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Local influence of cell viability on stretch-induced permeability of alveolar epithelial cell monolayers.

ABSTRACT: Ventilator induced lung injury (VILI), often attributed to over-distension of the alveolar epithelial cell layer, can trigger loss of barrier function. Alveolar epithelial cell monolayers can be used as an idealized in vitro model of the pulmonary epithelium, with cell death and tight junction disruption and permeability employed to estimate stretch-induced changes in barrier function. We adapted a method published for vascular endothelial permeability, compare its sensitivity with our previously published method, and determine the relationship between breeches in barrier properties after stretch and regions of cell death After 4-5 days in culture, primary rat alveolar epithelial cells seeded on plasma treated polydimethylsiloxane membrane coated with biotin-labeled fibronectin, or fibronectin alone were stretched in the presence of FITC-tagged streptavidin (biotin-labeled membrane) or BODIPY-ouabain. We found that the FITC-labeling method was a more sensitive indicator of permeability disruption, with significantly larger positively stained areas visible in the presence of stretch and with ATP production inhibitor Antimycin-A. Triple-stained images with Hoescht (nuclei), Ethidium Homodimer (EthD, damaged cell nuclei) and FITC (permeable regions) were used to determine that within permeable regions intact cells were positioned closer to damaged cells than in non-permeable regions. We concluded that local cell death may be an important contributor to barrier integrity.

SUBMITTER: Song MJ

PROVIDER: S-EPMC4779366 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Local influence of cell viability on stretch-induced permeability of alveolar epithelial cell monolayers.

Song M J MJ Davis C I CI Lawrence G G GG Margulies S S SS

Cellular and molecular bioengineering 20150708 1

Ventilator induced lung injury (VILI), often attributed to over-distension of the alveolar epithelial cell layer, can trigger loss of barrier function. Alveolar epithelial cell monolayers can be used as an idealized in vitro model of the pulmonary epithelium, with cell death and tight junction disruption and permeability employed to estimate stretch-induced changes in barrier function. We adapted a method published for vascular endothelial permeability, compare its sensitivity with our previousl ...[more]

PMID: 26958093

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Project description:The aim of the present study was to explore the potential role of SOX11 in the stretch‑induced mechanical injury to alveolar type 2 epithelial (AT2) cells. A cell stretch (CS) test was used to induce mechanical injury to primary cultured AT2 cells. Wound healing, adhesion, cell viability assays and flow cytometry were performed to evaluate the migration, adhesion, viability and apoptosis of AT2 cells. Changes in the invasive ability of AT2 cells were detected using a Transwell invasion assay. To further explore the underlying molecular mechanisms, reverse transcription‑quantitative PCR and western blot analysis were used to assess the expression levels of SOX11, FAK, Akt, caspase‑3/8, p65 and matrix metalloproteinase (MMP)7. Co‑immunoprecipitation (Co‑IP) and luciferase reporter assays were used to detect the interaction between SOX11 and FAK. CS reduced the invasion, migration and adhesion, and increased the apoptosis of AT2 cells. It also resulted in the downregulation of SOX11 and FAK expression in AT2 cells. The overexpression of SOX11 reversed these changes, whereas the knockdown of SOX11 aggravated the deterioration of the aforementioned biological behaviors and the apoptosis of the AT2 cells following CS. The overexpression of SOX11 upregulated the FAK and Akt expression levels, and downregulated caspase‑3/8 expression, whereas the silencing of SOX11 reversed these changes following CS. Furthermore, the effects of SOX11 overexpression were inhibited by FAK antagonism. The results of Co‑IP demonstrated that SOX11 and FAK were bound together, and that the expression of FAK was significantly increased in the SOX11 overexpression group. Luciferase assays revealed that the luciferase activity and the mRNA expression of FAK were significantly increased following transfection with pcDNA SOX11 and pGL3 FAK promoter. Co‑IP and luciferase assays revealed that SOX11 directly regulated the expression of FAK. On the whole, the present study demonstrates that the downregulated expression of SOX11 and FAK are involved in the stretch‑induced mechanical injury to AT2 cells. The overexpression of SOX11 significantly alleviates AT2 cell injury through the upregulation of FAK and Akt, and the inhibition of apoptosis. These findings suggest that the activation of SOX11 and FAK may be potential preventive and therapeutic options for ventilator‑induced lung injury.

Project description:Alcohol use disorders (AUDs), including alcohol abuse and dependence, have been linked to the development of acute lung injury (ALI). Prior clinical investigations suggested an association between AUDs and abnormal alveolar epithelial permeability mediated through pulmonary oxidative stress that may partially explain this relationship. We sought to determine if correcting pulmonary oxidative stress in the setting of AUDs would normalize alveolar epithelial permeability in a double-blinded, randomized, placebo-controlled trial of Protandim, a nutraceutical reported to enhance antioxidant activity. We randomized 30 otherwise healthy AUD subjects to receive directly observed inpatient oral therapy with either Protandim (1,350 mg/day) or placebo. Subjects underwent bronchoalveolar lavage (BAL) and blood sampling before study drug administration and after 7 days of therapy; all AUD subjects completed the study protocol without adverse events. BAL total protein was measured at each timepoint as an indicator of alveolar epithelial permeability. In subjects with AUDs, before study drug initiation, BAL total protein values were not significantly higher than in 11 concurrently enrolled controls (P = 0.07). Over the 7-day study period, AUD subjects did not exhibit a significant change in BAL total protein, regardless of their randomization to Protandim {n = 14, -2% [intraquartile range (IQR), -56-146%]} or to placebo [n = 16, 77% (IQR -20-290%); P = 0.19]. Additionally, among those with AUDs, no significant changes in BAL oxidative stress indexes, epithelial growth factor, fibroblast growth factor, interleukin-1β, or interleukin-10 were observed regardless of drug type received. Plasma thiobarbituric acid reactive substances, a marker of lipid peroxidation, decreased significantly over time among AUD subjects randomized to placebo (P < 0.01). These results suggest that Protandim for 7 days in individuals with AUDs who are newly abstinent does not alter alveolar epithelial permeability. However, our work demonstrates the feasibility of safely conducting clinical trials that include serial bronchoscopies in a vulnerable population at risk for acute lung injury.

Dataset Information

Local influence of cell viability on stretch-induced permeability of alveolar epithelial cell monolayers.

Publications

Local influence of cell viability on stretch-induced permeability of alveolar epithelial cell monolayers.

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