Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction

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 (> or =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 > or =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.

FIG. 1

Effects of LPS fractions on transendothelial ¹⁴C-BSA flux. (A) Transendothelial ¹⁴C-BSA flux across monolayers was assayed after exposure for 6 h to increasing concentrations of either the lipid A fraction or the O-specific polysaccharide fraction derived from E. coli O111:B4 LPS. Each bar represents mean (± SE) transendothelial ¹⁴C-BSA flux. Pretreatment baseline ¹⁴C-BSA flux across monolayers exposed to either lipid A or O-specific polysaccharide fractions as well as ¹⁴C-BSA flux across naked filters are also shown. ∗, significantly increased compared with simultaneous medium control. n, number of monolayers studied. (B) EC monolayers were assayed for transendothelial ¹⁴C-BSA flux immediately after 6-h exposures to medium alone, PMB, LPS, or LPS (10 ng/ml) coadministered with increasing concentrations of PMB. Mean (± SE) pretreatment baseline ¹⁴C-BSA flux is also shown ∗, significantly increased compared to medium control; ∗∗, significantly decreased compared to LPS. n, number of monolayers studied.

FIG. 2

Dose-dependent effects of ENP on LPS-induced barrier dysfunction. Baseline barrier function was determined for all monolayers prior to treatment. Transendothelial ¹⁴C-BSA flux across monolayers was assayed after exposure for 6 h to medium, ENP, LPS, or LPS coadministered with increasing concentrations of ENP. Each bar represents mean (± SE) transendothelial ¹⁴C-BSA flux. ∗, significantly increased compared to medium control; ∗∗, significantly decreased compared to LPS alone. n, number of monolayers studied.

FIG. 3

Effect of ENP on LPS-induced tyrosine phosphorylation of a 66-kDa EC protein. For Western blot analysis of protein tyrosine phosphorylation, EC monolayers were exposed to medium, ENP (1.0 μg/ml), LPS (100 ng/ml), or LPS coadministered with ENP for 1 h. The EC lysates were resolved by SDS-PAGE, transferred to PVDF, and probed for phosphotyrosines. Molecular weights (in thousands) are indicated by arrows on the left. The blot is representative of three separate experiments.

FIG. 4

Effect of ENP on phosphotyrosine-containing proteins in LPS-exposed EC. EC monolayers grown on filters were exposed for 1 h to medium (A), ENP (1.0 μg/ml; B), LPS (100 ng/ml; C), or LPS coadministered with ENP (D). The monolayers were fixed, probed with FITC-conjugated antiphosphotyrosine antibody, and photographed through an epifluorescence microscope. Arrows indicate phosphotyrosine signal at intercellular boundaries. Magnification, ×600.

FIG. 5

Effects of ENP on LPS-induced changes in the F- and G-actin pools. For G- and F-actin measurements, monolayers were exposed for 6 h to medium, ENP, LPS, or LPS coadministered with ENP. (A) For the F-actin studies, monolayers were fixed, permeabilized, incubated with NBD-phallicidin, and extracted with methanol. The extracts were spectrofluorimetrically assayed, and F-actin concentrations were expressed as mean (± SE) fluorescent units per milligram of total EC protein. ∗, significantly decreased compared to medium control; ∗∗, significantly increased compared to LPS alone but not significantly decreased compared to medium alone. (B) For quantitation of the G-actin pool, EC were permeabilized and the G-actin-containing supernatants were tested in the DNase I inhibition assay standardized to pure G-actin. Each bar represents mean (± SE) G-actin expressed. ∗, significantly increased compared to medium control; ∗∗, significantly decreased compared to LPS alone but not significantly increased compared to medium alone. n for each experimental group is indicated in each bar.

FIG. 6

ENP cross-protects against a wide variety of endotoxins. Transendothelial ¹⁴C-BSA flux was assayed immediately following 6-h exposures to medium (open bar), equivalent concentrations based on KDO content of LPS derived from E. coli O111:B4 (10 ng/ml), E. coli 055:B5, P. aeruginosa, K. pneumoniae, S. marcescens, or S. minnesota (cross-hatched bars), or these same LPS preparations coadministered with ENP (100 ng/ml) (gray bars). Each bar represents mean (± SE) transendothelial ¹⁴C-BSA flux. Baseline barrier function for all monolayers studied is also indicated. ∗, significantly decreased compared to LPS alone at P < 0.05 but not significantly increased compared to medium control.