane.

[学科分类] 生物化学/生物物理