Abstract
Stress-associated premature senescence (SAPS) is involved in retinal microvascular injury and diabetic retinopathy. We have investigated the role and mode of action of miR-34a in retinal endothelial cells senescence in response to glucidic stress. Human retinal microvascular endothelial cells (HuREC) were exposed to glucidic stress (high glucose (HG) = 25 mM d-glucose) and compared to cells exposed to normal glucose (NG = 5 mM) or the osmotic control l-glucose (LG = 25 mM). HG stimulation of HuREC increased the expression of miR-34a and induced cellular senescence. HG also increased the expression of p16ink4a and p21waf1, while decreasing the histone deacetylase SIRT1. These effects were associated with diminished mitochondrial function and loss of mitochondrial biogenesis factors (i.e., PGC-1α, NRF1, and TFAM). Transfection of the cells with miR-34a inhibitor (IB) halted HG-induced mitochondrial dysfunction and up-regulation of senescence-associated markers, whereas miR-34a mimic promoted cellular senescence and mitochondrial dysfunction. Moreover, HG lowered levels of the mitochondrial antioxidants TrxR2 and SOD2, an effect blunted by miR-34a IB, and promoted by miR-34a mimic. 3’-UTR (3’-untranslated region) reporter assay of both genes validated TrxR2 as a direct target of miR-34a, but not SOD2. Our results show that miR-34a is a key player of HG-induced SAPS in retinal endothelial cells via multiple pathways involved in mitochondrial function and biogenesis.
Highlights
Diabetic retinopathy (DR) is a potentially blinding complication characterized by progressive retinal neurovascular injury and consequent visual impairment [1,2]
To determine whether up-regulation miR-34a contributes by lower florescence intensity with TMREof and MitoTracker dyes.to HG-induced vascular senescence and increased reactive oxygen species (ROS) production, we examined the effects of inhibiting miR-34a activity by transfecting the withInhibition a specificPrevents antagomiR
34a inhibitor significantly reduced the number of SA- -Gal positive cells (p < 0.01; Figure 4A, B), and increased ROS production, we examined the effects of inhibiting miR-34a activity by transfecting their number remained higher than control (NG)
Summary
Diabetic retinopathy (DR) is a potentially blinding complication characterized by progressive retinal neurovascular injury and consequent visual impairment [1,2]. Recent studies by our group and others have shown that hyperglycemia accelerates vascular senescence through a process known as stress-associated premature senescence (SAPS) [3,4,5]. Antioxidants 2019, 8, 328 pathogenic mechanism appears to be associated with increased oxidative stress and epigenetic silencing of NAD+ -dependent histone deacetylase sirtuin-1 (silent mating type information regulation 2 homolog). Endothelial cells acquire senescence-like features, leading to expression of inflammatory cytokines and increased apoptosis [7,8,9]. Clinical and experimental evidence implicates epigenetic mechanisms, such as differential expression of microRNAs (miRs), in the pathogenic processes involved in diabetes-induced retinal neurovascular injury [10,11]
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