Epigenetic modifications in hyperhomocysteinemia; potential role in diabetic retinopathy and age-related macular degeneration

To study Hyperhomocysteinemia (HHcy)-induced epigenetic modifications as potential mechanisms of blood retinal barrier (BRB) dysfunction, retinas isolated from three- week-old mice with elevated level of Homocysteine (Hcy) due to lack of the enzyme cystathionine β-synthase (cbs-/-, cbs+/- and cbs+/+), human retinal endothelial cells (HRECs), and human retinal pigmented epithelial cells (ARPE-19) treated with or without Hcy were evaluated for (1) histone deacetylases (HDAC), (2) DNA methylation (DNMT), and (3) miRNA analysis. Differentially expressed miRNAs in mice with HHcy were further compared with miRNA analysis of diabetic mice retinas (STZ) and miRNAs within the exosomes released from Hcy-treated RPEs. Differentially expressed miRNAs were further evaluated for predicted target genes and associated pathways using Ingenuity Pathway Analysis. HHcy significantly increased HDAC and DNMT activity in HRECs, ARPE-19, and cbs mice retinas, whereas inhibition of HDAC and DNMT decreased Hcy-induced BRB dysfunction. MiRNA profiling detected 127 miRNAs in cbs+/- and 39 miRNAs in cbs-/- mice retinas, which were significantly differentially expressed compared to cbs+/+. MiRNA pathway analysis showed their involvement in HDAC and DNMT activation, endoplasmic reticulum (ER), and oxidative stresses, inflammation, hypoxia, and angiogenesis pathways. Hcy-induced epigenetic modifications may be involved in retinopathies associated with HHcy, such as agerelated macular degeneration and diabetic retinopathy.