Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis.
ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung.Using oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to alphavbeta3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-alphavbeta3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7.Our results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease.
Project description:Idiopathic pulmonary fibrosis (IPF), the most common interstitial lung disease, arises from transforming growth factor beta 1- (TGF?1-) induced aberrant fibroproliferation in response to epithelial injury. The TGF?1-) induced aberrant fibroproliferation in response to epithelial injury. The TGF. We first performed microarray data mining of previously published gene expression datasets to identify key gene signatures in IPF lung tissues. HYAL1 expression levels in IPF and normal lung tissues were then characterized using immunohistochemistry followed by real-time quantitative reverse transcription-PCR (qRT-PCR) and western blot analysis on isolated fibroblasts from fresh lung tissues of IPF and healthy donors. A human fetal lung fibroblast HFL-1 cell line, which was used in place of primary lung fibroblasts, was used to assess the proliferative or apoptotic effects associated with lentiviral-induced HYAL1 overexpression using CCK-8 cell proliferation assay and Annexin V-APC staining. The identification of potentially associated molecular pathways was performed using microarray analysis followed by qRT-PCR and western blot analysis. Lung tissue microarray data mining and immunohistochemistry revealed significantly downregulation of HYAL1 in IPF lung tissue. However, HYAL1 in IPF lung tissue. However, HYAL1 in IPF lung tissue. However, HYAL1 in IPF lung tissue. However, ?1-) induced aberrant fibroproliferation in response to epithelial injury. The TGF?1-) induced aberrant fibroproliferation in response to epithelial injury. The TGF. We showed that HYAL1 overexpression could prevent HFL-1 fibroproliferation. Furthermore, our findings suggest that transcriptional regulators and BMP receptor signaling may be involved in HYAL1 modulation in IPF therapy.HYAL1 in IPF lung tissue. However.
Project description:<h4>Rationale</h4>Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis.<h4>Objectives</h4>To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance.<h4>Methods</h4>Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed.<h4>Measurements and main results</h4>IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses.<h4>Conclusions</h4>The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.
Project description:OBJECTIVES:Matrix metalloproteinase-8 (MMP-8) promotes lung fibrotic responses to bleomycin in mice. Although prior studies reported that MMP-8 levels are increased in plasma and bronchoalveolar lavage fluid (BALF) samples from IPF patients, neither the bioactive forms nor the cellular sources of MMP-8 in idiopathic pulmonary fibrosis (IPF) patients have been identified. It is not known whether MMP-8 expression is dys-regulated in IPF leukocytes or whether MMP-8 plasma levels correlate with IPF outcomes. Our goal was to address these knowledge gaps. METHODS:We measured MMP-8 levels and forms in blood and lung samples from IPF patients versus controls using ELISAs, western blotting, and qPCR, and assessed whether MMP-8 plasma levels in 73 IPF patients correlate with rate of lung function decline and mortality. We used immunostaining to localize MMP-8 expression in IPF lungs. We quantified MMP-8 levels and forms in blood leukocytes from IPF patients versus controls. RESULTS:IPF patients have increased BALF, whole lung, and plasma levels of soluble MMP-8 protein. Active MMP-8 is the main form elevated in IPF lungs. MMP-8 mRNA levels are increased in monocytes from IPF patients, but IPF patients and controls have similar levels of MMP-8 in PMNs. Surprisingly, macrophages and airway epithelial cells are the main cells expressing MMP-8 in IPF lungs. Plasma and BALF MMP-8 levels do not correlate with decline in lung function and/or mortality in IPF patients. CONCLUSION:Blood and lung MMP-8 levels are increased in IPF patients. Active MMP-8 is the main form elevated in IPF lungs. Surprisingly, blood monocytes, lung macrophages, and airway epithelial cells are the main cells in which MMP-8 is upregulated in IPF patients. Plasma and BALF MMP-8 levels are unlikely to serve as a prognostic biomarker for IPF patients. These results provide new information about the expression patterns of MMP-8 in IPF patients.
Project description:Excessive extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF). Epithelial-mesenchymal transition, involving transition of alveolar epithelial cells (AECs) to pulmonary fibroblasts, appears to be an important contributory process to lung fibrosis. Although aberrant expression of microRNAs (miRs) is involved in a variety of pathophysiologic processes, the role of miRs in fibrotic lung diseases is less well understood. In the present study, we found that miR-200a, miR-200b, and miR-200c are significantly down-regulated in the lungs of mice with experimental lung fibrosis. Levels of miR-200a and miR-200c were reduced in the lungs of patients with IPF. miR-200 had greater expression in AECs than in lung fibroblasts, and AECs from mice with experimental pulmonary fibrosis had diminished expression of miR-200. We found that the miR-200 family members inhibit transforming growth factor-?1-induced epithelial-mesenchymal transition of AECs. miR-200 family members can reverse the fibrogenic activity of pulmonary fibroblasts from mice with experimental pulmonary fibrosis and from patients with IPF. Indeed, the introduction of miR-200c diminishes experimental pulmonary fibrosis in mice. Thus, the miR-200 family members participate importantly in fibrotic lung diseases and suggest that restoring miR-200 expression in the lungs may represent a novel therapeutic approach in treating pulmonary fibrotic diseases.
Project description:Idiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal disease. However, IPF treatment has been limited by the low drug delivery efficiency to lungs and dysfunctional "injured" type II alveolar epithelial cell (AEC II). Here, we present surface-engineered nanoparticles (PER NPs) loading astaxanthin (AST) and trametinib (TRA) adhered to monocyte-derived multipotent cell (MOMC) forming programmed therapeutics (MOMC/PER). Specifically, the cell surface is designed to backpack plenty of PER NPs that reach directly to the lungs due to the homing characteristic of the MOMC and released PER NPs retarget injured AEC II after responding to the matrix metalloproteinase-2 (MMP-2) in IPF tissues. Then, released AST can enhance synergetic effect of TRA for inhibiting myofibroblast activation, and MOMC can also repair injured AEC II to promote damaged lung regeneration. Our findings provide proof of concept for developing a strategy for cell-mediated lung-targeted delivery platform carrying dual combined therapies to reverse IPF.
Project description:Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF), a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized the HumanMethylation 27 array, which assays the DNA methylation level of 27,568 CpG sites across the genome, to compare the DNA methylation patterns of IPF fibroblasts (n?=?6) with those of nonfibrotic patient controls (n?=?3) and commercially available normal lung fibroblast cell lines (n?=?3). We found that multiple CpG sites across the genome are differentially methylated (as defined by P value less than 0.05 and fold change greater than 2) in IPF fibroblasts compared to fibroblasts from nonfibrotic controls. These methylation differences occurred both in genes recognized to be important in fibroproliferation and extracellular matrix generation, as well as in genes not previously recognized to participate in those processes (including organ morphogenesis and potassium ion channels). We used bisulfite sequencing to independently verify DNA methylation differences in 3 genes (CDKN2B, CARD10, and MGMT); these methylation changes corresponded with differences in gene expression at the mRNA and protein level. These differences in DNA methylation were stable throughout multiple cell passages. DNA methylation differences may thus help to explain a proportion of the differences in gene expression previously observed in studies of IPF fibroblasts. Moreover, significant variability in DNA methylation was observed among individual IPF cell lines, suggesting that differences in DNA methylation may contribute to fibroblast heterogeneity among patients with IPF. These results demonstrate that IPF fibroblasts exhibit global differences in DNA methylation that may contribute to the excessive fibroproliferation associated with this disease.
Project description:Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease pathologically characterized by loss of epithelial cells and activation of fibroblasts and myofibroblasts. The etiology of IPF remains unclear and the disease pathology is poorly understood with no known efficacious therapy. PM014 is an herbal extract that has been shown to have beneficial effects in pulmonary diseases, which are likely to exert anti-inflammatory bioactions. In the present study, we observed that bleomycin (BLM) caused increased inflammatory infiltration as well as collagen deposition in lungs of mice on day 14 after treatment. Administration of PM014 suppressed BLM-induced inflammatory responses and fibrotic changes in dose-dependent manner in mice. Additionally, we provided in vitro evidence suggesting that PM014 inhibited TGF-?1-induced epithelial-mesenchymal transition (EMT) and fibroblast activation in alveolar epithelial cells and human lung fibroblasts from healthy donor and IPF patients. PM014 appeared to target TGF-?1 signaling via Smad-dependent pathways and p38 mitogen-activated protein kinases (MAPKs) pathways. Taken together, our data suggest that PM014 administration exerts a protective effect against lung fibrosis and highlight PM014 as a viable treatment option that may bring benefits to patient with IPF.
Project description:Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by epithelial cell activation, expansion of the fibroblast population and excessive extracellular matrix accumulation. The mechanisms are incompletely understood but evidence indicates that the deregulation of several proteases contributes to its pathogenesis. Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that may promote migration and facilitate epithelial to mesenchymal transition (EMT), two critical processes in the pathogenesis of IPF. Thus, we hypothesized that over-expression of TMPRSS4 in the lung could promote the initiation and/or progression of IPF. In this study we first evaluated the expression and localization of TMPRSS4 in IPF lungs by real time PCR, western blot and immunohistochemistry. Then we examined the lung fibrotic response in wild-type and TMPRSS4 deficient mice using the bleomycin-induced lung injury model. We found that this protease is upregulated in IPF lungs, where was primarily expressed by epithelial and mast cells. Paralleling the findings in vivo, TMPRSS4 was expressed by alveolar and bronchial epithelial cells in vitro and unexpectedly, provoked an increase of E-cadherin. No expression was observed in normal human or IPF lung fibroblasts. The lung fibrotic response evaluated at 28 days after bleomycin injury was markedly attenuated in the haplodeficient and deficient TMPRSS4 mice. By morphology, a significant reduction of the fibrotic index was observed in KO and heterozygous mice which was confirmed by measurement of collagen content (hydroxyproline: WT: 164±21.1 ?g/lung versus TMPRSS4 haploinsufficient: 110.2±14.3 ?g/lung and TMPRSS4 deficient mice: 114.1±24.2 ?g/lung (p<0.01). As in IPF, TMPRSS4 was also expressed in epithelial and mast cells. These findings indicate that TMPRSS4 is upregulated in IPF lungs and that may have a profibrotic role.
Project description:Prior work has shown that transforming growth factor-? (TGF-?) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, ?-smooth muscle actin (?-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and ?-SMA; markers of mesenchymal cells. Addition of a TGF-? receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-? can mediate this process.
Project description:Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown etiology. The pathogenic mechanisms are unclear, but evidence indicates that aberrantly activated alveolar epithelial cells secrete a variety of mediators which induce the migration, proliferation and activation of fibroblasts and finally the excessive accumulation of extracellular matrix with the consequent destruction of the lung parenchyma. CC16 (approved symbol SCGB1A1), a putative anti-inflammatory protein produced by "club" cells in the distal airways, has not been evaluated in IPF lungs. In this study, we determined the serum and bronchoalveolar lavage (BAL) levels as well as the lung cell localization of this protein. Also, we explored the usefulness of serum levels of CC16 for the differential diagnosis of IPF (n = 85), compared with non-IPF interstitial lung diseases [chronic hypersensitivity pneumonitis (cHP; n = 85) and connective tissue diseases (CTD-ILD; n = 85)]. CC16 was significantly increased in serum and BAL fluids of IPF patients and was found not only in club cells but also in alveolar epithelial cells. When compared with non-IPF patients and controls, serum levels were significantly increased (p<0.0001). Sensitivity and specificity for CC16 (cut-off 41ng/mL) were 24% and 90%, positive predictive value 56% and negative predictive value 69%. These findings demonstrate that CC16 is upregulated in IPF patients suggesting that may participate in its pathogenesis. Although higher than the serum levels of non-IPF patients it shows modest sensitivity to be useful as a potential biomarker for the differential diagnosis.