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Site-specific and endothelial-mediated dysfunction of the alveolar-capillary barrier in response to lipopolysaccharides.

ABSTRACT: Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar-capillary barrier (ACB) in the lung. In this study, we analyse the site-specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co-cultures of H441 with endothelial cells cultured on Transwells® were treated with LPS from the apical or basolateral compartment. Barrier properties were analysed by the transepithelial electrical resistance (TEER), by transport assays, and immunostaining and assessment of tight junctional molecules at protein level. Furthermore, pro-inflammatory cytokines and immune-modulatory molecules were evaluated by ELISA and semiquantitative real-time PCR. Liquid chromatography-mass spectrometry-based proteomics (LS-MS) was used to identify proteins and effector molecules secreted by endothelial cells in response to LPS. In co-cultures treated with LPS from the basolateral compartment, we noticed a significant reduction of TEER, increased permeability and induction of pro-inflammatory cytokines. Conversely, apical treatment did not affect the barrier. No changes were noticed in H441 monoculture upon LPS treatment. However, LPS resulted in an increased expression of pro-inflammatory cytokines such as IL-6 in OEC and in turn induced the reduction of TEER and an increase in SP-A expression in H441 monoculture, and H441/OEC co-cultures after LPS treatment from basolateral compartment. LS-MS-based proteomics revealed factors associated with LPS-mediated lung injury such as ICAM-1, VCAM-1, Angiopoietin 2, complement factors and cathepsin S, emphasizing the role of epithelial-endothelial crosstalk in the ACB in ALI/ARDS.

SUBMITTER: Janga H

PROVIDER: S-EPMC5783864 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Site-specific and endothelial-mediated dysfunction of the alveolar-capillary barrier in response to lipopolysaccharides.

Janga Harshavardhan H Cassidy Liam L Wang Fanlu F Spengler Dietmar D Oestern-Fitschen Stefanie S Krause Martin F MF Seekamp Andreas A Tholey Andreas A Fuchs Sabine S

Journal of cellular and molecular medicine 20171205 2

Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar-capillary barrier (ACB) in the lung. In this study, we analyse the site-specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co-cultures of H441 with endothelial cells cultured on Transwells<sup>®</sup> were treated with LPS from the apical or basolateral compartment. Barrier propertie ...[more]

PMID: 29210175

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Project description:Acute lung injury (ALI) is an inflammatory disorder associated with reduced alveolar-capillary barrier function and increased pulmonary vascular permeability. Vasodilator-stimulated phosphoprotein (VASP) is widely associated with all types of modulations of cytoskeleton rearrangement-dependent cellular morphology and function, such as adhesion, shrinkage, and permeability. The present studies were conducted to investigate the effects and mechanisms by which tumor necrosis factor-alpha (TNF-α) increases the tight junction permeability in lung tissue associated with acute lung inflammation. After incubating A549 cells for 24 hours with different concentrations (0-100 ng/mL) of TNF-α, 0.1 to 8 ng/mL TNF-α exhibited no significant effect on cell viability compared with the 0 ng/mL TNF-α group (control group). However, 10 ng/mL and 100 ng/mL TNF-α dramatically inhibited the viability of A549 cells compared with the control group (*p<0.05). Monolayer cell permeability assay results indicated that A549 cells incubated with 10 ng/mL TNF-α for 24 hours displayed significantly increased cell permeability (*p<0.05). Moreover, the inhibition of VASP expression increased the cell permeability (*p<0.05). Pretreating A549 cells with cobalt chloride (to mimic a hypoxia environment) increased protein expression level of hypoxia inducible factor-1α (HIF-1α) (*p<0.05), whereas protein expression level of VASP decreased significantly (*p<0.05). In LPS-induced ALI mice, the concentrations of TNF-α in lung tissues and serum significantly increased at one hour, and the value reached a peak at four hours. Moreover, the Evans Blue absorption value of the mouse lung tissues reached a peak at four hours. The HIF-1α protein expression level in mouse lung tissues increased significantly at four hours and eight hours (**p<0.001), whereas the VASP protein expression level decreased significantly (**p<0.01). Taken together, our data demonstrate that HIF-1α acts downstream of TNF-α to inhibit VASP expression and to modulate the acute pulmonary inflammation process, and these molecules play an important role in the impairment of the alveolar-capillary barrier.

Dataset Information

Site-specific and endothelial-mediated dysfunction of the alveolar-capillary barrier in response to lipopolysaccharides.

Publications

Site-specific and endothelial-mediated dysfunction of the alveolar-capillary barrier in response to lipopolysaccharides.

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