Abstract
Purpose: :
To compare expression profiles in the retinal pigment epithelium derived from the macula (M) versus the mid–periphery (P1) and far–periphery (P2) in young human eyes, and to identify pathways or individual genes involved in phenotypic differences observed between these regions in several ophthalmic disorders.
Methods: :
Human eyes (age < 40 yrs) were obtained from the Dutch corneabank, within 24 hours postmortem. After freezing of the bulbi, fragments were cut containing central and peripheral parts of the bulbi. Sections were cut and used for histological evaluation and to isolate RPE cells using laser dissection microscopy. RNA was extracted, amplified and hybridized to custom designed oligonucleotide arrays enriched for sequences expressed in human retina and RPE (Agilent technologies). Data analysis was carried out using the Rosetta Resolver analysis package, GOstat and EASE. Real–time qPCR analyses were carried out to confirm the microarray data obtained.
Results: :
ANOVA analysis of the expression profiles M, P1 and P2 generated a list of 859 genes showing significant differences in expression. For 9 out of 13 selected genes (70%), the same significant result was also observed in the qPCR analysis. Analysis using GOstat and EASE resulted in the identification of an overrepresented group of genes linked to the gene–ontology term "extracellular matrix" (GO:0005737). This group contains genes encoding proteins known to be present in the Bruch’s membrane (collagens, laminin, fibrillin) but also a number of proteins that have a less well defined functional role, that may well be expressed in the Bruch’s membrane but also in the interphotoreceptor matrix (Sparc related proteins). Expression of these genes is generally most abundant in periphery. Furthermore, genes with a functional role in angiogenesis, or cell adhesion have been identified as well.
Conclusions: :
The gene expression changes observed for the structural proteins correlate well with the observed decrease in thickness of the Bruch’s membrane (BM) and thickness and integrity of the elastic lamina of the BM especially from the periphery towards the macula. However, the changes observed in genes encoding proteins involved in cell adhesion, ECM degradation and angiogenesis may reveal an interesing link with etiology of age related macular degeneration, diabetic retinopathy, vitreoretinopathy, and (hereditairy) retinal detachment.
Keywords: retinal pigment epithelium • gene microarray • topography