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Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase.

Maeda M, Hayashi T, Mizuno N, Hattori Y, Kuzuya M - PLoS ONE (2015)

Bottom Line: Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis.Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage.However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

ABSTRACT
Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-β-galactosidase (SA-β-gal) activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-β-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction.

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ROS and superoxide generation in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Intracellular ROS was measured by visualizing the use of the fluorescent probe CM-H2DCFDA. (B) Superoxide was detected via DHE and was analyzed using flow cytometry. (C) Expression of p22phox protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22phox siRNA effectively eliminated p22phox protein expression. (E) Transfection of p22phox siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22phox siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22phox siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *p<0.05; **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG.
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pone.0123169.g006: ROS and superoxide generation in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Intracellular ROS was measured by visualizing the use of the fluorescent probe CM-H2DCFDA. (B) Superoxide was detected via DHE and was analyzed using flow cytometry. (C) Expression of p22phox protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22phox siRNA effectively eliminated p22phox protein expression. (E) Transfection of p22phox siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22phox siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22phox siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *p<0.05; **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG.

Mentions: Our previous studies have demonstrated that increased ROS plays a critical role in endothelial cell senescence caused by high-glucose stimuli [2,6,7,8]. ROS production was significantly increased by both constant and intermittent high glucose concentrations. The extent of increased ROS production was not different between constant and intermittent high glucose (Fig 6A). When superoxide was measured using the oxidative fluorescent dye DHE, a higher increase in superoxide production was observed under intermittent high-glucose conditions compared with constant high-glucose conditions (Fig 6B). NADPH oxidase is one of the most important sources of superoxide in vascular cells and p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system [20, 25, 26]. Constant exposure to high glucose did not alter the expression level of p22phox; however, intermittent exposure to high glucose resulted in a striking up-regulation of p22phox expression (Fig 6C). To explore the involvement of p22phox up-regulation in the senescent action of glucose fluctuations in endothelial cells, we used siRNA to specifically ablate p22phox mRNA in HUVECs. Our transfection of p22phox siRNA effectively silenced the endothelial expression of p22phox compared with that of the negative control siRNA (Fig 6D). Under the transfection of p22phox siRNA, there was no difference in superoxide production between cells that were exposed to normal, constant high, or intermittent high glucose (Fig 6E). Furthermore, the increase of SA-β-gal activity observed in cells exposed to oscillating glucose was significant but less pronounced when p22phox siRNA was transfected (Fig 6F, compared with Fig 2A). Under the transfection of p22phox siRNA, there was no difference in DNA damage in AP site between cells that were exposed to normal, constant high, or intermittent high glucose (Fig 6G).


Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase.

Maeda M, Hayashi T, Mizuno N, Hattori Y, Kuzuya M - PLoS ONE (2015)

ROS and superoxide generation in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Intracellular ROS was measured by visualizing the use of the fluorescent probe CM-H2DCFDA. (B) Superoxide was detected via DHE and was analyzed using flow cytometry. (C) Expression of p22phox protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22phox siRNA effectively eliminated p22phox protein expression. (E) Transfection of p22phox siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22phox siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22phox siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *p<0.05; **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG.
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Related In: Results  -  Collection

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pone.0123169.g006: ROS and superoxide generation in HUVECs exposed to high glucose.NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Intracellular ROS was measured by visualizing the use of the fluorescent probe CM-H2DCFDA. (B) Superoxide was detected via DHE and was analyzed using flow cytometry. (C) Expression of p22phox protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22phox siRNA effectively eliminated p22phox protein expression. (E) Transfection of p22phox siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22phox siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22phox siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *p<0.05; **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG.
Mentions: Our previous studies have demonstrated that increased ROS plays a critical role in endothelial cell senescence caused by high-glucose stimuli [2,6,7,8]. ROS production was significantly increased by both constant and intermittent high glucose concentrations. The extent of increased ROS production was not different between constant and intermittent high glucose (Fig 6A). When superoxide was measured using the oxidative fluorescent dye DHE, a higher increase in superoxide production was observed under intermittent high-glucose conditions compared with constant high-glucose conditions (Fig 6B). NADPH oxidase is one of the most important sources of superoxide in vascular cells and p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system [20, 25, 26]. Constant exposure to high glucose did not alter the expression level of p22phox; however, intermittent exposure to high glucose resulted in a striking up-regulation of p22phox expression (Fig 6C). To explore the involvement of p22phox up-regulation in the senescent action of glucose fluctuations in endothelial cells, we used siRNA to specifically ablate p22phox mRNA in HUVECs. Our transfection of p22phox siRNA effectively silenced the endothelial expression of p22phox compared with that of the negative control siRNA (Fig 6D). Under the transfection of p22phox siRNA, there was no difference in superoxide production between cells that were exposed to normal, constant high, or intermittent high glucose (Fig 6E). Furthermore, the increase of SA-β-gal activity observed in cells exposed to oscillating glucose was significant but less pronounced when p22phox siRNA was transfected (Fig 6F, compared with Fig 2A). Under the transfection of p22phox siRNA, there was no difference in DNA damage in AP site between cells that were exposed to normal, constant high, or intermittent high glucose (Fig 6G).

Bottom Line: Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis.Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage.However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

ABSTRACT
Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-β-galactosidase (SA-β-gal) activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-β-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction.

Show MeSH
Related in: MedlinePlus