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Essentials Monocytes (Mo) transdifferentiate into endothelial cell-like (ECL) cells. Mo induce tissue factor (TF) expression and secretion in microvascular endothelial cells (mECs). TF interacts with Mo in a paracrine fashion, inducing their transdifferentiation into ECL cells. TF generates a positive feedback crosstalk between Mo and mECs that promotes angiogenesis. SUMMARY: Background Monocytes (Mo) increase neovascularization by releasing proangiogenic mediators and/or transdifferentiating into endothelial cell-like (ECL) cells. Recently, we have reported that Mo-microvascular endothelial cells (mECs) crosstalk induces mEC-tissue factor (TF) expression and promotes angiogenesis. However, the effect of TF on Mo remains unknown. Objective Here, we analyzed whether TF might exert angiogenic effects by inducing transdifferentiation of Mo. Methods Full-length TF (flTF) and alternatively spliced TF (asTF) were overexpressed in mECs, and their supernatants were added to Mo cultures. CD16 positivity and expression of vascular endothelial cell (VEC) markers in Mo were analyzed by fluorescence activated cell sorting. The capacity to form tube-like structures were visualized in three-dimensional cultures. Results In mECs flTF and asTF expression and release were increased in cultures with Mo-conditioned media. TF variants induced expansion of a CD16+ Mo subset and Mo transdifferentiation into ECL-cells expressing VEC markers that can form new microvessels. CD16+ Mo exposed to TF showed an increased expression of VE-cadherin, von Willebrand factor (VWF) and eNOS. Mo cultured with supernatants obtained from TF-silenced mECs did not transdifferentiate to ECL-cells or expressed VEC markers. Blocking ß1-integrin in Mo significantly blocked the effects of the TF variants. Conclusions Mo induce mECs to express and release TF, which drives CD16- Mo to transform into CD16+ Mo and to transdifferentiate into ECL-cells that can form new microvessels. Our results reveal a TF-mediated positive feedback between mECs and Mo that stimulates Mo differentiation and induces angiogenesis.

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BACKGROUND: Tissue factor (TF) is the most relevant physiological trigger of thrombosis contributing to the presentation of clinical ischemic events after plaque rupture. However, the role of human vascular smooth muscle cell (HVSMC) TF in vascular remodeling, restenosis and atherosclerosis is less known. We have hypothesized that TF contributes to atherosclerotic lesion formation, triggering smooth muscle cell migration through a specific yet unknown signaling pathway. OBJECTIVES: The aim of this study has been to investigate the signal transduction mechanism by which TF may contribute to the transition of resident static contractile HVSMC into a migrating cell that promotes atherosclerotic plaque progression. METHODS: We have used a system biology discovery approach with gene-engineered HVSMCs to identify genes/proteins involved in the TF-triggered effects in HVSMC obtained from the coronary arteries of human adult hearts. RESULTS: Analysis of wild-type HVSMC (TF(+) ) and TF(-) silenced HVSMC (TF(-) ) showed that TF is involved in the regulation of Wnt signaling and in the expression of downstream proteins that affect the atherosclerotic process. CONCLUSIONS: The 'in silico' analysis pointed to specific Wnt-pathway proteins that have been validated in cell culture and also have been found expressed in human advanced atherosclerotic plaques but not in early lesions. TF signals through Wnt to regulate coronary smooth muscle cell migration and vascular remodeling.

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BACKGROUND: Tissue factor (TF) is the most relevant physiological trigger of thrombosis. Additionally TF is a transmembrane receptor with cell signaling functions. OBJECTIVES: The aim of this study was to investigate TF subcellular localization, function and signaling in human coronary artery smooth muscle cell migration. METHODS: Coronary arteries and primary cultures of vascular smooth muscle cells (HVSMC) were obtained from human explanted hearts. Wound repair and Boyden chamber assays were used to measure migration in vitro. TF-pro-coagulant activity (TF-PCA) was measured in extracted cellular membranes. Analysis of TF distribution was performed by confocal microscopy. A nucleofector device was used for TF and protease activated receptor 2 (PAR2) silencing. mRNA levels were analyzed by RT-PCR. RESULTS: In migrating HVSMC TF translocates to the leading edge of the cells showing an intense patch-like staining in the lamellipodia. In the migrating front TF colocalizes with filamin (FLN) in the polarized lipid rafts. TF-PCA was increased in migrating cells. Silencing of the TF gene inhibits RSK-induced FLN-Ser-2152 phosphorylation, down-regulates CDC42, RhoA, and Rac1 protein expression and significantly inhibits cell migration. Silencing PAR2 also inhibits cell migration; however, silencing both TF and PAR2 induces a significantly higher effect on migration. Smooth muscle cells expressing TF have been identified in non-lipid-rich human coronary artery atherosclerotic plaques. CONCLUSIONS: TF translocates to the cell front in association with cytoskeleton proteins and regulates HVSMC migration by mechanisms dependent and independent of factor (F)VIIa/PAR2. These results extend the functional role of TF to smooth muscle cell trafficking in vessel wall remodeling.

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BACKGROUND: Tissue factor (TF) and its signaling mediators play a crucial role in angiogenesis. We have previously shown that TF-induced endothelial cell (EC) CCL2 release contributes to neovessel formation. OBJECTIVE: In this study, we have investigated the signaling pathways involved in TF-induced EC tube formation. METHODS: The human microvascular endothelial cell line (HMEC-1) cultured onto basement membrane-like gel (Matrigel) was used to study TF signaling pathways during neovessels formation. RESULTS: Inhibition of endogenous TF expression in ECs using siRNA resulted in inhibition of a stable tube-like structure formation in three-dimensional cultures, associated with a down-regulation of Akt activation, an increased phosphorylation of Raf at Ser(259) with a subsequent reduction of Raf kinase and a reduction of ERK1/2 phosphorylation ending up in Ets-1 transcription factor inhibition. Conversely, overexpression of TF resulted in an increase in tube formation and up-regulation of Akt protein. Moreover, immunoprecipitation of Akt and western blotting of the immunoprecipitates with anti-TF antibody revealed a direct interaction between TF and Akt. The effects of silencing TF were partially reversed by a PAR2 agonist that rescued tube formation, indicating that the TF-Akt pathway induces PAR2-independent effector signaling. Finally, enforced expression of Akt in TF-silenced ECs rescued tube formation in a Matrigel assay and induced Ets-1 phosphorylation. CONCLUSIONS: In EC, TF forms a complex with Akt activating Raf/ERK and Ets-1 signaling induces microvessel formation.

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BACKGROUND: The ability of nitrous compounds to donate nitric oxide (NO), an agent with vasodilating and inhibitory effects on platelet function, has been considered a useful pharmacologic strategy for cardiovascular complications. The purpose of this study was to investigate the effects of a new NO donor, LA419, on platelet interaction in an ex vivo model with human blood circulating through collagen-rich surfaces. MATERIALS AND METHODS: Platelet adhesive and cohesive function were analyzed by morphometric procedures after perfusion techniques. Treated blood was exposed to thrombogenic surfaces and platelet interactions were morphometrically evaluated. RESULTS: All the concentrations studied of LA419 (10 microM, 20 microM and 100 microM) reduced overall platelet interaction with a collagen surface (27.19 +/- 4.72; 25.52 +/- 3.52; and 23.44 +/- 3.01, P < 0.05, respectively, vs. 32.31 +/- 1.61% in the control). The antithrombotic effect was confirmed by results in cross-sectional studies performed in arterial vessels exposed to circulating blood. Values of thrombus and covered surface at 20 microM LA419 were, respectively, 13.67 +/- 4.97% and 19.01 +/- 5.89%; respect to controls 34.80 +/- 5.29% and 37.93 +/- 5.34% (P < 0.05). Moreover, LA419 reduced significantly thrombus area (88.45 +/- 21.97 microm(2); P < 0.05) with respect to controls (168.45 +/- 21.97 microm(2)) and thrombus height, from an average of 10.27 +/- 1.05 microm in nontreated blood to 7.16 +/- 0.6 microm in treated samples (P < 0.05). CONCLUSION: From the present data we can conclude that LA419 possesses a strong antiplatelet action, as demonstrated by its ability to significantly inhibit the interaction of platelet with highly thrombogenic collagen surfaces.

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BACKGROUND: Cyclooxygenase (COX)-2-selective non-steroidal anti-inflammatory drugs have been used for anti-inflammatory therapy. However, it has also been described that they may increase risk of cardiovascular events. OBJECTIVES: To study the effects of COX2 inhibitor rofecoxib on platelet function using in vitro tests. Results were compared with those obtained in a parallel experiment with acetyl salicylic acid (ASA). METHODS: Studies of platelet aggregation, using different agonists, were performed by a turbidimetric method. Adhesive and cohesive function of platelets were analyzed by perfusion techniques, treated blood was exposed to thrombogenic surfaces and platelet interaction was morphometrically evaluated. RESULTS: Twenty-five micro M of rofecoxib induced a prolonged lag time and a reduction in the percentage of aggregation when arachidonic acid, ADP or collagen were used as agonists. In perfusion studies with parallel chamber rofecoxib 50 microM and ASA 500 microM reduced overall platelet interaction with the collagen surface (17.4 +/- 3.7, P < 0.05; vs. 32.1 +/- 2.6%P < 0.05 and 17.9 +/- 2.4, vs. 31.9 +/- 3.24, P < 0.05, respectively). In studies performed on annular chambers, 25 micro M of rofecoxib reduced platelet interaction; values of the thrombus and covered surface were 17.4 +/- 4.5%; P < 0.05 and 21.1 +/- 4.1%; P < 0.05, respectively, vs. 30.4 +/- 7.5% and 33.5 +/- 6.5 in the control. ASA did also impair thrombus formation but differences did not reach the levels of statistical significance. Moreover, rofecoxib but not ASA reduced significantly thrombus height and thrombus area (7.4 +/- 0.5 microM; P < 0.005 and 96.0 +/- 21.2 microM(2); P < 0.05 vs. control 11.2 +/- 0.9 microM and 220.0 +/- 47.7 microM(2), respectively). CONCLUSION: We conclude that under our experimental conditions, rofecoxib diminished platelet aggregation induced by different agonists and inhibited platelet-mediated thrombogenesis in an in vitro model of thrombosis.

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BACKGROUND: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. OBJECTIVE: To investigate signaling through tyrosine-phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. METHODS: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL(-1)) and type I collagen (Col-I; 20 microg mL(-1)), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col-I-coated coverslips, using citrated whole blood samples perfused through a parallel-plate chamber. RESULTS AND CONCLUSIONS: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.

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Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.

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