Retinal pigment epithelium (RPE), a single layer of cells present between the retina and choroid in the eye, is vital for the normal functioning of the retina. Many of the degenerative,inflammatory and age-related diseases of the retina are associated with the degeneration and /or dysfunction of the RPE. We have developed a human RPE cell culture system,derived from adult donor eyes, and have used this as a model to investigate the various roles of RPE in the pathophysiology of retinal disorders. We focused our attention on growth factors and inflammatory cytokines, since these molecules are involved in many of the retinal disorders such as uveitis, age related macular degeneration (AMD), diabetic retinopathy and retinal detachments. If untreated, these disorders may lead to loss of visual function. Since the association of inflammation with retinal disorders (AMD) is now recognized as a key component, we evaluated the role of inflammatory mediators on the expression of VEGF, a known agent in vascular leakage, retinal and choroidal neovascularization in AMD. Inflammatory cytokines are produced in the retinal microenvironment by macrophages and other infilterating cells into the retina and choroid. Using GeneChip (Human Genome U133 plus array, Affymetrix),we evaluated the effects of inflammatory cytokines(IC mix=interferon-gamma, interleukin-1 and tumor necrosis factor-alpha)on human RPE cells by microarray analyses. This system provides genome-wide changes in the expresssion of most of the characterized human genes. IC mix significantly enhanced the expression of many of the cytokines and chemokines as well as growth factors such as vascular endothelial growth factors (VEGFs). It is important to note that VEGFs are associated with many of the blinding retinal disorders like ARMD. Microarray analysis revealed about 10 fold increase in the levels of VEGF-A and VEGF-C mRNA in HRPE cells treated with IC mix. We validated microarray results by studying the secretion of VEGFs proteins by HRPE. The secretion of VEGF-A and VEGF-C increased by 10 to 20 fold in HRPE cells treated with IC mix or with individual cytokines. Even at very low concentrations of IC, that mimic the patho-physiological conditions within retinal microenvironment, significant quantities of VEGFs are secreted by HRPE cells. Other regulators of angiogenesis such as angiopoietins, thrombospondins, endostatins and pigment epithelial derived factors were not effected by IC mix treatment. Our results showed close relationship between inflammatory events (IC mix)and choroidal neovascularization (VEGFs) in ARMD. Affymetrix microarray studies for gene expression profiling also surprisingly revealed the induction of interleukin-11 (IL-11) by transforming growth factor-beta(TGF-b). IL-11 acts as an anti-inflammatory agent by inhibiting the macrophage production of TNF-alpha and IL-1. IL-11 was also shown to have cytoprotective properties by inhibiting cellular death pathway known as apoptosis. We found that IFN-gamma down regulates IL-1 and TNF-alpha induced IL-11 secretion by RPE and choroidal cells. In contrast, TGF-b induced IL-11 secretion was not affected by IFN-gamma. We are evaluating the mechanisms of the regulation of expression of IL-11 and potential usefulness of IL-11 as an agent in the protection of retina from oxidative and inflammatory insults. Recent discoveries in cellular and molecular biology point to the microRNAs(miRNAs)as potential regulators of gene expression and as valuable tools in the development of ocular therapeutics. miRNAs are noncoding RNA oligonucleotides of 20-25 bases. So far only about 500 miRNA genes have been identified, and their products are processed both in nucleus and cytoplasm by a variety of enzymes to produce mature forms. miRNAs bind to 3'untranslated regions of messengerRNAs(mRNA)that may lead to mRNA degradation and/or inhibition of translation. Potential applications of miRNAs as tools in understanding and treatment of ocular diseases are being investigated. Using microRNA array analysis, we studied the regulation of miRNA expression in human RPE cells by inflammatory mediators. Initial results showed significant upregulation of mir-155 and mir-146 and these results have been validated by Real Time PCR analysis. We are currently investigating the role of mir-155 and mir-146 in the regulation of the expression of various cytokines, chemokines and growth factors associated with retinal disorders.
