The Role of Catalase in Pulmonary Fibrosis

Background

Catalase is preferentially expressed in bronchiolar and alveolar epithelial cells, and acts as an endogenous antioxidant enzyme in normal lungs. We thus postulated epithelial damage would be associated with a functional deficiency of catalase during the development of lung fibrosis.

Methods

The present study evaluates the expression of catalase mRNA and protein in human interstitial pneumonias and in mouse bleomycin-induced lung injury. We examined the degree of bleomycin-induced inflammation and fibrosis in the mice with lowered catalase activity.

Results

In humans, catalase was decreased at the levels of activity, protein content and mRNA expression in fibrotic lungs (n = 12) compared to control lungs (n = 10). Immunohistochemistry revealed a decrease in catalase in bronchiolar epithelium and abnormal re-epithelialization in fibrotic areas. In C57BL/6J mice, catalase activity was suppressed along with downregulation of catalase mRNA in whole lung homogenates after bleomycin administration. In acatalasemic mice, neutrophilic inflammation was prolonged until 14 days, and there was a higher degree of lung fibrosis in association with a higher level of transforming growth factor-β expression and total collagen content following bleomycin treatment compared to wild-type mice.

Conclusions

Taken together, these findings demonstrate diminished catalase expression and activity in human pulmonary fibrosis and suggest the protective role of catalase against bleomycin-induced inflammation and subsequent fibrosis.     

Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia

Transgenic (TG) human (h) extracellular superoxide dismutase (EC-SOD) targeted to type II cells protects postnatal newborn mouse lung development against hyperoxia by unknown mechanisms. Because alveolar development depends on timely proliferation of type II epithelium and differentiation to type I epithelium, we measured proliferation in bronchiolar and alveolar (surfactant protein C-positive) epithelium in air and 95% O2-exposed wild-type (WT) and TG hEC-SOD newborn mice at postnatal days 3, 5, and 7 (P3-P7), traversing the transition from saccular to alveolar stages. We found that TG hEC-SOD ameliorated the 95% O2-impaired bromodeoxyuridine uptake in alveolar and bronchiolar epithelium at P3, but not at P5 and P7, when overall epithelial proliferation rates were lower in air-exposed WT mice. Mouse EC-, CuZn-, and Mn-SOD expression were unaffected by hyperoxia or genotype. TG mice had less DNA damage than 95% O2-exposed WT mice at P3, measured by TdT-mediated dUTP nick end labeling (P < 0.05). Hyperoxia induced cell-cycle inhibitory protein p21cip/waf mRNA at P3, WT > TG, P = 0.06. 95% O2 impaired apical expression of type I cell alpha protein (T1alpha) in WT but not in TG mice at P3 and increased T1alpha in WT and TG mice at P7. Reducing the 95% O2-induced impairment of epithelial proliferation at a critical window of lung development was associated with protection against DNA damage and preservation of apical T1alpha expression at P3.     

TGF-beta 1 as an enhancer of Fas-mediated apoptosis of lung epithelial cells

Transforming growth factor-beta 1 (TGF-beta 1) has important roles in lung fibrosis and the potential to induce apoptosis in several types of cells. We previously demonstrated that apoptosis of lung epithelial cells induced by Fas ligation may be involved in the development of pulmonary fibrosis. In this study, we show that TGF-beta1 induces apoptosis of primary cultured bronchiolar epithelial cells via caspase-3 activation and down-regulation of cyclin-dependent kinase inhibitor p21. Concentrations of TGF-beta 1 that were not sufficient to induce apoptosis alone could enhance agonistic anti-Fas Ab or rFas ligand-mediated apoptosis of cultured bronchiolar epithelial cells. Soluble Fas ligand in the bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) also induced apoptosis of cultured bronchiolar epithelial cells that was significantly attenuated by anti-TGF-beta Ab. Otherwise, BALF from patients with hypersensitivity pneumonitis (HP) could not induce apoptosis on bronchiolar epithelial cells, despite its comparable amounts of soluble Fas ligand. The concentrations of TGF-beta 1 in BALF from patients with IPF were significantly higher compared with those in BALF from patients with HP or controls. Furthermore, coincubation with the low concentration of TGF-beta 1 and HP BALF created proapoptotic effects comparable with the IPF BALF. In vivo, the administration of TGF-beta 1 could enhance Fas-mediated epithelial cell apoptosis and lung injury via caspase-3 activation in mice. Our results demonstrate a novel role of TGF-beta 1 in the pathophysiology of pulmonary fibrosis as an enhancer of Fas-mediated apoptosis of lung epithelial cells.     

Stat3 is required for cytoprotection of the respiratory epithelium during adenoviral infection

The role of Stat3 in the maintenance of pulmonary homeostasis following adenoviral-mediated lung injury was assessed in vivo. Stat3 was selectively deleted from bronchiolar and alveolar epithelial cells in Stat3(DeltaDelta) mice. Although lung histology and function were unaltered by deletion of Stat3 in vivo, Stat3(DeltaDelta) mice were highly susceptible to lung injury caused by intratracheal administration of AV1-GFP, an early (E) region 1- and E3-deleted, nonproliferative adenovirus. Severe airspace enlargement, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in Stat3(DeltaDelta) mice as early as 1 day after exposure to the virus. Although surfactant protein A, B, and C content and surfactant protein-B mRNA expression in Stat3(DeltaDelta) mice were similar, TUNEL staining and caspase-3 were increased in alveolar type II epithelial cells of Stat3(DeltaDelta) mice after exposure to virus. RNA microarray analysis of type II epithelial cells isolated from Stat3(DeltaDelta) mice demonstrated significant changes in expression of numerous genes, including those genes regulating apoptosis, supporting the concept that the susceptibility of Stat3-deficient mice to adenovirus was related to the role of Stat3 in the regulation of cell survival. AV1-Bcl-x(L), an E1- and E3-deleted, nonproliferative adenovirus expressing the antiapoptotic protein Bcl-x(L), protected Stat3(DeltaDelta) mice from adenoviral-induced lung injury. Adenoviral infection of the lungs of Stat3-deficient mice was associated with severe injury of the alveolar and bronchiolar epithelium. Thus, Stat3 plays a critical cytoprotective role that is required for epithelial cell survival and maintenance of alveolar structures during the early phases of pulmonary adenoviral infection.     

Conditional expression of transforming growth factor-alpha in adult mouse lung causes pulmonary fibrosis

To determine whether overexpression of transforming growth factor (TGF)-alpha in the adult lung causes remodeling independently of developmental influences, we generated conditional transgenic mice expressing TGF-alpha in the epithelium under control of the doxycycline (Dox)-regulatable Clara cell secretory protein promoter. Two transgenic lines were generated, and following 4 days of Dox-induction TGF-alpha levels in whole lung homogenate were increased 13- to 18-fold above nontransgenic levels. After TGF-alpha induction, transgenic mice developed progressive pulmonary fibrosis and body weight loss, with mice losing 15% of their weight after 6 wk of TGF-alpha induction. Fibrosis was detected within 4 days of TGF-alpha induction and developed initially in the perivascular, peribronchial, and pleural regions but later extended into the interstitium. Fibrotic regions were composed of increased collagen and cellular proliferation and were adjacent to airway and alveolar epithelial sites of TGF-alpha expression. Fibrosis progressed in the absence of inflammatory cell infiltrates as determined by histology, without changes in bronchiolar alveolar lavage total or differential cell counts and without changes in proinflammatory cytokines TNF-alpha or IL-6. Active TGF-beta in whole lung homogenate was not altered 1 and 4 days after TGF-alpha induction, and immunostaining was not increased in the peribronchial/perivascular areas at all time points. Chronic epithelial expression of TGF-alpha in adult mice caused progressive pulmonary fibrosis associated with increased collagen and extracellular matrix deposition and increased cellular proliferation. Induction of pulmonary fibrosis by TGF-alpha was independent of inflammation or early activation of TGF-beta.     

Comparative proteomic analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and lung transplant donor lungs

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease for which no effective therapy exists to date. To identify the molecular mechanisms underlying IPF, we performed comparative proteome analysis of lung tissue from patients with sporadic IPF (n = 14) and human donor lungs (controls, n = 10) using two-dimensional gel electrophoresis and MALDI-TOF-MS. Eighty-nine differentially expressed proteins were identified, from which 51 were up-regulated and 38 down-regulated in IPF. Increased expression of markers for the unfolded protein response (UPR), heat-shock proteins, and DNA damage stress markers indicated a chronic cell stress-response in IPF lungs. By means of immunohistochemistry, induction of UPR markers was encountered in type-II alveolar epithelial cells of IPF but not of control lungs. In contrast, up-regulation of heat-shock protein 27 (Hsp27) was exclusively observed in proliferating bronchiolar basal cells and associated with aberrant re-epithelialization at the bronchiolo-alveolar junctions. Among the down-regulated proteins in IPF were antioxidants, members of the annexin family, and structural epithelial proteins. In summary, our results indicate that IPF is characterized by epithelial cell injury, apoptosis, and aberrant epithelial proliferation.     

Gamma-glutamyl transferase deficiency results in lung oxidant stress in normoxia

gamma-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGT(enu1) mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGT(enu1) lung. Glutathione content was barely decreased in GGT(enu1) lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGT(enu1) alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGT(enu1) lung. When GGT(enu1) mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.     

Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin

To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-kappa B activation in the respiratory epithelium using a mutant I kappa B-alpha construct that functioned as a dominant negative inhibitor of NF-kappa B translocation (dnI kappa B-alpha). We developed two lines of transgenic mice in which expression of dnI kappa B-alpha was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-kappa B was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-alpha, IL-1 beta, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-alpha within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-kappa B activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.     

The alpha-isoform of caveolin-1 is a marker of vasculogenesis in early lung development.

Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5' variant that will translate mainly into caveolin-1alpha and -beta isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1alpha localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1alpha is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1alpha is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1alpha is also expressed in alveolar Type I cells.     

Reduction of Smad3 accelerates re-epithelialization in a murine model of colitis

To determine the role of Smad3 in re-epithelialization and inflammation, experimental colitis was induced in Smad3 heterozygous mice and their wild-type littermates by single intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol. The area of epithelial deficiency was significantly reduced in the heterozygotes on the 4th-6th day after TNBS administration as compared to the controls although the number of inflammatory cells in the colonic mucosa in the heterozygotes and their wild-type littermates varied similarly throughout the course of colitis. Proliferation of the intestinal epithelium in the heterozygotes was significantly accelerated as compared to that in the wild-type controls on the 1st and 2nd days after TNBS administration. These results suggest that reduction of Smad3 significantly accelerates re-epithelialization of the intestinal mucosa without enhancing inflammation. Suppression of TGF-beta1 induction in the colonic mucosa of the heterozygotes may lead to a higher level of proliferation of intestinal epithelial cells.     

VEGF induces airway epithelial cell proliferation in human fetal lung in vitro

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen involved in normal and abnormal angiogenesis. VEGF mRNA and protein are abundant in distal epithelium of midtrimester human fetal lung. In the present study, we identified immunoreactivity for KDR, a major VEGF-specific receptor, in distal lung epithelial cells of human fetal lung tissue, suggesting a possible autocrine or paracrine regulatory role for VEGF in pulmonary epithelial cell growth and differentiation. Addition of exogenous VEGF to human fetal lung explants resulted in increased epithelium volume density and lumen volume density in the tissues, both morphometric parameters of tissue differentiation. Cellular proliferation demonstrated by bromodeoxyuridine uptake was prominent in distal airway epithelial cells and increased in the VEGF-treated explants. VEGF-treated explants also demonstrated increased surfactant protein (SP) A mRNA, SP-C mRNA, and SP-A protein levels compared with controls. However, SP-B mRNA levels were unaffected by VEGF treatment. [(3)H]choline incorporation into total phosphatidylcholine was increased by VEGF treatment, but incorporation into disaturated phosphatidylcholine was not affected by exogenous VEGF. Based on these observations, we conclude that VEGF may be an important autocrine growth factor for distal airway epithelial cells in the developing human lung.     

Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb

BACKGROUND: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. METHODS: In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. RESULTS: We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. CONCLUSION: This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.     

Protein Kinase D Is Increased and Activated in Lung Epithelial Cells and Macrophages in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.     

IL-12 reduces the severity of Sendai virus-induced bronchiolar inflammation and remodeling

The goal of this research was to determine whether differential pulmonary IL-12 gene expression controls susceptibility to Sendai virus-induced chronic airway inflammation and fibrosis in inbred rat strains. Sendai virus-resistant F344 rats and susceptible BN rats were studied from 1 to 14 days following virus inoculation. F344 rats had 3.4-fold higher IL-12 mRNA levels detected by real-time PCR in lung than BN rats as early as two days following inoculation. This increase in mRNA was associated at two days with increased total IL-12 protein and with a 2-fold increase in numbers of bronchiolar, OX-6-positive dendritic cells and an increased number of IL-12 p40-positive, bronchiolar macrophages and dendritic cells (p<0.05). Virus-susceptible BN rats treated with 3 mug of recombinant, mouse IL-12 intraperitoneally at the time of virus inoculation had a 22.1% decrease in severity of chronic bronchiolar inflammation and a 23.8% decrease in fibrosis compared to virus-inoculated BN rats treated with saline. IL-12 treatment induced increased IFN-gamma mRNA and protein expression after virus inoculation (p<0.05). The results demonstrate that there is differential pulmonary IL-12 gene expression between virus-susceptible and resistant rat strains and that IL-12 treatment can provide significant protection from virus-induced chronic airway inflammation and remodeling during early life.     

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by alpha-smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.     

siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway

Caveolin-1, the principal integral membrane protein of caveolae, has been implicated in regulating the structural integrity of caveolae, vesicular trafficking, and signal transduction. Although the functions of caveolin-1 are beginning to be explored in caveolin-1-/- mice, these results are confounded by unknown compensatory mechanisms and the development of pulmonary hypertension, cardiomyopathy, and lung fibrosis. To address the role of caveolin-1 in regulating lung vascular permeability, in the present study we used small interfering RNA (siRNA) to knock down caveolin-1 expression in mouse lung endothelia in vivo. Intravenous injection of siRNA against caveolin-1 mRNA incorporated in liposomes selectively reduced the expression of caveolin-1 by approximately 90% within 96 h of injection compared with wild-type mice. We observed the concomitant disappearance of caveolae in lung vessel endothelia and dilated interendothelial junctions (IEJs) as well as increased lung vascular permeability to albumin via IEJs. The reduced caveolin-1 expression also resulted in increased plasma nitric oxide concentration. The nitric oxide synthase inhibitor L-NAME, in part, blocked the increased vascular albumin permeability. These morphological and functional effects of caveolin-1 knockdown were reversible within 168 h after siRNA injection, corresponding to the restoration of caveolin-1 expression. Thus our results demonstrate the essential requirement of caveolin-1 in mediating the formation of caveolae in endothelial cells in vivo and in negatively regulating IEJ permeability.     

Lack of evidence for caveolin-1 and CD147 interaction before and after bleomycin-induced lung injury

Immunohistochemical and in vitro studies indicate that caveolin-1, which occurs abundantly in alveolar epithelial type I cells and microvascular endothelial cells of the lung, is selectively downregulated in the alveolar epithelium following exposure to bleomycin. Bleomycin is also known to enhance the expression levels of metalloproteinases and of the metalloproteinase inducer CD147/EMMPRIN in lung cells. Experimental in vitro data has showed that MMP-inducing activity of CD147 is under the control of caveolin-1. We studied the effects of bleomycin on the expression of caveolin-1, CD147 and metalloproteinases using an alveolar epithelial rat cell line R3/1 with properties of both alveolar type I and type II cells and explanted rat lung slices. In parallel, retrospective samples of bleomycin-induced fibrosis in rats and mice as well as samples of wild type and caveolin-1 knockout animals were included for immunohistochemical comparison with in vitro data. Here we report that treatment with bleomycin downregulates caveolin-1 and increases CD147 and MMP-2 and -9 expression/activity in R3/1 cells using RT-PCR, Western blot analysis, MMP-2 activity assay and immunocytochemistry. Immunofluorescence double labeling revealed that caveolin-1 and CD147 were not colocalized in vitro. The in vitro findings were confirmed through immunohistochemical studies of the proteins in paraffin embedded precision-cut rat lung slices and in fibrotic rat lung tissues. The caveolin-1-negative hyperplastic ATII cells exhibited enhanced immunoreactivity for CD147 and MMP-2. Caveolin-1-negative ATI cells of fibrotic samples were mostly CD147 negative. There were no differences in the pulmonary expression of CD147 between the normal and caveolin-1 deficient animals. The results demonstrate that bleomycin-induced lung injury is associated with an increase in CD147 expression and MMP activity, particularly in alveolar epithelial cells. In addition, our data exclude any functional interaction between CD147 and alveolar epithelial caveolin-1.     

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis

Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca^2 +-activated Cl^? channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl^? channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl^? channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.