Abstract
Purpose :
Activation of retinal matrix metalloproteinase-9 (MMP-9) in diabetes is associated with accelerated capillary cell apoptosis. We have shown that the MMP-9 promoter undergoes epigenetic modifications, and its DNA is hydroxymethylated. Diabetic patients also have increased circulating homocysteine levels, and hyperhomocysteinemia is now considered as a risk factor for the development of diabetic retinopathy. Our aim was to investigate the role of homocysteine in the development of diabetic retinopathy.
Methods :
Human retinal endothelial cells (HRECs) incubated in high glucose, in the presence or absence of 100μM homocysteine, were analyzed for the expression of MMP-9 and its tissue inhibitor Timp1, DNA methylation machinery, and cell apoptosis. DNA methylation status of MMP-9 and Timp1 promoters was determined by quantifying methylated cytosine (5mC) and 5-hydroxy methylcytosine (5hmC) using immuno-capture technique. The binding of DNA methyl transferase-1 (Dnmt1) and hydroxymethylation enzymes Ten-Eleven Translocase-2 (Tet2) at the promoters were investigated by chromatin immunoprecipitation technique. Similar studies were performed in the retinal microvessels (using hypotonic method) from streptozotocin-induced diabetic rats received intraperitoneal administration of homocysteine (100μg/ kg BW/day) for 8 weeks.
Results :
Supplementation of homocysteine in a high glucose medium synergistically increased MMP-9, decreased Timp1, and exacerbated endothelial cell apoptosis. Compared to high glucose alone, Dnmt and Tet were also further increased, and while MMP-9 promoter had increased 5hmC, Dnmt1 and Te2 binding, Timp1 promoter had increased 5mC. Similar changes in dynamic DNA methylation were observed at the promoters of MMP-9 and Timp1 in the retinal microvessels from diabetic rats that received homocysteine.
Conclusions :
Increased homocysteine in diabetic environment epigenetically modifies both MMP-9 and its tissue inhibitor, exacerbating MMP-9 activation and capillary cell apoptosis. Thus, managing hyperhomocysteinemia in diabetic patients could protect retinal capillary cells from undergoing accelerated apoptosis, and slow down the development of retinopathy.
This is a 2020 ARVO Annual Meeting abstract.