Cinzia Cordazzo

Role of NF-κB and PPAR-γ in lung inflammation induced by monocyte-derived microparticles

Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferator-activated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-&kgr;B transcriptional activity and inflammation. We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-&kgr;B activation and is modulated by PPAR-&ggr;. MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-&kgr;B translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR. Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-&kgr;B activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-&ggr; agonists, rosiglitazone and 15-deoxy-&Dgr;12,14-prostaglandin-J2. Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-&ggr; agonists were reversed by the specific antagonist, GW9662. Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-&kgr;B activation through a PPAR-&ggr; dependent pathway.

Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca2+ mobilization

ObjectivesMicroparticles are membrane vesicles shed by cells upon activation and apoptosis. Agonists capable of inducing microparticle generation include cytokines, bacterial products, P-selectin, histamine. Cigarette smoke extract has also been recognized as an agonist involved in microparticle generation with an apoptosis-dependent mechanism. We investigated the possibility that cigarette smoke extract induces the rapid generation of proinflammatory microparticles by human mononuclear cells with a calcium-dependent mechanism.Materials and methodsHuman mononuclear cells were exposed to cigarette smoke extract. [Ca2+]i mobilization was assessed with the fluorescent probe Fluo-4 NW. Microparticles were quantified with a prothrombinase assay and by flow cytometry. Normal human bronchial epithelial cells and A549 alveolar cells were incubated with cigarette smoke extract-induced microparticles and the generation of ICAM-1, IL-8, and MCP-1 was assessed by ELISA.ResultsExposure to cigarette smoke extract induced a rapid increase in [Ca2+]i mobilization. Microparticle generation was also increased. EGTA, verapamil and the calmodulin inhibitor, W-7, inhibited microparticle generation. Incubation of lung epithelial cells with cigarette smoke extract-induced microparticles increased the expression of proinflammatory mediators.ConclusionsExposure of mononuclear cells to cigarette smoke extract causes a rapid shedding of microparticles with a proinflammatory potential that might add to the mechanisms of disease from tobacco use.

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.

The mechanisms of nadroparin-mediated inhibition of proliferation of two human lung cancer cell lines.

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.

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.

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.