Abstract
High homocysteine is routinely observed in diabetic patients, and this non-protein amino acid is considered as an independent risk factor for diabetic retinopathy. Homocysteine biosynthesis from methionine forms S-adenosyl methionine (SAM), which is a major methyl donor critical in DNA methylation. Hyperhomocysteinemia is implicated in increased oxidative stress and activation of MMP-9, and in diabetic retinopathy, the activation of MMP-9 facilitates capillary cell apoptosis. Our aim was to investigate the mechanism by which homocysteine activates MMP-9 in diabetic retinopathy. Human retinal endothelial cells, incubated with/without 100 μM homocysteine, were analyzed for MMP-9 and its tissue inhibitor Timp1 expressions and interactions, and ROS levels. Timp1 and MMP-9 promoters were analyzed for methylated and hydroxymethylated cytosine levels (5mC and 5hmC respectively) by the DNA capture method, and DNA- methylating (Dnmt1) and hydroxymethylating enzymes (Tet2) binding by chromatin immunoprecipitation. The results were confirmed in retinal microvessels from diabetic rats receiving homocysteine. Homocysteine supplementation exacerbated hyperglycaemia-induced MMP-9 and ROS levels and decreased Timp1 and its interactions with MMP-9. Homocysteine also aggravated Dnmts and Tets activation, increased 5mC at Timp1 promoter and 5hmC at MMP-9 promoter, and suppressed Timp1 transcription and activated MMP-9 transcription. Similar results were obtained from retinal microvessels from diabetic rats receiving homocysteine. Thus, hyperhomocysteinemia in diabetes activates MMP-9 functionally by reducing Timp1-MMP-9 interactions and transcriptionally by altering DNA methylation-hydroxymethylation of its promoter. The regulation of homocysteine could prevent/slow down the development of retinopathy and prevent their vision loss in diabetic patients.
Highlights
Retinopathy is one of the major microvascular complications of diabetes
We investigated the effect of homocysteine on matrix metalloproteinase-9 (MMP-9)-Timp1 interactions and on the DNA methylation status of the MMP-9 and Timp1 promoters
Diabetes activates MMP-9 in the retina and its vasculature [6] and MMP-9 is activated by homocysteine [14,15]
Summary
Retinopathy is one of the major microvascular complications of diabetes. Cytosolic and mitochondrial free radicals are elevated in the retina and its vasculature, and matrix metalloproteinase-9 (MMP-9) is activated [2,3]. MMP-9 activation is an early event, followed by their translocation inside the mitochondria and damage of the mitochondrial membranes [4]. MMP-9 activity is regulated primarily by its tissue inhibitor, Timp, and Timp binding with MMP-9 catalytic domain blocks access of substrates to the active site of MMP-9. A fine balance between MMP-9 and Timp is important in regulating both MMP-9 proenzyme and MMP-9 activation [5]. While retinal MMP-9 expression is upregulated, that of Timp is downregulated [6,7]
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