Abstract
Purpose: :
Iris pigmental epithelial cells (IPE) have been used in transplantation studies to replace damaged or surgically removed RPE, but issues remain as to whether IPE can perform all known RPE functions. Previous study in our lab using DNA microarrays reveal significant differences in the gene expression profiles of native RPE versus IPE. Herein we studied mRNA gene expression of retinoid processing genes to determine possible differences in expression of these genes in RPE versus IPE.
Methods: :
Primary RPE and IPE from six human donors (age: 55–83 years) were collected within 48 hours of death and total RNA was isolated. Oligo primers were constructed with LC Primer Design. Real–time quantitative polymerase chain reaction (Roche Lightcycler) was used to study the expression levels of retinoid metabolisms genes and RT–PCR data were analyzed with Lightcycler3 Data Analysis software.
Results: :
The mRNA for numerous genes that are important in retinoid metabolism are expressed in high levels in RPE compared with only background levels in IPE, including retinal G protein coupled receptor (RGR), rhodopsin (RHO), retinal pigment epithelium derived rhodopsin homolog (RRH), retinol dehydrogenase 5 (RDH5), retinaldehyde binding protein 1 (RLBP1), arrestins (SAG), and RPE65. These proteins are essential for the visual cycle, since RHO/RRH, a blue and UV light–absorbing opsin, and RGR may act together as a stereospecific photoisomerase to generate 11–cis–retinal in the RPE; RDH5 constitutes a major enzyme to oxidize 11–cis–retinol to 11–cis–retinal in the RPE cells to complete the metabolic cycle for visual pigment regeneration; RLBP1 functions as an acceptor of 11–cis–retinol in the isomerization step of the visual cycle and as a substrate carrier for 11–cis–retinol dehydrogenase (e.g. RDH5); arrestins are regulators of the active state of G–protein–coupled receptors; and RPE65 is responsible for isomerization of all–trans–retinaldehyde to its photoactive 11–cis–retinaldehyde.
Conclusions: :
IPE do not express many of the genes known to be essential for the visual cycle, thus suggesting genetic modifications maybe necessary if IPE is used to replace damaged human RPE. Future studies will determine if subretinal transplantation of IPE will shift the gene expression profile of these cells to more closely match native RPE.
Keywords: retinal pigment epithelium • transcription • transplantation