Airway Epithelial Wound Repair Research Articles

P039 Lactonase benefits to counteract the impacts of Pseudomonas aeruginosa virulence on the early phases of airway epithelial wound repair in cystic fibrosis

P039 Lactonase benefits to counteract the impacts of Pseudomonas aeruginosa virulence on the early phases of airway epithelial wound repair in cystic fibrosis

Distinct Effects of Respiratory Viral Infection Models on miR-149-5p, IL-6 and p63 Expression in BEAS-2B and A549 Epithelial Cells.

Respiratory viruses cause airway inflammation, resulting in epithelial injury and repair. miRNAs, including miR-149-5p, regulate different pathological conditions. We aimed to determine how miR-149-5p functions in regulating pro-inflammatory IL-6 and p63, key regulators of airway epithelial wound repair, in response to viral proteins in bronchial (BEAS-2B) and alveolar (A549) epithelial cells. BEAS-2B or A549 cells were incubated with poly (I:C, 0.5 µg/mL) for 48 h or SARS-CoV-2 spike protein-1 or 2 subunit (S1 or S2, 1 μg/mL) for 24 h. miR-149-5p was suppressed in BEAS-2B challenged with poly (I:C), correlating with IL-6 and p63 upregulation. miR-149-5p was down-regulated in A549 stimulated with poly (I:C); IL-6 expression increased, but p63 protein levels were undetectable. miR-149-5p remained unchanged in cells exposed to S1 or S2, while S1 transfection increased IL-6 expression in BEAS-2B cells. Ectopic over-expression of miR-149-5p in BEAS-2B cells suppressed IL-6 and p63 mRNA levels and inhibited poly (I:C)-induced IL-6 and p63 mRNA expressions. miR-149-5p directly suppressed IL-6 mRNA in BEAS-2B cells. Hence, BEAS-2B cells respond differently to poly (I:C), S1 or S2 compared to A549 cells. Thus, miR-149-5p dysregulation may be involved in poly (I:C)-stimulated but not S1- or S2-stimulated increased IL-6 production and p63 expression in BEAS-2B cells.

Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze.

The airway epithelium of children with wheeze is characterized by defective repair that contributes to disease pathobiology. Dysregulation of developmental processes controlled by Notch has been identified in chronic asthma. However, its role in airway epithelial cells of young children with wheeze, particularly during repair, is yet to be determined. We hypothesized that Notch is dysregulated in primary airway epithelial cells (pAEC) of children with wheeze contributing to defective repair. This study investigated transcriptional and protein expression and function of Notch in pAEC isolated from children with and without wheeze. Primary AEC of children with and without wheeze were found to express all known Notch receptors and ligands, although pAEC from children with wheeze expressed significantly lower NOTCH2 (10-fold, p = 0.004) and higher JAG1 (3.5-fold, p = 0.002) mRNA levels. These dysregulations were maintained in vitro and cultures from children with wheeze displayed altered kinetics of both NOTCH2 and JAG1 expression during repair. Following Notch signaling inhibition, pAEC from children without wheeze failed to repair (wound closure rate of 76.9 ± 3.2%). Overexpression of NOTCH2 in pAEC from children with wheeze failed to rescue epithelial repair following wounding. This study illustrates the involvement of the Notch pathway in airway epithelial wound repair in health and disease, where its dysregulation may contribute to asthma development.

Bronchial epithelium repair by Esculentin-1a-derived antimicrobial peptides: involvement of metalloproteinase-9 and interleukin-8, and evaluation of peptides\u2019 immunogenicity

The airway epithelium is seriously damaged upon pulmonary Pseudomonas aeruginosa infection, especially in cystic fibrosis (CF) sufferers. Therefore, the discovery of novel anti-infective agents accelerating healing of infected injured tissues is crucial. The antipseudomonal peptides esculentin-1a(1–21)NH2 and its diastereomer Esc(1–21)-1c (Esc peptides) hold promise in this respect. In fact, they stimulate airway epithelial wound repair, but no mechanistic insights are available. Here we demonstrated that this process occurs through promotion of cell migration by an indirect activation of epidermal growth factor receptor mediated by metalloproteinases. Furthermore, we showed an increased expression of metalloproteinase 9, at both gene and protein levels, in peptide-treated bronchial epithelial cells with a functional or mutated form of CF transmembrane conductance regulator. In addition, the two peptides counteracted the inhibitory effect of Pseudomonas lipopolysaccharide (mimicking an infection condition) on the wound healing activity of the airway epithelium, and they enhanced the production of interleukin-8 from both types of cells. Finally, no immunogenicity was discovered for Esc peptides, suggesting their potential safety for clinical usage. Besides representing a step forward in understanding the molecular mechanism underlying the peptide-induced wound healing activity, these studies have contributed to highlight Esc peptides as valuable therapeutics with multiple functions.

IL-13 signaling through IL-13 receptor α2 mediates airway epithelial wound repair.

Asthma is an airway inflammatory disease characterized by epithelial barrier dysfunction and airway remodeling. Interleukin-13 (IL-13) is a pleiotropic cytokine shown to contribute to features of airway remodeling. We have previously demonstrated that IL-13 is an important mediator of normal airway epithelial repair and health. The role of IL-13 signaling via its receptor subunits (IL-13Rα1/IL-4Rα and IL-13Rα2) in airway epithelial repair and restoration of intact barrier function is not well understood and was investigated in this study using in vitro models. The blocking of IL-13 signaling via IL-13Rα2 significantly reduced airway epithelial repair by 24 h post-mechanical wounding in 1HAEo- cells. Expression and release of repair-mediating growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and subsequent activation of EGF receptor (EGFR) were also significantly reduced in response to wounding when IL-13Rα2 was blocked. Our data support that IL-13 signals via IL-13Rα2 to mediate normal airway epithelial repair via HB-EGF-dependent activation of EGFR. In human donor lung tissues, we observed that airway epithelium of asthmatics expressed significantly decreased levels of IL-13Rα2 and increased levels of IL-13Rα1 compared with nonasthmatics. Dysregulated expression of IL-13 receptor subunits in the airways of asthmatics may thus contribute to the epithelial barrier dysfunction observed in asthma.-Yang, S. J., Allahverdian, S., Saunders, A. D. R., Liu, E., Dorscheid, D. R. IL-13 signaling through IL-13 receptor α2 mediates airway epithelial wound repair.

CTNNAL1 inhibits ozone-induced epithelial-mesenchymal transition in human bronchial epithelial cells.

What is the central question of this study? What is the effect of catenin alpha-like 1 (CTNNAL1), an asthma-related epithelial adhesion molecule that plays a vital role in airway epithelial wound repair, on airway epithelial-mesenchymal transition? What is the main finding and its importance? CTNNAL1 inhibits ozone-induced airway epithelial-mesenchymal transition features, mediated by repressing the expression of Twist1 mRNA and reducing TGF-β1 levels. These findings contribute to our understanding of the pathology of airway EMT and may indicate a possible therapeutic target for airway remodelling in bronchial asthma. Epithelial-mesenchymal transition (EMT), a crucial event occurring during epithelial and mesenchymal repair, was reported to be a possible mechanism for airway remodelling. Our previous work showed that the expression of catenin alpha-like 1 (CTNNAL1) was down-regulated in the bronchial epithelial cells of asthmatic models and played a vital role in airway epithelial wound repair. The aim of this study was to investigate the effect of CTNNAL1 on airway EMT. Overexpression or silencing of CTNNAL1 in human bronchial epithelial cells was induced by stable transfection. CTNNAL1 was silenced in primary mouse airway epithelial cells with an effective siRNA vector. Cells were stressed by ozone for 4days at 30minday-1 to induce EMT. EMT features, changes in the function of co-cultured lung fibroblasts, changes in the expression of the transcriptional repressors Snail/Slug and Twist1/Twist2 and changes in the secretion of transforming growth factorβ1 (TGF-β1) were assayed in different cell lines with or without ozone exposure. Both ozone exposure and silencing of CTNNAL1 induced EMT features in airway epithelial cells. Functional changes in lung fibroblasts increased after co-culture with (ozone-stressed) CTNNAL1-silenced cells. Snail and Twist1 expression increased, and the level of TGF-β1 was enhanced. Conversely, CTNNAL1 overexpression reversed EMT features, repressed mRNA levels of Twist1 and reduced the secretion of TGF-β1, both alone and in combination with ozone exposure. Our results indicate that ozone exposure induces airway EMT and that CTNNAL1 inhibits ozone-induced airway EMT. CTNNAL1 may play a role in airway EMT by repressing the expression of Twist1 mRNA and reducing the level of TGF-β1.

Dimethylarginine dimethylaminohydrolase (DDAH) overexpression enhances wound repair in airway epithelial cells exposed to agricultural organic dust

Objective: Workers exposed to dusts from concentrated animal feeding operations have a high prevalence of pulmonary diseases. These exposures lead to chronic inflammation and aberrant airway remodeling. Previous work shows that activating cAMP-dependent protein kinase (PKA) enhances airway epithelial wound repair while activating protein kinase C (PKC) inhibits wound repair. Hog barn dust extracts slow cell migration and wound repair via a PKC-dependent mechanism. Further, blocking nitric oxide (NO) production in bronchial epithelial cells prevents PKA activation. We hypothesized that blocking an endogenous NO inhibitor, asymmetric dimethylarginine, by overexpressing dimethylarginine dimethylaminohydrolase mitigates the effects of hog dust extract on airway epithelial would repair.Materials/methods: We cultured primary tracheal epithelial cells in monolayers from both wild-type (WT) and dimethylarginine dimethylaminohydrolase overexpressing C57Bl/6 (DDAH1 transgenic) mice and measured wound repair using the electric cell impedance sensing system.Results: Wound closure in epithelial cells from WT mice occurred within 24 h in vitro. In contrast, treatment of the WT cell monolayers with 5% hog dust extract prevented significant NO-stimulated wound closure. In cells from DDAH1 transgenic mice, control wounds were repaired up to 8 h earlier than seen in WT mice. A significant enhancement of wound repair was observed in DDAH cells compared to WT cells treated with hog dust extract for 24 h. Likewise, cells from DDAH1 transgenic mice demonstrated increased NO and PKA activity and decreased hog dust extract-stimulated PKC.Discussion/conclusion: Preserving the NO signal through endogenous inhibition of asymmetric dimethylarginine enhances wound repair even in the presence of dust exposure.

Airway Epithelial Repair by a Prebiotic Mannan Derived from Saccharomyces cerevisiae.

In asthmatic airways, repeated epithelial damage and repair occur. No current therapy directly targets this process. We aimed to determine the effects of mannan derived from S. cerevisiae (SC-MN) on airway epithelial wound repair, in vitro. The presence of functional mannose receptors in bronchial epithelial cells was shown by endocytosis of colloidal gold-Man BSA via clathrin-coated pits in 16HBE cells. In primary normal human bronchial epithelial cells (NHBEC), SC-MN significantly facilitated wound closure. Treatment with SC-MN stimulated cell spreading as indicated by a significant increase in the average lamellipodial width of wound edge 16HBE cells. In addition, NHBEC treated with SC-MN showed increased expression and activation of Krüppel-like factors (KLFs) 4 and 5, transcription factors important in epithelial cell survival and regulation of epithelial-mesenchymal transition. We conclude that SC-MN facilitates wound repair in human bronchial epithelium, involving mannose receptors.

Role of mitochondrial hydrogen peroxide induced by intermittent hypoxia in airway epithelial wound repair in vitro

The airway epithelium acts as a frontline barrier against various environmental insults and its repair process after airway injury is critical for the lung homeostasis restoration. Recently, the role of intracellular reactive oxygen species (ROS) as transcription-independent damage signaling has been highlighted in the wound repair process. Both conditions of continuous hypoxia and intermittent hypoxia (IH) induce ROS. Although IH is important in clinical settings, the roles of IH-induced ROS in the airway repair process have not been investigated. In this study, we firstly showed that IH induced mitochondrial hydrogen peroxide (H2O2) production and significantly decreased bronchial epithelial cell migration, prevented by catalase treatment in a wound scratch assay. RhoA activity was higher during repair process in the IH condition compared to in the normoxic condition, resulting in the cellular morphological changes shown by immunofluorescence staining: round cells, reduced central stress fiber numbers, pronounced cortical actin filament distributions, and punctate focal adhesions. These phenotypes were replicated by exogenous H2O2 treatment under the normoxic condition. Our findings confirmed the transcription-independent role of IH-induced intracellular ROS in the bronchial epithelial cell repair process and might have significant implications for impaired bronchial epithelial cell regeneration.

Delay of airway epithelial wound repair in COPD is associated with airflow obstruction severity.

BackgroundAirway epithelium integrity is essential to maintain its role of mechanical and functional barrier. Recurrent epithelial injuries require a complex mechanism of repair to restore its integrity. In chronic obstructive pulmonary disease (COPD), an abnormal airway epithelial repair may participate in airway remodeling. The objective was to determine if airway epithelial wound repair of airway epithelium is abnormal in COPD.MethodsPatients scheduled for lung resection were prospectively recruited. Demographic, clinical data and pulmonary function tests results were recorded. Emphysema was visually scored and histological remodeling features were noted. Primary bronchial epithelial cells (BEC) were extracted and cultured for wound closure assay. We determined the mean speed of wound closure (MSWC) and cell proliferation index, matrix metalloprotease (MMP)-2, MMP-9 and cytokines levels in supernatants of BEC 18 hours after cell wounding. In a subset of patients, bronchiolar epithelial cells were also cultured for wound closure assay for MSWC analyze.Results13 COPD and 7 non COPD patients were included. The severity of airflow obstruction and the severity of emphysema were associated with a lower MSWC in BEC (p = 0.01, 95% CI [0.15-0.80]; p = 0.04, 95% CI [−0.77;-0.03] respectively). Cell proliferation index was decreased in COPD patients (19 ± 6% in COPD vs 27 ± 3% in non COPD, p = 0.04). The severity of COPD was associated with a lower level of MMP-2 (7.8 ± 2 105 AU in COPD GOLD D vs 12.8 ± 0.13 105 AU in COPD GOLD A, p = 0.04) and a lower level of IL-4 (p = 0.03, 95% CI [0.09;0.87]). Moreover, higher levels of IL-4 and IL-2 were associated with a higher MSWC (p = 0.01, 95% CI [0.17;0.89] and p = 0.02, 95% CI [0.09;0.87] respectively). Clinical characteristics and smoking history were not associated with MSWC, cell proliferation index or MMP and cytokines levels. Finally, we showed an association of the MSWC of bronchial and corresponding bronchiolar epithelial cells obtained from the same patients (p = 0.02, 95% CI [0.12;0.89]).ConclusionOur results showed an abnormal bronchial epithelial wound closure process in severe COPD. Further studies are needed to elucidate the contribution and the regulation of this mechanism in the complex pathophysiology of COPD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-014-0151-9) contains supplementary material, which is available to authorized users.

Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue

Airway damage and remodelling are important components of lung pathology progression in cystic fibrosis (CF). Although repair mechanisms are engaged to restore the epithelial integrity, these processes are obviously insufficient to maintain lung function in CF airways. Our aims were therefore to study how the basic cystic fibrosis transmembrane conductance regulator (CFTR) defect could impact epithelial wound healing and to determine if CFTR correction could improve it. Wound-healing experiments, as well as cell migration and proliferation assays, were performed to study the early phases of epithelial repair in human CF and non-CF airway cells. CFTR function was evaluated using CFTR small interferring (si)RNA and inhibitor GlyH101 in non-CF cells, and conversely after CFTR rescue with the CFTR corrector VRT-325 in CF cells. Wound-healing experiments first showed that airway cells from CF patients repaired slower than non-CF cells. CFTR inhibition or silencing in non-CF primary airway cells significantly inhibited wound closure. GlyH101 also decreased cell migration and proliferation. Interestingly, wild-type CFTR transduction in CF airway cell lines or CFTR correction with VRT-325 in CFBE-ΔF508 and primary CF bronchial monolayers significantly improved wound healing. Altogether our results demonstrated that functional CFTR plays a critical role in wound repair, and CFTR correction may represent a novel strategy to promote the airway repair processes in CF.

CFTR channels and wound healing. Focus on “Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair”

cystic fibrosis (CF) is caused by severe loss-of-function mutations in the CFTR chloride channel ([9][1]). The predominant life-limiting pathology in CF patients is recurrent bacterial infections in the airways, which is attributable at least in part to reduced mucociliary clearance by the

Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair

The role of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial wound repair was investigated using normal human bronchial epithelial (NHBE) cells and a human airway epithelial cell line (Calu-3) of serous gland origin. Measurements of wound repair were performed using continuous impedance sensing to determine the time course for wound closure. Control experiments showed that the increase in impedance corresponding to cell migration into the wound was blocked by treatment with the actin polymerization inhibitor, cytochalasin D. Time lapse imaging revealed that NHBE and Calu-3 cell wound closure was dependent on cell migration, and that movement occurred as a collective sheet of cells. Selective inhibition of CFTR activity with CFTR(inh)-172 or short hairpin RNA silencing of CFTR expression produced a significant delay in wound repair. The CF cell line UNCCF1T also exhibited significantly slower migration than comparable normal airway epithelial cells. Inhibition of CFTR-dependent anion transport by treatment with CFTR(inh)-172 slowed wound closure to the same extent as silencing CFTR protein expression, indicating that ion transport by CFTR plays a critical role in migration. Moreover, morphologic analysis of migrating cells revealed that CFTR inhibition or silencing significantly reduced lamellipodia protrusion. These findings support the conclusion that CFTR participates in airway epithelial wound repair by a mechanism involving anion transport that is coupled to the regulation of lamellipodia protrusion at the leading edge of the cell.

Sialyl Lewis X modification of the epidermal growth factor receptor regulates receptor function during airway epithelial wound repair

Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N-glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLe(x)) in airway epithelial repair. To examine whether an sLe(x) decoration of EGFR can modulate receptor function during AEC repair. Primary normal human bronchial epithelial (NHBE) cells were cultured in vitro. Co-localization of sLe(x) and EGFR was examined using confocal microscopy. Expressions of RNA and protein were analysed using RT-PCR and Western blotting. The final step in the synthesis of sLe(x) was catalysed by a specific alpha-1,3-fucosyltransferase (FucT-IV). To evaluate the role of sLe(x) in EGFR activation, a knockdown of the FucT-IV gene with small interfering RNA (siRNA) and an inhibitory anti-sLe(x) antibody (KM-93) was used. We demonstrated a co-localization of sLe(x) with EGFR on NHBE cells using confocal microscopy. Using a blocking antibody for sLe(x) after a mechanical injury, we observed a reduction in EGFR phosphorylation and epithelial repair following injury. FucT-IV demonstrates a temporal expression coordinate with epithelial repair. Down-regulation of FucT-IV expression in NHBE by specific siRNA suppressed sLe(x) expression. The use of FucT-IV siRNA significantly reduced phosphorylation of EGFR and prevented epithelial repair. An immunohistochemical analysis of human normal and asthmatic airways showed a significant reduction in sLe(x) and tyrosine-phosphorylated EGFR (pY(845)-EGFR) in the epithelium of asthmatic subjects compared with that of normal subjects. The present data demonstrate that sLe(x), in association with EGFR, in NHBE is coordinate with repair. This glycosylation is important in modulating EGFR activity to affect the repair of normal primary AEC.

Glycosylation and annexin II cell surface translocation mediate airway epithelial wound repair

Glycosylation of cell surface proteins can regulate multiple cellular functions. We hypothesized that glycosylation and expression of glycoproteins after epithelial injury is important in mediating repair. We report the use of an in vitro culture model of human airway epithelial cells (1HAEo(-)) to identify mediators of epithelial repair. We characterized carbohydrate moieties associated with repair by their interaction with the lectin from Cicer arietinum, chickpea agglutinin (CPA). Using CPA, we identified changes in cell surface glycosylation during wound repair. Following mechanical wounding of confluent monolayers of 1HAEo(-) cells, CPA staining increases on the cell surface of groups of cells in proximity to the wound edge. Blocking the CPA carbohydrate ligand inhibited wound repair highlighting the role of the CPA carbohydrate ligand in epithelial repair. Annexin II (AII), a calcium-dependent, membrane-associated protein, was identified as a protein associated with the CPA ligand. By membrane protein biotinylation and immunodetection, we have shown that following mechanical wounding, the presentation of AII on the cell surface increases coordinate with repair. Cell surface AII accumulates in proximity to the wound. Furthermore, translocation of AII to the cell surface is N-glycosylation dependent. We are the first to demonstrate that following injury, N-glycosylation events and AII presentation on the cell surface of airway epithelial cells are important mediators in repair.

Airway Epithelial Repair, Regeneration, and Remodeling after Injury in Chronic Obstructive Pulmonary Disease

In chronic obstructive pulmonary disease (COPD), exacerbations are generally associated with several causes, including pollutants, viruses, bacteria that are responsible for an excess of inflammatory mediators, and proinflammatory cytokines released by activated epithelial and inflammatory cells. The normal response of the airway surface epithelium to injury includes a succession of cellular events, varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even complete denudation of the basement membrane. The epithelium then has to repair and regenerate to restore its functions, through several mechanisms, including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. In COPD, the remodeling of the airway epithelium, such as squamous metaplasia and mucous hyperplasia that occur during injury, may considerably disturb the innate immune functions of the airway epithelium. In vitro and in vivo models of airway epithelial wound repair and regeneration allow the study of the spatiotemporal modulation of cellular and molecular interaction factors-namely, the proinflammatory cytokines, the matrix metalloproteinases and their inhibitors, and the intercellular adhesion molecules. These factors may be markedly altered during exacerbation periods of COPD and their dysregulation may induce remodeling of the airway mucosa and a leakiness of the airway surface epithelium. More knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to cytoprotective and regenerative therapeutics, allowing the reconstitution of a functional, well-differentiated airway epithelium in COPD.

Nitric Oxide Promotes Airway Epithelial Wound Repair through Enhanced Activation of MMP-9

The airway epithelium provides a protective barrier against inhaled environmental toxins and microorganisms, and epithelial injury initiates a number of processes to restore its barrier integrity, including activation of matrix metalloproteinases such as MMP-9 (92-kD gelatinase B). Airway epithelial cells continuously produce nitric oxide (NO), which has been linked to cell migration and MMP-9 regulation in several cell types, but the importance of epithelial NO in mediating airway epithelial repair or MMP-9 activation is unknown. Using primary or immortalized human bronchial epithelial cells, we demonstrate that low concentrations of NO promote epithelial cell migration and wound repair in an in vitro wound assay, which was associated with increased localized expression and activation of MMP-9. In addition, in HBE1 cells that were stably transfected with inducible NOS (NOS2), to mimic constitutive epithelial NOS2 expression in vivo, NOS inhibition decreased epithelial wound repair and MMP-9 expression. The stimulatory effects of NO on epithelial wound repair and MMP-9 expression were dependent on cGMP-mediated pathways and were inhibited by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase. Inhibition of cGMP-dependent protein kinase (PKG) attenuated NO-mediated epithelial wound closure, but did not affect MMP-9 expression. However, pharmacologic MMP inhibition and siRNA knockdown of MMP-9 expression demonstrated the contribution of MMP-9 to NO-mediated wound closure. Overall, our results demonstrate that NOS2-derived NO contributes to airway epithelial repair by both PKG-dependent and -independent mechanisms, and involves NO-dependent expression and activation of MMP-9.

Pseudomonas Aeruginosa Virulence Factors Delay Airway Epithelial Wound Repair by Altering the Actin Cytoskeleton and Inducing Overactivation of Epithelial Matrix Metalloproteinase–2

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5 α had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.

Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium.

Cell migration is the earliest mechanism involved in the wound repair process of the respiratory epithelium and could be potentially enhanced by growth factors. In the present work, we investigated the localisation of the hepatocyte growth factor (HGF) receptor (c-Met) during wound repair and evaluated the effect of recombinant HGF (rHGF) on cell migration by using an in vitro model of airway epithelial wound repair. By using immunohistochemical methods, we observed that the immunoreactivity of the c-Met proto-oncogene was increased in epithelial cells engaged in the process of tissue repair. The incubation of wounded cultures with increasing concentrations of rHGF (0.2, 2, 20, and 200 ng/ml) induced a significant (P < 0.02) dose-dependent effect on the wound repair index, with a maximum effect produced at 20 ng/ml (+31.3%). The cell migration speed reached 50.2 micrometer/h at this concentration, compared to 20.4 micrometer/h in the absence of rHGF. No significant effect on cell proliferation was observed in the repairing area in the presence of rHGF. These results suggest that rHGF is able to improve the wound repair process of the airway epithelium by increasing cell migration.