Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p

The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation

Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

<p>Abstract</p> <p>Background</p> <p>Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled <it>in vitro </it>by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.</p> <p>Results</p> <p>Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase.</p> <p>Conclusion</p> <p>Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis.</p

p75(NTR)-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia

The communication between vascular endothelial cells (ECs) and pericytes in the microvasculature is fundamental for vascular growth and homeostasis; however, these processes are disrupted by diabetes. Here we show that modulation of p75NTR expression in ECs exposed to high glucose activates transcription of miR-503, which negatively affects pericyte function. p75NTR activates NF-κB to bind the miR-503 promoter and upregulate miR-503 expression in ECs. NF-κB further induces activation of Rho kinase and shedding of endothelial microparticles carrying miR-503, which transfer miR-503 from ECs to vascular pericytes. The integrin-mediated uptake of miR-503 in the recipient pericytes reduces expression of EFNB2 and VEGFA, resulting in impaired migration and proliferation. We confirm operation of the above mechanisms in mouse models of diabetes, in which EC-derived miR-503 reduces pericyte coverage of capillaries, increased permeability and impaired post-ischaemic angiogenesis in limb muscles. Collectively, our data demonstrate that miR-503 regulates pericyte–endothelial crosstalk in microvascular diabetic complications

Semi-empirical LCAO analysis of the surface states of Cu The (1 1 0) surface

International audienceWe have determined a set of parameters for LCAO calculations of the electronic structure of Cu. These parameters give bands which agree very well with the experiment for the bulk as well as for the surface states of the three lowest Miller index surfaces. The detailed analysis of the results for the (1 1 0) surface is reported in this paper. The criteria used for determining the parameter values are briefly discussed. It is shown that a good fit of the bulk bands is not, in general, sufficient to guarantee good results for the surface states. © 2003 Elsevier Science B.V. All rights reserved

LCAO study of the Cu(110)-p(2 x 1)O surface

We have performed semi-empirical LCAO calculations of the electronic structure of the Cu(1 1 0)-p(2 x 1)O surface. This has been done accounting for the Cu-Cu interactions by means of a recently proposed set of parameters, which give very good results for the bulk as well as for the surfaces of lowest Miller indices. Furthermore, the O-O interactions, which have been neglected in the preceding similar studies, have been taken into account. The resulting surface bands are in very good agreement with the overall set of the available experimental data. Several issues concerning the physical properties of this surface are addressed in the present paper: the changes induced on the clean surface bands by the adsorption and the reconstruction; the arrangement of the Cu and O atoms in the added rows; the position of the p(y) antibonding band of the oxygen. In particular, we have found that the latter has an energy of -0.2 eV at the Y point. This result confirms an experimental indication in the same direction previously reported by Courths et al. [R. Courths, S. Hilfher, P. Kemkes, G. Wiesen, Surf. Sci. 376 (1997) 43

Electronic structure of the Cu (1 1 0) -p (2 × 1) O surface by the semi-empirical LCAO method

We have performed LCAO semi-empirical calculations for the Cu(110)-p(2 × 1)O surface. The Cu substrate has been described by means of a recently proposed set of parameters giving excellent results for the bulk bands as well as for the surfaces states of the clean surfaces. The O-Cu and the O-O interactions have been accounted for by parameters determined by fitting the available photoemission and inverse photoemission experimental data. The O-O interactions (which were neglected in preceding LCAO studies) have an important role and contribute to fix the position of the "third" antibonding oxygen induced band. Our calculations localize this band 0.2 eV under the Fermi level at the Ȳ point. The position of this band has not yet firmly established experimentally. However, some indications of its presence 0.18 eV under the Fermi level at the Ȳ point have been given by Courths et al. [Surf. Sci. 376 (1997) 43]. Other issues are also discussed, such as the displacement above the Fermi level of the surface state localized 0.4 eV below EF on the clean surface, or the relative position of the Cu and O atoms in the added chains. © 2004 Elsevier B.V. All rights reserved

Dynamique des phases adsorbées et condensées dans les corps micro-poreux

The dynamics of adsorbed and condensed phases in micro-porous materials. Two effects occurring at a solid-gas interface are the permanent condensation and evaporation of the molecules, and the movement of molecules at the surface of the solid due to thermal agitation. In order to study the second of these effects, it is necessary to induce a superficial flow by means of a temperature difference, or of a vapour-condensate equilibrium pressure difference. In order to bring the two-dimensional flow to within measuring range, the gas flow must be limited. This can only he done by using a homogeneously porous permeable solid with a large specific surface area. The rate of flow in the gaseous phase can be calculated if the texture of the solid has been studied beforehand with the aid of permanent gases. It is then possible to determine experimentally the part played by a flow in an adsorbed phase of the form Gs = f (T, [dc/dx], c, t).L'interface solide-gaz est le siège de deux phénomènes, la condensation et l'évaporation permanentes des molécules d'une part, le déplacement des molécules par agitation thermique à la surface du solide d'autre part. Pour étudier le deuxième phénomène, il est nécessaire de créer un écoulement superficiel sous l'effet soit d'une différence de température, soit d'une différence de pression d'équilibre vapeur-condensat. Pour que l'écoulement à deux dimensions soit mesurable, il est indispensable de limiter l'écoulement gazeux. La seule méthode consiste à mettre en oeuvre un solide perméable à porosité homogène, de grande surface spécifique. Le débit en phase gazeuse peut être calculé si la texture du solide a été étudiée au préalable au moyen de gaz permanents. Il devient possible de déterminer expérimentalement la participation d'un écoulement en phase adsorbée de la forme : Gg=f (T, dc/dx,c,t)Eyraud C., Sapet J. C., Eyraud P., Daneyrolle C., Corbet F. Dynamique des phases adsorbées et condensées dans les corps micro-poreux. In: Bulles et gouttes. La tension superficielle en hydraulique. Compte rendu des septièmes journées de l'hydraulique. Paris, 4, 5 et 6 juin 1962. Tome 2, 1963