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Enhancement of FAK alleviates ventilator-induced alveolar epithelial cell injury.

ABSTRACT: Mechanical ventilation induces lung injury by damaging alveolar epithelial cells (AECs), but the pathogenesis remains unknown. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is involved in cell growth and intracellular signal transduction pathways. This study explored the potential role of FAK in AECs during lung injury induced by mechanical ventilation. High-volume mechanical ventilation (HMV) was used to create a mouse lung injury model, which was validated by analysis of lung weight, bronchoalveolar lavage fluid and histological investigation. The expression of FAK and Akt in AECs were evaluated. In addition, recombinant FAK was administered to mice via the tail vein, and then the extent of lung injury was assessed. Mouse AECs were cultured in vitro, and FAK expression in cells under stretch was investigated. The effects of FAK on cell proliferation, migration and apoptosis were investigated. The results showed that HMV decreased FAK expression in AECs of mice, while FAK supplementation attenuated lung injury, reduced protein levels/cell counts in the bronchoalveolar lavage fluid and decreased histological lung injury and oedema. The protective effect of FAK promoted AEC proliferation and migration and prevented cells from undergoing apoptosis, which restored the integrity of the alveoli through Akt pathway. Therefore, the decrease in FAK expression by HMV is essential for injury to epithelial cells and the disruption of alveolar integrity. FAK supplementation can reduce AEC injury associated with HMV.

SUBMITTER: Fang M

PROVIDER: S-EPMC6962166 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Enhancement of FAK alleviates ventilator-induced alveolar epithelial cell injury.

Fang Mingxing M Liu Na N Yao Xiaoguang X Xu Tieling T Wang Zhiyong Z

Scientific reports 20200115 1

Mechanical ventilation induces lung injury by damaging alveolar epithelial cells (AECs), but the pathogenesis remains unknown. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is involved in cell growth and intracellular signal transduction pathways. This study explored the potential role of FAK in AECs during lung injury induced by mechanical ventilation. High-volume mechanical ventilation (HMV) was used to create a mouse lung injury model, which was validated by analys ...[more]

PMID: 31942012

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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:Mechanical ventilation is an essential lifesaving therapy in acute respiratory distress syndrome (ARDS) that may cause ventilator-induced lung injury (VILI) through a positive feedback between altered alveolar mechanics, edema, surfactant inactivation, and injury. Although the biophysical forces that cause VILI are well documented, a knowledge gap remains in the quantitative link between altered parenchymal structure (namely alveolar derecruitment and flooding), pulmonary function, and VILI. This information is essential to developing diagnostic criteria and ventilation strategies to reduce VILI and improve ARDS survival. To address this unmet need, we mechanically ventilated mice to cause VILI. Lung structure was measured at three air inflation pressures using design-based stereology, and the mechanical function of the pulmonary system was measured with the forced oscillation technique. Assessment of the pulmonary surfactant included total surfactant, distribution of phospholipid aggregates, and surface tension lowering activity. VILI-induced changes in the surfactant included reduced surface tension lowering activity in the typically functional fraction of large phospholipid aggregates and a significant increase in the pool of surface-inactive small phospholipid aggregates. The dominant alterations in lung structure at low airway pressures were alveolar collapse and flooding. At higher airway pressures, alveolar collapse was mitigated and the flooded alveoli remained filled with proteinaceous edema. The loss of ventilated alveoli resulted in decreased alveolar gas volume and gas-exchange surface area. These data characterize three alveolar phenotypes in murine VILI: flooded and non-recruitable alveoli, unstable alveoli that derecruit at airway pressures below 5 cmH2O, and alveoli with relatively normal structure and function. The fraction of alveoli with each phenotype is reflected in the proportional changes in pulmonary system elastance at positive end expiratory pressures of 0, 3, and 6 cmH2O.

Project description:BackgroundVentilator-induced lung injury (VILI) is caused by stretch stimulation and other factors related to mechanical ventilation (MV). NOD-like receptor protein 3 (NLRP3), an important innate immune component, is strongly associated with VILI. This study aimed to investigate the effect and mechanisms of aerobic exercise (EX) on VILI.MethodsTo test the effects of the PKC inhibitor bisindolylmaleimide I on PKC and NLRP3, male C57BL/6 mice (7 weeks old, 19 ~ 23 g) were randomly divided into four groups: control group(C), bisindolylmaleimide I-pretreated group(B), MV group, and bisindolylmaleimide I-pretreated + MV (B + MV) group. The mice were pretreated with bisindolylmaleimide I through intraperitoneal injection (0.02 mg/kg) 1 h before MV. MV was performed at a high tidal volume (30 ml/kg). To explore the ameliorative effect of EX on VILI, the mice were randomly divided into C group, MV group, EX group and EX + MV group and subjected to either MV or 5 weeks of EX training. After ventilation, haematoxylin-eosin (HE) staining and wet/dry weight ratio was used to assess lung pathophysiological changes. PKCɑ, P-PKCɑ, ASC, procaspase-1, caspase-1, pro-IL-1β, IL-1β, NLRP3 and occludin (tight junction protein) expression in lung tissues was determined by Western blotting. The level of IL-6 in alveolar lavage fluid was determined by ELISA.ResultsNLRP3, P-PKCɑ, and PKCɑ levels were inceased in MV group, but bisindolylmaleimide I treatment reversed these changes. Inhibition of PKC production prevented NLRP3 activation. Moreover, MV increased ASC, procaspase-1, caspase-1, pro-IL-1β, and IL1β levels and decreased occludin levels, but EX alleviated these changes. HE staining and lung injury scoring confirmed an absence of obvious lung injury in C group and EX group. Lung injury was most severe in MV group but was improved in EX + MV group. Overall, these findings suggest that MV activates the NLRP3 inflammasome by activating PKCɑ and inducing occludin degradation, while Exercise attenuates NLRP3 inflammasome and PKCɑ activation. Besides, exercise improves cyclic stretch-induced degradation of occludin.ConclusionPKC activation can increase the level of NLRP3, which can lead to lung injury. Exercise can reduce lung injury by inhibiting PKCɑ and NLRP3 activation. Exercise maybe a potential measure for clinical prevention of VILI.

Project description:BackgroundAdvanced glycation end product receptor (RAGE) acts as a receptor of pro-inflammatory ligands and is highly expressed in alveolar epithelial cells (AECs). Autophagy in AECs has received much attention recently. However, the roles of autophagy and RAGE in the pathogenesis of acute lung injury remain unclear. Therefore, this study aimed to explore whether RAGE activation signals take part in the dysfunction of alveolar epithelial barrier through autophagic death.MethodsAcute lung injury animal models were established using C57BL/6 and Ager gene knockout (Ager -/- mice) mice in this study. A549 cells and primary type II alveolar epithelial (ATII) cells were treated with siRNA to reduce Ager gene expression. Autophagy was inhibited by 3-methyladenine (3-MA). Lung injury was assessed by histopathological examination. Cell viability was estimated by cell counting kit-8 (CCK-8) assay. The serum and bronchoalveolar lavage fluid (BALF) levels of interleukin (IL)-6, IL-8 and soluble RAGE (sRAGE) were evaluated by Enzyme-linked immunosorbent assay (ELISA). The involvement of RAGE signals, autophagy and apoptosis was assessed using western blots, immunohistochemistry, immunofluorescence, transmission electron microscopy and TUNEL test.ResultsThe expression of RAGE was promoted by lipopolysaccharide (LPS), which was associated with activation of autophagy both in mice lung tissues and A549 cells as well as primary ATII cells. sRAGE in BALF was positively correlated with IL-6 and IL-8 levels. Compared with the wild-type mice, inflammation and apoptosis in lung tissues were alleviated in Ager-/- mice. Persistently activated autophagy contributed to cell apoptosis, whereas the inhibition of autophagy by 3-MA protected lungs from damage. In addition, Ager knockdown inhibited LPS-induced autophagy activation and attenuated lung injury. In vitro, knockdown of RAGE significantly suppressed the activation of LPS-induced autophagy and apoptosis of A549 and primary ATII cells. Furthermore, RAGE activated the downstream STAT3 signaling pathway.ConclusionRAGE plays an essential role in the pathogenesis of ATII cells injury. Our results suggested that RAGE inhibition alleviated LPS-induced lung injury by directly suppressing autophagic apoptosis of alveolar epithelial cells.

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Enhancement of FAK alleviates ventilator-induced alveolar epithelial cell injury.

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Enhancement of FAK alleviates ventilator-induced alveolar epithelial cell injury.

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