Intermittent High Glucose Implements Stress-Induced Senescence in Human Vascular Endothelial Cells: Role of Superoxide Production by NADPH Oxidase

Morihiko Maeda; Toshio Hayashi; Natsumi Mizuno; Yuichi Hattori; Masafumi Kuzuya

doi:10.1371/journal.pone.0123169

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 p16^INK4a , 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 p22^phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22^phox 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.

Citation: Maeda M, Hayashi T, Mizuno N, Hattori Y, Kuzuya M (2015) Intermittent High Glucose Implements Stress-Induced Senescence in Human Vascular Endothelial Cells: Role of Superoxide Production by NADPH Oxidase. PLoS ONE 10(4): e0123169. https://doi.org/10.1371/journal.pone.0123169

Academic Editor: Gabriele Saretzki, University of Newcastle, UNITED KINGDOM

Received: September 10, 2014; Accepted: February 24, 2015; Published: April 16, 2015

Copyright: © 2015 Maeda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper.

Funding: This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan No. 24590881. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Senescence of vascular endothelial cells is an important contributor to the pathogenesis of human atherosclerosis [1]. Numerous studies have shown that the senescent phenotype of endothelial cells can be transformed from anti-atherosclerotic to pro-atherosclerotic [2]. We have provided evidence that senescent endothelial cells are found in human atherosclerotic lesions but not in nonatherosclerotic lesions [3]. Diabetes mellitus is a well-documented, high-risk factor for the development of atherosclerosis [4]. The hallmark of diabetes is the presence of hyperglycemia, which can accelerate senescence in endothelial cells [5]. In several publications, we have demonstrated that high glucose can induce endothelial cell senescence [3,6,7,8]. In fact, we previously reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus [8].

Rapid fluctuations of glycemia, including postprandial hyperglycemia and interprandial hypoglycemia, may play a crucial role in the pathogenesis of diabetic cardiovascular complications [9,10]. In clinical practice, the intensive treatment goal targets minimizing the sharp fluctuations of blood glucose levels that occur in inadequately treated patients. Intermittent, rather than constant, hyperglycemia can induce an increased production of collagen by cultured mesangial cells [11]. Furthermore, intermittent high glucose has been demonstrated to exhibit more marked increases in endothelial cell apoptosis [12,13], endothelial expression of adhesion molecules [14], and vascular smooth muscle cell proliferation [15] than constant high glucose. Thus, glucose fluctuations appear to be more deleterious to vascular cell integrity than constant high glucose concentrations.

In the present study, we verified whether intermittent vs. constant exposure to high glucose might have different effects on cellular senescence in human vascular endothelial cells. We also evaluated how fluctuating glucose makes an impact on the changes in endothelial nitric oxide synthase (eNOS) and reactive oxygen species (ROS), both of which may play pivotal roles in endothelial cell senescence [2,3].

Materials and methods

Cell culture

Human umbilical venous endothelial cells (HUVECs) were purchased from Lonza (Walkersville, MD, USA) and were cultured in endothelial cell growth medium-2 until the start of the experiment. To exclude the insulin effect [6], the cells were cultured in modified endothelial cell growth medium-2 that lacked insulin-like growth factor-1 but contained 2% fetal bovine serum during the experimental period. According to our previous study [3], five- to seven-passage subconfluent cells were used in the experiments. The cells were harvested at subconfluence and were seeded into six-well plates. Subsequently, they were exposed to the experimental condition for 3 days. They were grouped as follows: (1) constant normal glucose medium (5.5 mM); (2) constant high glucose medium (22 mM); and (3) alternating normal and high glucose media every 12 h (Fig 1). In the constant normal or high glucose group, each fresh medium was provided every 24 h. Mannitol was used to rule out the effect of osmotic pressure [6]. In measurements of telomere length, we continued the culture with every 3 days changing the tissue culture medium up to 28 days.

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Fig 1. Research Design.

The endothelial cells were exposed to the experimental condition for 3 days. They were grouped as follows: (1) constant normal glucose medium (5.5 mM:NG); (2) constant high glucose medium (22 mM: HG); and (3) alternating normal and high glucose media every 12 h (N/HG).

https://doi.org/10.1371/journal.pone.0123169.g001

Measurement of cellular senescence

Cytochemical staining of senescence-associated-β-galactosidase (SA-β-gal) was performed using a Senescence Detection Kit (BioVision, Milpitas, CA, USA), as previously described, which showed the detailed effect of endothelial senescence including the growth curve [6–8]. SA-β-gal activity was measured using flow cytometry [6]. Immunoblot detection was used to identify the tumor suppressor p53, which is known to be a canonical inducer of cellular senescence [16]. SA-β-gal activity was measured using a microscope [8]. The cells were fixed for 10 min in 2% formaldehyde, 0.2% glutaraldehyde in PBS and were incubated for 12 hours at 37°C without CO₂ with fresh β-gal staining solution: 1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl₂, pH 6.0. The cells were counterstained with 4’6-diamidinophenylindole (DAPI; 0.2 mg/ml in 10 mM NaCl) for 10 min to count the total number of cells. The percentage of SA-β-gal-positive cells was determined by counting the number of blue cells within a sample of 1,000 cells.

Cellular senescence related proteins such as p21, p16^INK4a p16 and p53 were measured by western blotting. We used p21 (sc-397, SANTA CRUZ) rabbit polyclonal antibody (1:200), p16^INK4a(ab54210, abcam) mouse monoclonal antibody (1:500), p53 mouse monoclonal antibody as well asβ-actin(ab8226, abcam) mouse monoclonal (1:15,000) as first antibody. We also used anti-rabbit IgG, HRP-Linked antibody (#7074, Cell Signaling) for p21 (1:5,000), anti-mouse IgG, HRP-Linked antibody (#7076, Cell Signaling) for p16 (1:5,000), p53(1:5,000) and β-actin (1:10,000) as 2^nd antibody.

We further quantificated DNA Damage of endothelial cells by using QIAamp DNA Mini Kit (51304, QIAGEN) and DNA Damage Quantification Colorimetric Kit (#K253-25, Bio Vision). Apurinic/apyrimidinic(AP) sites are one of the major types of DNA lesions formed during the course of base excision and repair of oxidized, deaminated or alkylated bases. It has been estimated that about 2×10⁵ base lesions are generated per cell per day. The level of AP sites in cells can be good indicator of DNA lesion and repair against chemical damage and cellular senescence. This Kit utilizes the ARP (Aldehyde Reactive Probe) reagent that reacts specifically with an aldehyde group, which is the open ring from of the AP sites. After treating DNA containg AP sites with ARP reagents, AP sites are tagged with biotin resideues, which can be quantified using avidin-biotin assay followed by a colorimetric detection.

Human telomerase activity and length

Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA^PLUS kit (Roche Diagnostics, Mannheim, Germany) [6]. Telomere length was measured via fluorescence in situ hybridization using flow cytometry [6, 17]. For the assay standardization, we added an aliquot of a single batch of 1,301 cells (a sub-line of human T cell lymphoblast-like cell) to each sample as an internal control.

Western blot analysis

Western blotting was performed as described in our previous reports [7,8,18]. The membrane was blotted with the indicated antibodies and was processed using chemiluminescence. Namely, we used p21 (sc-397, SANT CRUZ) rabbit polyclonal (1:200), p16^INK4a(ab54210, abcam) mouse monoclonal (1:500) and β-actin(ab8226, abcam) mouse monoclonal antibodies (1:15,000) as 1^st antibodies and anti-rabbit IgG, HRP-Linked antibody (#7074, Cell Signaling) for p21 (1:5,000) anti-mouse IgG, HRP-Linked antibody (#7076, Cell Signaling) for p16 (1:5,000) and β-actin (1:10,000) antibodies as 2^nd antibodies.

DNA Damage Quantification

To detect DNA damage quantification, we used QIAamp DNA Mini Kit (51304, QIAGEN) And DNA Damage Quantification Colorimetric Kit (#K253-25, Bio Vision). Apurinic/apyrimidinic(AP) sites are one of the major types of DNA lesions formed during the course of base excision and repair of oxidized, deaminated or alkylated bases. It has been estimated that about 2×10⁵ base lesions are generated per cell per day. The level of AP sites in cells can be good indicator of DNA lesion and epair against chemical damage and cell aging. This Kit utilizes the ARP (Aldehyde Reactive Probe) reagent that reacts specifically with an aldehyde group, which is the open ring from of the AP sites. After treating DNA containing AP sites with ARP reagents, AP sites are tagged with biotin resideues, which can be quantified using avidin-biotin assay followed by a colorimetric detection.

Nitric oxide and metabolites of nitric oxide measurement

To detect nitric oxide (NO) production by HUVECs, we loaded HUVECs with 4,5-diaminofluorescein diacetate (DAF-2 DA, Cayman Chemical, San Diego, CA, USA). This membrane-permeable dye is hydrolyzed intracellularly by cytosolic esterases releasing DAF-2, which is converted in the presence of NO into a fluorescent product, DAF-2 triazole [19].

The metabolites of NO (NOx:NO2^- and NO3^-) in the medium were measured using the HPLC system to develop NOx measurement [20].

Analysis of ROS and superoxide generation

To detect intracellular ROS, including hydrogen peroxide, hydroxyl radical, and peroxynitrite, we loaded the endothelial cells using the fluorescent probe 5-(and-6)- chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) (Invitrogen, Carlsbad, CA, USA) to produce a final concentration of 10 mM for 45 min in phosphate buffered saline at 37°C. At the end of the experiment, the cells were rinsed with phosphate-buffered saline and were subsequently placed in the culture medium. Imaging was conducted using a Cell Observer microscope and AxoVision software (Carl Zeiss, Oberkochen, Germany) [18]. The production of superoxide anion was speculated from measurements using dihydroethidium (DHE) (Invitrogen) [21–23]. The cells were incubated using fluorescent probes at the concentration of 10 mM at 37°C for 30 min, and flow cytometry was performed [8].

Transfection of siRNAs

All small interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The nonsilencing control siRNA was used as a negative control. Scrambled siRNA-treated cells were also used as controls. The transfection of siRNA was performed in Opti-MEM I Reduced-Serum Medium using Lipofectamine RNAiMAX (Invitrogen) [24]. We confirmed the expression of a target gene by Western-blot. There are differences in the suppression ratio (namely reflecting the transfection efficiency) according to target genes. In siRNA of eNOS, the suppression ratio was approximately 99%. In siRNA of p22phox, it was 96.5%. There is a report that the transfection reagent (RNAiMAX), which we used in the present study, shows the high transfection efficiency in smooth muscle cell as adherent cell, too [25].

Statistics

The values are presented as the mean ± standard deviation (SD). One-way ANOVA followed by Tukey’s multiple comparison test was used, and p<0.05 was considered to be statistically significant.

Results

When HUVECs were exposed to constant high glucose, there was a significant increase in SA-β-gal activity, a widely used quantitative marker for aging in vitro. This effect was enhanced when the cells were exposed to intermittent high glucose concentrations (Fig 2A). Similarly, cytochemical staining SA-β-gal showed that the number of SA-β-gal positive cells showed that the number of SA-β-gal positive cells was induced by the high-glucose condition and was increased under the fluctuating-glucose condition (Fig 2B). SA-β-gal activity was not affected by osmolality, which was confirmed by the lack of effect of mannitol, as reported in our previous study [6].

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Fig 2. Effect of high glucose on SA-β-gal activity in HUVECs.

NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. To confirm the effect of glucose in HUVECs, NG, HG and N/HG were cultured with each Stimulus for 3 days. HUVECs were cultured with constant high glucose (HG) and intermittent glucose (N/HG) introduced in Fig 1. Namely, N/HG was stimulated twice with HG (at 4-hour intervals) for a total of 4 hours daily (9 a.m. to 11 a.m., 3 p.m. to 5 p.m.), and was cultured in NG in other time of the total 4-hour HG stimulation. (A) SA-β-gal activity was evaluated cytochemically. The values of the three independent experiments are mean ± S.D. **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG. (B) SA- β-gal-positive cells (blue) can be detected via cytochemical staining.

https://doi.org/10.1371/journal.pone.0123169.g002

Endothelial senescence related proteins such as p53, p21, p16^INK4a and were measured by western blotting (Fig 3A, 3B, 3C and 3D). Intermittent high glucose stimulus (IHG) increased these proteins significantly compared with the condition of normal glucose and tended to increase them than those by continuous high glucose stimuli. We further quantificated DNA damage of endothelial cells. DNA damage was observed by the stimuli of inthermittent high glucose (Fig 3E) as well as continuous high glucose stimuli. These results show that intermittent high glucose condition induced endothelial senescence, which starts as early as 3 days after the start of the stimuli.

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Fig 3. Effect of high glucose on p53, p21, p16^INKa, and DNA ladder on Apurinic/apyrimidinic (AP) sites.

HUVECs were cultured with constant high glucose (HG) and intermittent glucose (N/HG) introduced in Fig 1. Namely, N/HG was stimulated twice with HG (at 4-hour intervals) for a total of 4 hours daily (9 a.m. to 11 a.m., 3 p.m. to 5 p.m.), and was cultured in NG in other time of the total 4-hour HG stimulation. (A)-(D): Effect of high glucose on p53, p21, p16^INKa protein. (E): Effect of continuous and intermittent high glucose on endothelial DNA damage on Apurinic/apyrimidinic(AP) sites. *p<0.05; **p<0.01 vs. NG; #p<0.05 vs. HG. The values of the three independent experiments are mean ± S.D.

https://doi.org/10.1371/journal.pone.0123169.g003

The telomerase, a specialized ribonucleprotein transcriptase that is important for long-term eukaryotic cell proliferation and genomic stability to replenish the DNA at telomeres, was significantly decreased in a constant high-glucose environment (Fig 4A). Subsequently, telomere length, a likely index of biological aging, was shortened by 4 weeks, which we continued the culture with every 3 days changing the tissue culture medium up to 28 days. It suggested replicative senescence (Fig 4B). In contrast, both telomerase activity and telomere length remained unchanged after exposure of HUVECs to intermittent high glucose (Fig 4A and 4B).

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Fig 4. Effect of high glucose on telomerase activity and telomere length in HUVECs.

NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Telomerase activity was measured by the telomere repeat application protocol (trap) assay. The values of the three independent experiments are mean ± S.D. **p<0.01 vs. NG; ##p<0.01 vs. HG. (B) Telomere length was measured to evaluate the relationship to replicative senescence. The data shown represent the average of two independent experiments.

https://doi.org/10.1371/journal.pone.0123169.g004

We have previously shown that eNOS plays a pivotal role in the regulation of the senescence program in vascular endothelial cells [2,6,7,8]. Under constant or intermittent high glucose, however, eNOS total expression, Ser-1177 eNOS phosphorylation, and Thr-495 eNOS dephosphorylation levels were substantially similar to those under normal glucose (Fig 5A–5D). When we examined the basal NO levels from HUVECs using the fluorescent dye DAF-2 DA, no significant difference was found among the three groups (Fig 5E). Concentration of NOx; metabolites of NO, NO2^- + NO3^-, in culture medium also supports those data (Fig 5F).

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Fig 5. eNOS activity 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) Typical Western blots for total eNOS, Ser-1177 phosphorylated eNOS, and Thr-495 phosphorylated eNOS are shown. GAPDH served as the loading control. (B-D) Summary of quantification of densitometric measurement of the immunoblot data. Total eNOS expression and eNOS phosphorylation levels were normalized to GAPDH and total eNOS, respectively. (E) NO production by HUVECs, using the fluorescent dye DAF-2-DA. (F) The data of NOx (NO2^- and NO3^-), NO metabolites measured by HPLC.

https://doi.org/10.1371/journal.pone.0123169.g005

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 p22^phox 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 p22^phox; however, intermittent exposure to high glucose resulted in a striking up-regulation of p22^phox expression (Fig 6C). To explore the involvement of p22^phox up-regulation in the senescent action of glucose fluctuations in endothelial cells, we used siRNA to specifically ablate p22^phox mRNA in HUVECs. Our transfection of p22^phox siRNA effectively silenced the endothelial expression of p22^phox compared with that of the negative control siRNA (Fig 6D). Under the transfection of p22^phox 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 p22^phox siRNA was transfected (Fig 6F, compared with Fig 2A). Under the transfection of p22^phox 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).

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Fig 6. 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 p22^phox protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22^phox siRNA effectively eliminated p22^phox protein expression. (E) Transfection of p22^phox siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22^phox siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22^phox 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.

https://doi.org/10.1371/journal.pone.0123169.g006

Discussion

Endothelial cell dysfunction is identified to be an early culprit in causing the development of vascular complications in diabetes [26, 27]. Endothelial cell senescence causes endothelial dysfunction [1]. Thus, endothelial cell senescence may be involved in diabetes-related vascular disorders. Our recent studies have provided increasing evidence that high glucose accelerates endothelial cell senescence [3,6,7,8].

The present study showed that human vascular endothelial cells exposed to high glucose concentrations increased cell senescence, which was determined by measuring SA-β-gal activity. This evidence was more enhanced in cells that were exposed to intermediate rather than constant high glucose concentrations. Further, p16^INK4a and p21were increased by 3 days intermittent high glucose stimuli. Although increase of p53 was not large compared with those of p16^INK4a and p21, the pathway via p16INK4a may be main process of cellular senescence, on the other hands, p53 relates apoptosis more than senescence [28]. Our findings may suggest that glucose fluctuation appears to be more deleterious to endothelial cells than a constant high glucose concentration and it may accelerate endothelial senescence within even 3 days.

As reported earlier [6,8], telomerase activity was markedly decreased when HUVECs were exposed to a constant high glucose concentration, and thus, presumably resulted in shortened telomeres. However, telomere extension by the overexpression of telomerase does not affect stress-induced senescence [29] but prevents replicative senescence [30]. Therefore, the change in telomerase activity, subsequent to the change in telomere length induced by a constant high glucose concentration may represent replicative senescence. We studied telomere activity by 3 days observation, however, in measurements of telomere length, we continued the culture with every 3days changing the incubation fluid up to 28 days to observe significant change of telomere length. It needs much longer time to observe change of the telomere length compared with the change of telomerase activity. Intermittent high glucose did not alter telomerase activity and telomere length. Telomerase activity, subsequently telomere length might be affected by ROS as below. It is reported that shorter telomeres with high telomerase activity in unstimulated leukocytes were associated with reduced social support and greater early life adversity [30]. The effect of telomerase might be complicated.

We have shown a pivotal role of increased ROS in endothelial cell senescence caused by high glucose stimuli [3,6,7,8]. In this study, no difference in ROS production was found between the cells exposed to constant and intermittent high glucose. The detection of ROS was performed after staining HUVECs with CM-H₂DCFDA, which is now used for monitoring intracellular hydrogen peroxide, hydroxyl radical, and peroxynitrite production in cells. Thus, the enhanced cell senescent effect of glucose fluctuation cannot be attributed to the increased production of these radicals. When DHE, which is generally speculated to be specific for superoxide, was used as its probe, we found a larger production of superoxide in the cells incubated with oscillating glucose than in the cells with constant high glucose. There may be the limitation of the DHE method for the correct measurement of superoxide, however there are no perfect methods for that easily available except an ESR (electron spin resonance) method [21–23]. The endothelial expression of p22^phox, a critical component of the superoxide-generating NADH/NADPH oxidase system, was strikingly up-regulated under intermittent high-glucose conditions. Importantly, the ablation of p22^phox by siRNA abrogated the increase in superoxide production and undermined the implementation of endothelial cell senescence in a fluctuating-glucose environment. We thus suggest that glucose fluctuations display a more adverse effect on senescence in human vascular endothelial cells than constant high glucose concentrations, in part, by NADPH oxidase-derived superoxide overproduction. Nox2, referred to as gp91phox, likely plays a role, and the hetero-dimer with p22phox may be produced and may increase catalytic activity. Nox4 may play a role and may limit the actual catalytic activity [31, 32]. However, in this experiment, we did not evaluate the identification of the Nox2/ Nox4 ratio because our primary object was to investigate the effect of intermittent high glucose (models of impaired glucose tolerance and metabolic syndrome) on endothelial senescence. Similarly, mitochondria may produce ROS as well as NADPH oxidase, however, we could not investigate the source of ROS in detail in the present study.

Our recent study has demonstrated that the antisenescence effect of calcium-channel blockers in human endothelial cells is associated with increased eNOS activity [8], implying that eNOS activation is important in the regulation of the senescence program in endothelial cells by NO-mediated delay of cellular senescence [2]. The activity of eNOS is regulated by reciprocal phosphorylation of the activating site Ser-1177 and inhibiting site Thr-495, which affects NO bioavailability [33]. We found that both constant and intermittent glucose led to no changes in eNOS total expression, Ser-1177 eNOS phosphorylation, Thr-495 eNOS dephosphorylation, or NO release from control levels in HUVECs. This finding suggests that the baneful effect of glucose fluctuation on endothelial cell senescence cannot be attributed to a substantial alteration in eNOS activation. Several papers have reported the expression of iNOS by high glucose in endothelial cells; however, the glucose concentration in those papers was higher (33 mM or more) than those in our finding (22 mM)[34,35], and inducers of iNOS, such as lipopolysaccharides, were simultaneously used in several experiments. Other papers have reported no induction of iNOS by high glucose in the cultured endothelium [36]. In fact, we have observed iNOS in neighboring necrotic areas in atherosclerosis but not in the endothelium in diabetic patients [37]. Altogether, we believe that iNOS is not induced by high glucose. The NOx amount was not increased in the present study and we did not detect iNOS under similar conditions [3,7]. We speculate that one reason is the non-use of IGF in the culture medium to rule out the effect of insulin in our study [6].

The present results may be clinically relevant. Our findings support a prior clinical report that fluctuations in blood glucose levels cause endothelial dysfunction in diabetic patients [38]. Additionally, our findings might support the hypothesis that impaired glucose tolerance causes atherosclerosis via stress-induced senescence. Thus, glucose fluctuation in blood can be a risk predictor for the occurrence and the progression of cardiovascular diseases in diabetes. Appreciably, turning up highly promising management of postprandial hyperglycemic spikes is an important determinant of overall glucose control to prevent the onset and the development of hyperglycemic or diabetic cardiovascular complications.

Conclusion

Impaired glucose tolerance occurs frequently in the elderly and poses an independent risk for atherosclerosis. Endothelial cellular senescence precedes atherosclerosis. Constant high glucose levels increased SA-β-gal activity, a cellular senescence marker, and showed replicative senescence. This effect was more pronounced in intermittent high glucose, which demonstrated a stress-induced senescence. Intermittent, but not constant, high glucose strikingly up-regulated the expression of p22^phox, an NADPH oxidase component, increasing superoxide. Intermittent high glucose promotes vascular endothelial senescence and, in part, depends on superoxide overproduction.

Acknowledgments

The authors thank Toshio Fujimori for his technical assistance.

Author Contributions

Conceived and designed the experiments: MM TH. Performed the experiments: MM NM. Analyzed the data: MM TH. Contributed reagents/materials/analysis tools: MM TH YH MK. Wrote the paper: MM TH YH.

References

1. Minamino T, Komuro I. Vascular cell senescence: contribution to atherosclerosis, Circ Res. 2007;100: 15–26. pmid:17204661
- View Article
- PubMed/NCBI
- Google Scholar
2. Hayashi T, Yano K, Matsui-Hirai H, Yokoo H, Hattori Y, Iguchi A. Nitric oxide and endothelial cellular senescence. Pharmacol Ther. 2008;120: 333–339. pmid:18930078
- View Article
- PubMed/NCBI
- Google Scholar
3. Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, et al. Endothelial cellular senescence is inhibited by nitric oxide: Implications in atherosclerosis associated with menopause and diabetes, Proc Natl Acad Sci USA. 2006;103:17018–17023. pmid:17075048
- View Article
- PubMed/NCBI
- Google Scholar
4. Chait A, Bornfeldt KE. Diabetes and atherosclerosis: is there a role for hyperglycemia? J Lipid Res. 2009;50 (Suppl): S335–S339. pmid:19029122
- View Article
- PubMed/NCBI
- Google Scholar
5. Yokoi T, Fukuo K, Yasuda O, Hotta M, Miyazaki J, Takemura Y, et al. Apoptosis signal-regulating kinase 1 mediates cellular senescence induced by high glucose in endothelial cells. Diabetes 2006;55:1660–1665. pmid:16731828
- View Article
- PubMed/NCBI
- Google Scholar
6. Matsui-Hirai H, Hayashi T, Yamamoto S, Ina K, Maeda M, Kotani H, et al. Dose-dependent modulatory effects of insulin on glucose-induced endothelial senescence in vitro and in vivo: A relationship between telomeres and nitric oxide. J Pharmacol Exp Ther. 2011;337:591–599. pmid:21357660
- View Article
- PubMed/NCBI
- Google Scholar
7. Hayashi T, Kotani K, Yamaguchi T, Taguchi K, Iida M, Ina K, et al. Endothelial cellular senescence is inhibited by liver X receptor activation with an additional mechanism for its atheroprotection in diabetes. Proc Natl Acad Sci USA. 2014;111:1168–1173. pmid:24398515
- View Article
- PubMed/NCBI
- Google Scholar
8. Hayashi T, Yamaguchi T, Sakakibara Y, Taguchi K, Maeda M, Kuzuya M, et al. eNOS- dependent antisenscence effect of a calcium channel blocker in human endothelial cells. PLoS One 2014;9: e88391. pmid:24520379
- View Article
- PubMed/NCBI
- Google Scholar
9. Ceriello A. The emerging role of post-prandial hyperglycaemic spikes in the pathogenesis of diabetic complications, Diabet. Med. 1998;15:188–193. pmid:9545118
- View Article
- PubMed/NCBI
- Google Scholar
10. Monnier L, Mas E, Ginet C, Michel F, Villon L, Cristol J.P, et al. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA 2006; 295:1681–1687. pmid:16609090
- View Article
- PubMed/NCBI
- Google Scholar
11. Takeuchi A, Throckmorton DC, Brogden AP. Periodic high extracellular glucose enhances production of collagen III and IV by mesangial cells. Am. J. Physiol. 1995;268:F13–F18. pmid:7840239
- View Article
- PubMed/NCBI
- Google Scholar
12. Risso A, Mercuri F, Quagliaro L, Damante G, Ceriello A. Intermittent high glucose enhances apoptosis in human umbilical vein endothelial cells in culture. Am. J. Physiol. Endocrinol. Metab. 2001;281:E924–E930. pmid:11595647
- View Article
- PubMed/NCBI
- Google Scholar
13. Quagliaro L, Piconi L, Assaloni R, Martinelli L, Motz E, Ceriello A. Intermittent high glucose enhances apoptosis related to oxidative stress in human umbilical vein endothelial cells. The role of protein kinase C and NAD(P)H-oxidase activation. Diabetes 2003;52:2795–2804. pmid:14578299
- View Article
- PubMed/NCBI
- Google Scholar
14. Quagliaro L, Piconi L, Assaloni R, Ros Da, Maier A, Zuodar G, et al. Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: The distinct role of protein kinase C and mitochondrial superoxide production, Atherosclerosis 2005;183:259–267. pmid:16285992
- View Article
- PubMed/NCBI
- Google Scholar
15. Sun J, Xu Y, Dai Z, Sun Y. Intermittent high glucose enhances proliferation of vascular smooth muscle cells by upregulating osteopontin. Mol. Cell. Endocrinol. 2009;313:64–69. pmid:19723557
- View Article
- PubMed/NCBI
- Google Scholar
16. Salminen A, Kaarniranta K. Control of p53 and NF-kB signaling by WIP1 and MIF: role in cellular senescence and organismal aging, Cell Signal. 2011; 23:747–752. pmid:20940041
- View Article
- PubMed/NCBI
- Google Scholar
17. Spyridopoulos I, Haendeler J, Urbich C, Brummendorf TH, Oh H, Schneider MD, et al. Statins enhance migratory capacity by upregulation of the telomere repeat-binding factor TRF2 in endothelial progenitor cells. Circulation 2004; 110:3136–3142 pmid:15520325
- View Article
- PubMed/NCBI
- Google Scholar
18. Taguchi K, Sakata K, Ohashi W, Imaizumi T, Imura J, Hattori Y. Tonic inhibition by G protein-coupled receptor kinase 2 of Akt/endothelial nitric-oxide synthase signaling in human vascular endothelial cells under conditions of hyperglycemia with high insulin levels, J. Pharmacol. Exp. Ther. 2014; 349:199–208. pmid:24570070
- View Article
- PubMed/NCBI
- Google Scholar
19. Herrera M 1, Hong NJ, Ortiz PA, Garvin JL. Endothelin-1 inhibits thick ascending limb transport via Akt-stimulated nitric oxide production. J Biol Chem. 2009; 284:1454–60. pmid:19033447
- View Article
- PubMed/NCBI
- Google Scholar
20. Hayashi T, Juliet PA, Miyazaki A, Ignarro LJ, Iguchi A. High glucose downregulates the number of caveolae in monocytes through oxidative stress from NADPH oxidase: implications for atherosclerosis. Biochim Biophys Acta. 2007;1772:364–72. pmid:17240121
- View Article
- PubMed/NCBI
- Google Scholar
21. Nolly MB, Caldiz CI, Yeves AM. The signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium. J Mol Cell Cardiol. 2014;67:60–68. pmid:24355174
- View Article
- PubMed/NCBI
- Google Scholar
22. Han J, Tan P. Fuzi attenuates diabetic neuropathy in rats and protects Schwann cells from apoptosis induced by high glucose. PLoS One. 2014;9:e86539. pmid:24466139
- View Article
- PubMed/NCBI
- Google Scholar
23. Wanschel AC, Caceres VM. Cardioprotective mechanism of S-nitroso-N- acetylcysteine via S-nitrosated betadrenoceptor-2 in the LDLr-/- mice. Nitric Oxide. 2014;36:58–66. pmid:24333561
- View Article
- PubMed/NCBI
- Google Scholar
24. Hunt MA, Currie MJ, Robinson BA, Dachs GU. Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents. J Biomol Tech. 2010;21:66–72. pmid:20592869
- View Article
- PubMed/NCBI
- Google Scholar
25. Nabzdyk CS, Chun M, Pradhan L, Logerfo FW. High throughput RNAi assay optimization using adherent cell cytometry. J Transl Med 2011;9:48 pmid:21518450
- View Article
- PubMed/NCBI
- Google Scholar
26. Christ M, Bauersachs J, Liebetrau C, Heck CM, Günther A, Wehling M. Glucose increases endothelial-dependent superoxide formation in coronary arteries by NAD(P)H oxidase activation: attenuation by the 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor, Diabetes 2002; 51:2648–52. pmid:12145183
- View Article
- PubMed/NCBI
- Google Scholar
27. Sena CM, Pereira AM, Seiça R. Endothelial dysfunction—a major mediator of diabetic vascular disease, Biochim Biophys Acta. 2013;1832:2216–31. pmid:23994612
- View Article
- PubMed/NCBI
- Google Scholar
28. Chen J, Huang X, Halicka D, Brodsky S, Avram A, Eskander J, et al. Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53. Am J Physiol Heart Circ Physiol 2006;290: H1575–1586 pmid:16243918
- View Article
- PubMed/NCBI
- Google Scholar
29. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, et al. Extension of life-span by introduction of telomerase into normal human cells, Science 1998;279:349–52. pmid:9454332
- View Article
- PubMed/NCBI
- Google Scholar
30. Zalli A, Carvalho LA, Lin J, Hamer M, Erusalimsky JD, Blackburn EH, Steptoe A. Shorter telomeres with high telomerase activity are associated with raised allostatic load and impoverished psychosocial resources. Proc Natl Acad Sci U S A. 2014;111:4519–4524 pmid:24616496
- View Article
- PubMed/NCBI
- Google Scholar
31. Erusalimsky JD. Vascular endothelial senescence: from mechanisms to pathophysiology. J Appl Physiol 2009;106: 326–332 pmid:19036896
- View Article
- PubMed/NCBI
- Google Scholar
32. Erusalimsky JD, Skene C. Mechanisms of endothelial senescence Exp physiol 2008;94.3:299–304
- View Article
- Google Scholar
33. Park MH, Heo SJ, Park PJ, Moon SH, Sung SH, Jeon BT, et al. 6,6'-bieckol isolated from Ecklonia cava protects oxidative stress through inhibiting expression of ROS and proinflammatory enzymes in high-glucose-induced human umbilical vein endothelial cells. Appl Biochem Biotechnol. 2014;174:632–43. pmid:25086922
- View Article
- PubMed/NCBI
- Google Scholar
34. Zhang X, Fu Y, Xu X, Li M, Du L, Han Y, et al. PERK pathway are involved in NO-induced apoptosis in endothelial cells cocultured with RPE under high glucose conditions. Nitric Oxide. 2014;40:10–16. pmid:24813399
- View Article
- PubMed/NCBI
- Google Scholar
35. Liu XJ 1, Zhang ZD, Ma XC. High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway. Exp Ther Med. 2013;6:361–7. pmid:24137189
- View Article
- PubMed/NCBI
- Google Scholar
36. Guo X, Chen LW, Liu WL, Guo ZG. High glucose inhibits expression of inducible and constitutive nitric oxide synthase in bovine aortic endothelial cells. Acta Pharmacol Sin. 2000;21:325–8. pmid:11324460
- View Article
- PubMed/NCBI
- Google Scholar
37. Esaki T, Hayashi T, Muto E, Yamada K, Kuzuya M, Iguchi A. Expression of inducible nitric oxide synthase in T lymphocytes and macrophages of cholesterol-fed rabbits. Atherosclerosis. 1997;128:39–46 pmid:9051196
- View Article
- PubMed/NCBI
- Google Scholar
38. Torimoto K, Okada Y, Mori H, Tanaka. Relationship between fluctuations in glucose levels measured by continuous glucose monitoring and vascular endothelial dysfunction in type 2 diabetes mellitus, Cardiovasc. Diabetology 2013;12:1. pmid:23280391
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Minamino T, Komuro I. Vascular cell senescence: contribution to atherosclerosis, Circ Res. 2007;100: 15–26. pmid:17204661
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Hayashi T, Yano K, Matsui-Hirai H, Yokoo H, Hattori Y, Iguchi A. Nitric oxide and endothelial cellular senescence. Pharmacol Ther. 2008;120: 333–339. pmid:18930078
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, et al. Endothelial cellular senescence is inhibited by nitric oxide: Implications in atherosclerosis associated with menopause and diabetes, Proc Natl Acad Sci USA. 2006;103:17018–17023. pmid:17075048
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Chait A, Bornfeldt KE. Diabetes and atherosclerosis: is there a role for hyperglycemia? J Lipid Res. 2009;50 (Suppl): S335–S339. pmid:19029122
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Yokoi T, Fukuo K, Yasuda O, Hotta M, Miyazaki J, Takemura Y, et al. Apoptosis signal-regulating kinase 1 mediates cellular senescence induced by high glucose in endothelial cells. Diabetes 2006;55:1660–1665. pmid:16731828
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Matsui-Hirai H, Hayashi T, Yamamoto S, Ina K, Maeda M, Kotani H, et al. Dose-dependent modulatory effects of insulin on glucose-induced endothelial senescence in vitro and in vivo: A relationship between telomeres and nitric oxide. J Pharmacol Exp Ther. 2011;337:591–599. pmid:21357660
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Hayashi T, Kotani K, Yamaguchi T, Taguchi K, Iida M, Ina K, et al. Endothelial cellular senescence is inhibited by liver X receptor activation with an additional mechanism for its atheroprotection in diabetes. Proc Natl Acad Sci USA. 2014;111:1168–1173. pmid:24398515
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Hayashi T, Yamaguchi T, Sakakibara Y, Taguchi K, Maeda M, Kuzuya M, et al. eNOS- dependent antisenscence effect of a calcium channel blocker in human endothelial cells. PLoS One 2014;9: e88391. pmid:24520379
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Ceriello A. The emerging role of post-prandial hyperglycaemic spikes in the pathogenesis of diabetic complications, Diabet. Med. 1998;15:188–193. pmid:9545118
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Monnier L, Mas E, Ginet C, Michel F, Villon L, Cristol J.P, et al. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA 2006; 295:1681–1687. pmid:16609090
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Takeuchi A, Throckmorton DC, Brogden AP. Periodic high extracellular glucose enhances production of collagen III and IV by mesangial cells. Am. J. Physiol. 1995;268:F13–F18. pmid:7840239
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Risso A, Mercuri F, Quagliaro L, Damante G, Ceriello A. Intermittent high glucose enhances apoptosis in human umbilical vein endothelial cells in culture. Am. J. Physiol. Endocrinol. Metab. 2001;281:E924–E930. pmid:11595647
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Quagliaro L, Piconi L, Assaloni R, Martinelli L, Motz E, Ceriello A. Intermittent high glucose enhances apoptosis related to oxidative stress in human umbilical vein endothelial cells. The role of protein kinase C and NAD(P)H-oxidase activation. Diabetes 2003;52:2795–2804. pmid:14578299
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Quagliaro L, Piconi L, Assaloni R, Ros Da, Maier A, Zuodar G, et al. Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: The distinct role of protein kinase C and mitochondrial superoxide production, Atherosclerosis 2005;183:259–267. pmid:16285992
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Sun J, Xu Y, Dai Z, Sun Y. Intermittent high glucose enhances proliferation of vascular smooth muscle cells by upregulating osteopontin. Mol. Cell. Endocrinol. 2009;313:64–69. pmid:19723557
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Salminen A, Kaarniranta K. Control of p53 and NF-kB signaling by WIP1 and MIF: role in cellular senescence and organismal aging, Cell Signal. 2011; 23:747–752. pmid:20940041
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Spyridopoulos I, Haendeler J, Urbich C, Brummendorf TH, Oh H, Schneider MD, et al. Statins enhance migratory capacity by upregulation of the telomere repeat-binding factor TRF2 in endothelial progenitor cells. Circulation 2004; 110:3136–3142 pmid:15520325
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Taguchi K, Sakata K, Ohashi W, Imaizumi T, Imura J, Hattori Y. Tonic inhibition by G protein-coupled receptor kinase 2 of Akt/endothelial nitric-oxide synthase signaling in human vascular endothelial cells under conditions of hyperglycemia with high insulin levels, J. Pharmacol. Exp. Ther. 2014; 349:199–208. pmid:24570070
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Herrera M 1, Hong NJ, Ortiz PA, Garvin JL. Endothelin-1 inhibits thick ascending limb transport via Akt-stimulated nitric oxide production. J Biol Chem. 2009; 284:1454–60. pmid:19033447
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Hayashi T, Juliet PA, Miyazaki A, Ignarro LJ, Iguchi A. High glucose downregulates the number of caveolae in monocytes through oxidative stress from NADPH oxidase: implications for atherosclerosis. Biochim Biophys Acta. 2007;1772:364–72. pmid:17240121
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Nolly MB, Caldiz CI, Yeves AM. The signaling pathway for aldosterone-induced mitochondrial production of superoxide anion in the myocardium. J Mol Cell Cardiol. 2014;67:60–68. pmid:24355174
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Han J, Tan P. Fuzi attenuates diabetic neuropathy in rats and protects Schwann cells from apoptosis induced by high glucose. PLoS One. 2014;9:e86539. pmid:24466139
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Wanschel AC, Caceres VM. Cardioprotective mechanism of S-nitroso-N- acetylcysteine via S-nitrosated betadrenoceptor-2 in the LDLr-/- mice. Nitric Oxide. 2014;36:58–66. pmid:24333561
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Hunt MA, Currie MJ, Robinson BA, Dachs GU. Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents. J Biomol Tech. 2010;21:66–72. pmid:20592869
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Nabzdyk CS, Chun M, Pradhan L, Logerfo FW. High throughput RNAi assay optimization using adherent cell cytometry. J Transl Med 2011;9:48 pmid:21518450
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Christ M, Bauersachs J, Liebetrau C, Heck CM, Günther A, Wehling M. Glucose increases endothelial-dependent superoxide formation in coronary arteries by NAD(P)H oxidase activation: attenuation by the 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitor, Diabetes 2002; 51:2648–52. pmid:12145183
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Sena CM, Pereira AM, Seiça R. Endothelial dysfunction—a major mediator of diabetic vascular disease, Biochim Biophys Acta. 2013;1832:2216–31. pmid:23994612
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Chen J, Huang X, Halicka D, Brodsky S, Avram A, Eskander J, et al. Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53. Am J Physiol Heart Circ Physiol 2006;290: H1575–1586 pmid:16243918
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, et al. Extension of life-span by introduction of telomerase into normal human cells, Science 1998;279:349–52. pmid:9454332
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Zalli A, Carvalho LA, Lin J, Hamer M, Erusalimsky JD, Blackburn EH, Steptoe A. Shorter telomeres with high telomerase activity are associated with raised allostatic load and impoverished psychosocial resources. Proc Natl Acad Sci U S A. 2014;111:4519–4524 pmid:24616496
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Erusalimsky JD. Vascular endothelial senescence: from mechanisms to pathophysiology. J Appl Physiol 2009;106: 326–332 pmid:19036896
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Erusalimsky JD, Skene C. Mechanisms of endothelial senescence Exp physiol 2008;94.3:299–304
View Article
Google Scholar

[126] View Article

[127] Google Scholar

[ref33] 33. Park MH, Heo SJ, Park PJ, Moon SH, Sung SH, Jeon BT, et al. 6,6'-bieckol isolated from Ecklonia cava protects oxidative stress through inhibiting expression of ROS and proinflammatory enzymes in high-glucose-induced human umbilical vein endothelial cells. Appl Biochem Biotechnol. 2014;174:632–43. pmid:25086922
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref34] 34. Zhang X, Fu Y, Xu X, Li M, Du L, Han Y, et al. PERK pathway are involved in NO-induced apoptosis in endothelial cells cocultured with RPE under high glucose conditions. Nitric Oxide. 2014;40:10–16. pmid:24813399
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref35] 35. Liu XJ 1, Zhang ZD, Ma XC. High glucose enhances LPS-stimulated human PMVEC hyperpermeability via the NO pathway. Exp Ther Med. 2013;6:361–7. pmid:24137189
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref36] 36. Guo X, Chen LW, Liu WL, Guo ZG. High glucose inhibits expression of inducible and constitutive nitric oxide synthase in bovine aortic endothelial cells. Acta Pharmacol Sin. 2000;21:325–8. pmid:11324460
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref37] 37. Esaki T, Hayashi T, Muto E, Yamada K, Kuzuya M, Iguchi A. Expression of inducible nitric oxide synthase in T lymphocytes and macrophages of cholesterol-fed rabbits. Atherosclerosis. 1997;128:39–46 pmid:9051196
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref38] 38. Torimoto K, Okada Y, Mori H, Tanaka. Relationship between fluctuations in glucose levels measured by continuous glucose monitoring and vascular endothelial dysfunction in type 2 diabetes mellitus, Cardiovasc. Diabetology 2013;12:1. pmid:23280391
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Cell culture

Measurement of cellular senescence

Human telomerase activity and length

Western blot analysis

DNA Damage Quantification

Nitric oxide and metabolites of nitric oxide measurement

Analysis of ROS and superoxide generation

Transfection of siRNAs

Statistics

Results

Discussion

Conclusion

Acknowledgments

Author Contributions

References