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 age-related macular degeneration and diabetic retinopathy.

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-β-synthase is deficient (cbs −/−), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.