Alveolar Epithelial Cell Repair Research Articles

Therapeutic effects of fatty acid binding protein 1 in mice with pulmonary fibrosis by regulating alveolar epithelial regeneration

IntroductionIdiopathic pulmonary fibrosis is a progressive fibrotic lung disease with limited therapeutic options and high lethality, related to alveolar type II epithelial (ATII) cell dysregulation, the abnormal repair of alveolar...

Senescence of IPF Lung Fibroblasts Disrupt Alveolar Epithelial Cell Proliferation and Promote Migration in Wound Healing.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease marked by excessive accumulation of lung fibroblasts (LFs) and collagen in the lung parenchyma. The mechanisms that underlie IPF pathophysiology are thought to reflect repeated alveolar epithelial injury leading to an aberrant wound repair response. Recent work has shown that IPF-LFs display increased characteristics of senescence including growth arrest and a senescence-associated secretory phenotype (SASP) suggesting that senescent LFs contribute to dysfunctional wound repair process. Here, we investigated the influence of senescent LFs on alveolar epithelial cell repair responses in a co-culture system. Alveolar epithelial cell proliferation was attenuated when in co-culture with cells or conditioned media from, senescence-induced control LFs or IPF-LFs. Cell-cycle analyses showed that a larger number of epithelial cells were arrested in G2/M phase when co-cultured with IPF-LFs, than in monoculture. Paradoxically, the presence of LFs resulted in increased A549 migration after mechanical injury. Our data suggest that senescent LFs may contribute to aberrant re-epithelialization by inhibiting proliferation in IPF.

The Anti-fibrotic and Anti-inflammatory Potential of Bone Marrow-Derived Mesenchymal Stem Cells and Nintedanib in Bleomycin-Induced Lung Fibrosis in Rats.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive lung damage. Tyrosine kinase inhibitors are approved to treat people with IPF while bone marrow-derived mesenchymal stem cell therapy was previously suggested to inhibit pulmonary fibrosis through the alveolar epithelial cell repair. The present study aimed to evaluate the anti-inflammatory and anti-fibrotic effect of the bone marrow-derived mesenchymal stem cell (BM-MSC) therapy in comparison with nintedanib, a tyrosine kinase inhibitor, on improving survival in bleomycin-induced lung fibrosis in rats. Moreover, the combined therapy of BM-MSCs and nintedanib will be evaluated. In the present study, IPF was induced through intra-tracheal instillation of bleomycin (5mg/kg) in rats then treatments were administered 14days thereafter. Nintedanib (100mg/kg, I.P.) was administered daily for 28days, while BM-MSCs were injected once intravenously in tail vein in the dose 1 × 106cells/ml/rat. In the present study, both treatment regimens effectively inhibited lung fibrosis through several pathways, suppressing tumor growth factor-β (TGF-β)/SMAD3 expression which is considered the master signaling pathway. Nintedanib and BLM-MSCs exerted their anti-inflammatory effect through minimizing the expression of TNF-α and IL-6. In addition, the histopathological examination of the lung tissue showed a significant decrease in the alveolar wall thickening, in the inflammatory infiltrate, and in the collagen fiber deposition in response to either nintedanib or BM-MSC and their combination. In conclusion, the therapeutic pulmonary anti-fibrotic activity of nintedanib or BM-MSC is mediated through their anti-inflammatory properties and inhibition of SMAD-3/TGF-β expression.

Hypercapnia Alters Alveolar Epithelial Repair by a pH-Dependent and Adenylate Cyclase-Mediated Mechanism

Lung cell injury and repair is a hallmark of the acute respiratory distress syndrome (ARDS). Lung protective mechanical ventilation strategies in these patients may lead to hypercapnia (HC). Although HC has been explored in the clinical context of ARDS, its effect upon alveolar epithelial cell (AEC) wounding and repair remains poorly understood. We have previously reported that HC alters the likelihood of AEC repair by a pH-sensitive but otherwise unknown mechanism. Adenylate cyclase (AC) is an attractive candidate as a putative AEC CO2 sensor and effector as it is bicarbonate sensitive and controls key mediators of AEC repair. The effect of HC on AC activity and plasma membrane (PM) wound repair was measured in AEC type 1 exposed to normocapnia (NC, 40 Torr) or HC (80 Torr), ± tromethamine (THAM) or sodium bicarbonate (HCO3) ± AC probes in a micropuncture model of AEC injury relevant to ARDS. Intracellular pH and AC activity were measured and correlated with repair. HC decreased intracellular pH 0.56, cAMP by 37%, and absolute PM repair rate by 26%. Buffering or pharmacologic manipulation of AC reduced or reversed the effects of HC on AC activity (THAM 103%, HCO3 113% of NC cAMP, ns; Forskolin 168%, p < 0.05) and PM repair (THAM 87%, HCO3 108% of NC likelihood to repair, ns; Forskolin 160%, p < 0.01). These findings suggest AC to be a putative AEC CO2 sensor and modulator of AEC repair, and may have implications for future pharmacologic targeting of downstream messengers of the AC-cAMP axis in experimental models of ARDS.

Specialized Pro-resolving Mediators Regulate Alveolar Fluid Clearance during Acute Respiratory Distress Syndrome.

Objective:Acute respiratory distress syndrome (ARDS) is an acute and lethal clinical syndrome that is characterized by the injury of alveolar epithelium, which impairs active fluid transport in the lung, and impedes the reabsorption of edema fluid from the alveolar space. This review aimed to discuss the role of pro-resolving mediators on the regulation of alveolar fluid clearance (AFC) in ARDS.Data Sources:Articles published up to September 2017 were selected from the PubMed, with the keywords of “alveolar fluid clearance” or “lung edema” or “acute lung injury” or “acute respiratory distress syndrome”, and “specialized pro-resolving mediators” or “lipoxin” or “resolvin” or “protectin” or “maresin” or “alveolar epithelial cells” or “aspirin-triggered lipid mediators” or “carbon monoxide and heme oxygenase” or “annexin A1”.Study Selection:We included all relevant articles published up to September 2017, with no limitation of study design.Results:Specialized pro-resolving mediators (SPMs), as the proinflammatory mediators, not only upregulated epithelial sodium channel, Na,K-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporins levels, but also improved Na,K-ATPase activity to promote AFC in ARDS. In addition to the direct effects on ion channels and pumps of the alveolar epithelium, the SPMs also inhibited the inflammatory cytokine expression and improved the alveolar epithelial cell repair to enhance the AFC in ARDS.Conclusions:The present review discusses a novel mechanism for pulmonary edema fluid reabsorption. SPMs might provide new opportunities to design “reabsorption-targeted” therapies with high degrees of precision in controlling ALI/ARDS.

Gefitinib and Erlotinib Lead to Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Independent of Epidermal Growth Factor Receptor in A549 Cells.

Gefitinib and erlotinib are anticancer agents, which inhibit epidermal growth factor receptor (EGFR) tyrosine kinase. Interstitial lung disease (ILD) occurs in patients with non-small cell lung cancer receiving EGFR inhibitors. In the present study, we examined whether gefitinib- and erlotinib-induced lung injury related to ILD through endoplasmic reticulum (ER) stress, which is a causative intracellular mechanism in cytotoxicity caused by various chemicals in adenocarcinomic human alveolar basal epithelial cells. These two EGFR inhibitors increased Parkinson juvenile disease protein 2 and C/EBP homologous protein mRNA expressions, and activated the eukaryotic initiation factor (eIF) 2α/activating transcription factor 4 pathway without protein kinase R-like ER kinase activation in A549 cells. Gefitinib and erlotinib caused neither ER stress nor cell death; however, these agents inhibited cell growth via the reduction of cyclin-D1 expression. Tauroursodeoxycholic acid, which is known to suppress eIF2α phosphorylation, cancelled the effects of EGFR inhibitors on cyclin-D1 expression and cell proliferation in a concentration-dependent manner. The results of an EGFR-silencing study using siRNA showed that gefitinib and erlotinib affected eIF2α phosphorylation and cyclin-D1 expression independent of EGFR inhibition. Therefore, the inhibition of cell growth by these EGFR inhibitors might equate to impairment of the alveolar epithelial cell repair system via eIF2α phosphorylation and reduced cyclin-D1 expression.

Modulation of TRIM72 Alters the Repair Capacity of Lung Epithelial Cells

The molecular mechanisms for alveolar epithelial cell plasma membrane wounding and repair are largely unknown. Previous studies identified TRIM72 (also known as mitsugumin 53) from striated muscle tissues and linked its physiological functions in these tissues to membrane repair, cell proliferation, and glucose metabolism. Here, using biochemical and immunochemical assays, we have characterized TRIM72 expression in lung tissue and cells, and investigated the physiological function of TRIM72 in the repair of alveolar epithelial cells. In vivo injury of alveolus resident cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled by postinjury administration of propidium iodide and quantified in optical slices of subpleural alveoli. Primary alveolar epithelial cells were isolated to study plasma membrane wounding and repair in vitro, using a dual-dye incorporation assay following glass bead–induced damage to separately quantify injured versus repaired cells. Our results show that TRIM72 protein was expressed in alveolar epithelia of mouse, rat, and human lungs. High tidal volume ventilation of trim72 knockout mice caused significantly more fatal damage of alveolus cells as compared with the wild-type mice. Strikingly, injury estimates in trim72 overexpressor lungs were significantly lower than in the wild-type. The in vitro cell wounding assay further revealed that absence of TRIM72 led to significant repair defects in alveolar epithelial cells, whereas the total number of wounded cells (repaired plus nonrepaired) was similar in the wild-type and trim72 knockout groups. The cellular function of TRIM72 in the lung may be further linked to caveolin 1, as physical and physiological interactions were identified between these two proteins. These data suggest an important role for TRIM72 in the repair of alveolar epithelial cells under plasma membrane stress failure and potential reversal of cell repair defects by TRIM72 overexpression.

TRIM72 is required for effective repair of alveolar epithelial cell wounding.

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.

Effect of inhaled arsenic trioxide and moxa leaf oil on eosinophil in asthmatic guinea pigs

Objective The experiment was designed to observe the effect of inhaled arsenic trioxide（As2O3）and in combination with inhaled moxa leaf oil on eosinophils（EOS）and lung tissue’s morphology in asthmatic guinea pigs, and to explore its mechanism of relieving wheezing. Methods The ovalbumin（OVA）-induced asthmatic guinea pig model was established. Sixty guinea pigs were randomly divided into As2O3 atomization inhalation group B1 As2O3 low dose group：2.0 mg/（kg·d）, B2 As2O3 high dose group：4.0 mg/（kg·d）, combined treatment group C1 low dose group：2.0 mg/（kg·d）＋moxa leaf oil 0.05 ml, C2 high dose group： 4.0 mg/（kg·d）＋moxa leaf oil 0.10 ml, saline aerosol inhalation group（group A） asthma model group）and normal control group（group D）according to random permutation table method. After 7 days medicated, the EOS in bronchoalveolar lavage fluid（BALF）were compared. Guinea pig lung, heart, liver and kidney pathological specimens were prepared and the change of the lung tissue’s inflammation and heart, liver and kidney tissue were investigated. Lung tissue’s electron microscopy specimens were prepared and the apoptosis of acidophilic cells and repair of alveolar epithelial cells were observed. Results Compared with asthma model group’s EOS number[（58.08±19.01）×105], the difference in As2O3 low dose group、As2O3 high dose group、combined treatment low dose group、combined treatment high dose group and normal control＆amp;nbsp;group[（26.37±1.12）×105, （11.50±1.61）×105, （14.16±4.88）×105, （5.03±1.66）×105 and（0.35±0.16）×105, respectively] were statistically significant（P<0.01）; there was statistical difference between As2O3 low dose group and As2O3 high dose group（P<0.01）;There was statistical difference between As2O3 low dose group and combined treatment low dose group（P<0.01）;There was statistical difference between As2O3 high dose group and combined treatment high dose group（P<0.05）. Compared with combined treatment high dose group, difference in As2O3 low dose group、As2O3 high dose group and combined treatment low dose group was statistically significant（P<0.05）. The difference in B1, B2, C1, C2 and D group was statistically significant compared with group A （P<0.01）. The difference in B1 and B2, B1 and C1 group was statistically significant （P<0.01）. The difference between B2 and C2 group was statistically significant（P<0.05）. The difference in C2 group was statistically significant compared with group B1, B2 and C1（P<0.05）. Conclusion EOS abnormal apoptosis was an important pathogenesis of bronchial asthma. The mechanism of arsenic trioxide anti-asthma was that arsenic trioxide could reduce acidophil infiltration and promote acidophilic cell apoptosis. The mechanism of moxa leaf oil anti-asthma was it could reduce acidophil infiltration. Smaller doses of the combination of inhaled As2O3 and moxa leaf oil was safe and effective to achieve the effect of relieving wheezing and the combined use had synergy. Key words: Arsenic trioxide; Moxa leaf oil; Inhaled; Bronchial asthma; Guinea pig; Eosinophile

Activation of Wnt/β-catenin signaling promotes lung epithelial repair

Background: Pulmonary emphysema is characterized by airspace enlargement and impaired alveolar repair capacity. Recently, we reported that the Wnt/β-catenin pathway represent as a potential target to promote survival and repair of alveolar epithelial cells in emphysema.

Impact of oxidative stress on epithelial repair and alveolar oedema, within in vitro model of lung injury

Repair of lung injury is a complex process including epithelial growth, alveolar clearance and down‐regulation of inflammatory response. The impacts of oxidative stress in this process are still unknown. To study this question, we developed an in vitro model of lung injury to characterise the molecular and cellular mechanisms of distal lung epithelial repair. Alveolar epithelial cells in primary culture were submitted to oxidative stress with bleomycin (B : 12,5–150mU/ml) or DMNQ (D : 2,5–15μM). Bleomycin and DMNQ inhibit the epithelial repair (24–48h) following a mechanical injury in a time and concentration dependent manner (p&lt;0,0001). These agents also decrease by 60% and 80% respectively the cell migration in Boyden's chamber (p&lt;0,0001) and decrease cell proliferation measured as thymidine 3H incorporation by 95% and 70% (p&lt;0,0001). These results show that oxidative stress has an impact on alveolar epithelial cell repair by inhibiting the cell migration and proliferation processes. The impact of bleomycin on the ionic transport was studied in Ussing chamber. The treatment decreases by 37% the total as well as amiloride‐sensitive transepithelial current (p&lt;0,05). Basolateral permeabilisation in presence of a Na gradient shows that the epithelial Na channel (ENaC) is not involved in this decrease. The oxidative stress therefore in addition to inhibiting epithelial repair also decreases the Na transport involved in lung liquid clearance.Supported by RSR‐FRSQ, CCFF, CIHR.

Fibroblast growth factor-10 attenuates H2O2-induced alveolar epithelial cell DNA damage: role of MAPK activation and DNA repair.

Fibroblast growth factor-10 (FGF-10), an alveolar epithelial cell (AEC) mitogen that is critical for lung development, may promote AEC repair. We determined whether FGF-10 attenuates H2O2-induced, A549 and rat alveolar type II cell DNA damage. We show that FGF-10 prevents H2O2-induced DNA damage assessed by an alkaline elution, ethidium bromide fluorescence as well as by a comet assay. Mitogen-activated protein kinase inhibitors abolished the protective effect of FGF-10 against H2O2-induced DNA damage yet had no effect on H2O2-induced DNA damage. A Grb2-SOS inhibitor (SH3 binding peptide), an Ras inhibitor (farnesyl transferase inhibitor 277), and an Raf-1 inhibitor (forskolin) each prevented FGF-10- and H2O2-induced A549 cell ERK1/2 phosphorylation. Also, FGF-10 and H2O2 each induced negligible ERK1/2 phosphorylation in Ras dominant-negative (N17) cells. Inhibitors of Ras and Raf-1 blocked the protective effect of FGF-10 against H2O2-induced DNA damage but had no effect on H2O2-induced DNA damage. Furthermore, cold conditions and aphidicolin, an inhibitor of DNA polymerase-alpha, -delta, and -epsilon, each blocked the protective effects of FGF-10, suggesting a role for DNA repair. We conclude that FGF-10 attenuates H2O2-induced AEC DNA damage by mechanisms that involve activation of Grb2-SOS/Ras/RAF-1/ERK1/2 pathway and DNA repair.

Role of MMP-2 in Alveolar Epithelial Cell Repair after Bleomycin Administration in Rabbits

Matrix metalloproteinases (MMPs) have been implicated in the pathological processes of interstitial lung diseases. However, underlying mechanisms, particularly for activity levels and distribution of activated MMP-2 in the disease process, are yet to be elucidated. The present study investigated the immunolocalization of MMP-2, membrane type 1-matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase (TIMP)-2, p53, and Ki-67 in a rabbit model of bleomycin-induced pulmonary fibrosis. Gelatin zymography and in situ zymography were used to examine the activity and the localization of MMP-2. Furthermore, we performed Western blot and in situ hybridization for MT1-MMP, an activator for MMP-2. The total MMP-2 level estimated by gelatin zymography increased significantly at 3, 7, and 14 days after bleomycin administration, compared with controls. In the immunohistochemical study, immunoreaction for MMP-2 was strongest in alveolar epithelial cells among the cell populations. Swollen and/or elongated type II alveolar epithelial cells showed strong immunoreactions for MMP-2, MT1-MMP, and TIMP-2. After bleomycin administration, immunoreaction for p53 was observed in bronchiolar and alveolar epithelial cells. The proportion of p53-positive cells was high in epithelial cells from 1 to 14 days as MMP-2 levels were increased, suggesting that p53 may be responsible, at least in part, for the increase of MMP-2. The ratio of activated MMP-2 to total MMP-2 estimated by gelatin zymography increased significantly at 3, 7, 14, and 28 days after bleomycin treatment. In situ zymography revealed that type II alveolar epithelial cells degraded gelatin. An increased expression of MT1-MMP protein was observed by Western blot following administration of bleomycin. In situ hybridization demonstrated that type II alveolar epithelial cells gave intense signal for MT1-MMP mRNA. These results suggest that type II alveolar epithelial cells express MT1-MMP and activate MMP-2 on their cell surfaces, which may lead to the elongation and migration of alveolar epithelial cells in the repair process of bleomycin-induced pulmonary fibrosis.

Transforming growth factor-alpha enhances alveolar epithelial cell repair in a new in vitro model.

Alveolar epithelial type II cells are essential for regenerating an intact alveolar barrier after destruction of type I cells in vivo. The first objective of these experimental studies was to develop an in vitro model to quantify alveolar epithelial cell wound repair. The second objective was to investigate mechanisms of alveolar epithelial cell wound healing by studying the effects of serum and transforming growth factor-alpha (TGF-alpha) on wound closure. Primary cultures of rat alveolar type II cells were prepared by standard methods and grown to form confluent monolayers in 48 h. Then a wound was made by denuding an area (mean initial area of 2.1 +/- 0.6 mm2) of the monolayer. Re-epithelialization of the denuded area over time in the presence or absence of serum was measured using quantitative measurements from time-lapse video microscopy. The half time of wound healing was significantly enhanced in the presence of serum compared with serum-free conditions (2.4 +/- 0.2 vs. 17.4 +/- 0.8 h, P < 0.001). We then tested the hypothesis that TGF-alpha is an important growth factor for stimulating wound repair of alveolar epithelial cells. Exogenous addition of TGF-alpha in serum-free medium resulted in a significantly more rapid wound closure, and, furthermore, the addition of a monoclonal antibody to TGF-alpha in the presence of serum significantly decreased fourfold the rate of wound closure. Measurement of internuclear cell distance confirmed that both cell motility and cell spreading were responsible for closure of the wound. These data demonstrate that 1) the mechanisms of alveolar cell repair can be studied in vitro and that 2) TGF-alpha is a potent growth factor that enhances in vitro alveolar epithelial cell wound closure.