Group V secretory phospholipase A2 impairs endothelial protein C receptor-dependent protein C activation and accelerates thrombosis in vivo.

BACKGROUND: Endothelial protein C receptor (EPCR) must be bound to a molecule of phosphatidylcholine (PC) to be fully functional, i.e. to interact with protein C/activated protein C (APC) properly. PC can be replaced with other lipids, such as lysophosphatidylcholine or platelet-activating factor, by the action of group V secretory phospholipase A2 (sPLA2-V), an enzyme that is upregulated in a variety of inflammatory conditions. Studies in purified systems have demonstrated that the substitution of PC notably impairs EPCR function in a process called EPCR encryption. OBJECTIVES: To analyze whether sPLA2-V was able to regulate EPCR-dependent protein C activation in vivo, and its impact on thrombosis and the hemostatic system. METHODS: Mice were transfected with sPLA2-V by hydrodynamic gene delivery. The effects on thrombosis were studied with the laser carotid artery occlusion model, and APC generation capacity was measured with ELISA. Global hemostasis was analyzed with thromboelastometry. RESULTS: We found that sPLA2-V overexpression in mice significantly decreased their ability to generate APC. Furthermore, a murine carotid artery laser thrombosis model revealed that higher sPLA2-V levels were directly associated with faster artery thrombosis. CONCLUSIONS: sPLA2-V plays a thrombogenic role by impairing the ability of EPCR to promote protein C activation.

Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs.

AIMS: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. CONCLUSIONS: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.

Influence of the carbohydrate source on beta-glucan production and enzyme activities involved in sugar metabolism in Pediococcus parvulus 2.6.

The influence of carbohydrate source on growth, exopolysaccharide (EPS) production and on the activity of the enzymes implicated in energy generation and UDP-glucose synthesis in Pediococcus parvulus 2.6 was evaluated. The highest EPS production was obtained on glucose, while fructose was a poor substrate for EPS synthesis. HPLC and NMR analysis on monomer composition and structure of the EPS showed that this strain produced the same beta-glucan, regardless of the carbohydrate source. The alpha-phosphoglucomutase specific activities were dependent on the carbohydrate source and a high correlation between the activity of this enzyme and the amount of EPS was found in glucose- and maltose-grown cultures. alpha-UDP-glucose pyrophosphorylase activity, necessary for the activation of glucose, was very low, but significantly higher on glucose as sugar source. In vitro phosphorylation assays and transport activities showed that glucose is taken up by a proton motive force-dependent permease, while fructose is internalized by an inducible phosphotransferase system, which renders fructose-6-phosphate. The levels of 6-phosphofructokinase activity and alpha-phosphoglucomutase activities determined on fructose were higher and lower, than those found on glucose or maltose, respectively. This suggests that fructose-6-phosphate is mainly diverted to glycolysis and explains the low EPS synthesis on fructose. Results indicate that alpha-phosphoglucomutase and/or alpha-UDP-glucose pyrophosphorylase might be the bottlenecks for EPS biosynthesis, opening the field for metabolic-engineering strategies aimed to improve EPS production.

Xanthine oxidase contributes to lung leak in rats subjected to skin burn.

We found that rats subjected to thermal skin injury (skin burn) had increased serum xanthine oxidase (XO) activities, increased serum complement activation (decreased serum CH50 levels), increased erythrocyte (RBC) fragility, increased lung neutrophil accumulation, and increased lung leak compared to sham-treated rats. Treatment of rats with allopurinol (an XO inhibitor) not only decreased serum XO activity, but also decreased complement activation, RBC fragility, lung neutrophil accumulation, and lung leak abnormalities in rats subjected to skin burn. We conclude that XO may contribute to acute lung injury and a number of events associated with the development of acute lung leak following skin burn.

Local skin burn causes systemic (lung and kidney) endothelial cell injury reflected by increased circulating and decreased tissue factor VIII-related antigen.

Inasmuch as xanthine oxidase (XO)-derived O2* metabolites may contribute to vascular endothelial injury and Factor VIII antigen (F8Ag) is a component of endothelial cells, we hypothesized that XO-derived O2* might damage and cause distant organ endothelial cells to release F8Ag in rats subjected to skin burn. We found that serum F8Ag (ELISA) increased in the blood of rats subjected to skin burn (70 degrees C water to shaved dorsal skin for 30 seconds) but not in sham control rats (30 degrees C water). Coincidentally, F8Ag levels also decreased in lung and kidney tissue sections (immunofluorescent staining) of burned rats but not sham rats. Increases in circulating F8Ag levels and decreases in tissue F8Ag levels appeared to result from XO-derived O2* metabolites: F8Ag levels did not increase in the blood and did not decrease in the tissues of rats pretreated with allopurinol (a specific XO inhibitor, 50 mg/kg) or dimethylthiourea (DMTU) (a permeable O2* metabolite scavenger, 250 mg/kg). Lung injury as assessed by permeability studies (I125-albumin leak) paralleled changes in blood F8Ag levels in sham, burn, allopurinol-, and DMTU-treated groups. We conclude that skin burn causes a systemic vascular injury that can be inhibited by allopurinol or DMTU and is reflected by increased circulating and tissue decreased Factor VIII antigen levels. Release of Factor VIII antigen may serve as a valuable marker of distant organ injury in patients with skin burn.

Xanthine oxidase-derived oxygen radicals induce pulmonary edema via direct endothelial cell injury.

Pulmonary hypoperfusion/ischemia-reperfusion (I/R) may initiate ARDS (nonhydrostatic pulmonary edema). Endothelial damage via xanthine oxidase (XO)-derived oxygen radicals (O2*) may mediate I/R injury. We previously documented Factor VIII antigen (F8) as a marker for endothelial injury. The purpose of this study was to (1) document I/R-induced nonhydrostatic pulmonary edema, (2) identify whether XO or O2* mediates nonhydrostatic edema, and (3) identify the site of injury (? endothelium). Rat lungs were isolated, ventilated, and perfused (100 min, control, or 40 min at 37 degrees C, I (static vent.), + 60 min, R). Effluent was analyzed for F8 release (ELISA: data relative to control). Tungsten-fed rats had negligible lung XO vs rats fed standard diet (3.6 vs 34.5 mU/g, (P less than 0.05). Catalase (CAT) 50 micrograms/ml) was added to perfusate prior to R. Sectioned lungs were fluorescein anti-F8 photographed (IF) and qualitatively assessed. (Table: see text). We conclude that (1) pulmonary hypoperfusion (I/R) leads to nonhydrostatic pulmonary edema, and (2) the edema results in part from XO-generated O2* directed at the capillary endothelium.

Hydrogen peroxide mediates reperfusion injury in the isolated rat heart.

In an isolated, normothermic rat heart model (Langendorff, 37 degrees C), dimethylthiourea (DMTU) infusion only during reperfusion reduced both injury and measurable hydrogen peroxide (H2O2) concentrations after global ischemia. Cardiac function was assessed by measurement of ventricular developed pressure (DP). H2O2 was assessed using H2O2 dependent aminotriazole inactivation of myocardial catalase. Depletion of xanthine oxidase by two methods (tungsten or allopurinol inhibition) also improved recovery of function and H2O2 production. The results indicate that XO derived H2O2 contributes to myocardial reperfusion injury.

Xanthine oxidase produces hydrogen peroxide which contributes to reperfusion injury of ischemic, isolated, perfused rat hearts.

Three lines of investigation indicated that hydrogen peroxide (H2O2) from xanthine oxidase (XO) contributes to cardiac dysfunction during reperfusion after ischemia. First, addition of dimethylthiourea (DMTU), a highly permeant O2 metabolite scavenger (but not urea) simultaneously with reperfusion improved recovery of ventricular function as assessed by ventricular developed pressure (DP), contractility (+dP/dt), and relaxation rate (-dP/dt) in isolated Krebs-Henseleit-perfused rat hearts subjected to global normothermic ischemia. Second, hearts from rats fed tungsten or treated with allopurinol had negligible XO activities (less than 0.5 mU/g wet myocardium compared with greater than 6.0 mU/g in control hearts) and increased ventricular function after ischemia and reperfusion. Third, myocardial H2O2-dependent inactivation of catalase occurred after reperfusion following ischemia, but not after ischemia without reperfusion or perfusion without ischemia. In contrast, myocardial catalase did not decrease during reperfusion of ischemic hearts treated with DMTU, tungsten, or allopurinol.