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ASK1-ER stress pathway-mediated fibrotic-EV release contributes to the interaction of alveolar epithelial cells and lung fibroblasts to promote mechanical ventilation-induced pulmonary fibrosis.

ABSTRACT: Recent clinical research has revealed that mechanical ventilation (MV) can initiate pulmonary fibrosis and induce mechanical ventilation-induced pulmonary fibrosis (MVPF). However, the underlying mechanism remains largely uncharacterized. Based on a mouse model of MVPF and an alveolar epithelial cell cyclic strain model, the present study explores the possible mechanism of MVPF. Single-cell RNA-sequencing and EV RNA-sequencing analysis revealed that MV promoted apoptosis signal-regulating kinase 1 (ASK1)-mediated endoplasmic reticulum (ER) stress pathway activation and extracellular vesicle (EV) release from alveolar epithelial cells. Furthermore, the ASK1-ER stress pathway was shown to mediate mechanical stretch (MS)- or MV-induced EV release and lung fibroblast activation in vivo and in vitro. These processes were suppressed by ER stress inhibitors or by silencing ASK1 with ASK1- short hairpin RNA (shRNA). In addition, MVPF was suppressed by inhibiting ASK1 and ER stress in vivo. Therefore, the present study demonstrates that ASK1-ER stress pathway-mediated fibrotic-EV release from alveolar epithelial cells contributes to fibroblast activation and the initiation of pulmonary fibrosis during MV. The inhibited release of EVs targeting the ASK1-ER stress pathway might be a promising treatment strategy for MVPF.

SUBMITTER: Tang R

PROVIDER: S-EPMC9734805 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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ASK1-ER stress pathway-mediated fibrotic-EV release contributes to the interaction of alveolar epithelial cells and lung fibroblasts to promote mechanical ventilation-induced pulmonary fibrosis.

Tang Ri R Mei Shuya S Xu Qiaoyi Q Feng Jinhua J Zhou Yang Y Xing Shunpeng S He Zhengyu Z Gao Yuan Y

Experimental & molecular medicine 20221206 12

Recent clinical research has revealed that mechanical ventilation (MV) can initiate pulmonary fibrosis and induce mechanical ventilation-induced pulmonary fibrosis (MVPF). However, the underlying mechanism remains largely uncharacterized. Based on a mouse model of MVPF and an alveolar epithelial cell cyclic strain model, the present study explores the possible mechanism of MVPF. Single-cell RNA-sequencing and EV RNA-sequencing analysis revealed that MV promoted apoptosis signal-regulating kinase ...[more]

PMID: 36473935

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Project description:BackgroundSeptic shock is often associated with acute respiratory distress syndrome, a serious clinical problem exacerbated by improper mechanical ventilation. Ventilator-induced lung injury (VILI) can exacerbate the lung injury caused by acute respiratory distress syndrome, significantly increasing the morbidity and mortality. In this study, we asked the following questions: what is the effect of the lung position (dependent lung versus nondependent lung) on the rate at which VILI occurs in the normal lung? Will positive end-expiratory pressure (PEEP) slow the progression of lung injury in either the dependent lung or the nondependent lung?Materials and methodsSprague-Dawley rats (n = 19) were placed on mechanical ventilation, and the subpleural alveolar mechanics were measured with an in vivo microscope. Animals were placed in the lateral decubitus position, left lung up to measure nondependent alveolar mechanics and left lung down to film dependent alveolar mechanics. Animals were ventilated with a high peak inspiratory pressure of 45 cmH2O and either a low PEEP of 3 cmH2O or a high PEEP of 10 cmH2O for 90 minutes. Animals were separated into four groups based on the lung position and the amount of PEEP: Group I, dependent + low PEEP (n = 5); Group II, nondependent + low PEEP (n = 4); Group III, dependent + high PEEP (n = 5); and Group IV, nondependent + high PEEP (n = 5). Hemodynamic and lung function parameters were recorded concomitant with the filming of alveolar mechanics. Histological assessment was performed at necropsy to determine the presence of lung edema.ResultsVILI occurred earliest (60 min) in Group II. Alveolar instability eventually developed in Groups I and II at 75 minutes. Alveoli in both the high PEEP groups were stable for the entire experiment. There were no significant differences in arterial PO2 or in the degree of edema measured histologically among experimental groups.ConclusionThis open-chest animal model demonstrates that the position of the normal lung (dependent or nondependent) plays a role on the rate of VILI.

Project description:BackgroundPositive end-expiratory airway pressure (PEEP) is a potent component of management for patients receiving mechanical ventilation (MV). However, PEEP may cause the development of diaphragm remodeling, making it difficult for patients to be weaned from MV. The current study aimed to explore the role of PEEP in VIDD.MethodsEighteen adult male New Zealand rabbits were divided into three groups at random: nonventilated animals (the CON group), animals with volume-assist/control mode without/ with PEEP 8 cmH2O (the MV group/ the MV + PEEP group) for 48 h with mechanical ventilation. Ventilator parameters and diaphragm were collected during the experiment for further analysis.ResultsThere was no difference among the three groups in arterial blood gas and the diaphragmatic excursion during the experiment. The tidal volume, respiratory rate and minute ventilation were similar in MV + PEEP group and MV group. Airway peak pressure in MV + PEEP group was significantly higher than that in MV group (p < 0.001), and mechanical power was significantly higher (p < 0.001). RNA-seq showed that genes associated with fibrosis were enriched in the MV + PEEP group. This results were further confirmed on mRNA expression. As shown by Masson's trichrome staining, there was more collagen fiber in the MV + PEEP group than that in the MV group (p = 0.001). Sirius red staining showed more positive staining of total collagen fibers and type I/III fibers in the MV + PEEP group (p = 0.001; p = 0.001). The western blot results also showed upregulation of collagen types 1A1, III, 6A1 and 6A2 in the MV + PEEP group compared to the MV group (p < 0.001, all). Moreover, the positive immunofluorescence of COL III in the MV + PEEP group was more intense (p = 0.003). Furthermore, the expression of TGF-β1, one of the most potent fibrogenic factors, was upregulated at both the mRNA and protein levels in the MV + PEEP group (mRNA: p = 0.03; protein: p = 0.04).ConclusionsWe demonstrated that PEEP application for 48 h in mechanically ventilated rabbits will cause collagen deposition and fibrosis in the diaphragm. Moreover, activation of the TGF-β1 signaling pathway and myofibroblast differentiation may be the potential mechanism of this diaphragmatic fibrosis. These findings might provide novel therapeutic targets for PEEP application-induced diaphragm dysfunction.

Project description:ObjectivesAlthough mechanical ventilation is a life-saving measure for patients in respiratory failure, prolonged mechanical ventilation results in diaphragmatic weakness attributable to fiber atrophy and contractile dysfunction. Therefore, identifying the signaling pathways responsible for mechanical ventilation-induced diaphragmatic weakness is important. In this context, it is established that oxidative stress is required for mechanical ventilation-induced diaphragmatic weakness to occur. Numerous redox-sensitive signaling pathways exist in muscle including the transcription factor nuclear factor-κB. Although it has been suggested that nuclear factor-κB contributes to proteolytic signaling in inactivity-induced atrophy in locomotor muscles, the role that nuclear factor-κB plays in mechanical ventilation-induced diaphragmatic weakness is unknown. We tested the hypothesis that nuclear factor-κB activation plays a key signaling role in mechanical ventilation-induced diaphragmatic weakness and that oxidative stress is required for nuclear factor-κB activation.DesignCause and effect was determined by independently treating mechanically ventilated animals with either a specific nuclear factor-κB inhibitor (SN50) or a clinically relevant antioxidant (curcumin).Measurements and main resultsInhibition of nuclear factor-κB activity partially attenuated both mechanical ventilation-induced diaphragmatic atrophy and contractile dysfunction. Further, treatment with the antioxidant curcumin prevented mechanical ventilation-induced activation of nuclear factor-κB in the diaphragm and rescued the diaphragm from both mechanical ventilation-induced atrophy and contractile dysfunction.ConclusionsCollectively, these findings support the hypothesis that nuclear factor-κB activation plays a significant signaling role in mechanical ventilation-induced diaphragmatic weakness and that oxidative stress is an upstream activator of nuclear factor-κB. Finally, our results suggest that prevention of mechanical ventilation-induced oxidative stress in the diaphragm could be a useful clinical strategy to prevent or delay mechanical ventilation-induced diaphragmatic weakness.

Project description:Ventilator-induced lung injury (VILI) impacts clinical outcomes in acute respiratory distress syndrome (ARDS), which is characterized by neutrophil-mediated inflammation and loss of alveolar barrier function. Recent epidemiological studies suggest that smoking may be a risk factor for the development of ARDS. Because alveolar type II cells are central to maintaining the alveolar epithelial barrier during oxidative stress, mediated in part by neutrophilic inflammation and mechanical ventilation, we hypothesized that exposure to cigarette smoke and mechanical strain have interactive effects leading to the activation of and damage to alveolar type II cells.To determine if cigarette smoke increases susceptibility to VILI in vivo, a clinically relevant rat model was established. Rats were exposed to three research cigarettes per day for two weeks. After this period, some rats were mechanically ventilated for 4 hours. Bronchoalveolar lavage (BAL) and differential cell count was done and alveolar type II cells were isolated. Proteomic analysis was performed on the isolated alveolar type II cells to discover alterations in cellular pathways at the protein level that might contribute to injury. Effects on levels of proteins in pathways associated with innate immunity, oxidative stress and apoptosis were evaluated in alveolar type II cell lysates by enzyme-linked immunosorbent assay. Statistical comparisons were performed by t-tests, and the results were corrected for multiple comparisons using the false discovery rate.Tobacco smoke exposure increased airspace neutrophil influx in response to mechanical ventilation. The combined exposure to cigarette smoke and mechanical ventilation significantly increased BAL neutrophil count and protein content. Neutrophils were significantly higher after smoke exposure and ventilation than after ventilation alone. DNA fragments were significantly elevated in alveolar type II cells. Smoke exposure did not significantly alter other protein-level markers of cell activation, including Toll-like receptor 4; caspases 3, 8 and 9; and heat shock protein 70.Cigarette smoke exposure may impact ventilator-associated alveolar epithelial injury by augmenting neutrophil influx. We found that cigarette smoke had less effect on other pathways previously associated with VILI, including innate immunity, oxidative stress and apoptosis.

Project description:IntroductionAcute respiratory distress syndrome causes a heterogeneous lung injury, and without protective mechanical ventilation a secondary ventilator-induced lung injury can occur. To ventilate noncompliant lung regions, high inflation pressures are required to 'pop open' the injured alveoli. The temporal impact, however, of these elevated pressures on normal alveolar mechanics (that is, the dynamic change in alveolar size and shape during ventilation) is unknown. In the present study we found that ventilating the normal lung with high peak pressure (45 cmH(2)0) and low positive end-expiratory pressure (PEEP of 3 cmH(2)O) did not initially result in altered alveolar mechanics, but alveolar instability developed over time.MethodsAnesthetized rats underwent tracheostomy, were placed on pressure control ventilation, and underwent sternotomy. Rats were then assigned to one of three ventilation strategies: control group (n = 3, P control = 14 cmH(2)O, PEEP = 3 cmH(2)O), high pressure/low PEEP group (n = 6, P control = 45 cmH(2)O, PEEP = 3 cmH(2)O), and high pressure/high PEEP group (n = 5, P control = 45 cmH(2)O, PEEP = 10 cmH(2)O). In vivo microscopic footage of subpleural alveolar stability (that is, recruitment/derecruitment) was taken at baseline and than every 15 minutes for 90 minutes following ventilator adjustments. Alveolar recruitment/derecruitment was determined by measuring the area of individual alveoli at peak inspiration (I) and end expiration (E) by computer image analysis. Alveolar recruitment/derecruitment was quantified by the percentage change in alveolar area during tidal ventilation (%I - E Delta).ResultsAlveoli were stable in the control group for the entire experiment (low %I - E Delta). Alveoli in the high pressure/low PEEP group were initially stable (low %I - E Delta), but with time alveolar recruitment/derecruitment developed. The development of alveolar instability in the high pressure/low PEEP group was associated with histologic lung injury.ConclusionA large change in lung volume with each breath will, in time, lead to unstable alveoli and pulmonary damage. Reducing the change in lung volume by increasing the PEEP, even with high inflation pressure, prevents alveolar instability and reduces injury. We speculate that ventilation with large changes in lung volume over time results in surfactant deactivation, which leads to alveolar instability.

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ASK1-ER stress pathway-mediated fibrotic-EV release contributes to the interaction of alveolar epithelial cells and lung fibroblasts to promote mechanical ventilation-induced pulmonary fibrosis.

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ASK1-ER stress pathway-mediated fibrotic-EV release contributes to the interaction of alveolar epithelial cells and lung fibroblasts to promote mechanical ventilation-induced pulmonary fibrosis.

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