Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: Concurrent F‐actin depolymerization and new actin synthesis

Abstract

Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: 1) transendothelial 14C-albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in 14C-albumin flux (P < 0.001) and intercellular gap formation at > or = 2-6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (+/- SE) G-actin (micrograms/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization.

Bacterial Lipopolysaccharide Disrupts Endothelial Monolayer Integrity and Survival Signaling Events through Caspase Cleavage of Adherens Junction Proteins

“…DISCUSSION In this report, EC monolayers cultured on ECM-coated filters were used in two parallel experimental systems to demonstrate comparable dose and time requirements for LPS-induced increments in transendothelial [ 14 C]BSA flux and EC detachment. Our present and previous studies as well as studies by other investigators have demonstrated that LPS concentrations of Ն10 ng/ml and LPS exposure times of Ն2 h are required to induce a wide range of EC responses including actin reorganization, intercellular gap formation, loss of barrier function, and EC detachment (8,9,(13)(14)(15). Although this 2-h stimulus-toresponse lag time is sufficient for new protein synthesis to occur, neither LPS-induced barrier dysfunction (8) nor EC detachment (15) was blocked by prior protein synthesis inhibition.…”

“…EC Culture-Bovine pulmonary artery ECs (American Type Culture Collection, Rockville, MD) were cultured in Dulbecco's modified Eagle's medium (Sigma) enriched with 20% fetal bovine serum (FBS) (HyClone Laboratories), L-glutamine (5 mM), nonessential amino acids, and vitamins in the presence of penicillin (50 units/ml) and streptomycin (50 g/ml) (Sigma) as described (8,9). Only cells from passages two through seven were studied.…”

Suppression of endotoxin-induced inflammation by taxol

“…LPS challenge of cultured endothelial cells results in the loss of monolayer integrity and paracellular gap formation [14,15]. Microtubule stabilisation by taxol may interfere with LPS effects on cell-cell junctions, since recent data suggest that microtubules and adherence junctions interact dynamically [16,17].…”

“…Microtubule stabilisation by taxol may interfere with LPS effects on cell-cell junctions, since recent data suggest that microtubules and adherence junctions interact dynamically [16,17]. Conversely, in LPS-challenged cells, endothelial junctions may be weakened due to contractile forces generated by cytoskeletal motor proteins [14,18]. Microtubule stabilisation with taxol may affect the contractile cytoskeleton in a way that preserves cell-cell junctions.…”

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

“…A structure-function relationship appears to exist between EC actin organization/shape and barrier function. Agents that disrupt actin microfilaments increase endothelial permeability, previous F-actin stabilization protects against this increase, and established mediators of permeability induce actin reorganization (Shasby et al, 1982;Bussolino et al, 1987;Goldblum et al, 1993). In EC, F-actin is arranged into both central transcytoplasmic cables and a peripheral band (Wong and Gotlieb, 1986).…”