Abstract
Abstract: :
Purpose: In the aging eye there is an increase of apoptosis of the RPE cells solely in the macula zone. Furthermore, the subsequent retinal degeneration in AMD occurs predominantly in the macula whereas the peripheral retina virtually remains intact. Both RPE and choroid are discussed to play a central role in this degeneration process. The different properties of central versus peripheral RPE/choroid may be, at least in part, due to differences in gene expression. Therefore, we quantified the mRNA expression of important genes in RPE/choroid isolated from the macular area and the periphery of human eyes in order to compare their different expression. Methods: The posterior segment of human donor eyes were radially dissected by four cuts. RPE/choroid squares of 4 by 10 mm² were cut out and immediately lysed in RNA lysis buffer. After RNA preparation and oligo–d(T) primed reverse transcription the analyzed genes were quantified, using SybrGreen I –based Real Time RT–PCR. For normalizing the highly expressed gene GAPDH (glyceraldehydes–3–phosphate dehydrogenase) and the lower expressed gene PBGD (porphobilinogen deaminase) were used. Results: The spatial mRNA expression in human RPE/choroid of more than twenty genes were analyzed. Normalization with both calibration genes resulted in comparable mean normalized expression data. The analyzed genes include those important for phagocytosis (aVß5–integrin receptor, CD36/scavanger receptor), integrity of choriocapillaries (VEGF, PEDF), retinoid metabolism (11–cis retinal dehydrogenase), RPE–characterizing genes (RPE65, tyrosinase), neurothrophic factors (BDNF, GDNF), and other genes functioning in signal transduction (G3BP, PIG–B), transcription regulation (NFI–B2, KE03) and imflammation (Cox–2, leukotriene–A4 hydrolase). Neurothropic factors and phagocytosis related genes were up regulated more than twice in the macula compared to the periphery. RPE cell characterizing genes like RPE65, tyrosinase, and 11–cis retinal dehydrogenase as well as PEDF and transcription regulatory genes, were down regulated in the macula to 20% to 50% of the level in the periphery. Conclusions: The spatial quantification of gene expression in normal human RPE/choroid uncovers the importance of the action of different genes in distinct regions of the retina. A further comparison with the corresponding gene expression in pathologically changed eyes (e.g. AMD) may elucidate the influence of specific gene activity that contributes to the onset or progression of degenerative retinal diseases.
Keywords: age–related macular degeneration • gene/expression • retinal pigment epithelium