Rationale: Extracellular matrix (ECM) is a dynamic tissue that contributes to organ integrity and function, and its regulation of cell phenotype is a major aspect of cell biology. However, standard in vitro culture approaches are of unclear physiologic relevance because they do not mimic the compositional, architectural, or distensible nature of a living organ. In the lung, fibroblasts exist in ECM-rich interstitial spaces and are key effectors of lung fibrogenesis.
Objectives: To better address how ECM influences fibroblast phenotype in a disease-specific manner, we developed a culture system using acellular human normal and fibrotic lungs.
Methods: Decellularization was achieved using treatment with detergents, salts, and DNase. The resultant matrices can be sectioned as uniform slices within which cells were cultured.
Measurements and Main Results: We report that the decellularization process effectively removes cellular and nuclear material while retaining native dimensionality and stiffness of lung tissue. We demonstrate that lung fibroblasts reseeded into acellular lung matrices can be subsequently assayed using conventional protocols; in this manner we show that fibrotic matrices clearly promote transforming growth factor-β–independent myofibroblast differentiation compared with normal matrices. Furthermore, comprehensive analysis of acellular matrix ECM details significant compositional differences between normal and fibrotic lungs, paving the way for further study of novel hypotheses.
Conclusions: This methodology is expected to allow investigation of important ECM-based hypotheses in human tissues and permits future scientific exploration in an organ- and disease-specific manner.
extracellular matrix; lung fibrosis; fibroblast
The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII–like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.
Xanthomas are rare bone tumors that occur more often in the appendicular skeleton and typically appear radiographically benign, with a narrow zone of transition and a sclerotic rim. We report the case of a 57-year-old woman with hyperlipidemia presenting with bilateral shoulder pain after minor trauma. Radiographic and histopathologic investigation demonstrated intraosseous xanthoma with atypical features, including multifocality, a wide zone of transition and pathologic fractures-characteristics more commonly associated with aggressive lesions such as multiple myeloma or metastasis. The diagnosis, imaging, and histological appearance of xanthoma of bone are reviewed.
Xanthoma; Hyperlipidemia; Pathologic fracture
Schistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion.
First trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses.
Exposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women.
Soluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn.
Approximately 40 million women of childbearing age suffer from schistosome infection globally at any given time. Multiple studies in rodent models, as well as a few reports in humans, suggest that schistosome infection results in poor pregnancy outcomes. We have previously shown that antigens released from schistosome eggs result in a pronounced pro-inflammatory response in syncytialized third trimester trophoblasts. Herein, we examine the effect of schistosome egg antigens on a first trimester trophoblast cell line, an accepted model for early placental development. Not only is the pro-inflammatory response recapitulated in this model system, but we also observed a decrease in migration and invasion of trophoblast cells after exposure to these antigens. Both migration and invasion are key aspects in early placental development, and inadequate invasion has been implicated in pregnancy-related diseases such as growth restriction and preeclampsia. This study is the first to examine the impact of schistosome antigens on early placental development, and may have implications for the subsequent health of both the pregnancy and the child.
Fibroblasts perform critical functions during the normal host response to tissue injury, but the inappropriate accumulation and persistent activation of these cells results in the development of tissue fibrosis. The mechanisms accounting for the aberrant accumulation of fibroblasts during fibrotic repair are poorly understood, although evidence supports a role for fibroblast resistance to apoptosis as a contributing factor. We have shown that TGF-β1 and endothelin-1 (ET-1), soluble mediators implicated in fibrogenesis, promote fibroblast resistance to apoptosis. Moreover, we recently found that ET-1 induced apoptosis resistance in normal lung fibroblasts through the upregulation of survivin, a member of the Inhibitor of Apoptosis (IAP) protein family. In the current study, we sought to determine the role of survivin in the apoptosis resistance of primary fibroblasts isolated from the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF), a fibrotic lung disease of unclear etiology for which there is no definitive therapy. First, we examined survivin expression in lung tissue from patients with IPF and found that there is robust expression in the fibroblasts residing within fibroblastic foci (the “active” lesions in IPF which correlate with mortality). Next, we show that survivin expression is increased in fibroblasts isolated from IPF lung tissue compared to cells from normal lung tissue. Consistent with a role in fibrogenesis, we demonstrate that TGF-β1 increases survivin expression in normal lung fibroblasts. Finally, we show that inhibition of survivin enhances susceptibility of a subset of IPF fibroblasts to apoptosis. Collectively, these findings suggest that increased survivin expression represents one mechanism contributing an apoptosis-resistant phenotype in IPF fibroblasts.
Myofibroblast; Idiopathic Pulmonary Fibrosis; Inhibitor of Apoptosis; Lung Fibrosis; Fas
Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and – independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.
Fibrosis; Rho-kinase; Inhibitor of Apoptosis; Mesenchymal Cell; Focal Adhesion Kinase; AKT
Hematopoietic stem cell transplant patients are susceptible to infection despite cellular reconstitution. In a murine model of syngeneic bone marrow transplantation (BMT), we previously reported that BMT mice have impaired host defense against Pseudomonas aeruginosa pneumonia due to overproduction of (PG)E2 in lung. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an effector in the PGE2 signaling pathway that negatively regulates alveolar macrophage (AM) phagocytosis and bacterial killing. Therefore, examined whether overproduction of PGE2 after BMT inhibits AM host defense by up-regulating PTEN phosphatase activity. We found that PTEN activity is elevated in BMT AMs in response to increased PGE2 signaling and that pharmacological inhibition of PTEN activity in BMT AMs fully restores phagocytosis of serum-opsonized P. aeruginosa but only partially restores phagocytosis of nonopsonized P. aeruginosa. In wild-type mice transplanted with myeloid-specific conditional PTEN knockout (PTEN CKO) bone marrow, bacterial clearance is improved after challenge with P. aeruginosa pneumonia. Furthermore, PTEN CKO BMT AMs display improved TNF-α production and enhanced phagocytosis and killing of serum-opsonized P. aeruginosa despite overproduction of PGE2. However, AM phagocytosis of nonopsonized P. aeruginosa is only partially restored in the absence of PTEN after BMT. This may be related to elevated AM expression of IL-1 receptor–associated kinase (IRAK)-M, a molecule previously identified in the PGE2 signaling pathway to inhibit AM phagocytosis of nonopsonized bacteria. These data suggest that PGE2 signaling up-regulates IRAK-M independently of PTEN and that these molecules differentially inhibit opsonized and nonopsonized phagocytosis of P. aeruginosa.
pneumonia; lung; neutrophil; eicosanoids; bacteria
Previous research in our laboratory has demonstrated that repeated intranasal exposure to Aspergillus fumigatus conidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper TH2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the TH2 response has begun to resolve. TH17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged with A. fumigatus conidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4+ CD25+ FoxP3+ cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving TH2-type inflammation to repeated inhalation of fungal conidia.
Pulmonary arterial remodeling is a pathological process seen in a number of clinical disease states, driven by inflammatory cells and mediators in the remodeled artery microenvironment. In murine models, Th2 cell-mediated immune responses to inhaled antigens, such as purified Aspergillus allergen, have been reported to induce remodeling of pulmonary arteries. We have previously shown that repeated intranasal exposure of healthy C57BL/6 mice to viable, resting Aspergillus fumigatus conidia leads to the development of chronic pulmonary inflammation and the coevolution of Th1, Th2, and Th17 responses in the lungs. Our objective was to determine whether repeated intranasal exposure to Aspergillus conidia would induce pulmonary arterial remodeling in this mixed Th inflammatory microenvironment. Using weekly intranasal conidial challenges, mice developed robust pulmonary arterial remodeling after eight exposures (but not after two or four). The process was partially mediated by CD4+ T cells and by interleukin-4 (IL-4) production, did not require eosinophils, and was independent of gamma interferon (IFN-γ) and IL-17. Furthermore, remodeling could occur even in the presence of strong Th1 and Th17 responses. Rather than serving an anti-inflammatory function, IL-10 was required for the development of the Th2 response to A. fumigatus conidia. However, in contrast to previous studies of pulmonary arterial remodeling driven by the A. fumigatus allergen, viable conidia also stimulated pulmonary arterial remodeling in the absence of CD4+ T cells. Remodeling was completely abrogated in IL-10−/− mice, suggesting that a second, CD4+ T cell-independent, IL-10-dependent pathway was also driving pulmonary arterial remodeling in response to repeated conidial exposure.
Background. Schistosomes infect 200 million individuals annually and cause significant hepatic fibrosis in up to 20%. Little is known regarding the mechanisms of schistosome-associated hepatic fibrosis in humans, and few biomarkers for risk of fibrosis have been identified.
Methods. We treated 611 Schistosoma japonicum–infected Filipinos with praziquantel (PZQ) and performed ultrasound to quantify hepatic fibrosis at baseline and 12 months after PZQ treatment. We developed a multiplexed assay (FibroPlex) that quantifies predictors and effect modifiers of fibrosis. We measured FibroPlex analytes produced by peripheral blood mononuclear cells stimulated with schistosome egg antigen 4 weeks after PZQ treatment and related these levels to risk of fibrosis 1 year after PZQ treatment.
Results. After adjusting for potential confounders, including baseline grade of fibrosis, individuals with detectable tissue inhibitor of matrix-metalloprotease–1 (TIMP-1) had a 3.5-fold greater risk of fibrosis 1 year after PZQ treatment, compared with individuals with undetectable levels (odds ratio, 3.48; 95% confidence interval, 1.41–8.43; P = .007).
Discussion Because TIMP-1 inhibits most matrix metalloproteases, which are responsible for collagen degradation, these data suggest that schistosome-associated hepatic fibrosis results, in part, from excessive inhibition of collagen remodeling. These data further suggest that TIMP-1 is a promising biomarker for assessing risk of hepatic fibrosis in schistosomiasis and, potentially, other infectious and noninfectious causes of liver disease.
In order to make quantitative statements regarding behavior patterns in animals, it is important to establish whether new observations are statistically consistent with the animal's equilibrium behavior. For example, traumatic stress from the presence of a telemetry transmitter may modify the baseline behavior of an animal, which in turn can lead to a bias in results. From the perspective of information theory such a bias can be interpreted as the amount of information gained from a new measurement, relative to an existing equilibrium distribution. One important concept in information theory is the relative entropy, from which we develop a framework for quantifying time-dependent differences between new observations and equilibrium. We demonstrate the utility of the relative entropy by analyzing observed speed distributions of Pacific bluefin tuna, recorded within a 48-hour time span after capture and release. When the observed and equilibrium distributions are Gaussian, we show that the tuna's behavior is modified by traumatic stress, and that the resulting modification is dominated by the difference in central tendencies of the two distributions. Within a 95% confidence level, we find that the tuna's behavior is significantly altered for approximately 5 hours after release. Our analysis reveals a periodic fluctuation in speed corresponding to the moment just before sunrise on each day, a phenomenon related to the tuna's daily diving pattern that occurs in response to changes in ambient light.
Fibronectin (FN)1, a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for Extra Type III Domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome ten (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-β, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten−/− fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten−/− cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten−/− cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.
Fibronectin; alternative splicing; PTEN; fibrosis; fibroblast; rapamycin
Aspergillus fumigatus, a ubiquitous airborne fungus, can cause invasive infection in immunocompromised individuals but also triggers allergic bronchopulmonary aspergillosis in a subset of otherwise healthy individuals repeatedly exposed to the organism. This study addresses a critical gap in our understanding of the immunoregulation in response to repeated exposure to A. fumigatus conidia. C57BL/6 mice were challenged intranasally with A. fumigatus conidia weekly, and leukocyte composition, activation, and cytokine production were examined after two, four, and eight challenges. Approximately 99% of A. fumigatus conidia were cleared within 24 h after inoculation, and repeated exposure to A. fumigatus conidia did not result in hyphal growth or accumulation of conidia with time. After 2 challenges, there was an early influx of neutrophils and regulatory T (Treg) cells into the lungs but minimal inflammation. Repeated exposure promoted sustained expansion of the draining lymph nodes, while the influx of eosinophils and other myeloid cells into the lungs peaked after four exposures and then decreased despite continued A. fumigatus challenges. Goblet cell metaplasia and low-level fibrosis were evident during the response. Repeated exposure to A. fumigatus conidia induced T cell activation in the lungs and the codevelopment by four exposures of TH1, TH2, and TH17 responses in the lungs, which were maintained through eight exposures. Changes in CD4 T cell polarization or Treg numbers did not account for the reduction in myeloid cell numbers later in the response, suggesting a non-T-cell regulatory pathway involved in dampening inflammation during repeated exposure to A. fumigatus conidia.
Fibroblast migration plays an important role in the normal wound healing process; however, dysregulated cell migration may contribute to the progressive formation of fibrotic lesions in the diseased condition. To examine the role of focal-adhesion-kinase (FAK)-related non-kinase (FRNK) in regulation of fibrotic lung fibroblast migration, we examined cell migration, FRNK expression, and activation of focal adhesion kinase (FAK) and Rho GTPase (Rho and Rac) in primary lung fibroblasts derived from both idiopathic pulmonary fibrosis (IPF) patients and normal human controls. Fibrotic (IPF) lung fibroblasts have increased cell migration when compared to control human lung fibroblasts. FRNK expression is significantly reduced in IPF lung fibroblasts, while activation of FAK, Rho and Rac are increased in IPF lung fibroblasts. Endogenous FRNK expression is inversely correlated with FAK activation and cell migration rate in IPF lung fibroblasts. Forced exogenous FRNK expression abrogates the increased cell migration, and blocked the activation of FAK and Rho GTPase (Rho and Rac), in IPF lung fibroblasts. These data for the first time provide evidence that downregulation of endogenous FRNK plays a role in promoting cell migration through FAK and Rho GTPase in fibrotic IPF lung fibroblasts.
focal adhesion kinase; FAK-related non-kinase; fibroblast; migration; fibrosis; lung
Rationale: Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process.
Objectives: To determine if a targeted injury to type II cells would result in pulmonary fibrosis.
Methods: A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was administered to these animals to specifically target the type II epithelium for injury. Lung fibrosis was assessed by histology and hydroxyproline measurement.
Measurements and Main Results: Transgenic mice treated with diphtheria toxin developed an approximately twofold increase in their lung hydroxyproline content on Days 21 and 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction.
Conclusions: The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in idiopathic pulmonary fibrosis and the corresponding areas of scarring.
diphtheria toxin; lung; collagen; scarring
Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor–1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1–/– mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1–/– mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.
Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-β1 (TGF-β1) is a well-known inducer of myofibroblast differentiation. TGF-β1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E2 (PGE2) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE2 does so by interfering with TGF-β1 signaling is unknown. Thus we examined the effects of PGE2 in the presence and absence of TGF-β1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE2 does not interfere with TGF-β1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE2 has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE2 treatment diminishes TGF-β1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE2 stimulation alone do not always mirror the effects of PGE2 in the presence of TGF-β1, indicating that the context for EP2 signaling is different in the presence of TGF-β1. Taken together, our results demonstrate that PGE2 has the potential to limit TGF-β1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.
fibrosis; eicosanoid; lung; signal transduction
Idiopathic pulmonary fibrosis (IPF) is a progressive, usually fatal, form of interstitial lung disease characterized by failure of alveolar re-epithelialization, persistence of fibroblasts/myofibroblasts, deposition of extracellular matrix, and distortion of lung architecture which ultimately results in respiratory failure. Clinical IPF is associated with a histopathological pattern of usual interstitial pneumonia (UIP) on surgical lung biopsy. Therapy for this disease with glucocorticoids and other immunomodulatory agents is largely ineffective and recent trials of newer anti-fibrotic agents have been disappointing. While the inciting event(s) leading to the initiation of scar formation in UIP remain unknown, recent advances in our understanding of the mechanisms underlying both normal and aberrant wound healing have shed some light on pathogenetic mechanisms that may play significant roles in this disease. Unlike other fibrotic diseases of the lung, such as those associated with collagen vascular disease, occupational exposure, or chemotherapeutic agents, UIP is not associated with a significant inflammatory response; rather, dysregulated epithelial–mesenchymal interactions predominate. Identification of pathways crucial to fibrogenesis might offer potentially novel therapeutic targets to slow or halt the progression of IPF. This review focuses on evolving concepts of cellular and molecular mechanisms in the pathogenesis of UIP/IPF.
pulmonary fibrosis; myofibroblast; extracellular matrix; alveolar epithelial cell; fibrogenesis
Following a fibrogenic stimulus, the hepatic stellate cell (HSC) transforms from a quiescent to an activated cell type associated with increased proliferation, collagen and smooth muscle α-actin (αSMA) expression. Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN), a tumor suppressor phosphatase, has been shown to play a role in several nonmalignant diseases. Here, we investigated the role of PTEN during HSC activation.
Rat HSCs 2 days after isolation were transduced with adenoviruses expressing either the wild-type (WT) or a dominant negative form of PTEN, and culture-associated activation of HSCs, including morphological changes, expression of αSMA and α1(I) collagen, and cell proliferation, were evaluated. Apoptosis of HSCs was detected by measuring activity of caspase 3/7. Phosphorylation status of Akt, p70S6K, and Erk was detected by Western blotting.
Overexpression of WT-PTEN inhibited phenotypic changes were associated with HSC activation, including morphological changes, expression of αSMA and α1(I) collagen, and HSC proliferation, including cyclin D1 expression. WT-PTEN expression also induced apoptosis in HSCs with increased caspase 3/7 activity. Expression of WT-PTEN also caused decreased activation of Akt, p70S6K, and Erk signaling pathways.
Taken together, these findings show that PTEN represents an important negative regulator for transactivation of HSCs. This may have important implications for the design of therapeutic strategies to prevent the progression of liver fibrosis.
Hepatic stellate cells; Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN); Liver fibrosis; Smooth muscle α-actin (αSMA); Collagen
Rationale: Tissue fibrosis is considered a dysregulated wound-healing response. Fibronectin containing extra type III domain A (EDA) is implicated in the regulation of wound healing. EDA-containing fibronectin is deposited during wound repair, and its presence precedes that of collagen.
Objectives: To investigate the role of EDA-containing fibronectin in lung fibrogenesis.
Methods: Primary lung fibroblasts from patients with idiopathic pulmonary fibrosis or from patients undergoing resection for lung cancer were assessed for EDA-containing fibronectin and α-smooth muscle actin (α-SMA) expression. Mice lacking the EDA domain of fibronectin and their wild-type littermates were challenged with the bleomycin model of lung fibrosis. Primary lung fibroblasts from these mice were assayed in vitro to determine the contribution of EDA-containing fibronectin to fibroblast phenotypes.
Measurements and Main Results: Idiopathic pulmonary fibrosis lung fibroblasts produced markedly more EDA-containing fibronectin and α-SMA than control fibroblasts. EDA-null mice failed to develop significant fibrosis 21 days after bleomycin challenge, whereas wild-type controls developed the expected increase in total lung collagen. Histologic analysis of EDA-null lungs after bleomycin showed less collagen and fewer α-SMA–expressing myofibroblasts compared with that observed in wild-type mice. Failure to develop lung fibrosis in EDA-null mice correlated with diminished activation of latent transforming growth factor (TGF)-β and decreased lung fibroblast responsiveness to active TGF-β in vitro.
Conclusions: The data show that EDA-containing fibronectin is essential for the fibrotic resolution of lung injury through TGF-β activation and responsiveness, and suggest that EDA-containing fibronectin plays a critical role in tissue fibrogenesis.
fibrosis; fibronectin; TGF-β; myofibroblast
Angiogenesis is crucial for tumor biology. There are many mechanisms by which tumors induce angiogenesis. We hypothesize that each individual tumor develops a unique mechanism to induce angiogenesis, and that activation of a particular angiogenic pathway suppresses the evolution of alternative pathways. We characterized 168 human non–small cell lung cancer (NSCLC) specimens for levels of angiogenic factors (angiogenic CXC chemokines, basic fibroblast growth factor, and vascular endothelial growth factor). We also induced lung tumor formation in A/J mice by injecting the tobacco carcinogen NNK. We dissected individual lung tumors and measured expression of angiogenic factors from three distinct families using real-time PCR. Finally, we controlled the angiogenic milieu using in vivo models to determine the resultant phenotype of the angiogenic factors expressed by NSCLC cells. Human tumors displayed marked variation in the expression of angiogenic factors. Individual mouse tumors, even from within the same mouse, displayed variability in their pattern of expression of angiogenic factors. In a sponge model of angiogenesis using murine lung cancer cells, implanting LLC cells with an angiogenic factor suppressed the expression of other angiogenic factors in implanted sponges. This suppressive effect was not seen in vitro. We conclude that lung cancer tumors evolve a unique and dominant angiogenic phenotype. Once an angiogenic pathway is activated, it may allow for tumor growth to proceed in the absence of a selection pressure to activate a second pathway.
angiogenesis; mouse model; chemokines; cytokines; carcinogen
Transforming growth factor-β (TGF-β) is a prototypical tumour-suppressor cytokine with cytostatic and pro-apoptotic effects on most target cells; however, mechanisms of its pro-survival/anti-apoptotic signalling in certain cell types and contexts remain unclear. In human lung fibroblasts, TGF-β1 is known to induce myofibroblast differentiation in association with the delayed activation of focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Here, we demonstrate that FAK and AKT are independently regulated by early activation of SMAD3 and p38 MAPK, respectively. Pharmacologic or genetic approaches that disrupt SMAD3 signalling block TGF-β1-induced activation of FAK, but not AKT; in contrast, disruption of early p38 MAPK signalling abrogates AKT activation, but does not alter FAK activation. TGF-β1 is able to activate AKT in cells expressing mutant FAK or in cells treated with an RGD-containing peptide that interferes with integrin signalling, inhibits FAK activation and induces anoikis (apoptosis induced by loss of adhesion signalling). TGF-β1 protects myofibroblasts from anoikis, in part, by activation of the PI3K-AKT pathway. Thus, TGF-β1 co-ordinately and independently activates the FAK and AKT protein kinase pathways to confer an anoikis-resistant phenotype to myofibroblasts. Activation of these pro-survival/anti-anoikis pathways in myofibroblasts likely contributes to essential roles of TGF-β1 in tissue fibrosis and tumour-promotion.
Focal Adhesion Kinase; Protein kinase B; SMAD proteins; p38 MAPK; Transforming growth factor-beta; Apoptosis; Anoikis; Fibroblasts