Epigenetic Regulation of Chemokine Signaling in Age-Related Macular Degeneration

Decoding critical disease mechanisms for therapeutic exploitation is often made possible through the identification of specific molecular markers, a method which has been successfully translated with several hematologic and soft tissue cancers. With age- related macular degeneration (AMD), the discovery of such a molecular signature that is specific for abnormal retinal pigment epithelium (RPE) or choroidal endothelial cells (CECs) would enable the detection of early disease and serve as a promising therapeutic target. No such entity had been reported until recent data reported from my mentor’s lab that chemokine receptor 3 (CCR3), a cell surface G protein coupled chemokine receptor predominantly found on leukocytes and best known for its role in promoting eosinophil and mast cell trafficking in allergic response [1], was expressed in human CECs only in the context of CNV due to AMD [2]. This finding has revealed a potentially important phenotypic switch in CECs that may help decipher the disease mechanisms by which AMD leads to the formation of CNV. More recently, an independent group found increased expression of a CCR3 ligand, eotaxin-1, both in the RPE and sera of patients with early and intermediate dry AMD [3]. However, the initiating molecular events that lead to this altered chemokine expression remain unknown. New insights into epigenetic derangements and chromatin remodeling have garnered great interest as a potent biologic regulatory system that may serve as a critical aging mechanism. Interestingly, promoter studies and experiments in aging Th2 cells suggested that CCR3 expression is controlled by chromatin remodeling [4, 5]. Such gene-specific chromatin modifications have been found to be important in regulating cell function based on differential expression of gene classes [6]. Histone deacetylases (HDAC) are a unique control of the chromatin remodeling process and have become a novel target for the epigenetic regulation of critical pathways in aging and neurodegenerative diseases [7] including AMD [8]. Preliminary unpublished data from my laboratory demonstrates the human RPE possesses an HDAC1 regulated mechanism of eotaxin-3 expression which is also the most abundant of the eotaxins that we have measured in advanced dry AMD (unpublished data). While HDAC inhibitors are reported to suppress neovascularization in various mouse models [9, 10] and are being employed off-label in the treatment various retinal dystrophies and AMD, the effects HDAC inhibitors on chemokine expression in the chorioretinal microenvironment are very unclear. The proposed work will utilize the latest techniques in molecular biology to quantitatively assess expression levels of various HDAC classes in dry AMD compared to age-matched controls and study HDAC mediated regulation and inhibition of important chemokine signaling axes. Specific Aim 1: Determine expression levels of HDACs in retina, RPE and choroid from eyes with dry AMD compared to age-matched controls using massively parallel sequencing, western blotting, and immunofluorescence. Rationale: There is data to suggest that HDAC expression may change with age; however, no study has systematically evaluated levels of this important epigenetic modifier in dry AMD compared to age-matched controls using advanced molecular techniques. In this aim, I will rigorously study current tissue archives and freshly harvested eyes from patients with dry AMD and appropriate controls to quantitate levels of the various HDAC classes. Specific Aim 2: Study the effects of enforced expression and inhibition of HDACs on specific cytokines including eotaxins and VEGF-A in RPE in vivo in mouse models of dry AMD and in vitro in primary human RPE isolates. Rationale: HDACs modulate the expression of various genes. Preliminary data suggest that HDAC inhibition leads to increased eotaxin-3 in human RPE isolates, similar to RPE from advanced dry AMD. Here, I will study the effects of HDAC expression and inhibition on specific cytokines known to be involved in the pathogenesis and progression of AMD using massively parallel sequencing, multiplex bead assays, and mass-spectrometry based proteomics.