Cristina Balia

Angiotensin II induces the generation of procoagulant microparticles by human mononuclear cells via an angiotensin type 2 receptor-mediated pathway

INTRODUCTION Microparticles are small vesicles shed by cells upon activation and during apoptosis which participate in physiologically relevant phenomena, including blood coagulation. Intracellular calcium mobilization is one of the mechanisms of microparticle generation during cell activation. Because the renin-angiotensin system has been proposed as a link between hypertension and increased thrombotic risk, we investigated whether angiotensin II upregulates the generation of procoagulant microparticles by human mononuclear cells. MATERIALS AND METHODS Human mononuclear cells were exposed to angiotensin II for 15min. Intracellular calcium concentration was assessed by a Fluo 4 based kit. The supernatants were analyzed for both microparticle content, with a commercially available kit based on phosphatidylserine analysis, and microparticle-associated tissue factor, with a one-stage clotting assay. RESULTS Intracellular calcium concentration is increased upon exposure of mononuclear cells to angiotensin II. Incubation with angiotensin II stimulates microparticles release; microparticle-associated tissue factor is also upregulated. The effect is inhibited by an angiotensin receptor type 2 antagonist (PD123319) and not by two angiotensin type 1 antagonists (Losartan and Olmesartan). CONCLUSIONS Angiotensin receptor 2-mediated upregulation of tissue factor-bearing, procoagulant microparticle generation represents a novel mechanism linking the renin-angiotensin system to thrombosis.

Leptin induces the generation of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells.

BACKGROUND Obesity is linked to increased thrombotic risk. Circulating leptin concentration correlates with body mass index. Microparticles are small (.05-1 μm) vesicles shed by activated and apoptotic cells, involved in numerous pathophysiologically relevant phenomena including blood coagulation and thrombosis. We tested the hypothesis that leptin induces the shedding of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells, and investigated the intracellular mechanisms leading to microparticle release upon incubation with leptin. METHODS Peripheral blood mononuclear cells were isolated from healthy donors. Cells were incubated with leptin in the presence or in the absence of a phospholipase C inhibitor, U73122, a calmodulin inhibitor, W-7, and three inhibitors of mitogen activated protein kinases. Microparticle generation was assessed as phosphatidylserine concentration with a prothrombinase assay and by cytofluorimetric analysis. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Intracellular calcium concentration was assessed by a fluorescent probe. RESULTS Leptin increased intracellular calcium mobilization and stimulated the generation of tissue factor-bearing MP by peripheral blood mononuclear cells, as assessed by phosphatidylserine quantification, clotting tests and flow-cytometry. U73122, PD98059 (an extracellular signal-regulated kinase1/2 inhibitor), and W-7, significantly inhibited leptin-induced MP release. SB203580 (a p38 inhibitor), and SP600125 (a c-Jun N-terminal kinase inhibitor) had no effect. CONCLUSION Leptin-induced generation of procoagulant microparticles might represent a link between obesity and atherothrombotic risk. Inhibition of leptin-induced microparticle generation might prove a viable strategy for the reduction of such risk in obese individuals.

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 (1h) 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.

Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae

The tumour-suppressor gene BRCA2 has been demonstrated to be involved in maintenance of genome integrity by affecting DNA double-strand break repair and homologous recombination. Protein-truncating mutations in BRCA2 predispose women to early onset breast and ovarian cancers and account for 15-30% of familial breast cancer risk. In contrast, the human cancer risk due to missense mutations, intronic variants, and in-frame deletions and insertions in the BRCA2 gene, called unclassified variants, has not been determined. Here, we want to define if the yeast Saccharomyces cerevisiae is a good model to study the role of BRCA2 in DNA recombination and repair and to characterise the unclassified BRCA2 missense variants. Therefore, we expressed the wild-type BRCA2 in yeast and determined the effect of BRCA2 on yeast homologous recombination, methyl methanesulphonate (MMS)-induced Rad51 and Rad52 foci and MMS sensitivity. The expression of BRCA2 induces a high increase in both intra- and inter-recombination events and confers a higher MMS resistance as compared with the negative control. This may suggest that BRCA2 gets involved in DNA repair pathways in yeast. Moreover, the expression of BRCA2 did not affect the number of cells carrying Rad51 or Rad52 nuclear foci. Finally, we aimed to investigate if yeast could be reliable system to set up a functional assay to distinguish a mutated protein from a neutral polymorphism. Therefore, we have expressed two neutral (M1915T and A2951T) and one pathogenic variant (G2748D) in yeast and checked the effect on recombination. The neutral M1915T variant increased intra-chromosomal recombination by almost 2-fold and the other neutral A2975T variant increased intra-chromosomal recombination 2.5-fold as compared with the control. On the other end, the pathogenic variant G2748D did not increase intra- and inter-chromosomal recombination in yeast and, consequently, confers a phenotype very different from the wild-type BRCA2. Moreover, we have also evaluated whether the expression of the selected unclassified variants affects homologous recombination in yeast. Results indicated that the expression of the selected BRCA2 variants differentially affects yeast recombination suggesting that yeast could be a very promising genetic system to characterise BRCA2 missense variants.

High glucose potentiates and renin-angiotensin blockade downregulates LPS-induced 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 (ANG) II, the effector arm of the Renin Angiotensin System (RAS). The RAS, in turn, plays a pathophysiological role in diabetes, a proinflammatory state to which elevated glucose, the disease hallmark, contributes by activating key signalling pathways and increasing the cellular content of RAS components. AIMS To evaluate the effect of high glucose concentrations on TF antigen (Ag) expression and procoagulant activity (PCA) in lipopolysaccharide(LPS)-primed human mononuclear cell(MNC)s and to test whether pharmacological RAS blockade modifies that pattern. METHODS LPS-activated MNCs exposed to increasing D-glucose (from 5.5 to 50mM) in absence or presence of aliskiren, a renin inhibitor, zofenopril, an ANG converting enzyme inhibitor, and olmesartan, an ANGII type I receptor blocker. PCA was assessed by one-stage clotting assay and TF antigen expression by ELISA. RESULTS Increasing ambient glucose (range 5.5-50mM) potentiated LPS-induced PCA and TF Ag expression. Aliskiren, zofenopril and olmesartan downregulated those responses but the efficacy of the former decreased by ascending drug concentration while both zofenopril and olmesartan showed an opposite behaviour. TF Ag expression modulation by RAS blockade was stronger in 50 than 5mM ambient glucose. CONCLUSIONS High glucose potentiates the procoagulant action of LPS in human MNCs and RAS blockers downregulate that response possibly as a reflection of the underlying involvement of the system in that mechanism.

Effect of the overexpression of BRCA2 unclassified missense variants on spontaneous homologous recombination in human cells.

Breast Cancer 2 gene (BRCA2) mutation carriers have a 45% chance of developing breast cancer and a 11% risk of developing ovarian cancer by the age of 70. While hundreds of BRCA2-truncating mutations have been associated with an increased cancer risk in carriers, the contribution of unclassified variants (UCVs) to cancer risk remains largely undefined. BRCA2-defective cells show a high degree of chromosome instability. Although a functional assay based on the BRCA2 capability to stimulate DSB-induced homologous recombination (HR) as a way to classify UCVs has been proposed, so far no data are available concerning the effect of BRCA2 UCVs on spontaneous HR. In this study, we proposed a novel functional HR-based assay that determines the effect of the transient overexpression of the BRCA2 variant on spontaneous HR. This assay will help one in the difficult task of classifying UCVs, and it will give more information on how BRCA2 may induce genome instability and on the basic mechanism of BRCA2-induced tumourigenesis. We chose 11 BRCA2 UCVs not previously described or classified in other articles, and distributed along the entire BRCA2-coding region. They are as follows: G173V, D191V, S286P, M927V, T1011R, L1019V, N1878K, S2006R, R2108C, G2353R and V3091I. Basically, because the expression of BRCA2wt and the neutral variants did not increase spontaneous HR, we classified the variants G173V, S286P, M927V, T1011R and L1019V as HR-negative and presumed that they were not pathogenic. The HR-positive variants, D191V, N1878K, S2006R, R2108C, G2353R, and V3091I, which increased HR as much as the cancer-associated variant G2748D, could probably be classified as pathogenic. We observed that all our variants in the C-terminus of the protein behaved differently from the wt, suggesting a role for this protein region in spontaneous HR.

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.

CD18-mediated adhesion is required for the induction of a proinflammatory phenotype in lung epithelial cells by mononuclear cell-derived extracellular vesicles

Abstract Extracellular vesicles are submicron vesicles that upregulate the synthesis of proinflammatory mediators by lung epithelial cells. We investigated whether these structures adhere to lung epithelial cells, and whether adhesion is a prerequisite for their proinflammatory activity. Extracellular vesicles were generated by stimulation of normal human mononuclear cells with the calcium ionophore A23187, and labelled with carboxyfluorescein diacetate succinimidyl ester. Adhesion of vesicles to monolayers of immortalized bronchial epithelial (16HBE) and alveolar (A549) cells was analyzed by fluorescence microscopy. The role of candidate adhesion receptors was evaluated with inhibitory monoclonal antibodies and soluble peptides. The synthesis of proinflammatory mediators was assessed by ELISA. Transmission electron microscopy confirmed the generation of closed vesicles with an approximate size range between 50 and 600 nm. Adhesion of extracellular vesicles to epithelial cells was upregulated upon stimulation of the latter with tumor necrosis factor‐&agr;. Adhesion was blocked by an anti‐CD18 antibody, by peptides containing the sequence RGD and, to a lesser extent, by an antibody to ICAM‐1. The same molecules also blocked the upregulation of the synthesis of interleukin‐8 and monocyte chemotactic protein‐1 induced by extracellular vesicles. CD18‐mediated adhesion of extracellular vesicles is a prerequisite for their proinflammatory activity. HighlightsMonocytic extracellular vesicles upregulate proinflammatory mediators.CD18‐mediated adhesion to target lung epithelial cells is necessary.An RGD‐containing peptide and an anti‐ICAM1 antibody inhibit the phenomenon.

CD18-mediated adhesion is required for lung inflammation induced by mononuclear cell-derived extracellular vesicles

Extracellular vesicles are submicron vesicles that upregulate the synthesis of proinflammatory mediators by lung epithelial cells. We investigated whether these structures adhere to lung epithelial cells, and whether adhesion is a prerequisite for their proinflammatory activity. Extracellular vesicles were generated by stimulation of normal human mononuclear cells with the calcium ionophore A23187, and labelled with carboxyfluorescein diacetate succinimidyl ester. Adhesion of vesicles to monolayers of immortalized bronchial epithelial cells (16HBE) and alveolar cells (A549) was analysed by fluorescence microscopy. The role of candidate adhesion receptors was evaluated with inhibitory monoclonal antibodies and soluble peptides. The synthesis of proinflammatory mediators was assessed by ELISA. Transmission electron microscopy confirmed the generation of closed vesicles with an approximate size range between 50 and 600 nm. Adhesion of extracellular vesicles to epithelial cells was minimal in baseline conditions and was upregulated upon stimulation of the latter with tumour necrosis factor-α. Adhesion was blocked by an anti-CD18 antibody and by peptides containing the sequence RGD. The same molecules also blocked the upregulation of the synthesis of interleukin-8 and monocyte chemotactic protein-1 induced by extracellular vesicles. Summary statement Extracellular vesicles upregulate the synthesis of proinflammatory mediators by lung epithelial cells. CD18-mediated adhesion to target cells is required for this proinflammatory effect and might represent a target for anti-inflammatory therapy.