Janusz Witowski

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.

Toxicity of Free Radicals to Mesothelial Cells and Peritoneal Membrane

We studied the toxicity of free radicals to human mesothelial cells in vitro and to the peritoneal membrane of rats during peritoneal dialysis. Free radicals cause damage to mesothelial cells as measured by release of cytosolic markers such as 86Rb and lactate dehydrogenase. Vitamin E neutralized the toxic effect of free radicals in vitro. Human mesothelial cells exposed over 6 h to a mixture of essential and nonessential amino acids in medium are more vulnerable to the cytotoxic effect of free radicals than control cells exposed to medium alone. Cells exposed previously to glucose or glycerol are less vulnerable than controls. In rats free radicals generated intraperitoneally by a xanthine-xanthine oxidase system induce changes in peritoneal permeability similar to those observed during peritonitis: loss of ultrafiltration, increased glucose absorption from the dialysate and augmented transperitoneal loss of albumin. In addition lipids in the peritoneum became peroxidated. The addition of vitamin E to the peritoneal fluid with xanthine-xanthine oxidase prevents peroxidation of lipids and the subsequent loss of ultrafiltration. Our results show that free radicals may exert a potentially toxic effect on the peritoneal membrane during peritonitis. In such circumstances the addition of free radical scavenger to the dialysis fluid may preserve intact structure and function of peritoneum.

Relation of salivary antioxidant status and cytokine levels to clinical parameters of oral health in pregnant women with diabetes.

OBJECTIVEnBoth pregnancy and diabetes are thought to predispose to the impairment of oral health. As saliva contributes to oral homeostasis, we have characterised its properties and flow rate in pregnant women with or without diabetes.nnnDESIGNnUnstimulated whole mixed saliva was collected from 63 women in the first trimester of pregnancy and analysed for the concentration of selected antioxidants, cytokines, and growth factors.nnnRESULTSnPregnant women with diabetes were found to have markedly increased indexes of caries activity, plaque formation, gingival and periodontal status, as well as increased salivary antioxidant capacity and pro-inflammatory cytokine levels. These changes were more pronounced in patients with long-term disease and systemic diabetic complications, but only partly correlated with the level of blood glycated haemoglobin. Of the cytokines examined, salivary VEGF and HGF concentrations in diabetic pregnant women correlated in a positive and negative manner, respectively, with the prevalence of caries. Moreover, VEGF levels in this group correlated inversely with the probing depth and clinical attachment levels. All such associations did not occur in healthy individuals. In contrast, the salivary pH and flow rate correlated inversely with several parameters of caries and plaque formation irrespectively of whether the pregnant women were diabetic or not.nnnCONCLUSIONSnDiabetes in pregnant women significantly changes saliva properties, which may contribute to accelerated deterioration of the oral status in this population.

New Insights into the Biology of Peritoneal Mesothelial Cells: The Roles of Epithelial-to- Mesenchymal Transition and Cellular Senescence

Ultrafiltration failure due to dysfunction of the peritoneum as a dialyzing organ is a major clinical limitation of peritoneal dialysis. It is increasingly clear that mesothelial cells play an important role in fibrogenesis and vasculopathy that underlie peritoneal membrane dysfunction. New and extensively studied aspects of peritoneal mesothelial cell biology include epithelial-to-mesenchymal transition and cellular senescence. We discuss the potential significance of these processes for the peritoneal membrane function.

Human peritoneal fibroblasts are a potent source of neutrophil-targeting cytokines : a key role of IL-1β stimulation

Polymorphonuclear leukocyte (PMN) infiltration is a cardinal feature of peritonitis. CXC chemokine ligands 1 and 8 (CXCL1 and CXCL8), and the cytokine granulocyte colony-stimulating factor (G-CSF) are the key mediators of PMN accumulation. Increasing evidence points to an important role of human peritoneal fibroblasts (HPFB) in the response of the peritoneum to infection. We have examined the synthesis of PMN-targeting cytokines by HPFB exposed to intraperitoneal milieu as represented by peritoneal dialysate effluent (PDE) from patients undergoing peritoneal dialysis. PDE obtained during peritonitis, but not during infection-free periods, significantly increased production of CXCL1, CXCL8, and G-CSF by HPFB. The effect was largely blocked by antibodies to interleukin-1β (IL-1β), whereas neutralization of tumor necrosis factor-α (TNFα) had no major effect. Similar pattern of inhibition was observed when HPFB were exposed to conditioned media from endotoxin-stimulated peritoneal macrophages. Significance of IL-1β stimulation was further shown in experiments with recombinant cytokines. Compared with TNFα, exposure of HPFB to recombinant IL-1β resulted in a much higher release of PMN-targeting cytokines. The assessment of mRNA degradation revealed that the IL-1β-induced transcripts of CXCL1, CXCL8, and G-CSF were more stable compared with those induced by TNFα. These data indicate that HPFB can be a significant source of PMN-targeting cytokines when stimulated with IL-1β in the inflamed peritoneum.

Impaired response to oxidative stress in senescent cells may lead to accumulation of DNA damage in mesothelial cells from aged donors

The accumulation of 8-hydroxy-2-deoxyguanosine (8-OH-dG) exemplifies oxidative DNA injury, which is strongly implicated in ageing. We show that human peritoneal mesothelial cells (HPMCs) from donors >75 years have lower proliferative capacity but increased 8-OH-dG content compared with cells from individuals <25 years. We detected a positive relationship between the donors age and the 8-OH-dG level in early-passage HPMCs, and an inverse relationship between those 8-OH-dG levels and subsequent replicative lifespan of HPMCs (n=30). In early-passage cells from donors >75 years, the repair of oxidant-induced 8-OH-dG was delayed compared to cells from donors <25 years. This was coupled with prolonged removal of reactive oxygen species and faster decline in superoxide dismutase activity. Similar effects were observed in HPMCs rendered senescent in vitro. These results indicate that increased 8-OH-dG levels in HPMCs from aged individuals may reflect the in vivo presence of senescent cells with increased vulnerability to oxidative stress-induced DNA damage.

Correlation between the donor age and the proliferative lifespan of human peritoneal mesothelial cells in vitro: is TGF-β1 a link?

In the present study we have examined the relationship between calendar age of the donor and the proliferative lifespan and TGF-beta1 production by human peritoneal mesothelial cells (HPMC) in culture. The experiments were performed on primary omentum-derived HPMC isolated from patients undergoing elective abdominal surgery. There was an inverse relationship between the calendar age of the tissue donor and the replicative lifespan of HPMC in vitro (n=49, r=-0.3991, p<0.005). There was also a positive correlation between the donors age and the magnitude of TGF-beta1 production by first-passage HPMC (n=28, r=0.5400, p<0.004). In turn, the TGF-beta1 levels correlated inversely with the proliferative lifespan of HPMC in vitro (n=28, r=-0.4671, p<0.02). These findings indicate that the reduced proliferative capacity of HPMC isolated from older donors may be associated with increased TGF-beta1 release, which may, in turn, result from the age-related accumulation of senescent HPMC in the peritoneum.

Interpretation of elevated serum VEGF concentrations in patients with myocardial infarction

Serum has been considered an unsuitable medium for measurements of circulating vascular endothelial growth factor (VEGF) since platelets release significant quantities of VEGF during clotting. Nevertheless, the assessment of platelet-derived VEGF may be important in patients with acute coronary syndromes characterized by intraluminal thrombosis. The present study aimed at identifying the factors that impact on the interpretation of serum VEGF concentrations in patients with ST-elevation myocardial infarction (STEMI). VEGF was measured in 106 patients with STEMI and correlated with clinical and angiographic parameters. Serum VEGF levels were significantly higher in patients with STEMI than in healthy controls. Although the average number of platelets did not differ between the groups, the patients with STEMI, but not the controls, exhibited a significant correlation between serum VEGF levels and platelet counts. Stratification of patients according to different criteria revealed that VEGF concentrations were particularly elevated shortly (<3h) after the onset of chest pain in those patients who had occluding thrombi graded as large (3-4) on a TIMI scale. These data demonstrate that high levels of serum VEGF detected early in the course of STEMI may derive from activated platelets and may characterize patients with extensive intracoronary thrombosis.

Effect of Phosphatidylcholine on the Function of Human Mesothelial Cells in vitro

We tested the hypothesis that phosphatidylcholine (PC) molecules present in the dialysis solution may interact with the mesothelial cell membrane and modify its function. In vitro experiments were performed on human mesothelial cells (HMC) in culture. PC decreased proliferation of HMC when used at concentrations of 200 mg/l and higher. PC was also cytotoxic to HMC as measured by the release of lactate dehydrogenase from their cytosol. Cells exposed to PC had a diminished capacity for taking up 86Rb from medium. PC decreased the fibrinolytic properties of HMC and increased their procoagulant activity. Our results suggest that the positive short-term effect of the addition of PC to the dialysis solution (i.e., an increase in ultrafiltration) may be over-shadowed by its deleterious action on HMC membrane.

Biomarker research to improve clinical outcomes of peritoneal dialysis: consensus of the European Training and Research in Peritoneal Dialysis (EuTRiPD) network

Peritoneal dialysis (PD) therapy substantially requires biomarkers as tools to identify patients who are at the highest risk for PD-related complications and to guide personalized interventions that may improve clinical outcome in the individual patient. In this consensus article, members of the European Training and Research in Peritoneal Dialysis Network (EuTRiPD) review the current status of biomarker research in PD and suggest a selection of biomarkers that can be relevant to the care of PD patients and that are directly accessible in PD effluents. Currently used biomarkers such as interleukin-6, interleukin-8, exxa0vivo-stimulated interleukin-6 release, cancer antigen-125, and advanced oxidation protein products that were collected through a Delphi procedure were first triaged for inclusion as surrogate endpoints in a clinical trial. Next, novel biomarkers were selected as promising candidates for proof-of-concept studies and were differentiated into inflammation signatures (including interleukin-17, M1/M2 macrophages, and regulatory T cell/T helper 17), mesothelial-to-mesenchymal transition signatures (including microRNA-21 and microRNA-31), and signatures for senescence and inadequate cellular stress responses. Finally, the need for defining pathogen-specific immune fingerprints and phenotype-associated molecular signatures utilizing effluents from the clinical cohorts of PD patients and omics technologies and bioinformatics-biostatistics in future joint-research efforts was expressed. Biomarker research in PD offers the potential to develop valuable tools for improving patient management. However, for all biomarkers discussed in this consensus article, the association of biological rationales with relevant clinical outcomes remains to be rigorously validated in adequately powered, prospective, independent clinical studies.