Histone Deacetylase Expression and Inhibition in Age Related Macular Degeneration

Abstract

Purpose: The role of histone deacetylases (HDACs) in the postnatal control of cell survival and gene expression remains unclear. Reports of improved retinal function in retinitis pigmentosa and age-related macular degeneration (AMD) led us to investigate the effects of specific HDAC inhibition on the retinal pigment epithelium (RPE) in vitro and in vivo and analyze HDAC expression in situ.

Methods: Eyes with dry AMD and controls (n=3) were examined by immunohistochemistry for HDAC expression. Total RNA was harvested from RPE/choroid (n=3) and interrogated with Nanostring™ and real-time PCR. Wild-type (Wt) C57BL/6J mouse eyes (n=3) were evaluated with HDAC immunofluorescence. Mice (n=6) were imaged by fundus photography 1 week after intravitreous injection of HDAC inhibitors (suberoylanilide hydroxamic acid (SAHA), trichostatin-A (TSA)) and vehicle (DMSO). ZO-1 immuno-stained RPE/choroid flatmounts (n=3) were analyzed by fluorescent microscopy. RPE/choroid tissue (n=3) was harvested at 48 hours and analyzed by targeted PCR arrays (Taqman®). Primary human RPE (hRPE) isolates (n=6, individual donors) were treated with various HDAC inhibitors. Short-interfering RNAs targeting HDACs were also evaluated in vitro. Cell viability assays and targeted PCR arrays were performed. Statistical analyses were performed with non-parametric tests (Mann-Whitney U) with significance determined at P<0.05.

Results: HDAC-2 was down-regulated in eyes with AMD compared to controls (-1.45±0.23, P<0.05) and expressed in mouse RPE and in hRPE. Intravitreous injection of Class I/II HDAC inhibitors led to focal atrophy of RPE as observed on fundus imaging and ZO-1 immunofluorescence. HDAC inhibition in hRPE resulted in upregulated cell death and wide-spread changes in the cytokine expression profile. Similar disruptions in pro-inflammatory gene expression were observed in the mouse model and with specific targeting of HDAC-2 via RNA interference in vitro.

Conclusions: We found that Class I/II HDAC inhibition resulted in increased RPE cell death and inflammatory cytokine expression. Down-regulation of HDAC-2 was observed in RPE from eyes with AMD suggesting a role for this important epigenetic mechanism in the pathogenesis of this disease.

Keywords: 412 age-related macular degeneration • 533 gene/expression • 701 retinal pigment epithelium