Abstract
Purpose :
Preliminary data demonstrate significant down-regulation of HDAC1/2 expression in the RPE of eyes with atrophic age-related macular degeneration (AMD). RPE cytotoxicity and patterned inflammatory gene expression were observed after Class I HDAC inhibition in vitro and in vivo. In this study, we investigated the cellular effects of a potent HDAC1/2 inhibitor, romidepsin, on RPE cell viability and interrogated unique signatures of acetyl-histone marks.
Methods :
Short term and long-term (>21d) primary human RPE (hRPE) isolates (n=3, Lonza) and ARPE19 cultures were exposed to a dose range of romidepsin (0.1/0.5/1 ng/mL, Selleck). Cell viability was assessed using MTS assay (AQueous One, Promega, measurement of trans-epithelial resistance (TER) and evaluation of tight-junction by ZO-1 immunofluorescence. Levels of histone acetylation (H3Ac/H4Ac) were measured by Western Blotting while specific acetyl-histone marks were evaluated by multiplex assay (EpiGentek). Statistical analyses were performed with ANOVA and Mann-Whitney U test.
Results :
Romidepsin induced a dose-dependent loss of RPE cell viability by MTS assay (P<0.05) in both short-term and long-term RPE cultures with decreased TER (P<0.05) and degradation of ZO-1 tight junctions. Western blotting revealed significantly upgregulated H3ac and H4ac levels after romidepsin treatment (12/24h, P<0.05). Multiplex assays identified a specific acetyl-histone signature with elevated levels of H3k14ac, H3k18ac, H4k8ac and H4k16ac.
Conclusions :
Class I HDAC expression is critical for RPE homeostasis and cell viability. Targeted inhibition of HDAC1/2 with romidepsin leads to loss of cell viability, decreased TER, disorganized tight junctions in long-term hRPE cultures. There is robust upregulation of acetylated H3/H4 with a specific signature of acetyl-histone marks that may be critical in the epigenetic regulation of RPE cell death. These histone marks may be important in signaling pathways of RPE degeneration that occur during progression of atrophic AMD and may serve as novel targets for AMD therapeutics.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.