Catherine Pasquier

Reactive oxygen species activate focal adhesion kinase, paxillin and p130cas tyrosine phosphorylation in endothelial cells.

Reactive oxygen species (ROS), particularly hydroxyl radical (HO*), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 microM) and herbimycin A (0.9 microM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120-130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO*. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 microM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO*, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.

Focal Adhesion Kinase Regulation by Oxidative Stress in Different Cell Types

Focal adhesion kinase (FAK) is a tyrosine kinase ubiquitously expressed in cells. It was initially shown to be the initiator of focal adhesion formation in adherent cells, after its binding to integrins which induce its autophosphorylation. However, it can be also activated by a great variety of other stimuli able to act on different intracellular signaling. Reactive oxygen species (ROS), which have been shown to act as external or internal cell stimuli, induce tyrosine phosphorylation of FAK. Its autophosphorylation is followed by a submembranous localization which is crucial for many of the biological roles of FAK, including cell spreading, cell migration, cell proliferation, and prevention of apoptosis. It plays an important role in development of tumor cells, its regulation could be thus a way of impairing cell proliferation in cancer. We describe in this review the structure, activity, and functions of FAK in different cells and how ROS are able, like other stimuli, to induce its phosphorylation and modification of cell morphology and structure. The link between ROS and FAK activation could explain the role of ROS in mediating cell proliferation, cell migration, or apoptosis.

Anti-proteinase-3 (PR3) antibodies (C-ANCA) recognize various targets on the human umbilical vein endothelial cell (HUVEC) membrane

Numerous studies suggest that C‐ANCA are directly pathogenic in vasculitis by activating leucocytes (oxidative burst, enzyme release, endothelial cytotoxicity, etc.). We and others have shown that C‐ANCA can also directly activate HUVEC, but the precise target on HUVEC is unknown. We show in this study that C‐ANCA recognize various targets on the HUVEC membrane (different from PR3 in our model), leading to secondary cell activation. Polyclonal affinity‐purified C‐ANCA recognized targets on the unfixed endothelial membrane in fluorescent ELISA, flow cytometry, and immunoprecipitation studies. C‐ANCA did not react with Fcγ receptors. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) experiments showed that HUVEC did not express PR3. The targets of polyclonal and monoclonal anti‐PR3 antibodies on the endothelial membrane were not the same. Some epitopes were lost after trypsin–EDTA digestion and formaldehyde fixation of cells, whereas anti‐PR3 targeted unfixed HUVEC. This suggests that anti‐PR3 react with the endothelial membrane and recognize conformational epitopes shared with PR3. Endothelial cells may thus participate in the inflammation associated with Wegeners granulomatosis and contribute to the emergence of clinical manifestations.

Oxidative damage to lysozyme by the hydroxyl radical: comparative effects of scavengers

The hydroxyl radical (OH.) is a highly-damaging reactive oxygen species, given its high reactivity and the consequent generation of secondary free radicals. This study was aimed at determining the qualitative and quantitative aspects of OH. scavenging by pentoxifylline (Ptx, a methylxanthine), uric acid and thymine on the OH.-induced alterations of a protein, lysozyme. Lysozyme was inactivated by OH. with a yield of 6.5 mol OH./mol lysozyme; moreover, SDS-PAGE showed a loss of native lysozyme (14.4 kDa), the presence of dimer and trimer aggregates and characteristic fragmentation. Tryptophan fluorescence was lost before aggregation became detectable in terms of bityrosine formation. Increasing concentrations of OH. scavengers gave increasing protection of lysozyme activity. Although all three compounds scavenge OH. with high rate constants, their effects were different: uric acid and Ptx prevented aggregation and preserved enzyme activity, whereas thymine preserved activity but did not prevent aggregation. These differences appear to be related to the formation of reducing secondary radicals, underlining the importance of this mechanism in the effects of scavengers.

Gamma and Pulse Radiolysis Study of Pentoxifylline, a Methylxanthine

Pentoxifylline (Ptx) is a tri-substituted purine with anti-inflammatory properties which are thought to be due, in part, to oxygen radical scavenging. This paper reports an investigation of the reaction of Ptx with the hydroxyl radical [OH.], superoxide anion, azide radical and hydrogen peroxide generated by pulse and gamma-radiolysis, which was carried out to determine the scavenging properties of Ptx towards oxygen radicals. The superoxide anion, azide radical and hydrogen peroxide did not react with Ptx, whereas OH. reacted rapidly. In gamma-radiolysis, the action of OH. on Ptx at pH 7.4 gave rise to an end-product separated by high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry as C-8-OH-Ptx (yield 0.12 x 10(-6) mol J-1). The reaction of Ptx with OH. after pulse radiolysis at pH 7-7.4 occurred with a rate constant of (7.7 +/- 1.0) x 10(9) mol-1 s-1, forming time-dependent transient radicals. The initial spectrum (2 microseconds after the pulse) showed three maxima (310, 338 and 500 nm). A decrease in the absorbance around 500 nm and an increase around 310 nm reflected a first-order reaction, suggesting a unimolecular rearrangement. It was shown by redox titration that at least two OH-adducts were formed, one with reducing and the other with oxidizing properties. These results suggest that the reducing radical may be (C-8-OH-Ptx)(.).

Mechanism of lysozyme inactivation and degradation by iron

The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.

12 – Redox Regulation of Cell Adhesion Processes

This chapter discusses the role of oxidants and antioxidants in the regulation of cell adhesion processes as well as the molecular mechanisms of such regulation. Cell adhesion processes play a major role in the regulation of immune functions as well as of other vital biological processes, such as embryogenesis, cell growth, differentiation, and wound repair. Cell adhesion, which is a multistep process including rolling, firm attachment, and transmigration of leukocytes, is mediated by several classes of cell adhesion molecules. Cell adhesion molecule expression and adhesive properties of cells are modified greatly in several diseased conditions involving redox imbalances, such as cancer, atherosclerosis, diabetes, chronic inflammation, and ischemiareperfusion injury. Several stimuli such as cytokines, chemokines, and phorbol ester are known to activate the expression and function of cell adhesion molecules. Both these evidences indicate that the overall cell adhesion process is redox regulated—direct activation of cell adhesion processes by oxidants and inhibitory action of antioxidants on cell adhesion molecule expression and function.

24 – Reactive Oxygen Species Increase Neutrophil Adherence to Endothelial Cells and Activate Tyrosine Phosphorylation of Cytoskeleton Proteins

Publisher Summary nThis chapter focuses on the involvement of adhesion molecules in the increased adherence of neutrophils to reactive oxygen species (ROS)-stimulated endothelial cells, on the oxygen species responsible for this adhesion, and on the intracellular-signaling pathway leading to cytoskeleton modification by ROS. Oxidative stress, defined as an increase in the production of ROS, such as superoxide anion (O2·-), hydrogen peroxide (H202), and hydroxyl radical (HO·), has been related to reperfusion injury in the heart and other organs. Several laboratories have shown that reperfusion of ischemic tissues leads to a marked local increase in the number of polymorphonuclear neutrophils (PMN) adhering to endothelial cells. The adherence of PMN to endothelial cells is related to many factors, especially adhesion molecules; the latter include intercellular adhesion molecule (ICAM-1), E-, and P-selectins on the endothelium, and L-selectin, β2 integrins, and carbohydrates such as sialyl-Lewis x on PMN. Endothelial cells also express platelet-activating factor (PAF), which binds to specific receptors on PMN. A large part of ones knowledge on the interaction of the endothelium with PMN is derived from in vitro studies using cultured endothelial cells isolated from human umbilical cord vein (HUVEC) and isolated PMN. Although these models are not strictly identical to in vivo conditions, they have shown that after exposure to histamine or thrombin, endothelial cells express P-selectin (GMP-140) and PAF within minutes.