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

Abstract

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.

Faulty homocysteine recycling in diabetic retinopathy

“…Homocysteine conversion to SAM serves as a methyl donor for DNA methylation, and DNA methyl transferases (Dnmts) are redox-sensitive enzymes [11,34]. The role of DNA methylation in the regulation of CBS and MTHFR gene transcripts in diabetic retinopathy was determined.…”

“…Although hyperglycemia is considered as the main instigator of its development, systemic factors including hyperlipidemia and blood pressure are also intimately associated with the development of diabetic retinopathy [35]. Nondiabetic normal population generally has > 15 μM plasma homocysteine, but in diabetic patients, they can go as high as 50-100 μM [10,11]. High homocysteine in diabetic patients is associated with increased macular thickness without macular edema [36], and in patients with retinopathy, high homocysteine is considered to act as a common link through which other systemic factors could exert their deleterious effect on the progression of diabetic retinopathy [6,37].…”

“…High plasma homocysteine levels are associated with endothelial dysfunction, and in diabetic patients, with many complications including nephropathy, cardiomyopathy and neuropathy [7][8][9]. Studies using genetically manipulated mice that can accumulate homocysteine have suggested a role for homocysteine in diabetic retinopathy; these animals have impaired visual function and damaged blood retinal barrier [10,11]. Homocysteine was also shown to induce mitochondrial dysfunction, and in retinal ganglion cells, it was implicated in the dysregulation of mitochondrial dynamics [12].…”

Homocysteine Induces Inflammation in Retina and Brain

“…However, the underlying mechanisms of HHcy-induced barrier dysfunction need further investigation. Recently, we reported endoplasmic reticulum (ER) stress [23], oxidative stress [19], and epigenetic modification [24] as possible mechanisms, however, major questions remain regarding the role of inflammation in this process.…”

“…We used microglia as the main cell activated in inflammation in addition to HRECs and RPE, which play crucial roles in inner and outer BRB dysfunction in DR and AMD, respectively. Different concentrations of Hcy were also used to evaluate the effect of various levels of HHcy on the induction of inflammation in different cells and in different disease models, as reported in our previous studies [19,20,24].…”