RPE Cytotoxicity and Caspase Activation after Treatment with Valproic Acid

Abstract

Purpose: Age-related macular degeneration (AMD) is a progressive disease of the retina, retinal pigment epithelium (RPE) and choroid. Recent studies suggest that the HDAC inhibitor valproic acid (VPA) has neuroprotective properties and potential therapeutic efficacy in retinal degeneration. Here, we show that intravitreous delivery of VPA in a mouse model and treatment of human cell cultures induces RPE cytotoxicity. HDACs have become a novel target in aging and neurodegenerative diseases including AMD. An improved understanding of their regulation and pharmacologic inhibition in the eye is essential prior to translation to clinical studies.

Methods: Primary human RPE (hRPE) isolates (n=6, individual single donors) were treated with VPA (1mM/4mM) or vehicle (PBS). Cell viability assays (SYTOX®, Invitrogen), caspase-3 fluorometric quantification (DEVD-AFC, Biovision) and targeted PCR arrays (Taqman®, Life) were performed. Wild-type C57BL/6J mice (n=6-8) were evaluated by fundus photography at baseline and 7 days after intravitreous injection of VPA (1/10μg). RPE/choroid flatmounts (n=4-6) were immuno-stained for the intercellular junction marker, zonula occludens-1 (ZO-1) and examined by fluorescent microscopy. RPE/choroid tissue (n=3) was harvested at 48 hours and analyzed by targeted PCR arrays.

Results: Exposure of primary hRPE isolates to the HDAC inhibitor VPA led to a two fold increase in cell death (48 hours) and to a 4 fold upregulation of caspase 3 (P<0.05) at 12 hours compared to vehicle. Intravitreous injection of VPA induced frank RPE loss, deep hypo-pigmented lesions in the fundus image and severe disruption of ZO-1 staining on RPE flatmounts. Targeted PCR arrays revealed wide-spread disruption in pro-inflammatory cytokine gene expression in both human and mouse samples.

Conclusions: Intravitreous injection in C57BL/6J mice or treatment of primary hRPE isolates with VPA induced cytotoxicity in a dose-dependent fashion with increased caspase-3 activation. Widely altered cytokine expression was observed with VPA and current studies in our laboratory have focused on the specific HDACs that are contributory to the inflammatory response.

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium • 615 neuroprotection