Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells

The replicative life span of cells in culture is thought to be determined by the gradually rising pool of senescent cells rather than by the simultaneous loss of proliferative capacity by all cells in the population. We found that early-passage cultures of human peritoneal mesothelial cells (HPMCs) contained a significant fraction of senescent-like cells. Furthermore, early-passage populations with a high percentage of senescent cells had a reduced subsequent life span in culture compared with populations consisting of the same number of apparently young cells but containing no senescent cells. The exposure of early-passage HPMCs to the conditioned medium from cultures containing senescent cells resulted in the retardation of growth and the induction of senescence-associated beta-galactosidase (SA-beta-Gal). This effect could be partly reduced by neutralizing TGF-beta1 activity. The timely treatment with N-tert-butyl-alpha-phenylnitrone (PBN) reduced oxidative stress, the number of early senescent cells, TGF-beta1 secretion, and ultimately extended the population life span. The effect was evident only when PBN was introduced at a very early, but not at a late, phase of tissue culture history. These results indicate that a sudden onset of senescence in early-passage HPMCs is related to oxidative stress and may influence the replicative life span of the population as a whole.     

Mitochondrial dysfunction is a possible cause of accelerated senescence of mesothelial cells exposed to high glucose

High glucose has been found to accelerate cell senescence in vitro. The exact mechanism of this effect is, however, still poorly understood. In this paper we show that human peritoneal mesothelial cells (HPMCs) propagated under high (30 mM) glucose were characterized by higher density of DNA double-strand breaks than cells exposed to standard (5 mM) glucose concentration. Under both low and high glucose conditions, the vast majority of DNA damage localized to non-telomeric regions of the genome. Moreover, exposure to high glucose resulted in increased accumulation of lipofuscin, increased production of superoxides and peroxides as well as reduced mitochondrial membrane potential and increased mitochondrial mass. Treatment of cells with the free radical scavenger PBN partially rescued the premature senescence caused by high glucose. Together, these results indicate that high glucose may accelerate senescence of HPMCs by impairing mitochondrial function, resulting in overproduction of reactive oxygen species and extensive DNA damage.     

Up-regulation of NDRG2 in senescent lens epithelial cells contributes to age-related cataract in human

Background

Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer''s disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract.

Methodology/Principal Findings

Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H₂O₂ to simulate senescence. After being exposed to 50 µM H₂O₂ for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50–200 µM H₂O₂ for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses.

Conclusions/Significance

NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.     

The bystander effect contributes to the accumulation of senescent cells in vivo

Senescent cells accumulate with age in multiple tissues and may cause age‐associated disease and functional decline. In vitro, senescent cells induce senescence in bystander cells. To see how important this bystander effect may be for accumulation of senescent cells in vivo, we xenotransplanted senescent cells into skeletal muscle and skin of immunocompromised NSG mice. 3 weeks after the last transplantation, mouse dermal fibroblasts and myofibres displayed multiple senescence markers in the vicinity of transplanted senescent cells, but not where non‐senescent or no cells were injected. Adjacent to injected senescent cells, the magnitude of the bystander effect was similar to the increase in senescence markers in myofibres between 8 and 32 months of age. The age‐associated increase of senescence markers in muscle correlated with fibre thinning, a widely used marker of muscle aging and sarcopenia. Senescent cell transplantation resulted in borderline induction of centrally nucleated fibres and no significant thinning, suggesting that myofibre aging might be a delayed consequence of senescence‐like signalling. To assess the relative importance of the bystander effect versus cell‐autonomous senescence, we compared senescent hepatocyte frequencies in livers of wild‐type and NSG mice under ad libitum and dietary restricted feeding. This enabled us to approximate cell‐autonomous and bystander‐driven senescent cell accumulation as well as the impact of immunosurveillance separately. The results suggest a significant impact of the bystander effect for accumulation of senescent hepatocytes in liver and indicate that senostatic interventions like dietary restriction may act as senolytics in immunocompetent animals.     

Oxidative stress and innate immunity status in chickens exposed to high dose of ascorbic acid

The effects of high dose ascorbic acid (10 000 mg· kg^–1 in the diet) and the transition metal on the presence of oxidative stress in the internal organs of growing chicks, as well as on the innate immune system status, were investigated. Supplementation with a high dose of ascorbic acid had pro‐inflammatory effects on the intestinal mucosa, and lysozyme levels were decreased significantly in all organs studied. High‐dose ascorbic acid caused an imbalance between prooxidative and antioxidative activities and was associated with the generation of semiquinone radicals. We observed that ascorbic acid increased iron and cadmium absorption. When a high dose of ascorbic acid was applied, elevated kidney and intestinal mucosa iron concentrations were observed. The amount of free malondialdehyde in the above organs has increased as well. These data have important implications for the mechanism of the oxidative stress development under the influence of high dose of ascorbic acid, indicating the importance of the side reactions of the mitochondrial electron transport chain with the formation of semiquinone radicals and the role of transition metals in this process. Copyright © 2013 John Wiley & Sons, Ltd.     

Oxidative stress contributes to vascular endothelial dysfunction in heart failure

Congestive heart failure (HF) is characterized by inadequate nitric oxide (NO) production in the vasculature. Because NO is degraded by oxygen radicals, we hypothesized that NO is degraded faster in HF from inadequate peripheral arterial antioxidant reserves. HF was induced in male Sprague-Dawley rats by left coronary artery ligation. Vascular endothelial function was evaluated by measuring the NO-mediated vasorelaxation response to acetylcholine (ACh; 10(-9)-10(-4) M) in excised aortas. This was repeated with the free radical generator pyrogallol (20 microM) and again with pyrogallol and superoxide dismutase (SOD; 60 U/ml). Aortic and myocardial SOD activity was also determined. ACh-induced vasorelaxation was reduced in HF (n = 9) compared with normal control rats (n = 11; P < 0.001). Pyrogallol further reduced vasorelaxation in HF: 74 +/- 11% at 10(-4) M ACh versus 58 +/- 10% in normal control rats (P < 0.004). There was a trend (P = 0.06) toward reduced SOD activity in HF aortas. In conclusion, altered NO-dependent vasorelaxation in HF is in part due to excessive degradation of NO and is likely related to reduced vascular SOD activity.     

HDACs and the senescent phenotype of WI-38 cells

Background  

Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells.     

CXCR7 contributes to the aggressive phenotype of cholangiocarcinoma cells

Development of cholangiocarcinoma (CCA) is dependent on a cross-talk with stromal cells, which release different chemokines including CXCL12, that interacts with two different receptors, CXCR4 and CXCR7. The aim of the present study was to investigate the role of CXCR7 in CCA cells. CXCR7 is overexpressed by different CCA cell lines and in human CCA specimens. Knock-down of CXCR7 in HuCCT-1 cells reduced migration, invasion, and CXCL12-induced adhesion to collagen I. Survival of CCA was also reduced in CXCR7-silenced cells. The ability of CXCL12 to induce cell migration and survival was also blocked by CCX733, a CXCR7 antagonist. Similar effects of CXCR7 activation were observed in CCLP-1 cells and in primary iCCA cells. Enrichment of tumor stem-like cells by a 3D culture system resulted in increased CXCR7 expression compared to cells grown in monolayers, and genetic knockdown of CXCR7 robustly reduced sphere formation both in HuCCT-1 and in CCLP-1 cells. In HuCCT-1 cells CXCR7 was found to interact with β-arrestin 2, which was necessary to mediate CXCL12-induced migration, but not survival. In conclusion, CXCR7 is widely expressed in CCA, and contributes to the aggressive phenotype of CCA cells, inducing cell migration, invasion, adhesion, survival, growth and stem cell-like features. Cell migration induced by CXCR7 requires interaction with β-arrestin 2.     

Low in vivo brain glucose consumption and high oxidative stress in accelerated aging

The validity of the free radical theory of aging has been recently questioned. Our aim was to test whether there is oxidative stress in tissues critically involved in accelerated aging (senescence-accelerated mice, SAM) and whether this correlates with lower glucose consumption in vivo and behavioural tests. Positron emission tomography shows that brains of old SAM-prone animals consume less glucose than young ones. Behavioural characteristics, mitochondrial peroxide production, and damage in both the central nervous system and bone marrow stem cells also indicate that SAM-prone animals age faster than SAM-resistant ones. Our results support the role of the free radical theory of aging in critical tissues involved in aging and that this correlates with glucose consumption.     

Oxidative stress contributes to muscle atrophy in chronic kidney disease patients

Abstract

Objectives

Patients with chronic kidney disease have impaired muscle metabolism, resulting in muscle atrophy. Oxidative stress has previously been identified as a significant contributor to muscle atrophy in other populations, but the contribution in chronic kidney disease is unknown. The aim of this study was to investigate the association between oxidative stress, grip strength, and lean mass in patients with chronic kidney disease.

Methods

This is a cross-sectional study of 152 participants with stage 3 or 4 chronic kidney disease. Outcome measures include grip strength, lean mass, plasma total F2-isoprostanes, inflammation, peak oxygen uptake, and standard clinical measures.

Results

Thirty four (22.4%) chronic kidney disease patients had elevated oxidative stress levels (plasma F2-isoprostanes >250 pg/ml), with 82% of patients below age-predicted grip strength normative values. There was a significant negative association between plasma F2-isoprostanes and grip strength (r = ?0.251) and lean mass (r = ?0.243). There were no associations with inflammation markers. Multiple linear regression identified plasma F2-isoprostanes as a significant predictor of grip strength independent of other predictors: sex, diabetes status, body mass index, body fat percent, and phosphate (adjusted r² = 69.5, P < 0.001).

Discussion

Plasma F2-isoprostanes were independently associated with reduced strength in chronic kidney disease patients.     

Reduction in flippase activity contributes to surface presentation of phosphatidylserine in human senescent erythrocytes

Mature human erythrocytes circulate in blood for approximately 120 days, and senescent erythrocytes are removed by splenic macrophages. During this process, the cell membranes of senescent erythrocytes express phosphatidylserine, which is recognized as a signal for phagocytosis by macrophages. However, the mechanisms underlying phosphatidylserine exposure in senescent erythrocytes remain unclear. To clarify these mechanisms, we isolated senescent erythrocytes using density gradient centrifugation and applied fluorescence‐labelled lipids to investigate the flippase and scramblase activities. Senescent erythrocytes showed a decrease in flippase activity but not scramblase activity. Intracellular ATP and K⁺, the known influential factors on flippase activity, were altered in senescent erythrocytes. Furthermore, quantification by immunoblotting showed that the main flippase molecule in erythrocytes, ATP11C, was partially lost in the senescent cells. Collectively, these results suggest that multiple factors, including altered intracellular substances and reduced ATP11C levels, contribute to decreased flippase activity in senescent erythrocytes in turn to, present phosphatidylserine on their cell membrane. The present study may enable the identification of novel therapeutic approaches for anaemic states, such as those in inflammatory diseases, rheumatoid arthritis, or renal anaemia, resulting from the abnormally shortened lifespan of erythrocytes.     

Oxidative stress response in two representative bacteria exposed to atrazine

Bacteria are present extensively in the environment. Investigation of their antioxidant properties will be useful for further study on atrazine stress tolerance of bacteria and the defense mechanism of antioxidant enzymes against atrazine or other triazine herbicides. Superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and total antioxidant capacity (T-AOC) from one Gram-negative representative strain Escherichia coli K12 and one Gram-positive representative strain Bacillus subtilis B19, respectively, were tested for response to atrazine stress. The results indicated that SOD, CAT, GST and T-AOC were induced upon exposure to atrazine. The growth of two bacteria was better in the absence than in the presence of atrazine, indicating that atrazine can decrease bacterial growth. The changes of enzyme activities indicate the presence of oxidative stress. Oxidative stress induced by atrazine may be due to imbalance of redox potential in bacterial cells, which leads to bacterial metabolic disorder.     

Inhibitory effect of acetylsalicylic acid on matrix metalloproteinase - 2 activity in human endothelial cells exposed to high glucose

Matrix metalloproteinases play a major role in the process of angiogenesis, an important feature of diabetes complications, cancer or rheumatoid arthritis. High glucose concentrations were reported to augment metalloproteinase-2 secretion in some cell types. In the present study we investigated the influence of acetylsalicylic acid on metalloproteinase- 2 secretion and expression in endothelial cells cultured for one week in high glucose conditions (25 mM and 33 mM). Metalloproteinase-2 activity was evidenced by gel zymography, the protein was identified by Western blotting, and the gene expression was quantitated by RT-PCR. The results indicated a marked inhibitory effect of acetylsalicylic acid at gene expression level (approximately 43%) and also at secretion level in samples of conditioned media (approximately 30%) and cellular homogenates (approximately 70%). This may suggest that acetylsalicylic acid could have a beneficial effect in preventing the angiogenic process that appears in diabetes complications.     

Human-specific LAIR2 contributes to the high invasiveness of human extravillous trophoblast cells

The placenta is a temporary vital organ for intra-uterine development and growth. The anatomical structure of the placenta has evolved substantially, resulting in broad inter-species diversity. In particular, human placental extravillous trophoblast cells (EVTs) have evolved aggressive features, although the mechanism underlying this aggressiveness remains elusive. In the present study, we compared the human and mouse homologous gene databases and obtained 2272 human-specific genes, 807 of which are expressed in the placenta according to the UniGene database. Using the human trophoblast cell line HTR8/SVneo, we further verified the expression and function of one of these genes, the leukocyte-associated immunoglobulin-like receptor 2 (LAIR2). This gene shows increased expression during pregnancy and its reduced expression is associated with pregnancy complications. Although LAIR2 was expressed in the human placenta villus and decidua in the first trimester of pregnancy, it was not expressed in mouse tissues. Knockdown of LAIR2 markedly improved cell viability and inhibited the invasive ability of HTR8/SVneo cells. These data suggest that species-specific genes are pivotal to the evolution of a more aggressive human placenta to match the physiological demands of human development. Further investigation is required to obtain evidence on the function of LAIR2 and other specific genes in the placenta, providing insight on the mechanism, properties, and possible applications of this in humans.