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 inflammatory, infectious and other diseases of the retina are associated with the degeneration and /or dysfunction of the RPE. We have developed a human RPE cell culture system 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 transforming growth factor-beta (TGF-β), since TGF-β is involved in retinal disorders of proliferative, degenerative, inflammatory and infectious etiology. Age related macular degeneration (ARMD), proliferative vitreoretinopathy (PVR) and retinal detachments (RD) are the leading causes of visual impairment. We evaluated the effects of TGF-β on human RPE cells by microarray analyses by using GeneChip (Human Genome U133 plus array, Affymetrix). This system provides genome-wide changes in the expresssion of most of the characterized human genes. TGF-β significantly enhanced the expression of many of the extracellular matrix proteins (ECM) such as collagens, fibronectin, thrombospondin and chondritin sulfate proteoglycans as well as many growth factors such as vascular endothelial growth factors (VEGF) and platelet derived growth factors (PDGF). It is important to note that ECM and growth factors are associated with many of the blinding retinal disorders like ARMD, PVR, RD. We are using microarray analysis to identify the expression of extracellular macromolecules, not known to be present in the retina,and their possible role in retinal pathophysiology.? ? In order to study the role of ECM components and their regulation by TGF-β, we generated a mouse model by conditionally deleting TGF-β signaling in the neurons (cKO) by crossing TGF-β receptor I (TGF-β RI) floxed mice with nestin-Cre mice. The retinal detachments, seen in almost all of the cKO mice at birth, are at the RPE / photoreceptor layer junction of the neurosensory retina (NSR). The immunoassays for chondroitin-6-sulfate showed a very weak reaction in cKO mice in contrast to intense positive staining in the photoreceptor layer in wild-type mice. Studies with our cKO model suggest that the lack of functional TGF-β RI in retinal cells leads to decreased levels of chondroitin sulfate proteoglycans in the subretinal space and photoreceptor layers. This, in turn, causes retinal detachment due to the loss of adhesion of the NSR to RPE. ? ? We have previously reported that TGF-β is a potent stimulator of VEGF and PDGF expression by human RPE cells. Since inflammation in retinal disorders (ARMD) is now recognized as a key component, we evaluated the role of inflammatory mediators on the expression of VEGF, a known agent in retinal neovascularization in ARMD. Our results showed that Interferon-γ (IFN-γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) enhance significantly the secretion of VEGF-A and VEGF-C by RPE and choroidal cells. IFN-γ, IL-1, TNF-α and other cytokines are produced in the retinal microenvironment by macrophages and other infilterating cells into the retina and choroid. We are evaluating the mechanisms of the regulation of VEGF-A, VEGF-C and other angiogenic factors(angiogenin and pigment epithelial derived growth factor) by inflammatory mediators to delineate the involvement of inflammatory processes in ARMD and other retinal neovascularization disorders.? ? Affymetrix microarray studies for gene expression profiles also surprisingly revealed the induction of interleukin-11 (IL-11) by TGF-β. IL-11 acts as an anti-inflammatory agent by inhibiting the macrophage production of TNF-α and IL-1. IL-11 was also shown to have cytoprotective properties by inhibiting cellular death pathway known as apoptosis. We found that IFN-γ dowwn regulates IL-1 and TNF-α induced IL-11 secretion by RPE and choroidal cells. In contrast, TGF-β induced IL-11 secretion was not affected by IFN-γ. 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. miRNAs are noncoding RNA oligonucleotides of 20-25 bases. So far only about 500 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.
Showing the most recent 10 out of 15 publications
