Phospholipase A2 Activation by Poultry Particulate Matter is Mediated Through Extracellular Signal-Regulated Kinase in Lung Epithelial Cells: Regulation of Interleukin-8 Release

The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A₂ (cPLA₂) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA₂ in A549 cells, which was attenuated by AACOCF₃ (cPLA₂ inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA₂ serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA₂. The poultry PM-induced cPLA₂ serine phosphorylation and IL-8 release were attenuated by AACOCF₃, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA₂ knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA₂ activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.     

Regulation of hyperoxia-induced NADPH oxidase activation in human lung endothelial cells by the actin cytoskeleton and cortactin

Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47(phox), a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47(phox) to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47(phox). In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells.     

Prostaglandin E2 Induces Oncostatin M Expression in Human Chronic Wound Macrophages through Axl Receptor Tyrosine Kinase Pathway

Monocytes and macrophages (m) are plastic cells whose functions are governed by microenvironmental cues. Wound fluid bathing the wound tissue reflects the wound microenvironment. Current literature on wound inflammation is primarily based on the study of blood monocyte-derived macrophages, cells that have never been exposed to the wound microenvironment. We sought to compare pair-matched monocyte-derived macrophages with m isolated from chronic wounds of patients. Oncostatin M (OSM) was differentially overexpressed in pair-matched wound m. Both PGE(2) and its metabolite 13,14-dihydro-15-keto-PGE(2) (PGE-M) were abundant in wound fluid and induced OSM in wound-site m. Consistently, induction of OSM mRNA was observed in m isolated from PGE(2)-enriched polyvinyl alcohol sponges implanted in murine wounds. Treatment of human THP-1 cell-derived m with PGE(2) or PGE-M caused dose-dependent induction of OSM. Characterization of the signal transduction pathways demonstrated the involvement of EP4 receptor and cAMP signaling. In human m, PGE(2) phosphorylated Axl, a receptor tyrosine kinase (RTK). Axl phosphorylation was also induced by a cAMP analogue demonstrating interplay between the cAMP and RTK pathways. PGE(2)-dependent Axl phosphorylation led to AP-1 transactivation, which is directly implicated in inducible expression of OSM. Treatment of human m or mice excisional wounds with recombinant OSM resulted in an anti-inflammatory response as manifested by attenuated expression of endotoxin-induced TNF-α and IL-1β. OSM treatment also improved wound closure during the early inflammatory phase of healing. In summary, this work recognizes PGE(2) in the wound fluid as a potent inducer of m OSM, a cytokine with an anti-inflammatory role in cutaneous wound healing.     

Eicosanoid Signaling and Vascular Dysfunction: Methylmercury-Induced Phospholipase D Activation in Vascular Endothelial Cells

Mercury, especially methylmercury (MeHg), is implicated in the etiology of cardiovascular diseases. Earlier, we have reported that MeHg induces phospholipase D (PLD) activation through oxidative stress and thiol-redox alteration. Hence, we investigated the mechanism of the MeHg-induced PLD activation through the upstream regulation by phospholipase A₂ (PLA₂) and lipid oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) in the bovine pulmonary artery endothelial cells (BPAECs). Our results showed that MeHg significantly activated both PLA₂ (release of [³H]arachidonic acid, AA) and PLD (formation of [³²P]phosphatidylbutanol) in BPAECs in dose- (0–10 μM) and time-dependent (0–60 min) fashion. The cPLA₂-specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃), significantly attenuated the MeHg-induced [³H]AA release in ECs. MeHg-induced PLD activation was also inhibited by AACOCF₃ and the COX- and LOX-specific inhibitors. MeHg also induced the formation of COX- and LOX-catalyzed eicosanoids in ECs. MeHg-induced cytotoxicity (based on lactate dehydrogenase release) was protected by PLA₂-, COX-, and LOX-specific inhibitors and 1-butanol, the PLD-generated PA quencher. For the first time, our studies showed that MeHg activated PLD in vascular ECs through the upstream action of cPLA₂ and the COX- and LOX-generated eicosanoids. These results offered insights into the mechanism(s) of the MeHg-mediated vascular endothelial cell lipid signaling as an underlying cause of mercury-induced cardiovascular diseases.     

C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.     

Hyperglycemic oxoaldehyde, glyoxal, causes barrier dysfunction, cytoskeletal alterations, and inhibition of angiogenesis in vascular endothelial cells: aminoguanidine protection

Vascular endothelium is vulnerable to the attack of glucose-derived oxoaldehydes (glyoxal and methylglyoxal) during diabetes, through the formation of advanced glycation end products (AGEs). Although aminoguanidine (AG) has been shown to protect against the AGE-induced adverse effects, its protection against the glyoxal-induced alterations in vascular endothelial cells (ECs) such as cytotoxicity, barrier dysfunction, and inhibition of angiogenesis has not been reported and we investigated this in the bovine pulmonary artery ECs (BPAECs). The results showed that glyoxal (1–10 mM) significantly induced cytotoxicity and mitochondrial dysfunction in a dose- and time-dependent (4–12 h) fashion in ECs. Glyoxal was also observed to significantly inhibit EC proliferation. The study also revealed that glyoxal induced EC barrier dysfunction (loss of trans-endothelial electrical resistance), actin cytoskeletal rearrangement, and tight junction alterations in BPAECs. Furthermore, the results revealed that glyoxal significantly inhibited in vitro angiogenesis on the Matrigel. For the first time, this study demonstrated that AG significantly protected against the glyoxal-induced cytotoxicity, barrier dysfunction, cytoskeletal rearrangement, and inhibition of angiogenesis in BPAECs. Therefore, AG appears as a promising protective agent in the treatment of AGE-induced vascular endothelial alterations and dysfunction during diabetes, presumably by blocking the reactivity of the sugar-derived dicarbonyls such as glyoxal and preventing the formation of AGEs.     

Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1x10(3)M(-1)s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.     

Feldspar Tunneling by Fungi along Natural Productivity Gradients

Recently, it was hypothesized that ectomycorrhizas hyphae are involved in mineral tunneling. We evaluated the role of ectomycorrhizas in mineral weathering and the ecosystem influx of basic cations by correlating mineral tunnel density to ectomycorrhizas density in two forest productivity gradients. The gradients, two gentle slopes in northern Sweden, are the result of groundwater movement and are characterized by reduced productivity upslope due to lower nitrogen availability. As expected, ectomycorrhizas density in the O horizon was higher upslope, where nitrogen availability was lower and where the vegetation was dominated by ectomycorrhizas plant species. We consistently found that tunnel formation in mineral grains was more intense in nutrient-poor sites, indicating a higher contribution of fungi to ecosystem influx of potassium and calcium. ectomycorrhizas density was positively correlated with feldspar tunnel density in the upper 2 cm of the E horizon. This suggests that ectomycorrhizas are involved in mineral tunneling. We discuss the possible involvement of ericoid mycorrhizas and saprotrophic fungi in feldspar tunneling and the role of the weathering status of mineral grains as additional factors controlling mineral funneling.