scholarly journals Endotoxin-Neutralizing Protein Protects against Endotoxin-Induced Endothelial Barrier Dysfunction

1998 ◽  
Vol 66 (4) ◽  
pp. 1400-1407 ◽  
Author(s):  
Douglas D. Bannerman ◽  
Michael J. Fitzpatrick ◽  
Dell Y. Anderson ◽  
Apurba K. Bhattacharjee ◽  
Thomas J. Novitsky ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Phosphorylation ◽  
Lipid A ◽  
Protein A ◽  
Endothelial Barrier ◽  
Barrier Dysfunction ◽  
Recombinant Peptide ◽  
Gram Negative ◽  
Actin Organization ◽  
Cell Monolayers

ABSTRACT Bacterial lipopolysaccharide induces tyrosine phosphorylation of paxillin, actin reorganization, and opening of the transendothelial paracellular pathway through which macromoles flux. In this study, lipid A was shown to be the bioactive portion of the lipopolysaccharide molecule responsible for changes in endothelial barrier function. We then studied whether endotoxin-neutralizing protein, a recombinant peptide that is derived from Limulus antilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of 14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers. In the presence of serum, a 6-h exposure to lipopolysaccharide (10 ng/ml) increased transendothelial 14C-albumin flux compared to the simultaneous media control. Coadministration of endotoxin-neutralizing protein (≥10 ng/ml) with lipopolysaccharide (10 ng/ml) protected against lipopolysaccharide-induced barrier dysfunction. This protection was dose dependent, conferring total protection at endotoxin-neutralizing protein/lipopolysaccharide ratios of ≥10:1. Similarly, endotoxin-neutralizing protein was capable of blocking the lipopolysaccharide-induced endothelial cell responses that are prerequisite to barrier dysfunction, including tyrosine phosphorylation of paxillin and actin depolymerization. Finally, endotoxin-neutralizing protein cross-protected against lipopolysaccharide derived from diverse gram-negative bacteria. Thus, endotoxin-neutralizing protein offers a novel therapeutic intervention for the vascular endothelial dysfunction of gram-negative sepsis and its attendant endotoxemia.

Abstract 20531: C-Abl Mediated Tyrosine Phosphorylation of Paxillin is Essential for LPS-Induced Endothelial Barrier Dysfunction and Acute Lung Injury

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Panfeng Fu ◽  
Anne E Cress ◽  
Ting Wang ◽  
Joe G Garcia ◽  
Viswanathan Natarajan
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Human Lung ◽  
Adherens Junctions ◽  
Endothelial Barrier ◽  
Barrier Dysfunction ◽  
Endothelial Barrier Dysfunction ◽  
Lps Challenge

Paxillin, a multi-domain scaffold-adapter focal adhesion (FA) protein, plays an important role in facilitating protein networking and efficient signaling transduction. Paxillin is phosphorylated at multiple serine/threonine and tyrosine residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and the acute respiratory distress syndrome (ARDS) remains unclear. In this study, we used paxillin-specific siRNA and site-specific non-phosphorylatable mutants of paxillin to abrogate the function of paxillin, both in vitro and in vivo, to determine its role in the regulation of lung endothelial permeability and ARDS. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (ECs) resulted in paxillin accumulation at focal adhesions, enhanced tyrosine phosphorylation of paxillin at Y31 and Y118, and significant endothelial barrier dysfunction. However no significant changes in Y181 phosphorylation by LPS challenge was observed. Paxillin silencing (siRNA) attenuated LPS-induced endothelial barrier dysfunction and dissociation of VE-cadherin from adherens junctions. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase and not by Src or focal adhesion kinase (FAK) in human lung microvascular ECs. Furthermore, down-regulation of c-Abl (siRNA) significantly reduced LPS-mediated endothelial barrier dysfunction. Transfection of human lung microvascular ECs with paxillin Y31, Y118 and Y31/Y118 mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization at adherens junctions. In vivo, knockdown of paxillin with siRNA in mouse lungs ameliorated LPS-induced pulmonary protein leak and lung inflammation. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

scholarly journals Endotoxin protein: a B-cell mitogen and polyclonal activator of C3H/HeJ lymphocytes.

1976 ◽  
Vol 144 (3) ◽  
pp. 821-827 ◽  
Author(s):  
B M Sultzer ◽  
G W Goodman
Keyword(s):  
B Cell ◽  
Lipid A ◽  
Protein A ◽  
Cell Wall Protein ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Lipopolysaccharide Endotoxin ◽  
Polyclonal Activator ◽  
A Cell ◽  
Cell Mitogen

A cell wall protein that is ordinarily complexed to the lipopolysaccharide endotoxin in gram-negative bacteria has been separated by the use of aqueous phenol. The protein is active as a B-cell mitogen and polyclonal activator of murine lymphocytes including the C3H/HeJ strain which is a nonresponder to lipoplysaccharide or lipid A.

scholarly journals Insulin Sensitivity Is Retained in Mice with Endothelial Loss of Carcinoembryonic Antigen Cell Adhesion Molecule 1

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2093
Author(s):  
Harrison T. Muturi ◽  
Saja S. Khuder ◽  
Hilda E. Ghadieh ◽  
Emily L. Esakov ◽  
Hyelim Noh ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Insulin Sensitivity ◽  
Visceral Obesity ◽  
Matrix Metalloproteases ◽  
Endothelial Barrier ◽  
Barrier Dysfunction ◽  
Insulin Transport ◽  
Metabolic Role ◽  
Normal Glucose ◽  
Cell Adhesion Molecule 1

CEACAM1 regulates endothelial barrier integrity. Because insulin signaling in extrahepatic target tissues is regulated by insulin transport through the endothelium, we aimed at investigating the metabolic role of endothelial CEACAM1. To this end, we generated endothelial cell-specific Ceacam1 null mice (VECadCre+Cc1fl/fl) and carried out their metabolic phenotyping and mechanistic analysis by comparison to littermate controls. Hyperinsulinemic-euglycemic clamp analysis showed intact insulin sensitivity in VECadCre+Cc1fl/fl mice. This was associated with the absence of visceral obesity and lipolysis and normal levels of circulating non-esterified fatty acids, leptin, and adiponectin. Whereas the loss of endothelial Ceacam1 did not affect insulin-stimulated receptor phosphorylation, it reduced IRS-1/Akt/eNOS activation to lower nitric oxide production resulting from limited SHP2 sequestration. It also reduced Shc sequestration to activate NF-κB and increase the transcription of matrix metalloproteases, ultimately inducing plasma IL-6 and TNFα levels. Loss of endothelial Ceacam1 also induced the expression of the anti-inflammatory CEACAM1-4L variant in M2 macrophages in white adipose tissue. Together, this could cause endothelial barrier dysfunction and facilitate insulin transport, sustaining normal glucose homeostasis and retaining fat accumulation in adipocytes. The data assign a significant role for endothelial cell CEACAM1 in maintaining insulin sensitivity in peripheral extrahepatic target tissues.

VEGF stimulates tyrosine phosphorylation of β-catenin and small-pore endothelial barrier dysfunction

1999 ◽  
Vol 277 (5) ◽  
pp. H2038-H2049 ◽  
Author(s):  
Alex W. Cohen ◽  
José M. Carbajal ◽  
Richard C. Schaeffer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Endothelial Barrier ◽  
Vegf Receptor ◽  
Barrier Dysfunction ◽  
Transport Pathways ◽  
Endothelial Barrier Dysfunction ◽  
Herbimycin A ◽  
Selective Permeability ◽  
Small Pore

The purpose of this study was to test the hypothesis that tyrosine phosphorylation signaling events and protein kinase C (PKC) activation mediate vascular endothelial growth factor-A165(VEGF)-induced endothelial cell (EC) proliferation and barrier dysfunction in bovine pulmonary artery EC monolayers. A size-selective permeability assay showed that VEGF stimulated a delayed, prolonged (6–45 h), concentration-dependent (50–200 ng/ml, ∼1–4 nM) increase in the number of predominantly small-“pore” transport pathways (<60 Å) across EC monolayers. The tyrosine kinase inhibitor herbimycin A (HA) and the selective PKC inhibitor bisindolylmaleimide (BIM) prevented this phenomenon. After 6–24 h, VEGF-treated monolayers displayed an HA- and BIM-sensitive reorganization of β-catenin adherens junctions with fingerlike projections and the loss of β-catenin at sites of small paracellular hole formation. HA and BIM prevented the VEGF-induced increase in EC growth. HA blocked the VEGF-induced rapid and prolonged (10 min–45 h) increases in the phosphotyrosine (PY) contents of VEGF receptor 2, phospholipase C-γ1, paxillin, and β-catenin as well as ∼140- and 128- to 117-kDa proteins, whereas BIM inhibited only the tyrosine phosphorylation of β-catenin. These data suggest that VEGF initiates increased EC growth and chronic, small-pore endothelial barrier dysfunction by PY signaling through β-catenin that depends on PKC.

scholarly journals Atrial natriuretic peptide protects against Staphylococcus aureus-induced lung injury and endothelial barrier dysfunction

2011 ◽  
Vol 110 (1) ◽  
pp. 213-224 ◽  
Author(s):  
Junjie Xing ◽  
Nurgul Moldobaeva ◽  
Anna A. Birukova
Keyword(s):  
Staphylococcus Aureus ◽  
Lung Inflammation ◽  
Endothelial Barrier ◽  
Protective Effects ◽  
Barrier Dysfunction ◽  
Gram Positive ◽  
Gram Negative ◽  
Endothelial Barrier Dysfunction

Lung inflammation and alterations in endothelial cell (EC) permeability are key events to development of acute lung injury (ALI). Protective effects of atrial natriuretic peptide (ANP) have been shown against inflammatory signaling and endothelial barrier dysfunction induced by gram-negative bacterial wall liposaccharide. We hypothesized that ANP may possess more general protective effects and attenuate lung inflammation and EC barrier dysfunction by suppressing inflammatory cascades and barrier-disruptive mechanisms shared by gram-negative and gram-positive pathogens. C57BL/6J wild-type or ANP knockout mice (Nppa−/−) were treated with gram-positive bacterial cell wall compounds, Staphylococcus aureus-derived peptidoglycan (PepG) and/or lipoteichoic acid (LTA) (intratracheal, 2.5 mg/kg each), with or without ANP (intravenous, 2 μg/kg). In vitro, human pulmonary EC barrier properties were assessed by morphological analysis of gap formation and measurements of transendothelial electrical resistance. LTA and PepG markedly increased pulmonary EC permeability and activated p38 and ERK1/2 MAP kinases, NF-κB, and Rho/Rho kinase signaling. EC barrier dysfunction was further elevated upon combined LTA and PepG treatment, but abolished by ANP pretreatment. In vivo, LTA and PepG-induced accumulation of protein and cells in the bronchoalveolar lavage fluid, tissue neutrophil infiltration, and increased Evans blue extravasation in the lungs was significantly attenuated by intravenous injection of ANP. Accumulation of bronchoalveolar lavage markers of LTA/PepG-induced lung inflammation and barrier dysfunction was further augmented in ANP−/− mice and attenuated by exogenous ANP injection. These results strongly suggest a protective role of ANP in the in vitro and in vivo models of ALI associated with gram-positive infection. Thus ANP may have important implications in therapeutic strategies aimed at the treatment of sepsis and ALI-induced gram-positive bacterial pathogens.

scholarly journals Asef mediates HGF protective effects against LPS-induced lung injury and endothelial barrier dysfunction

2015 ◽  
Vol 308 (5) ◽  
pp. L452-L463 ◽  
Author(s):  
Fanyong Meng ◽  
Angelo Meliton ◽  
Nurgul Moldobaeva ◽  
Gokhan Mutlu ◽  
Yoshihiro Kawasaki ◽  
...  
Keyword(s):  
Lung Injury ◽  
Adhesion Molecule ◽  
Endothelial Barrier ◽  
Intercellular Adhesion Molecule ◽  
Protective Effects ◽  
Nucleotide Exchange ◽  
Intercellular Adhesion Molecule 1 ◽  
Barrier Dysfunction ◽  
Gram Negative ◽  
Anti Inflammatory

Increased vascular endothelial permeability and inflammation are major pathological mechanisms of pulmonary edema and its life-threatening complication, the acute respiratory distress syndrome (ARDS). We have previously described potent protective effects of hepatocyte growth factor (HGF) against thrombin-induced hyperpermeability and identified the Rac pathway as a key mechanism of HGF-mediated endothelial barrier protection. However, anti-inflammatory effects of HGF are less understood. This study examined effects of HGF on the pulmonary endothelial cell (EC) inflammatory activation and barrier dysfunction caused by the gram-negative bacterial pathogen lipopolysaccharide (LPS). We tested involvement of the novel Rac-specific guanine nucleotide exchange factor Asef in the HGF anti-inflammatory effects. HGF protected the pulmonary EC monolayer against LPS-induced hyperpermeability, disruption of monolayer integrity, activation of NF-kB signaling, expression of adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and production of IL-8. These effects were critically dependent on Asef. Small-interfering RNA-induced downregulation of Asef attenuated HGF protective effects against LPS-induced EC barrier failure. Protective effects of HGF against LPS-induced lung inflammation and vascular leak were also diminished in Asef knockout mice. Taken together, these results demonstrate potent anti-inflammatory effects by HGF and delineate a key role of Asef in the mediation of the HGF barrier protective and anti-inflammatory effects. Modulation of Asef activity may have important implications in therapeutic strategies aimed at the treatment of sepsis and acute lung injury/ARDS-induced gram-negative bacterial pathogens.

scholarly journals Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides

2011 ◽  
Vol 301 (6) ◽  
pp. L836-L846 ◽  
Author(s):  
Kelly S. Schweitzer ◽  
Hadi Hatoum ◽  
Mary Beth Brown ◽  
Mehak Gupta ◽  
Matthew J. Justice ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cell ◽  
P38 Mapk ◽  
Barrier Function ◽  
Rho Kinase ◽  
Endothelial Barrier ◽  
Kinase Activation ◽  
Cell Monolayers ◽  
Neutral Sphingomyelinase ◽  
Soluble Components

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.

scholarly journals Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides

1994 ◽  
Vol 303 (2) ◽  
pp. 407-411 ◽  
Author(s):  
J F Van Iwaarden ◽  
J C Pikaar ◽  
J Storm ◽  
E Brouwer ◽  
J Verhoef ◽  
...  
Keyword(s):  
Lipid A ◽  
Magnetic Beads ◽  
Protein A ◽  
Surfactant Protein ◽  
Biologically Active ◽  
Surfactant Protein A ◽  
Carbohydrate Binding ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

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