Katarzyna Korybalska

Accelerated senescence of human peritoneal mesothelial cells exposed to high glucose : the role of TGF-β1

Cellular senescence can be activated in response to noxious environmental stimuli. A senescent-like phenotype has been detected in the peritoneal mesothelium of mice exposed to high intraperitoneal glucose. We have sought to examine whether high glucose (HG) can induce the senescence program in human peritoneal mesothelial cells (HPMC) in vitro. Senescence of omentum-derived HPMC was induced by serial passages. Cells were cultured in media containing either 5 mM glucose, 30 mM glucose, or 5 mM glucose and 25 mM mannitol (M) for osmotic control. Compared with HPMC cultured in low glucose, the growth rate of cells exposed to HG was significantly decreased so that the cells reached fewer population doublings before entering senescence. Exposure to HG led to increased expression of senescence-associated β-galactosidase (SA-β-Gal) and of the cell cycle inhibitors p21Waf1 and p27Kip1. Late-passage HPMC exposed to HG displayed marked hypertrophy and released increased amounts of fibronectin and TGF-β1. These effects were absent from HPMC treated with equimolar M. Exposure of early-passage HPMC to exogenous recombinant TGF-β1 induced a senescence marker SA-β-Gal in a dose-dependent manner and mimicked other senescence-associated alterations induced by HG. The addition of anti-TGF-β1 neutralizing antibody partially reduced the activation of HG-induced SA-β-Gal. These results indicate that chronic exposure to elevated glucose may result in TGF-β1-mediated accelerated senescence of HPMC in vitro, which may hypothetically contribute to the peritoneal membrane dysfunction during peritoneal dialysis in vivo.

Senescent Peritoneal Mesothelial Cells Promote Ovarian Cancer Cell Adhesion : The Role of Oxidative Stress-Induced Fibronectin

Adhesion of ovarian cancer cells to the peritoneal mesothelium is a key step in the malignant progression of the disease. In an in vitro study, we showed that the adherence of ovarian cancer cells (of the OVCAR-3, SKOV-3, and A2780 cell lines) to senescent human omentum-derived peritoneal mesothelial cells (HOMCs) was greater than to early passage cells. The process was mediated primarily by the increased interaction of the alpha5beta1 integrin on cancer cells with HOMC-associated fibronectin (FN). In comparison with early passage HOMCs, senescent cells exhibited increased FN mRNA expression levels and produced significantly more FN. To assess the effect of senescence-associated oxidative stress on FN release, HOMCs were rendered senescent by exposure to an oxidant, tert-butyl hydroperoxide. Treatment with tert-butyl hydroperoxide resulted in a significant increase in HOMC FN mRNA and protein expression levels. The effect of oxidative stress on FN synthesis was found to be mediated by transforming growth factor-beta1, whose signaling pathway was controlled at upstream and downstream levels by p38 MAPK. The activity of p38 MAPK increased markedly in senescent HOMCs. Treatment of HOMCs with antioxidants significantly attenuated senescence-associated increases in p38 MAPK activity, production of both transforming growth factor-beta1 and FN, and ovarian cancer cell adhesion. These data indicate that oxidative stress that accompanies senescence may increase FN production by HOMCs and thus facilitate binding and dissemination of ovarian cancer cells.

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

OBJECTIVE Both 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. DESIGN Unstimulated 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. RESULTS Pregnant 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. CONCLUSIONS Diabetes in pregnant women significantly changes saliva properties, which may contribute to accelerated deterioration of the oral status in this population.

Recovery of Senescent Endothelial Cells From Injury

Percutaneous coronary intervention is increasingly performed in elderly patients. Because the procedure is associated with endothelial cell (EC) denudation, we compared recovery of young and old ECs from scratch injuries inflicted in culture. Although senescent ECs displayed markedly reduced potential to proliferate and migrate, they repopulated the wounds as fast as young cells. Morphometric analysis revealed that senescent cells were significantly larger and as a result far fewer senescent cells managed to cover the lesion. Compared with young EC, senescent cells displayed increased expression of senescence-associated β-galactosidase, nitric oxide synthase (eNOS), and AKT kinase, and secreted increased amounts of growth factors (VEGF, TGF-β), cytokines (IL-6, IL-8, MCP-1), adhesion molecules (sICAM-1), and matrix proteins (fibronectin). This secretory phenotype rather than the rate of wound closure per se may contribute to unfavorable vascular remodeling in the elderly undergoing coronary catheterization.

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.

Endothelial cell growth response to stimulation with serum from patients with acute ST-elevation myocardial infarction.

Acute ST-elevation myocardial infarction (STEMI) is associated with increased levels of angiogenic mediators [1,2] thought to derive both from the ischemic myocardium [3] and from activated platelets within intra-luminal thrombi [4]. Since their impact on the endothelium in vivo is poorly defined, we have attempted to mimic the diseased environment by incubating endothelial cells in vitro in the presence of serum from patients with STEMI (Fig. 1). Such an approach has previously been used in studies on inflammatory bowel disease [5], colorectal cancer [6], and ageing [7]. We measured the endothelial cell growth index in culture and then related it – without any assumptions – to a number of demographic, clinical, laboratory, and angiographic criteria. Subsequent post-hoc analyses formed a basis of this hypothesis-generating investigation. The study protocol was approved by the institutional ethics committee. All patients with STEMI underwent primary percutaneous coronary intervention (PCI) within 12 hours after the onset of chest pain. Only patientswith bare-metal stents, without prior statin therapy, and without co-existing neoplastic or inflammatory diseases were included in the analysis. Healthy regular blood donors with no prior treatment with statins served as the controls. Endothelial cell proliferation was found to be significantly greater following exposure to serum from patients with STEMI (n=106; median age 57, 71% men) than from healthy blood donors (n=50; median age 48 years, 84% men). The growth response clearly discriminated the two populations (Fig. 2). The difference was not related to a higher proportion of youngermen in the controls since the sub-analysis of 47 individuals from each group carefully matched for age and sex revealed exactly the same pattern and similar level of significance (not shown). Theproliferation index in STEMIpatients appeared to bemorewidely distributed than in control subjects. As a result, a number of patients had the proliferation index as low as the controls. In order to determine whether the magnitude of growth response differentiated the STEMI patients in any way, they were categorised into two groups according to whether their proliferation index was below or above the 25th percentile. This cut-off point was selected so that the patients with significant overlappingwith the interquartile range of the controls were classified as thosewith lowproliferation index (Fig. 2). All the remaining

Angiogenesis-Related Proteins - Their Role in the Pathogenesis and Treatment of Inflammatory Bowel Disease

The etiology of inflammatory bowel disease (IBD) is still not fully understood. Angiogenesis is one of the crucial phenomena sustaining chronic inflammation in the gastrointestinal tract. It has been also shown that the most potent anti-inflammatory drugs--anti-tumor necrosis factor alpha antibodies down-regulate intestinal angiogenesis, what is believed to contribute to their therapeutic efficacy. There are many proteins engaged in gastrointestinal angiogenesis, including pro-inflammatory cytokines, vascular growth factors, and adhesion molecules. Several of them are considered to be promising molecular targets for new drugs--monoclonal antibodies or fusion proteins. Here, we review new data highlighting the key role of proteins that regulate immune-mediated angiogenesis in IBD, like vascular endothelial growth factor, hypoxia inducible factor, angiopoietins, or basic fibroblast growth factor. We present the molecular mechanisms regulating the pathological proangiogenic activity in inflammatory conditions in IBD. We also discuss how new anti-cytokine regimens affect the function of angiogenesis-related proteins.

Salivary fingerprint of simple obesity

HighlightsSaliva of obese individuals contains increased concentrations of several mediators.TNF‐R1 and serpin A12 discriminate obesity with high sensitivity and specificity.Their levels correlate with plaque build‐up and inferior gingival status.Obesity, even without comorbidities, leads to distinct changes in the saliva. Background: The nature of a link between poor oral health and obesity is not fully understood. It is also unclear if saliva contributes to it and whether the properties of saliva change as a result of an increase in body mass or rather as a consequence of obesity‐associated comorbidities. This pilot study was undertaken in an attempt to determine if salivary biomarkers can identify obesity per se. Methods: Whole mixed saliva was analysed for 16 soluble parameters covering 4 categories (inflammation, oxidative stress, endothelial dysfunction, adipokines). In the discovery group, 19 obese and 25 non‐obese women matched for age, with similar hygiene habits, with no comorbidities and not taking any medication known to affect saliva secretion were analysed. In the validation group, a cohort of no‐preselected 81 individuals (34 obese) were analysed. Results: Individuals with obesity had significantly higher salivary concentrations of several cytokines and adipokines, of which TNF‐R1, serpin A12 and PAI‐1 were identified as parameters discriminating between obese and non‐obese subjects with the highest sensitivity and specificity. Conclusions: Obesity per se leads to distinct changes in the concentration of several parameters in saliva. These findings may have diagnostic implications for distinguishing the effects of obesity and obesity‐linked comorbidities on oral health.

Trefoil factor-3 is not a useful marker of mucosal healing in Crohn's disease treated with anti-TNF-α antibodies

AIM To evaluate whether repeated serum measurements of trefoil factor-3 (TFF-3) can reliably reflect mucosal healing (MH) in Crohns disease (CD) patients treated with anti-tumor necrosis factor-α (anti-TNF-α) antibodies. METHODS Serum TFF-3 was measured before and after anti-TNF-α induction therapy in 30 CD patients. The results were related to clinical, biochemical and endoscopic parameters. MH was defined as a ≥ 50% decrease in Simple Endoscopic Score for Crohns disease (SES-CD). RESULTS SES-CD correlated significantly with CD clinical activity and several standard biochemical parameters (albumin, leukocyte and platelet counts, C-reactive protein, erythrocyte sedimentation rate, fibrinogen). In contrast, SES-CD did not correlate with TFF-3 (P = 0.54). Moreover, TFF-3 levels did not change significantly after therapy irrespectively of whether the patients achieved MH or not. Likewise, TFF-3 did not correlate with changes in fecal calprotectin, which has been proposed as another biochemical marker of mucosal damage in CD. CONCLUSION Serum TFF-3 is not a convenient and reliable surrogate marker of MH during therapy with TNF-α antagonists in CD.

Oral Health Status of Patients with Lysosomal Storage Diseases in Poland

Patients with lysosomal storage diseases (LSDs) suffer from physical and mental disabilities, which together with poor access to professional care may lead to impaired oral health. This cross-sectional case-control study characterized the status of oral health in patients with LSDs in Poland. Thirty-six children and young adults with various forms of LSDs were examined. The data were compared with those from age- and sex-matched healthy controls. Exemplary cases were presented to highlight typical problems in oral care associated with LSDs. When possible, saliva was collected and analyzed for total protein, inflammatory mediators, and antioxidant status. Generally, patients with LSDs had significantly higher prevalence of caries, inferior gingival status, and inadequate oral hygiene. The severity of oral health impairment in mucopolysaccaridoses, the most common LSD in Poland, was similar to that seen in patients with mannosidoses or Pompe disease. Saliva could be collected only from few less handicapped patients. In MPS, it did not appear to differ significantly from the controls, but in patients with Pompe disease it contained lower concentrations of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1), but higher levels of tumor necrosis factor receptors 1 and 2 (TNF-R1, TNF-R2) and myeloperoxidase (MPO). In conclusion, Polish patients with LSDs have an inadequate level of oral hygiene and substantially deteriorated oral health.