Search

Alveolar type II (ATII) cell apoptosis and depressed fibrinolysis that promotes alveolar fibrin deposition are associated with acute lung injury (ALI) and the development of pulmonary fibrosis (PF). We therefore sought to determine whether p53-mediated inhibition of urokinase-type plasminogen activator (uPA) and induction of plasminogen activator inhibitor-1 (PAI-1) contribute to ATII cell apoptosis that precedes the development of PF. We also sought to determine whether caveolin-1 scaffolding domain peptide (CSP) reverses these changes to protect against ALI and PF. Tissues as well as isolated ATII cells from the lungs of wild-type (WT) mice with BLM injury show increased apoptosis, p53, and PAI-1, and reciprocal suppression of uPA and uPA receptor (uPAR) protein expression. Treatment of WT mice with CSP reverses these effects and protects ATII cells against bleomycin (BLM)-induced apoptosis whereas CSP fails to attenuate ATII cell apoptosis or decrease p53 or PAI-1 in uPA-deficient mice. These mice demonstrate more severe PF. Thus p53 is increased and inhibits expression of uPA and uPAR while increasing PAI-1, changes that promote ATII cell apoptosis in mice with BLM-induced ALI. We show that CSP, an intervention targeting this pathway, protects the lung epithelium from apoptosis and prevents PF in BLM-induced lung injury via uPA-mediated inhibition of p53 and PAI-1.

DNA methylation is a key mechanism for repression of gene expression, including that of α-smooth muscle actin (α-SMA) gene expression in fibroblasts. However, the trans-acting factors that interact with the methylated α-SMA gene to regulate its expression have not been identified. Using gel shift and chromatin immunoprecipitation (ChIP) assays, methyl CpG binding protein 2 (MeCP2) was shown to bind to the α-SMA gene. Suppression of MeCP2 gene expression by siRNA or its deficiency in lung fibroblasts isolated from MeCP2 knockout mice caused significant reduction of α-SMA gene expression. In contrast, transient transfection of MeCP2 expression plasmid into fibroblasts enhanced α-SMA gene expression. Moreover, in vivo studies revealed that compared to their wild type littermates, MeCP2-deficient mice exhibited significantly decreased alveolar wall thickness, inflammatory cell infiltration, interstitial collagen deposition, and myofibroblast differentiation in response to endotracheal injection of bleomycin. Thus, MeCP2 is essential for myofibroblast differentiation and pulmonary fibrosis., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and frequently fatal form of interstitial lung disease for which there are no proven drug therapies. The pathogenesis of IPF is complex and the urokinase-type plasminogen activator (uPA)/plasminogen system participates in the repair process. The balance between the activating enzyme uPA, and its inhibitor PAI-1, is a critical determinant of the amount of scar development that follows., Objective: To address the role of urokinase in the pathogenesis of pulmonary fibrosis and its implications for therapy., Methods: We reviewed a spectrum of therapeutic strategies and focused on fibrinolytic and anticoagulant drugs for IPF patients., Results/conclusion: There is currently a search for new pharmacotherapeutic agents that may modulate the fibrogenic pathways in IPF. Either blocking PAI-1 or using uPA itself may be a promising new therapeutic strategy.

Idiopathic pulmonary fibrosis is a chronic, progressive and often fatal form of interstitial lung disease. It is characterized by injury with loss of lung epithelial cells and abnormal tissue repair, resulting in abnormal accumulation of fibroblasts and myofibroblasts, deposition of extracellular matrix and distortion of lung architecture, leading to respiratory failure. This lethal lung disorder continues to pose major clinical challenges as an effective therapeutic regimen has yet to be developed. In this report, therapeutic strategies are reviewed, including the use of antifibrotic agents, inhibition of cytokines, leukotrienes and cytokines receptors, and molecular targeting of specific signaling pathways during fibrotic processes and angiogenesis. This article examines the body of evidence supporting present therapies and reviews the newer agents being tested in patients with idiopathic pulmonary fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and often fatal form of interstitial lung disease. It is characterized by injury with loss of lung epithelial cells and abnormal tissue repair, resulting in replacement of normal functional tissue, abnormal accumulation of fibroblasts and myofibroblasts, deposition of extracellular matrix, and distortion of lung architecture which results in respiratory failure. Despite improvements in the diagnostic approach to IPF and active research in recent years, the molecular mechanisms of the disease remain poorly understood. This highly lethal lung disorder continues to pose major clinical challenges since an effective therapeutic regimen has yet to be identified and developed. For example, a treatment modality has been based on the assumption that IPF is a chronic inflammatory disease, yet most available anti-inflammatory drugs are not effective in treating it. Hence researchers are now focusing on understanding alternative underlying mechanisms involved in the pathogenesis of IPF in the hope of discovering potentially new pharmaceutical targets. This paper will focus on lung tissue repair, regeneration, remodeling, and cell types that may be important to consider in therapeutic interventions and includes a more detailed discussion of the potential targets of current therapeutic attack in pulmonary fibrosis. The discovery that adult bone marrow stem cells can contribute to the formation of differentiated cell types in other tissues, especially after injury, implies that they have the potential to participate in tissue remodeling, and perhaps regeneration. The current promise of the use of adult stem cells for tissue regeneration, and the belief that once irreversibly damaged tissue could be restored to a normal functional capacity using stem cell-based therapy, suggests a novel approach for treatment of diverse chronic diseases. However this optimism is tempered by current evidence that the pathogenesis of pulmonary fibrosis may involve the recruitment of bone marrow-derived fibroblasts, which are the key contributors to the pathogenesis of this chronic progressive disorder. Nevertheless, stem cell-related therapies are widely viewed as promising treatment options for patients suffering from various types of pulmonary diseases. Gender mismatched bone marrow or lung transplant recipients serve as natural populations in which to study the role of bone marrow-derived stem cells in recovery from pulmonary diseases. Understanding the mechanism of recruitment of stem cells to sites of injury, and their involvement in tissue repair, regeneration, and remodeling may offer a novel therapeutic target for developing more effective treatments against this fatal disorder. This article reviews the new concepts in the pathogenesis, current and future treatment options of pulmonary fibrosis, and the recent advances regarding the roles of stem cells in lung tissue repair, regeneration, and remodeling.

Pulmonary fibrosis is characterized by inflammation, genesis of myofibroblasts, and abnormal tissue repair. Despite extensive research, its pathogenesis remains incompletely understood. Previously, the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) was found to be a key regulator of myofibroblast differentiation in vitro, and to be involved in the acute phase and inflammatory responses. In an attempt to test the role of C/EBPbeta in the development of pulmonary fibrosis, experiments using C/EBPbeta null mice and their wild-type littermates were conducted. Our findings indicated that, compared to wild-type mice, animals deficient in C/EBPbeta showed significantly reduced fibrotic lesions and collagen deposition in the lung upon endotracheal injection of bleomycin. Further studies on the mechanisms by which C/EBPbeta regulates fibrosis indicated that knockout of C/EBPbeta attenuates inflammatory cytokine expression in bleomycin-treated mice. The reduced alpha-smooth muscle actin gene expression in either isolated lung fibroblasts or lung tissue from bleomycin or saline-treated C/EBPbeta deficient mice suggests that C/EBPbeta regulates myofibroblast differentiation during fibrosis. Consistent with this finding, cells from C/EBPbeta deficient mice exhibited higher proliferative rates than those from wild-type mice. These data suggest that C/EBPbeta plays an essential role in pulmonary fibrosis and that this role appears to be multifactorial with respect to cytokine expression, cell differentiation, and proliferation., (Copyright (c) 2006 Pathological Society of Great Britain and Ireland.)

Eotaxin-1/CCL11 and its receptor CCR3 are involved in recruitment of eosinophils to diverse tissues, but their role in eosinophil recruitment in pulmonary fibrosis is unclear. The present study examined the pulmonary expression of CCL11 and CCR3 during bleomycin (blm)-induced lung injury and determined their importance in the recruitment of inflammatory cells and the development of lung fibrosis. In mice, blm induced a marked pulmonary expression of CCL11 and CCR3. Immunostaining for CCR3 revealed that this receptor was not only expressed by eosinophils but also by neutrophils. CCL11-deficient (CCL11(-/-)) mice developed significantly reduced pulmonary fibrosis. Expression of profibrotic cytokines such as transforming growth factor-beta1 was diminished in the absence of CCL11. Furthermore, increased lung expression of CCL11 significantly enhanced blm-induced lung fibrosis and production of profibrotic cytokines. These effects were also associated with an increase of eosinophil and neutrophil pulmonary infiltration. In contrast, mice treated with neutralizing CCR3 antibodies developed significantly reduced pulmonary fibrosis, eosinophilia, neutrophilia, and expression of profibrotic cytokines. Together, these data suggest that CCL11 and CCR3 are important in the pulmonary recruitment of granulocytes and play significant pathogenic roles in blm-induced lung fibrosis.

The role of gender and sex hormones is unclear in host response to lung injury, inflammation, and fibrosis. To examine gender influence on pulmonary fibrosis, male and female rats were given endotracheal injections of either saline or bleomycin. Female rats showed higher mortality rates and more severe fibrosis than did male rats, as indicated by higher levels of lung collagen deposition and fibrogenic cytokine expression. To clarify the potential role of female sex hormones in lung fibrosis, female rats were ovariectomized and treated with either estradiol or vehicle plus endotracheal injections of either saline or bleomycin. The results showed diminished fibrosis in the ovariectomized, bleomycin-treated rats without hormone replacement. Estradiol replacement restored the fibrotic response to that of the intact female mice in terms of lung collagen deposition and cytokine expression, which was accompanied by higher plasma estradiol levels. Furthermore, fibroblasts from bleomycin-treated rats exhibited increased responsiveness to estradiol treatment, causing dose-dependent increases in procollagen 1 and transforming growth factor-beta1 mRNA expression relative to untreated controls. Taken together these findings suggest that female mice may have an exaggerated response to lung injury relative to male mice because of female sex hormones, which have direct fibrogenic activity on lung fibroblasts. This may provide a mechanism for a hormonally mediated intensification of pulmonary fibrosis.

In general, there are two types of animal models: natural and experimental. Because there are no natural models for pulmonary fibrosis, an experimental model that reproduces key aspects of the human disease would be useful for the study of this form of lung disease, the natural history of which is not always known. To date, a variety of animal models have been used to investigate mechanisms of pulmonary and other types of fibrosis, including the intratracheal instillation of bleomycin, fluorescein isothiocyanate, or particulate matter, such as silica and asbestos. This chapter will describe two commonly used techniques, namely bleomycin and silica-induced, which have been developed for the study of pulmonary fibrosis in animal models.

Pulmonary fibrosis is characterized by lung inflammation and abnormal tissue repair, resulting in the replacement of normal functional tissue with an abnormal accumulation of fibroblasts and deposition of collagen in the lung. This process involves cellular interactions via a complex cytokine-signaling mechanism and heightened collagen gene expression, ultimately resulting in its abnormal collagen deposition in the lung. Our current understanding of the pathogenesis of pulmonary fibrosis suggests that in addition to inflammatory cells, the fibroblast and signaling events that mediate fibroblast proliferation and myofibroblasts, play important roles in the diverse processes that constitute fibrosis. Increasing knowledge of cytokine biology, cytokine-signaling and cell matrix interactions have shed some light on the genesis of pulmonary fibrosis; however, the importance of inflammation in pulmonary fibrosis remains controversial. This remains true because the inflammatory component is variable at the time of diagnosis, and the most potent anti-inflammatory drugs that have been widely used in the treatment of pulmonary fibrosis do not seem to interfere with the fibrotic disease progression. Pulmonary fibrosis is a highly lethal disorder, which continues to pose major clinical challenges because an effective therapeutic regimen is yet to be determined. This review summarizes recent progress in understanding the molecular mechanisms of pulmonary fibrosis, and includes a more detailed discussion of the potential points of therapeutic attack in pulmonary fibrosis. In addition, a detailed discussion is presented regarding each of the potential therapies which have emerged from the animal models of pulmonary fibrosis, and which have been developed through advances in cellular and molecular biology.

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.

Eosinophils are known to express cytokines capable of promoting fibrosis. Interleukin-5 (IL-5) is important in regulating eosinophilopoiesis, eosinophil recruitment and activation. Lung IL-5 expression is elevated in pulmonary fibrosis, wherein the eosinophil is a primary source of fibrogenic cytokines. To determine the role of IL-5 in pulmonary fibrosis, the effects of anti-IL-5 antibody were investigated in a model of bleomycin-induced pulmonary fibrosis. Fibrosis was induced in mice by endotracheal bleomycin treatment. Animals were also treated with either anti-IL-5 antibody or control IgG. Lungs were then analyzed for fibrosis, eosinophil influx, chemotactic activity, and cytokine expression. The results show that a primary chemotactic activity at the height of eosinophil recruitment is IL-5. Furthermore, anti-IL-5 antibody caused significant reduction in lung eosinophilia, cytokine expression, and fibrosis. These findings taken together suggest an important role for IL-5 in pulmonary fibrosis via its ability to regulate eosinophilic inflammation, and thus eosinophil-dependent fibrogenic cytokine production.

Pulmonary fibrosis is commonly characterized by inflammation of the alveolar wall, leading to derangement of normal alveolar architecture, and interstitial as well as intra-alveolar fibrosis. The process involves cellular interactions via a complex cytokine network and heightened collagen gene expression with abnormal deposition in the lung. Recent studies have identified a myriad of cytokines with potential roles in pulmonary fibrosis. Based on in vivo antibody neutralization studies, important roles for tumor necrosis á (TNFá), macrophage inflammatory protein 1á (MIP-1á) and transforming growth factor (TGF), have been established. The recent demonstration that the eosinophil is a major source for several of these key pro-fibrogenic cytokines during the early stages of fibrosis, strongly suggest a role for the eosinophil in pulmonary fibrosis. In vitro, eosinophils can elaborate factors capable of stimulating fibroblast proliferation, and their presence in lungs undergoing many forms of pulmonary fibrosis has been well documented. Further support for a role for eosinophils in pulmonary fibrosis are suggested by clinical data showing a correlation between lung eosinophil count and a poor prognosis and decreased responsiveness to therapy. This review will focus on the recent findings, which suggest novel potential roles for the eosinophil in the pathogenesis of pulmonary fibrosis.

Eosinophils are primary sources of fibrogenic cytokines in lung fibrosis, and interleukin (IL)-5 is important in their differentiation, proliferation, recruitment and activation. To investigate the potential role of this cytokine, lung IL-5 expression was examined in a murine model of bleomycin-induced pulmonary fibrosis. Analysis of lung RNA showed significant increases in lung IL-5 mRNA content between days 3 and 14 after induction of lung injury, which decreased toward control levels after day 21. In situ hybridization revealed essentially no detectable IL-5 mRNA expression before day 3, but showed elevated expression in mononuclear cells and eosinophils between days 3 and 14, localized within areas of active fibrosis. After 21 days, the intensity and number of IL-5-expressing cells significantly declined. Immunostaining with anti-IL-5 antibodies confirmed the predominant IL-5 expression by mononuclear cells and eosinophils in areas of active fibrosis. The kinetics of increase in the number of cells expressing significant IL-5 mRNA in lung sections paralleled that for IL-5 mRNA expression in whole-lung homogenates. These results demonstrate for the first time that IL-5 is upregulated in this murine model and suggest a novel role for this cytokine in pulmonary fibrosis via its ability to recruit and activate eosinophils.

Despite abundant evidence documenting the importance of TNF-alpha in the pathogenesis of pulmonary fibrosis, its actual role has not been fully elucidated. Recent observations also indicate that eosinophils found in fibrotic lung express elevated levels of cytokines known to be important in lung fibrosis. These findings suggest a possible role for TNF-alpha in eosinophil recruitment and cytokine expression in this disease. To examine this hypothesis, pulmonary fibrosis was induced in mice by endotracheal bleomycin treatment, and separate groups of animals were also treated with either anti-TNF-alpha Ab or control serum. On days 7 and 14 post-bleomycin treatment, lungs were harvested and analyzed for fibrosis, cytokine expression, and eosinophil influx. Anti-TNF-alpha caused a significant reduction in lung fibrosis, as indicated by a reduction in hydroxyproline content, which was accompanied by suppression of lung TGF-beta1, IL-5, and JE mRNA expression. Examination of tissue sections revealed a significant reduction in lung eosinophils and overall cellularity by anti-TNF-alpha treatment without a significant effect on the number of lung macrophages. The number of IL-5-expressing cells was also significantly reduced by anti-TNF-alpha treatment. Since IL-5 is important in eosinophil differentiation, activation, and recruitment, these findings suggest a novel mechanism by which TNF-alpha could mediate pulmonary fibrosis via induction of IL-5-mediated eosinophil recruitment and fibrogenic cytokine production. Since these eosinophil-derived cytokines include JE/monocyte chemotactic factor-1 and TGF-beta1, this cytokine networking orchestrated by TNF-alpha could, in turn, amplify the inflammatory response and drive the progression to fibrosis and end-stage lung disease.

The emergence of the myofibroblast phenotype (characterized by alpha-smooth muscle actin expression) in wound healing and in tissues undergoing fibrosis is thought to be responsible for the increased contractility of the affected tissues. In bleomycin-induced pulmonary fibrosis, the myofibroblast is also responsible for the observed increase in collagen gene expression. To evaluate further these phenotypic changes in lung fibroblasts, contractile and other phenotypic properties of fibroblasts isolated from lungs of rats with bleomycin-induced fibrosis were compared with those of normal rats using in vitro models. Pulmonary fibrosis was induced in rats by endotracheal injection on day 0, and 7 and 14 days later the animals were sacrificed and lung fibroblasts isolated. Using immunofluorescence, < 10% of fibroblasts from control animals express alpha-smooth muscle actin when cultured as a monolayer. In contrast, 19% and 21% of cells from day 7 and day 14 bleomycin-treated animals, respectively, expressed this actin and with greater intensity than in control lung cells. This increase in actin expression was associated with enhanced contractility when evaluated using a three-dimensional cell culture model consisting of fibroblast-populated collagen gels. This enhanced contractility was abolished by treatment with antibody to transforming growth factor-beta (TGF-beta), whereas exogenous TGF-beta 1 and serum-stimulated contraction of control lung fibroblasts. TGF-beta 1 gene expression was greater in cells from bleomycin-treated animals than those from control lungs. These results show that cells with the myofibroblast phenotype are more abundant in fibrotic lung, and that these cells possess greater contractile capacity in vitro at least partly by virtue of their enhanced endogenous TGF-beta 1 gene expression.

Recent studies indicate that monocyte chemoattractant protein-1 (MCP-1) may play an important role in pulmonary inflammation. In vitro studies show that a number of cell types are capable of producing MCP-1. In this study, MCP-1 expression in lungs of rats with bleomycin (BLM)-induced pulmonary fibrosis is examined to evaluate its cellular origin and potential role in pathogenesis. Lung fibrosis was induced in male Fisher 344 rats by endotracheal injection on day 0. On selected days after injection, lungs were harvested for in situ and Northern hybridization analyses for MCP-1 mRNA expression, immunochemical and histochemical analyses for MCP-1 protein expression, and identification of cell type. Northern analysis revealed significant elevation in lung MCP-1 mRNA expression beginning on day 3 post-BLM treatment, increasing to a peak on day 7, and then decreasing toward control levels after day 21. In situ hybridization combined with histochemical staining with chromotrope 2R indicate that most of the cells expressing MCP-1 mRNA at these time points are primarily eosinophils. A few scattered reactive fibroblasts, some mononuclear cells, epithelial cells, and cells of certain blood vessel walls also express this mRNA. Increased MCP-1 protein expression also was found to be predominantly within and adjacent to eosinophils. The eosinophils expressing this mRNA were found predominantly within areas of active fibrosis. The kinetics of increase in the number of cells expressing significant MCP-1 mRNA in lung sections paralleled that for MCP-1 mRNA expression, as assessed by Northern analysis. These results, for the first time, demonstrate that MCP-1 is up-regulated significantly in this rat animal model, and that infiltrating eosinophils represent the major cellular source for this increased MCP-1 expression.

Background: Endotracheal bleomycin administration in rats and other animal species causes rapid development of pulmonary fibrosis, characterized by a transiently increased number of contractile, filament-laden parenchymal cells and increased lung collagen synthesis and deposition. However, the identity and source of the cells that actively synthesize collagen and other extracellular matrix and their relationship to the altered lung structure and function remain uncertain., Experimental Design: In this study, the cells expressing alpha 1(I) and alpha 2(I) procollagen genes were identified and their localization analyzed in control and bleomycin-treated rat lungs at different time points, by in situ and Northern hybridization analyses., Results: In control lungs, only a few scattered fibroblasts with weak expression of the alpha 1(I) procollagen gene were localized exclusively in the adventitia of the primary and tertiary bronchi, as well as major blood vessels. At day 3 after bleomycin treatment, scattered interstitial cells with significantly increased alpha 1(I) and alpha 2(I) procollagen gene expression were observed in the adventitia of bronchioles, terminal bronchioles, and adjacent small blood vessels. At days 7 and 14, there was a dramatic increase in the number of interstitial cells expressing large amounts of alpha 1(I) procollagen messenger RNA in these areas and extending to the lung parenchyma. This was followed on days 21 and 28 by significant decreases in procollagen gene expression and the number of cells with increased collagen gene expression. Most of the cells with enhanced collagen gene expression were arrayed in a disorganized fashion and were localized mainly around bronchioles, terminal bronchioles, and adjacent small blood vessels as well as in the irregularly distributed fibrotic foci, some submesothelial areas, and injured parenchyma. Northern blot analysis was consistent with the above in situ hybridization observation of the kinetics of collagen gene expression., Conclusions: The results indicate that in this rat fibrosis model, increased numbers of the interstitial cells with high expression of type I procollagen genes are derived primarily from the fibroblasts in the adventitia of bronchioles, terminal bronchioles, and adjacent blood vessels, as well as the submesothelial region. This then can result in further expansion to adjacent parenchyma and alveolar areas.