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Role of Oxidative Stress in Epigenetic Modification of Matrix Metalloproteinase-9 in the Development of Diabetic Retinopathy
was 8,65±1.39%. The control group consisted of healthy individuals: Mongoloids with absence of family history for diabetes (n = 34, mean age 30,3 ± 1,3 years). Serum and hemolysate used as material for the study. Spectrophotometric and fluorometric methods used. Results: In Mongoloid patients the diene conjugates level increased compared with the control group has been found (1.34times higher , p<0,01). Antioxidant status in T1DM Mongoloids was characterized by a statistically significant increased blood total antioxidant activity (1.54-times higher, p<0,001) and a slight decreased superoxide dismutase activity (p<0,01) comparing to control group. Conclusion: Changes in the system of POL-AOD in Mongoloidpatients are characterized by low intensity, which is probably due to the increased activity of the antioxidant defense system. These data are confirmed by clinical course of type 1 diabetes mellitus with a rare development of complications in Mongoloids of Eastern Siberia compared to average performance in all population and figures of European one.
doi: 10.1016/j.freeradbiomed.2016.10.463 423 Role of Oxidative Stress in Epigenetic Modification of Matrix Metalloproteinase-9 in the Development of Diabetic Retinopathy Renu A Kowluru1 1 Wayne State University, Detroit, USA In the pathogenesis of diabetic retinopathy, damaged retinal mitochondria accelerate apoptosis of retinal capillary cells, and regulation of oxidative stress by manipulating mitochondrial superoxide dismutase (Sod2) protects mitochondrial homeostasis and prevents the development of diabetic retinopathy. We have shown that diabetes activates retinal matrix metalloproteinase-9, and activated MMP-9 damages the mitochondria. Recent studies have shown that the machinery responsible for epigenetic modifications is also altered in diabetes, and a dynamic DNA methylation process plays an important role in regulation of retinal MMP-9 transcription. The aim of this study is to investigate the role of oxidative stress in retinal MMP-9 transcription in diabetes. Methods: The effect of regulation of mitochondrial superoxide on DNA methylation of MMP-9 promoter was investigated in retinal endothelial cells incubated in the presence or absence of a MnSOD mimetic- MnTBAP, by quantifying the levels of 5 methyl cytosine (5mC) and hydroxyl-methyl cytosine (5hmC). The binding of DNA methylating and hydroxymenthylating enzymes (Dnmts and Tets, respectively) at MMP-9 promoter (by chromatin immunoprecipitation) and mitochondrial homeostasis (cytochrome c leakage in cytosol and mtDNA transcription) were also evaluated. The in vitro results were confirmed in the retina of diabetic mice overexpressing Sod2. Results: Addition of MnTBAP ameliorated glucose-induced decrease in 5mC levels and increase in Dnmt1 binding at the MMP9 promoter. In the same cell preparations, MnTBAP also ameliorated alterations in 5hmC levels and Tet binding, regulated MMP-9 transcription and prevented mitochondrial damage. Similarly, mice overexpressing Sod2 were protected from diabetesinduced alteration in MMP-9 promoter methylation and its transcription, and mitochondrial dysfunction. Conclusions: Thus, oxidative stress regulation by chemical/genetic approaches maintains retinal mitochondrial homeostasis by ameliorating epigenetic modifications of the MMP-9 promoter.
S176
doi: 10.1016/j.freeradbiomed.2016.10.464 424 Advanced Glycation End Products Inhibition Restores Sphingolipid Rheostat in Mice Liver and Prevents Diet-Induced Insulin Resistance Alessia Sofia Cento1, Debora Nigro1, Fausto Chiazza1, Federica Dal Bell1, Francesco Romaniello1, Massimo Collino1, Claudio Medana1, Manuela Aragno1, and Raffaella Mastrocola1 1 University of Turin, Italy The maintenance of sphingolipid rheostat by interconversion of ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) is relevant for the control of several metabolic pathways. In particular, accumulation of Cer and S1P in response to cellular stress interferes with insulin signalling and mitochondrial metabolism. Emerging evidence has highlighted a role for Advanced Glycation End Products (AGEs) in the imbalance of the sphingolipid rheostat. We and others have recently demonstrated the accumulation of AGEs in tissues of diet-induced insulinresistant mice and their involvement in altered lipid synthesis and mitochondrial dysfunction. We, thus, investigated the impact of a high trans fat diet on the sphingolipid rheostat in mice liver and whether the administration of the anti-AGEs compound pyridoxamine could prevent insulin resistance onset by restoring the Cer/Sph/S1P equilibrium. C57Bl/6J mice were fed a standard diet (SD) or a 60% trans fat diet (HFD) for 10 weeks. Two subgroups received pyridoxamine (150 mg/kg/day) in the drinking water. The pyridoxamine administration efficiently prevented insulin resistance, obesity, AGEs accumulation and RAGE hyperexpression induced by the HFD. HFD also induced a dramatic reduction of the liver Sph levels, paralleled by the increase in Cer levels, assessed by an UPLC/TQMS system, and the induction of the S1P receptors. Interestingly, pyridoxamine administration prevented the Cer increase by reducing Cer synthase expression and significantly inhibited S1P receptors by reducing S1P kinase levels, completely reverting Sph depletion. The present data indicate for the first time that the inhibition of AGEs is effective in the maintenance of the sphingolipid rheostat through the modulation of the involved enzymes, and this could contribute to the prevention of insulin resistance observed in the pyridoxamine-treated HFD mice. Therefore, it could be of relevance to further investigate on the predictive value of Cer/Sph/S1P imbalance for insulin resistance onset and on pyridoxamine supplementation as preventive strategy.
doi: 10.1016/j.freeradbiomed.2016.10.465 425 A Potential Role of Nox1 Isoform of NADPH Oxidase in the Development of Nonalcoholic Steatohepatitis Misaki Matsumoto1, Jia Zhang1, Xueqing Zhang1, and Chihiro YabeNishimura1 1 Kyoto Prefectural University of Medicine, Japan Oxidative stress is known to play a critical role in the development of nonalcoholic steatohepatitis. To clarify the source of oxidative stress, we examined the expression of NADPH oxidase isoforms in the liver of high fat diet (HFD)-fed mice. The mRNA expression of
SfRBM / SFRRI 2016
doi: 10.1016/j.freeradbiomed.2016.10.463 423 Role of Oxidative Stress in Epigenetic Modification of Matrix Metalloproteinase-9 in the Development of Diabetic Retinopathy Renu A Kowluru1 1 Wayne State University, Detroit, USA In the pathogenesis of diabetic retinopathy, damaged retinal mitochondria accelerate apoptosis of retinal capillary cells, and regulation of oxidative stress by manipulating mitochondrial superoxide dismutase (Sod2) protects mitochondrial homeostasis and prevents the development of diabetic retinopathy. We have shown that diabetes activates retinal matrix metalloproteinase-9, and activated MMP-9 damages the mitochondria. Recent studies have shown that the machinery responsible for epigenetic modifications is also altered in diabetes, and a dynamic DNA methylation process plays an important role in regulation of retinal MMP-9 transcription. The aim of this study is to investigate the role of oxidative stress in retinal MMP-9 transcription in diabetes. Methods: The effect of regulation of mitochondrial superoxide on DNA methylation of MMP-9 promoter was investigated in retinal endothelial cells incubated in the presence or absence of a MnSOD mimetic- MnTBAP, by quantifying the levels of 5 methyl cytosine (5mC) and hydroxyl-methyl cytosine (5hmC). The binding of DNA methylating and hydroxymenthylating enzymes (Dnmts and Tets, respectively) at MMP-9 promoter (by chromatin immunoprecipitation) and mitochondrial homeostasis (cytochrome c leakage in cytosol and mtDNA transcription) were also evaluated. The in vitro results were confirmed in the retina of diabetic mice overexpressing Sod2. Results: Addition of MnTBAP ameliorated glucose-induced decrease in 5mC levels and increase in Dnmt1 binding at the MMP9 promoter. In the same cell preparations, MnTBAP also ameliorated alterations in 5hmC levels and Tet binding, regulated MMP-9 transcription and prevented mitochondrial damage. Similarly, mice overexpressing Sod2 were protected from diabetesinduced alteration in MMP-9 promoter methylation and its transcription, and mitochondrial dysfunction. Conclusions: Thus, oxidative stress regulation by chemical/genetic approaches maintains retinal mitochondrial homeostasis by ameliorating epigenetic modifications of the MMP-9 promoter.
S176
doi: 10.1016/j.freeradbiomed.2016.10.464 424 Advanced Glycation End Products Inhibition Restores Sphingolipid Rheostat in Mice Liver and Prevents Diet-Induced Insulin Resistance Alessia Sofia Cento1, Debora Nigro1, Fausto Chiazza1, Federica Dal Bell1, Francesco Romaniello1, Massimo Collino1, Claudio Medana1, Manuela Aragno1, and Raffaella Mastrocola1 1 University of Turin, Italy The maintenance of sphingolipid rheostat by interconversion of ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) is relevant for the control of several metabolic pathways. In particular, accumulation of Cer and S1P in response to cellular stress interferes with insulin signalling and mitochondrial metabolism. Emerging evidence has highlighted a role for Advanced Glycation End Products (AGEs) in the imbalance of the sphingolipid rheostat. We and others have recently demonstrated the accumulation of AGEs in tissues of diet-induced insulinresistant mice and their involvement in altered lipid synthesis and mitochondrial dysfunction. We, thus, investigated the impact of a high trans fat diet on the sphingolipid rheostat in mice liver and whether the administration of the anti-AGEs compound pyridoxamine could prevent insulin resistance onset by restoring the Cer/Sph/S1P equilibrium. C57Bl/6J mice were fed a standard diet (SD) or a 60% trans fat diet (HFD) for 10 weeks. Two subgroups received pyridoxamine (150 mg/kg/day) in the drinking water. The pyridoxamine administration efficiently prevented insulin resistance, obesity, AGEs accumulation and RAGE hyperexpression induced by the HFD. HFD also induced a dramatic reduction of the liver Sph levels, paralleled by the increase in Cer levels, assessed by an UPLC/TQMS system, and the induction of the S1P receptors. Interestingly, pyridoxamine administration prevented the Cer increase by reducing Cer synthase expression and significantly inhibited S1P receptors by reducing S1P kinase levels, completely reverting Sph depletion. The present data indicate for the first time that the inhibition of AGEs is effective in the maintenance of the sphingolipid rheostat through the modulation of the involved enzymes, and this could contribute to the prevention of insulin resistance observed in the pyridoxamine-treated HFD mice. Therefore, it could be of relevance to further investigate on the predictive value of Cer/Sph/S1P imbalance for insulin resistance onset and on pyridoxamine supplementation as preventive strategy.
doi: 10.1016/j.freeradbiomed.2016.10.465 425 A Potential Role of Nox1 Isoform of NADPH Oxidase in the Development of Nonalcoholic Steatohepatitis Misaki Matsumoto1, Jia Zhang1, Xueqing Zhang1, and Chihiro YabeNishimura1 1 Kyoto Prefectural University of Medicine, Japan Oxidative stress is known to play a critical role in the development of nonalcoholic steatohepatitis. To clarify the source of oxidative stress, we examined the expression of NADPH oxidase isoforms in the liver of high fat diet (HFD)-fed mice. The mRNA expression of
SfRBM / SFRRI 2016