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. Age-related macular degeneration (AMD) is a slow progressive disorder that may take few years or decades to develop in to full blown disease leading to loss of vision. Association of Vascular Endothelial Growth Factors (VEGFs), also known as vascular permeability factor (leakage of fluids from blood and lymphatic vessels), with AMD is proven beyond doubt. Current therapies for AMD include anti-VEGF agents (antibodies, small molecules), which are very expensive and needs repeated injections into the back of the eye. For early intervention / prevention of AMD related pathologies, use of nutraceuticals as safe alternatives needs to be explored. We found resveratrol (3,5,4-trihydroxystilbene, RSV), a polyphenolic phytoalexin present naturally in grapes and many plant products, down-regulate the production of VEGFs enhanced by inflammatory cytokines in RPE cells. This inhibitory effect of RSV on VEGFs secretion was observed at a wide range of cytokine concentrations (indicators of inflammation) that mimics intraocular pathological conditions. Under these conditions, RSV had no significant effects on anti-angiogenic molecules such as endostatin and pigment epithelial derived factor. Our initial studies show that RSV acts as anti-angiogenic moleule by inhibiting VEGFs secretion by RPE and possibly other retinal cells. Currently, a large number of general population especially in USA and Europe are using RSV and / or other sources of RSV such as grapes, wine, grape seed extract as a dietary supplement because of many beneficial effects of RSV including life extension. Some nutraceutical companies are marketing natural, synthetic, modified RSV or their combination formulations as over the counter products. We suggest regular use of RSV or other products containing RSV at higher doses may have beneficial effects in preventing / slowing down of neovascularization, a major event associated with AMD. 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.
