Differential regulation of endothelial cell permeability by high and low doses of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine.

The generation of phospholipid oxidation products in atherosclerosis, sepsis, and lung pathologies affects endothelial barrier function, which exerts significant consequences on disease outcomes in general. Our group previously showed that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (OxPAPC) at low concentrations increases endothelial cell (EC) barrier function, but decreases it at higher concentrations. In this study, we determined the mechanisms responsible for the pulmonary endothelial cell barrier dysfunction induced by high OxPAPC concentrations. OxPAPC at a range of 5-20 µg/ml enhanced EC barriers, as indicated by increased transendothelial electrical resistance. In contrast, higher OxPAPC concentrations (50-100 µg/ml) rapidly increased EC permeability, which was accompanied by increased total cell protein tyrosine (Tyr) phosphorylation, phosphorylation at Tyr-418, the activation of Src kinase, and the phosphorylation of adherens junction (AJ) protein vascular endothelial cadherin (VE-cadherin) at Tyr-731 and Tyr-658, which was not observed in ECs stimulated with low OxPAPC doses. The early tyrosine phosphorylation of VE-cadherin was linked to the dissociation of VE-cadherin-p120-catenin/ß-catenin complexes and VE-cadherin internalization, whereas low OxPAPC doses promoted the formation of VE-cadherin-p120-catenin/ß-catenin complexes. High but not low doses of OxPAPC increased the production of reactive oxygen species (ROS) and protein oxidation. The inhibition of Src by PP2 and ROS production by N-acetyl cysteine inhibited the disassembly of VE-cadherin-p120-catenin complexes, and attenuated high OxPAPC-induced EC barrier disruption. These results show the differential effects of OxPAPC doses on VE-cadherin-p120-catenin complex assembly and EC barrier function. These data suggest that the rapid tyrosine phosphorylation of VE-cadherin and other potential targets mediated by Src and ROS-dependent mechanisms plays a key role in the dissociation of AJ complexes and EC barrier dysfunction induced by high OxPAPC doses.

Lipoxin A4 counterregulates GM-CSF signaling in eosinophilic granulocytes.

Eosinophils are granulated leukocytes that are involved in many inflammation-associated pathologies including airway inflammation in asthma. Resolution of eosinophilic inflammation and return to homeostasis is in part due to endogenous chemical mediators, for example, lipoxins, resolvins, and protectins. Lipoxins are endogenous eicosanoids that demonstrate antiinflammatory activity and are synthesized locally at sites of inflammation. In view of the importance of lipoxins (LXs) in resolving inflammation, we investigated the molecular basis of LXA(4) action on eosinophilic granulocytes stimulated with GM-CSF employing the eosinophilic leukemia cell line EoL-1 as well as peripheral blood eosinophils. We report herein that LXA(4) (1-100 nM) decreased protein tyrosine phosphorylation in EoL-1 cells stimulated with GM-CSF. Additionally, the expression of a number of GM-CSF-induced cytokines was inhibited by LXA(4) in a dose-dependent manner. Furthermore, using a proteomics approach involving mass spectrometry and immunoblot analysis we identified 11 proteins that were tyrosine phosphorylated after GM-CSF stimulation and whose phosphorylation was significantly inhibited by LXA(4) pretreatment. Included among these 11 proteins were alpha-fodrin (nonerythroid spectrin) and actin. Microscopic imaging showed that treatment of EoL-1 cells or blood eosinophils with GM-CSF resulted in the reorganization of actin and the translocation of alpha-fodrin from the cytoplasm to the plasma membrane. Importantly, alpha-fodrin translocation was prevented by LXA(4) but actin reorganization was not. Thus, the mechanism of LXA(4) action likely involves prevention of activation of eosinophilic granulocytes by GM-CSF through inhibition of protein tyrosine phosphorylation and modification of some cytoskeletal components.

Bronchoalveolar pepsin, bile acids, oxidation, and inflammation in children with gastroesophageal reflux disease.

INTRODUCTION: Gastroesophageal reflux has been suggested as an underlying cause of chronic lung disease. The aim of this study was to assess the value of pepsin and bile acids, both components of GI secretions, in the lungs of children with chronic lung diseases as possible markers for gastroesophageal reflux disease and their relation to oxidation and inflammation. MATERIALS AND METHODS: BAL was performed in 96 children with different chronic lung diseases. Gastroesophageal reflux was analyzed by two-channel, 24-h esophageal pH measurements. Lung pepsin and bile acids were measured in BAL enzymatically, interleukin (IL)-8 by enzyme-linked immunosorbent assay, and protein carbonyls by slot blot immunoassay. RESULTS: Sixty-five of the 96 children (68%) had an extensive proximal acidic reflux index. Children with reflux had higher pepsin concentrations in their BAL fluid (BALF), compared to children without reflux despite low specificity. No differences were observed for bile acids. Percentages of neutrophils, levels of protein carbonyls, and levels of IL-8 in BALF correlated with the number of proximal reflux events. CONCLUSIONS: Pulmonary microaspiration as demonstrated by pepsin detection in BALF is common in children with chronic lung diseases, suggesting that gastroesophageal reflux may contribute significantly to the disease pathogenesis. BALF pepsin concentration correlates positively with the number of proximal reflux events. Protein oxidation in BALF is higher in children with extensive proximal acidic reflux, suggesting that pulmonary microaspirations contribute to lung damage.

Oxidative changes of bronchoalveolar proteins in cystic fibrosis.

Chronic bacterial infection and severe, polymorphonuclear neutrophil-dominated endobronchial inflammation are characteristic hallmarks of cystic fibrosis (CF) lung disease. The free radicals generated can be deleterious for structure and function of many proteins. The goal of this study was to investigate the degree of oxidation of pulmonary epithelial lining fluid proteins. BAL fluid (BALF) from 55 children with CF and from 11 patients in a control group were investigated by dot-blot assay for content and by two-dimensional electrophoresis and Western blotting for the pattern of distribution of oxidized proteins. The highest level of oxidative stress, as assessed by the level of protein carbonyls, was found in patients with FEV1 < 80% of predicted or with highly elevated neutrophil counts. Compared to control subjects without lung disease, CF patients with normal lung function and CF patients with a normal neutrophil count in their BALF had significantly higher protein carbonyl levels. The extent of protein oxidation was directly related to the neutrophil granulocyte count and inversely to lung function. Our data support the hypothesis that oxidative damage of pulmonary proteins during chronic and excessive neutrophilic endobronchial inflammation may contribute to the decline of lung function in CF patients.

Oxidative damage to surfactant protein D in pulmonary diseases.

Surfactant protein D is an important innate host defence molecule that has been shown to interact with a variety of pathogens and to play a role in surfactant homeostasis. The aim of this study was to examine the influence of oxidation on surfactant protein D in different lung diseases. Bronchoalveolar lavage fluids (BALFs) from patients with different grade of protein oxidation were examined for changes in the primary chain and the quaternary structure of surfactant protein D. Significant changes of quaternary surfactant protein-D (SP-D) structure were detected under oxidative conditions in vitro and in vivo. The functional capacity of surfactant protein D to agglutinate bacteria was impaired by oxidation. We conclude that surfactant protein D is an important target of free radicals generated in the lungs. Host defence may be impaired due to the oxidation of surfactant protein D and may contribute to the suppurative lung diseases like cystic fibrosis (CF).

Agglutination of Pseudomonas aeruginosa by surfactant protein D.

Surfactant protein D (SP-D) is part of the innate host defense system, and may bind and agglutinate invading microorganisms to enhance their removal. The ability of bronchoalveolar lavage (BAL) fluid to agglutinate bacteria and the relationship to its SP-D content are of interest and not yet known. A micromethod on slides was used to assess the agglutination of Pseudomonas aeruginosa by recombinant SP-D and native human BAL fluid. The SP-D-induced agglutination was blocked by calcium depletion, alkaline pH, or the presence of maltose. Twenty-three of 30 BAL fluids from outpatients carrying a chronic tracheostoma clearly agglutinated P. aeruginosa, which was completely inhibited by maltose. The extent of the agglutination correlated weakly to the concentration of SP-D in the BAL fluid, but not to that of SP-A. The functional property, i.e., the agglutination of P. aeruginosa by BAL fluid, was characterized and appeared related in part to the concentration of SP-D. Additional factors, such as the multimeric organization of SP-D, are likely to contribute to the agglutination of microorganisms by BAL or other body fluids. The assay presented will allow the systematic evaluation of small-volume samples for SP-D agglutinating ability from subjects with various lung diseases.

Fragmented oxidation products define barrier disruptive endothelial cell response to OxPAPC.

Excessive concentrations of oxidized phospholipids (OxPL), the products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (PAPC) oxidation have been detected in atherosclerosis, septic inflammation, and acute lung injury (ALI) and have been shown to induce vascular barrier dysfunction. In contrast, oxidized PAPC (OxPAPC) at low concentrations exhibit potent barrier protective effects. The nature of such biphasic effects remains unclear. We tested the hypothesis that barrier-disruptive effects of high OxPAPC doses on endothelial cell (EC) monolayer are defined by fragmented products of PAPC oxidation (lysophosphatidyl choline [lyso-PC], 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-phosphatidylcholine [POVPC], 1-palmitoyl-2-glutaroyl-sn-glycero-phosphatidylcholine [PGPC]), whereas barrier enhancing effects are mediated by full length oxidated PAPC products and may be reproduced by single compounds contained in the OxPAPC such as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphatidyl choline (PEIPC). All 3 fragmented OxPAPC products increased EC permeability in a dose-dependent manner, whereas PEIPC decreased it and reversed barrier disruptive effects of lyso-PC, POVPC, and PGPC monitored by measurements of transendothelial electrical resistance. Immunofluorescence staining and western blot analysis showed that PGPC mimicked the cytoskeletal remodeling and tyrosine phosphorylation of adherens junction (AJ) protein vascular endothelial (VE)-cadherin leading to EC barrier dysfunction induced by high OxPAPC concentrations. Barrier-disruptive effects of PGPC were abrogated by reactive oxygen species (ROS) inhibitor, N-acetyl cysteine, or Src kinase inhibitor, PP-2. The results of this study show that barrier disruptive effects of fragmented OxPAPC constituents (lyso-PC, POVPC, PGPC) are balanced by barrier enhancing effects of full length oxygenated products (PEIPC). These data strongly suggest that barrier disruptive effects of OxPAPC at higher concentrations are dictated by predominant effects of fragmented phospholipids such as PGPC, which promote ROS-dependent activation of Src kinase and VE-cadherin phosphorylation at Tyr(658) and Tyr(731) leading to disruption of endothelial cell AJs.

A role for VEGFR2 activation in endothelial responses caused by barrier disruptive OxPAPC concentrations.

INTRODUCTION: Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion. Vascular endothelial growth factor receptor-2 (VEGFR2) is involved in the OxPAPC-induced EC inflammatory activation. This study examined a role of VEGFR2 in barrier dysfunction caused by high concentrations of OxPAPC and evaluated downstream signaling mechanisms resulting from the effect of OxPAPC in EC from pulmonary and systemic circulation. METHODS: EC monolayer permeability in human pulmonary artery endothelial cells (HPAEC) and human aortic endothelial cells (HAEC) was monitored by changes in transendothelial electrical resistance (TER) across EC monolayers. Actin cytoskeleton was examined by immunostaining with Texas Red labeled phalloidin. Phosphorylation of myosin light chains (MLC) and VE-Cadherin was examined by Western blot and immunofluorescence techniques. The role of VEGFR2 in OxPAPC-induced permeability and cytoskeletal arrangement were determined using siRNA-induced VEGFR2 knockdown. RESULTS: Low OxPAPC concentrations (5-20 µg/ml) induced a barrier protective response in both HPAEC and HAEC, while high OxPAPC concentrations (50-100 µg/ml) caused a rapid increase in permeability; actin stress fiber formation and increased MLC phosphorylation were observed as early as 30 min after treatment. VEGFR2 knockdown dramatically decreased the amount of MLC phosphorylation and stress fiber formation caused by high OxPAPC concentrations with modest effects on the amount of VE-cadherin phosphorylation at Y(731). We present evidence that activation of Rho is involved in the OxPAPC/VEGFR2 mechanism of EC permeability induced by high OxPAPC concentrations. Knockdown of VEGFR2 did not rescue the early drop in TER but prevented further development of OxPAPC-induced barrier dysfunction. CONCLUSIONS: This study shows that VEGFR2 is involved in the delayed phase of EC barrier dysfunction caused by high OxPAPC concentrations and contributes to stress fiber formation and increased MLC phosphorylation.

Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation.

Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRbeta with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRbeta. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-alpha. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.

Improvement of alveolar glutathione and lung function but not oxidative state in cystic fibrosis.

Chronic neutrophilic inflammation leads to oxidative damage, which may play an important role in the pathogenesis of cystic fibrosis lung disease. Bronchoalveolar lavage levels of the antioxidant glutathione are diminished in patients with cystic fibrosis. Here we evaluated the effects of glutathione aerosol on lower airway glutathione levels, lung function, and oxidative status. Pulmonary deposition of a radiolabeled monodisperse aerosol generated with a Pari LC Star nebulizer (Allergy Asthma Technology, Morton Grove, IL) connected to an AKITA inhalation device (Inamed, Gauting, Germany) was determined in six patients. In 17 additional patients bronchoalveolar lavage fluid was assessed before and after 14 days of inhalation with thrice-daily doses of 300 or 450 mg of glutathione. Intrathoracic deposition was 86.3 +/- 1.4% of the emitted dose. Glutathione concentration in lavage 1 hour postinhalation was increased three- to fourfold and was still almost doubled 12 hours postinhalation. FEV(1) transiently dropped after inhalation but increased compared with pretreatment values after 14 days (p < 0.001). This improvement was not related to the lavage content of oxidized proteins and lipids, which did not change with treatment. These results show that, using a new inhalation device with high efficacy, glutathione treatment of the lower airways is feasible. Reversion of markers of oxidative injury may need longer treatment, higher doses, or different types of antioxidants.