Renin Angiotensin System-mediated modulation of inflammation-induced Tissue Factor expression

Background: An intricate cross-talk connects Tissue Factor (TF) to inflammation and is amplified by locally active angiotensin(ang)II, that stimulates TF expression. High glucose may activate pro-inflammatory pathways and increase glucose-mediated angII production. Aim: To evaluate the effect of Renin Angiotensin System (RAS) blockade on TF expression in peripheral blood mononuclear cells (PBMCs) exposed to normal glucose (NG) and high glucose (HG), and stimulated by lipopolysaccharide (LPS), a well known pro-inflammatory agent. Methods: PBMCs, from healthy donors were LPS-stimulated and exposed to NG and HG. TF mRNA levels, antigen expression and pro-coagulant activity were assessed by Real Time-PCR, ELISA and 1-stage clotting assay respectively, in absence or presence of pharmacological intervention. Results: LPS stimulation increased TF expression in NG, an effect down-regulated by Aliskiren, a direct renin inhibitor, zofenopril, an angiotensin converting enzyme inhibitor, olmesartan, an angII type1 receptor (AT1R) blocker and Compound21, an highly selective angII type 2 receptor (AT2R) agonist. As compared with NG, HG amplified the LPS-stimulation of TF expression, an effect that Aliskiren, Zofenopril and Compound21 down- regulated in HG with a similar behavior to that in NG, while OLM activity was potentiated in HG. Conclusions: Our data disclose tight connections between the apparently unrelated domains of innate immunity and metabolic and cardiovascular regulation, expanding the restrictive view of angII as a peptide mainly involved in the control of blood pressure and water and salt homeostasis

Don’t call It smart: working from home during the pandemic crisis

The recent COVID-19 pandemic and related social distancing measures have significantly changed worldwide employment conditions. In developed economies, institutions and organizations, both public and private, are called upon to reflect on new organizational models of work and human resource management, which - in fact - should offer workers sufficient flexibility in adapting their work schedules remotely to their personal (and family) needs. This study aims to explore, within a Job Demands-Resources framework, whether and to what extent job demands (workload and social isolation), organizational job resources (perceived organizational support), and personal resources (self-efficacy, vision about the future and commitment to organizational change) have affected workers’ quality of life during the pandemic, taking into account the potential mediating role of job satisfaction and perceived stress. Using data from a sample of 293 workers, we estimate measurement and structural models, according to the Item Response Theory and the Path analysis frameworks, which allow us to operationalize the latent traits and study the complex structure of relationships between the latent dimensions. We inserted in the model as control variables, the socio-economic and demographic characteristics of the respondents, with particular emphasis on gender differences and the presence and age of children. The study offers insights into the relationship between remote work and quality of life, and the need to rethink human resource management policies considering the opportunities and critical issues highlighted by working full-time remotely

miR-19a and miR-20a and tissue factor expression in activated human peripheral blood mononuclear cells

Background and Aims. To investigate the behaviour of miR-19a and miR-20a, two microRNAs involved in posttranscriptional modulation of TF expression in peripheral blood mononuclear cells (PBMCs) exposed to high glucose (HG) and lipopolysaccharide (LPS), and to evaluate the involvement of angiotensin II in that process. Methods. TF Procoagulant Activity (PCA, one-stage clotting assay), antigen (Ag, ELISA), and miR-19a and miR-20a levels (specific TaqMan® MicroRNA Assays) were evaluated in PBMCs exposed to high glucose (HG, 50 mM), LPS (100 ng/mL), and Olmesartan (OLM, 10−6 M), an angiotensin II type 1 receptor antagonist. Results. HG increased TF expression and decreased both miRs as compared to control glucose conditions (11.1 mM). In HG-activated PBMCs, LPS stimulated TF expression and downregulated miR-20a, an effect reverted by OLM (10−6 M); miR-19a expression was unchanged by LPS in both CG and HG conditions. Conclusions. miR-19a and miR-20a are inhibited by inflammatory stimuli active on TF expression and their response differs by the stimulus under investigation; angiotensin II may participate in that mechanism

The effect of high glucose on the inhibitory action of C21, a selective AT2R agonist, of LPS-stimulated tissue factor expression in human mononuclear cells

Background: Intimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (AT) II, the effector arm of the Renin Angiotensin System (RAS). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated peripheral blood mononuclear cell(PBMC)s implying the existence of ATII type 2 receptor (AT2R)s whose stimulation attenuates inflammation-mediated procoagulant responses. High glucose, by activating key signalling pathways and increasing the cellular content of RAS components, augments TF expression and potentiates the inhibitory effect of AT1R antagonists. It is unknown, however, the impact of that stimulus on AT2R-mediated TF inhibition, an information useful to understand more precisely the role of that signal transduction pathway in the inflammation-mediated coagulation process. TF antigen (ELISA), procoagulant activity (PCA, 1-stage clotting assay) and TF-mRNA (real-time polymerase chain reaction) were assessed in PBMCs activated by LPS, a pro-inflammatory and procoagulant stimulus, exposed to either normal (N) or HG concentrations (5.5 and 50 mM respectively). Results: HG upregulated TF expression, an effect abolished by BAY 11-7082, a NFκB inhibitor. C21 inhibited LPS-stimulated PCA, TFAg and mRNA to an extent independent of glucose concentration but the response to Olmesartan, an AT1R antagonist, was quite evidently potentiated by HG. Conclusions: HG stimulates LPS-induced TF expression through mechanisms completely dependent upon NFkB activation. Both AT2R-stimulation and AT1R-blockade downregulate inflammation-mediated procoagulant response in PBMCs but HG impacts differently on the two different signal transduction pathway

Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cells

Background Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar. Methods Experiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient. Results Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action. Conclusions GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation

Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells.Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution.Particulate matter induces a dose- and time- dependent, rapid (1 h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm).Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity

Demand of Long-Term Care and benefit eligibility across European countries

In the context of an unprecedented aging process, the role of domiciliary care for older adults is becoming increasingly essential. In order to design effective and proactive policies of formal elderly-care, it is crucial to understand how vulnerable elderly individuals would adjust their informal long-term care utilization to changes in the formal-care provision. Although theoretical frameworks have been proposed, showing that a positive relationship could arise when the elderly exhibit an excess demand of care, empirical evidence is scant, due to the lack of credible instruments to account for the endogenous nature of formal-care decisions. We propose a novel instrument, an index that capture individuals’ eligibility status to the LTC domiciliary programmes implemented in their own nation or region. That is, a dummy variable - being eligible or not - which is grounded on the LTC regulation context at national or regional level, but still has individual within region variation due to differences in health conditions and vulnerability assessment. We estimate an IV two-part model using a representative sample of the over 60 population for non-institutionalised individuals in Austria, Germany, France and Belgium. Our results, which are robust to a number of different specifications, point at the lack of crowding-out of the informalby the formal-care, thus suggesting the existence of a substantial unmet demand of LTC among the elderly

Breast Cancer Susceptibility: the role of BRCA2 in Homologous Recombination.

Abstract Breast cancer is the most frequently diagnosed cancer in women and a small proportion of breast cancer cases, in particular those arising at a young age, is attributable to highly penetrant autosomal dominant genes, as BRCA1 and BRCA2. Heterozygous germline protein truncating mutations in the tumour suppressor gene BRCA2 predispose carriers to breast and ovarian cancer. The influence of unclassified variants (UCVs), on cancer risk and on gene function has not been determined. As BRCA2 mutant cells exhibit defective Double Strand Break Repair (DSBR) by HR we want to study the effect of BRCA2 wt and its mutated forms expression on spontaneous HR in two model systems; yeast is a good genetic model system to investigate factors affecting HR as demonstrated by Caligo et al., in 2008, and HeLa G1 human cell line have a recombination substrate useful to investigate HR in a clonogenic assay (Ciotta C. et al. 1998). We have selected 11 BRCA2 UCVs (G173V, D191V, S286P, M927V, T1011R, L1019V, N1878K, S2006R, R2108C, G2353R and V3091I) to test their effect on HR. Moreover we have chosen a pathogenic control (G2748D) and three neutral controls (H372N, M1915T and A2951T). BRCA2 wt increase spontaneous HR in yeast while the variants T1011R and S2006R behave differently to the wt reducing intra and inter- recombination as the pathogenic control G2748D. The overexpression of BRCA2 wt increases spontaneous HR in HeLa G1 cells as evaluated by a clonogenic assay and the variants D191V, N1878K, S2006R, R2108C, G2353R, V3091I behave in a different way respect the wt as the pathogenic control G2748D. In conclusion our data suggested that BRCA2 is deeper involved in spontaneous HR both in yeast and mammalian cells but the mechanisms that regulate its role have to be clarified. We observed that the transgene expression of BRCA2 wt increase HR and BRCA2 variants could affect the levels of HR