Cellular FXIII in Human Macrophage-Derived Foam Cells.

Macrophages express the A subunit of coagulation factor XIII (FXIII-A), a transglutaminase which cross-links proteins through Nε-(γ-L-glutamyl)-L-lysyl iso-peptide bonds. Macrophages are major cellular constituents of the atherosclerotic plaque; they may stabilize the plaque by cross-linking structural proteins and they may become transformed into foam cells by accumulating oxidized LDL (oxLDL). The combination of oxLDL staining by Oil Red O and immunofluorescent staining for FXIII-A demonstrated that FXIII-A is retained during the transformation of cultured human macrophages into foam cells. ELISA and Western blotting techniques revealed that the transformation of macrophages into foam cells elevated the intracellular FXIII-A content. This phenomenon seems specific for macrophage-derived foam cells; the transformation of vascular smooth muscle cells into foam cells fails to induce a similar effect. FXIII-A containing macrophages are abundant in the atherosclerotic plaque and FXIII-A is also present in the extracellular compartment. The protein cross-linking activity of FXIII-A in the plaque was demonstrated using an antibody labeling the iso-peptide bonds. Cells showing combined staining for FXIII-A and oxLDL in tissue sections demonstrated that FXIII-A-containing macrophages within the atherosclerotic plaque are also transformed into foam cells. Such cells may contribute to the formation of lipid core and the plaque structurization.

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

Silencing Osteopontin Expression Inhibits Proliferation, Invasion and Induce Altered Protein Expression in Melanoma Cells.

Osteopontin (OPN) is a multifunctional phosphoprotein that is expressed in different types of cancers, including melanoma. OPN overexpression is associated with tumor progression and metastasis formation; however, the role of OPN in cell invasion and metastasis formation is not completely understood. In this study we aimed to define OPN expression in melanoma tissues and cell lines and investigate the effect of OPN expression on cell proliferation and invasion after inhibiting OPN expression with small interfering RNA (siRNA). OPN gene expression was determined by qRT-PCR, while protein expression was examined using a Proteome Profiler Oncology Array. siRNA-mediated OPN knockdown led to decreased OPN expression in melanoma cell lines, which was associated with decreased cell proliferation and invasion. Proteome profile analysis revealed significantly different protein expression between the original and transfected cell lines. The altered expression of the differently expressed proteins was validated at the mRNA level. Furthermore, OPN-specific siRNA was able to reduce OPN expression and inhibit the invasiveness of melanoma cells. Our results revealed for the first time that silencing the OPN gene influences proliferation and invasion of melanoma cells by effecting EGFR, tenascin C, survivin, galectin-3 and enolase 2 expression. To predict protein-protein interactions along with putative pathways we used STRING analysis for the differentially expressed proteins. These proteins formed multiple clusters, including extracellular matrix organization, regulation of angiogenesis, cell death and cell migration, PI3K-Akt, MAPK and focal adhesion signaling pathways. Taken together these data suggest that OPN might be an ideal target for drug development and therapies.

Exploring the cross-sectional association between outdoor recreational facilities and leisure-time physical activity: the role of usage and residential self-selection.

BACKGROUND: The availability of outdoor recreational facilities is associated with increased leisure-time physical activity (PA). We investigated how much of this association is attributable to selection effects, and explored whether usage of recreational facilities was an explanatory mechanism. METHODS: We analysed data from 5199 participants in the SPOTLIGHT survey residing in five European urban regions. Adults completed a survey and a Google Street View-based virtual audit was conducted to objectively measure the availability of outdoor recreational facilities in the residential neighbourhood. We used negative binomial GEE models to examine the association between objective and subjective availability of outdoor recreational facilities and leisure-time PA, and explored whether this association was attenuated after adjustment for socioeconomic status and preference for neighbourhoods with recreational facilities (as indicators of self-selection). We examined whether reported use of recreational facilities was associated with leisure-time PA (as explanatory mechanism), and summarized the most important motivations for (not) using recreational facilities. RESULTS: Subjective - but not objective - availability of outdoor recreational facilities was associated with higher levels of total leisure-time PA. After adjustment for self-selection (which attenuated the association by 25%), we found a 25% difference in weekly minutes of total leisure-time PA between individuals with and without self-reported availability of outdoor recreational facilities. For our study population, this translates to about 28 min per week. Participants who reported outdoor recreational facilities to be present but indicated not to use them (RR = 1.19, 95% CI = 1.03;1.22), and those reporting outdoor recreational facilities to be present and to use them (RR = 1.33, 95% CI = 1.22, 1.45) had higher levels of total leisure-time PA than those who reported outdoor recreational facilities not to be present. Proximity to outdoor recreational facilities was the most important motivation for use. CONCLUSION: The modest attenuation in the association between availability of outdoor recreational facilities and self-reported leisure-time PA suggests that individuals' higher activity levels may be due more to the perceived availability of outdoor recreational facilities than to self-selection. The use of these facilities seemed to be an important underlying mechanism, and proximity was the main motivator for using recreational facilities.

Lack of interest in physical activity - individual and environmental attributes in adults across Europe: The SPOTLIGHT project.

A considerable proportion of European adults report little or no interest in physical activity. Identifying individual-level and environmental-level characteristics of these individuals can help designing effective interventions and policies to promote physical activity. This cross-sectional study additionally explored associations between level of interest and physical activity, after controlling for other individual and environmental variables. Measures of objective and perceived features of the physical environment of residence, self-reported physical activity and other lifestyle behaviors, barriers towards physical activity, general health, and demographics were obtained from 5205 European adults participating in the 2014 online SPOTLIGHT survey. t-Tests, chi-square tests, and generalized estimating equations with negative binomial log-link function were conducted. Adults not interested in physical activity reported a higher BMI and a lower self-rated health, were less educated, and to a smaller extent female and less frequently employed. They were more prone to have less healthy eating habits, and to perceive more barriers towards physical activity. Only minor differences were observed in environmental attributes: the non-interested were slightly more likely to live in neighborhoods objectively characterized as less aesthetic and containing more destinations, and perceived as less functional, safe, and aesthetic. Even after controlling for other individual and environmental factors, interest in physical activity remained a significant correlate of physical activity, supporting the importance of this association. This study is among the first to describe characteristics of individuals with reduced interest in physical activity, suggesting that (lack of) interest is a robust correlate of physical activity in several personal and environmental conditions.

Exploring the mediating role of energy balance-related behaviours in the association between sleep duration and obesity in European adults. The SPOTLIGHT project.

Sleep restriction is a risk factor for weight gain and obesity. Few studies have formally investigated the mediating role of energy balance-related behaviours in the sleep - obesity association. The aim of this study was to explore the mediating role of physical activity, sedentary behaviours and dietary habits in the association of sleep duration with obesity in adults in five European urban regions. Data on self-reported sleep duration, energy balance-related behaviours, height and weight and other covariates were collected between February and September 2014 from participants to the SPOTLIGHT survey (N=5900, mean age 52years). Participants were recruited from 60 urban neighbourhoods in Belgium, France, Hungary, the Netherlands and the United Kingdom. Multilevel logistic regression analyses were used to assess the associations of sleep duration, energy balance-related behaviours and obesity and mediating effects were calculated using MacKinnon's product-of-coefficients method. Results indicated that a 1h increase in sleeping time was associated with a 14% lower likelihood of being obese (OR=0.86, 95%CI=0.80; 0.93). Only work-related sedentary behaviour was identified as a statistically significant mediator in the association between sleep duration and obesity for the total sample, and youngest and oldest age group. We did not find evidence for a mediating role of dietary habits and physical activities.

Lifestyle correlates of overweight in adults: a hierarchical approach (the SPOTLIGHT project).

BACKGROUND: Obesity-related lifestyle behaviors usually co-exist but few studies have examined their simultaneous relation with body weight. This study aimed to identify the hierarchy of lifestyle-related behaviors associated with being overweight in adults, and to examine subgroups so identified. METHODS: Data were obtained from a cross-sectional survey conducted across 60 urban neighborhoods in 5 European urban regions between February and September 2014. Data on socio-demographics, physical activity, sedentary behaviors, eating habits, smoking, alcohol consumption, and sleep duration were collected by questionnaire. Participants also reported their weight and height. A recursive partitioning tree approach (CART) was applied to identify both main correlates of overweight and lifestyle subgroups. RESULTS: In 5295 adults, mean (SD) body mass index (BMI) was 25.2 (4.5) kg/m2, and 46.0 % were overweight (BMI ≥25 kg/m2). CART analysis showed that among all lifestyle-related behaviors examined, the first identified correlate was sitting time while watching television, followed by smoking status. Different combinations of lifestyle-related behaviors (prolonged daily television viewing, former smoking, short sleep, lower vegetable consumption, and lower physical activity) were associated with a higher likelihood of being overweight, revealing 10 subgroups. Members of four subgroups with overweight prevalence >50 % were mainly males, older adults, with lower education, and living in greener neighborhoods with low residential density. CONCLUSION: Sedentary behavior while watching television was identified as the most important correlate of being overweight. Delineating the hierarchy of correlates provides a better understanding of lifestyle-related behavior combinations which may assist in targeting preventative strategies aimed at tackling obesity.

Mediating role of energy-balance related behaviors in the association of neighborhood socio-economic status and residential area density with BMI: The SPOTLIGHT study.

OBJECTIVES: This study aimed to examine the mediating effects of energy-balance related behaviors on the association of neighborhood socio-economic status (SES) and neighborhood residential area density (RAD) with body mass index (BMI). METHODS: In total, 6037 adults from four neighborhood types (high SES/high RAD, high SES/low RAD, low SES/high RAD, and low SES/low RAD) in five Mid-European urban regions completed an online survey asking about their energy-balance related behaviors (physical activity [PA], sedentary behavior, and dietary behavior), determinants of these behaviors and their body weight and height. MacKinnon's product-of-coefficients test was used to assess mediating effects. RESULTS: Transport-related PA, leisure-time PA and vegetable intake seemed to mediate the association between neighborhood type and BMI. Residents from low SES/low RAD neighborhoods reported less transport-related PA, less leisure-time PA and less vegetable intake than high SES/high RAD residents, and these behaviors (i.e. transport-related PA, leisure-time PA and vegetable intake) were related to having a higher BMI. CONCLUSION: The association between neighborhood type and BMI can be explained, at least in part, by energy-balance related behaviors.

Obesity-related behaviours and BMI in five urban regions across Europe: sampling design and results from the SPOTLIGHT cross-sectional survey.

OBJECTIVES: To describe the design, methods and first results of a survey on obesity-related behaviours and body mass index (BMI) in adults living in neighbourhoods from five urban regions across Europe. DESIGN: A cross-sectional observational study in the framework of an European Union-funded project on obesogenic environments (SPOTLIGHT). SETTING: 60 urban neighbourhoods (12 per country) were randomly selected in large urban zones in Belgium, France, Hungary, the Netherlands and the UK, based on high or low values for median household income (socioeconomic status, SES) and residential area density. PARTICIPANTS: A total of 6037 adults (mean age 52 years, 56% female) participated in the online survey. OUTCOME MEASURES: Self-reported physical activity, sedentary behaviours, dietary habits and BMI. Other measures included general health; barriers and motivations for a healthy lifestyle, perceived social and physical environmental characteristics; the availability of transport modes and their use to specific destinations; self-defined neighbourhood boundaries and items related to residential selection. RESULTS: Across five countries, residents from low-SES neighbourhoods ate less fruit and vegetables, drank more sugary drinks and had a consistently higher BMI. SES differences in sedentary behaviours were observed in France, with residents from higher SES neighbourhoods reporting to sit more. Residents from low-density neighbourhoods were less physically active than those from high-density neighbourhoods; during leisure time and (most pronounced) for transport (except for Belgium). BMI differences by residential density were inconsistent across all countries. CONCLUSIONS: The SPOTLIGHT survey provides an original approach for investigating relations between environmental characteristics, obesity-related behaviours and obesity in Europe. First descriptive results indicate considerable differences in health behaviours and BMI between countries and neighbourhood types.

Factor XIII-A is involved in the regulation of gene expression in alternatively activated human macrophages.

Factor XIII subunit A (FXIII-A) is one of the most overrepresented genes that is expressed during the alternative activation of macrophages. Based on its substrate profile and its cellular localisation, FXIII-A is thought to function as an intracellular/intranuclear transglutaminase. Our aim was to find role for the intracellular FXIII-A by comparing the microarray profiles of alternatively activated monocyte-derived macrophages. Microarray analyses of FXIII-A-deficient patients and healthy controls were evaluated, followed by functional clustering of the differentially expressed genes. After a 48-hour differentiation in the presence of interleukin 4 (IL4), 1,017 probes out of the 24,398 expressed in macrophages from FXIII-A- deficient samples were IL4 sensitive, while only 596 probes were IL4 sensitive in wild-type samples. Of these genes, 307 were induced in both the deficient and the wild-type macrophages. Our results revealed that FXIII-A has important role(s) in mediating gene expression changes in macrophages during alternative activation. Functional clustering of the target genes carried out using Cytoscape/BiNGO and Ingenuity Pathways Analysis programs showed that, in the absence of FXIII-A, the most prominent differences are related to immune functions and to wound response. Our findings suggest that functional impairment of macrophages at the level of gene expression regulation plays a role in the wound healing defects of FXIII-A-deficient patients.

Increased genotoxic susceptibility of breast epithelial cells to ethylene oxide.

This study was carried out with the aim of elucidating the organ-specific effects of ethylene oxide in comparison with the sensitivity of cells from different tissues. An increased incidence of leukemia and lymphoma has been observed in workers exposed to ethylene oxide. However, contradictory findings exist regarding its ability to induce other tumor types, such as breast cancer. We characterized the genotoxicity of ethylene oxide by means of the alkaline version of comet assay in in vitro systems, in order to investigate the hypothesized role of this substance in the development of breast cancer. For this study, we used primary and secondary cultures of lymphoblasts (well-known target cells of the genotoxicity of ethylene oxide), breast epithelial cells (hypothesized target), peripheral blood lymphocytes (cells commonly used in biomonitoring), and of keratinocytes and cervical epithelial cells. DNA damage was measured and expressed as tail DNA, tail length, and tail moment. In the concentration range 0-100 microM, ethylene oxide induced a dose-dependent increase of DNA damage in the investigated cell types without notable cytotoxicity. A statistically significant increase of DNA damage could be observed after treatment with 20 microM ethylene oxide in lymphoblasts (51% increase of tail moment over the background), breast epithelial cells (26% increase) and peripheral lymphocytes (71% increase). In keratinocytes (5% increase) and cervical epithelial cells (5% increase) significant DNA damage could not be detected at this dose, but at higher concentrations (50-100 microM), such an increase was observed. These results are indicative of an increased sensitivity of breast epithelial cells towards genotoxic insults of ethylene oxide. Our observations provide additional data to evaluate the hypothesis that exposure to ethylene oxide may play a role in breast cancer, and the findings may contribute to the development of screening tests for monitoring an early response to genotoxic insults in occupational settings.

Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis.

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.