Reduced peribronchial fibrosis in allergen-challenged MMP-9-deficient mice

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that are responsible for the degradation of the extracellular matrix during tissue remodeling. We have used a mouse model of allergen-induced airway remodeling to determine whether MMP-9 plays a role in airway remodeling. MMP-9-deficient and wild-type (WT) mice were repetitively challenged intranasally with ovalbumin (OVA) antigen to develop features of airway remodeling including peribronchial fibrosis and increased thickness of the peribronchial smooth muscle layer. OVA-challenged MMP-9-deficient mice had less peribronchial fibrosis and total lung collagen compared with OVA-challenged WT mice. There was no reduction in mucus expression, smooth muscle thickness, or airway responsiveness in OVA-challenged MMP-9-deficient compared with OVA-challenged WT mice. OVA-challenged MMP-9-deficient mice had reduced levels of bronchoalveolar lavage (BAL) regulated on activation, normal T cell expressed, and secreted (RANTES), as well as reduced numbers of BAL and peribronchial eosinophils compared with OVA-challenged WT mice. There were no significant difference in levels of BAL eotaxin, thymus- and activation-regulated chemokine (TARC), or macrophage-derived chemokine (MDC) in OVA-challenged WT compared with MMP-9-deficient mice. Overall, this study demonstrates that MMP-9 may play a role in mediating selected aspects of allergen-induced airway remodeling (i.e., modest reduction in levels of peribronchial fibrosis) but does not play a significant role in mucus expression, smooth muscle thickness, or airway responsiveness.     

Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer

Dynamic remodeling of the extracellular matrix (ECM) is essential for development, wound healing and normal organ homeostasis. Life-threatening pathological conditions arise when ECM remodeling becomes excessive or uncontrolled. In this Perspective, we focus on how ECM remodeling contributes to fibrotic diseases and cancer, which both present challenging obstacles with respect to clinical treatment, to illustrate the importance and complexity of cell-ECM interactions in the pathogenesis of these conditions. Fibrotic diseases, which include pulmonary fibrosis, systemic sclerosis, liver cirrhosis and cardiovascular disease, account for over 45% of deaths in the developed world. ECM remodeling is also crucial for tumor malignancy and metastatic progression, which ultimately cause over 90% of deaths from cancer. Here, we discuss current methodologies and models for understanding and quantifying the impact of environmental cues provided by the ECM on disease progression, and how improving our understanding of ECM remodeling in these pathological conditions is crucial for uncovering novel therapeutic targets and treatment strategies. This can only be achieved through the use of appropriate in vitro and in vivo models to mimic disease, and with technologies that enable accurate monitoring, imaging and quantification of the ECM.     

B lymphocytes in renal interstitial fibrosis

Fibrosis is defined as an excessive deposition of extracellular matrix (ECM), which leads to the destruction of organ structure and impairment of organ function. Fibrosis occurs not only in kidney but also in lung, liver, heart, and skin. Common pathways of fibrosis are thought to exist. Renal interstitial fibrosis is a complex process that involves multiple molecular signaling and multiple cellular components, in which B cells appear to be one of the emerging important players. B cells may affect fibrosis through cytokine production and through interaction with other cells including fibroblasts, macrophages and T cells. This review summarizes recent research findings of B cells in fibrosis and provides an insight of how the future therapeutics of fibrosis could be developed from a B-cell point of view.     

Gene therapy for tissue regeneration

Tissue repair and regeneration are the normal biological responses of many different tissues in the body to injury. During the healing process, profound changes occur in cell composition and extracellular matrix (ECM) formation. Fibroblasts and equivalent reparative cells migrate to the wounded area and subsequently proliferate. These cells and reparative cells from the surrounding tissue are responsible for the rapid repair which results in tissue regeneration. Growth factors, one of which is transforming growth factor-beta (TGF-beta), stimulate fibroblasts and smooth muscle cells to proliferate and synthesize ECM proteins. This process of early repair provides a rapid way to restore new tissue and mechanical integrity. This early tissue repair process is normally followed by involution, which requires the production and activation of proteases, tissue maturation and remodeling, reorganization and finally regeneration. Alternately, failure to replace the critical components of the ECM, including elastin and basement membrane, results in abnormal regeneration of the epithelial cell layer. Although remodeling should occur during healing, provisional repair may be followed by excessive synthesis and deposition of collagen, which results in irreversible fibrosis and scarring. This excessive fibrosis which occurs in aberrant healing is at least in part mediated by persistent TGF-beta. Because of the central role of collagen in the wound healing process, the pharmacological control of collagen synthesis has been of paramount importance as a possible way to abrogate aberrant healing and prevent irreversible fibrosis. Fibrosis is an abnormal response to tissue injury.     

Smad3 null mice develop airspace enlargement and are resistant to TGF-beta-mediated pulmonary fibrosis

Transforming growth factor-beta 1 plays a key role in the pathogenesis of pulmonary fibrosis, mediating extracellular matrix (ECM) gene expression through a series of intracellular signaling molecules, including Smad2 and Smad3. We show that Smad3 null mice (knockout (KO)) develop progressive age-related increases in the size of alveolar spaces, associated with high spontaneous presence of matrix metalloproteinases (MMP-9 and MMP-12) in the lung. Moreover, transient overexpression of active TGF-beta 1 in lungs, using adenoviral vector-mediated gene transfer, resulted in progressive pulmonary fibrosis in wild-type mice, whereas no fibrosis was seen in the lungs of Smad3 KO mice up to 28 days. Significantly higher levels of matrix components (procollagen 3A1, connective tissue growth factor) and antiproteinases (plasminogen activator inhibitor-1, tissue inhibitor of metalloproteinase-1) were detected in wild-type lungs 4 days after TGF-beta 1 administration, while no such changes were seen in KO lungs. These data suggest a pivotal role of the Smad3 pathway in ECM metabolism. Basal activity of the pathway is required to maintain alveolar integrity and ECM homeostasis, but excessive signaling through the pathway results in fibrosis characterized by inhibited degradation and enhanced ECM deposition. The Smad3 pathway is involved in pathogenic mechanisms mediating tissue destruction (lack of repair) and fibrogenesis (excessive repair).     

Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers

In acute and chronic lung disease, widespread disruption of tissue architecture underlies compromised pulmonary function. Pulmonary fibroblasts have been implicated as critical effectors of tissue-destructive extracellular matrix (ECM) remodeling by mobilizing a spectrum of proteolytic enzymes. Although efforts to date have focused on the catabolism of type I collagen, the predominant component of the lung interstitial matrix, the key collagenolytic enzymes employed by pulmonary fibroblasts remain unidentified. Herein, membrane type-1 matrix metalloprotease (MT1-MMP) is identified as the dominant and direct-acting protease responsible for the type I collagenolytic activity mediated by both mouse and human pulmonary fibroblasts. Furthermore, MT1-MMP is shown to be essential for pulmonary fibroblast migration within three-dimensional (3-D) hydrogels of cross-linked type I collagen that recapitulate ECM barriers encountered in the in vivo environment. Together, these findings demonstrate that MT1-MMP serves as a key effector of type I collagenolytic activity in pulmonary fibroblasts and earmark this pericellular collagenase as a potential target for therapeutic intervention.     

Epithelium-fibroblast interactions in response to airway inflammation

Dramatic changes to the architecture of the airway walls have been commonly described in the airways of patients with asthma, cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Much research has focused on how airway inflammation drives these structural changes, particularly in terms of the mechanisms/mediators that are involved, and a number of parallels are observed between the disease phenotypes. For example, the increased deposition of extracellular matrix (ECM) at focal sites in the airway wall is seen in asthma and all interstitial lung diseases that involve fibrosis. In addition, increased expression of a number of well characterized cytokines and growth factors, such as TGF-beta and epidermal growth factor (EGF) have been demonstrated in these diseases. However, the role of the lesser-known cytokines, including the leukaemia inhibitory factor (LIF) and other members of the IL-6 family of cytokines in the pathogenesis of airway remodelling and fibrosis is largely unknown. However, the use of genetic manipulation in vivo and more specific inhibitors/antibodies in vitro has now provided increasing evidence to support the hypothesis that a complex interaction exists between these cytokines, ECM and integrins in regulating the function of both epithelial cells and fibroblasts.     

PPAR-gamma agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is characterized by alterations in fibroblast phenotypes resulting in excessive extracellular matrix accumulation and anatomic remodeling. Current therapies for this condition are largely ineffective. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily, the activation of which produces a number of biological effects, including alterations in metabolic and inflammatory responses. The role of PPAR-gamma as a potential therapeutic target for fibrotic lung diseases remains undefined. In the present study, we show expression of PPAR-gamma in fibroblasts obtained from normal human lungs and lungs of patients with idiopathic interstitial pneumonias. Treatment of lung fibroblasts and myofibroblasts with PPAR-gamma agonists results in inhibition of proliferative responses and induces cell cycle arrest. In addition, PPAR-gamma agonists, including a constitutively active PPAR-gamma construct (VP16-PPAR-gamma), inhibit the ability of transforming growth factor-beta1 to induce myofibroblast differentiation and collagen secretion. PPAR-gamma agonists also inhibit fibrosis in a murine model, even when administration is delayed until after the initial inflammation has largely resolved. These observations indicate that PPAR-gamma is an important regulator of fibroblast/myofibroblast activation and suggest a role for PPAR-gamma ligands as novel therapeutic agents for fibrotic lung diseases.     

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.     

Opposite effect of corticosteroids and long-acting beta(2)-agonists on serum- and TGF-beta(1)-induced extracellular matrix deposition by primary human lung fibroblasts

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation and major structural lung tissue changes including increased extracellular matrix (ECM) deposition. Inhaled corticosteroids and long-acting beta(2)-agonists (LABA) are the basic treatment for both diseases, but their effect on airway remodeling remains unclear. In this study, we investigated the effect of corticosteroids and LABA, alone or in combination, on total ECM and collagen deposition, gene expression, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels by primary human lung fibroblasts. In our model, fibroblasts in 0.3% albumin represented a non-inflammatory condition and stimulation with 5% FCS and/or TGF-beta(1) mimicked an inflammatory environment with activation of tissue repair. FCS (5%) increased total ECM, collagen deposition, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels. In 0.3% albumin, corticosteroids reduced total ECM and collagen deposition, involving the glucocorticoid receptor (GR) and downregulation of collagen, heat shock protein 47 (Hsp47), and Fli1 mRNA expression. In 5% FCS, corticosteroids increased ECM deposition, involving upregulation of COL4A1 and CTGF mRNA expression. LABA reduced total ECM and collagen deposition under all conditions partly via the beta(2)-adrenergic receptor. In combination, the drugs had an additive effect in the presence or absence of TGF-beta(1) further decreasing ECM deposition in 0.3% albumin whereas counteracting each other in 5% FCS. These data suggest that the effect of corticosteroids, but not of LABA, on ECM deposition by fibroblasts is altered by serum. These findings imply that as soon as airway inflammation is resolved, long-term treatment with combined drugs may beneficially reduce pathological tissue remodeling.     

The extracellular matrix: an active or passive player in fibrosis?

Fibrosis is characterized by excessive accumulation of collagen and other extracellular matrix (ECM) components, and this process has been likened to aberrant wound healing. The early phases of wound healing involve the formation of a provisional ECM containing fibrin, fibrinogen, and fibronectin. Fibroblasts occupy this matrix and proliferate in response to activators elaborated by leukocytes that have migrated into the wound and are retained by the ECM. This coincides with the appearance of the myofibroblast, a specialized form of fibroblast whose differentiation is primarily driven by cytokines, such as transforming growth factor-β (TGF-β), and by mechanical tension. When these signals are reduced, as when TGF-β secretion is reduced, or as in scar shrinkage, myofibroblasts undergo apoptosis, resulting in a collagen-rich, cell-poor scar. Retention of myofibroblasts in fibrosis has been described as the result of imbalanced cytokine signaling, especially with respect to levels of activated TGF-β. ECM components can regulate myofibroblast persistence directly, since this phenotype is dependent on extracellular hyaluronan, tenascin-C, and the fibronectin splice variant containing the "extra domain A," and also, indirectly, through retention of TGF-β-secreting cells such as eosinophils. Thus the ECM is actively involved in both cellular and extracellular events that lead to fibrosis. Targeting components of the ECM as cells respond to injury and inflammatory stimuli holds promise as a means to avoid development of fibrosis and direct the wound-healing process toward reestablishment of a healthy equilibrium.     

Multiple beta 1 integrins mediate enhancement of human airway smooth muscle cytokine secretion by fibronectin and type I collagen

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with interstitial collagen and fibronectin are major pathological features of airway remodeling in asthma. We have previously shown that these ECM components confer enhanced ASM proliferation in vitro, but their action on its newly characterized secretory function is unknown. Here, we examined the effects of fibronectin and collagen types I, III, and V on IL-1beta-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1beta-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that alpha2beta1, alpha5beta1, alphavbeta1, and alphavbeta3 integrins were required for up-regulation of IL-1beta-dependent ASM secretory responses by fibronectin, while alpha2beta1 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1beta-dependent ASM secretory responses through a beta1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma.     

Methods in studying ECM degradation

Almost all tissues in our body contain specific cells associated with the tissue itself, and an extracellular matrix (ECM) that consists of a variety of proteins of which the bulk is formed by different types of collagens, glycoproteins and proteoglycans. The ECM plays a pivotal role in numerous processes not only related to the mechanical properties of a tissue, but also in modulating cellular activity. For a proper functioning of a tissue remodeling of the ECM is essential. Some connective tissues are characterized by a very rapid turnover (e.g. periodontal ligament) whereas others hardly show signs of turnover (e.g. cartilage). In all situations degradation of the ECM constituents occur. Under certain conditions, especially during a pathological situation, a high level of degradation may take place. In other situations matrix synthesis and deposition outstrips breakdown, leading to a fibrosis. In order to obtain information on the level of degradation of the different ECM components, various methods have been employed. A number of these methods will be discussed in this article.     

Regulatory mechanisms of TGF‐β1‐induced fibrogenesis of human alveolar epithelial cells

Pulmonary fibrosis is characterized by an extensive activation of fibrogenic cells and deposition of extracellular matrix (ECM). Transforming growth factor (TGF)‐β1 plays a pivotal role in the pathogenesis of pulmonary fibrosis, probably through the epithelial‐ to‐mesenchymal transition (EMT) and ECM production. The present study investigates potential mechanism by which TGF‐β1 induces EMT and ECM production in the fibrogenesis of human lung epithelial cells during pulmonary fibrosis. The expression of EMT phenotype and other proteins relevant to fibrogenesis were measured and the cell bio‐behaviours were assessed using Cell‐IQ Alive Image Monitoring System. We found that TGF‐β1‐induced EMT was accompanied with increased collagen I deposition, which may be involved in the regulation of connective tissue growth factor (CTGF) and phosphoinositide 3‐kinase (PI3K) signalling pathway. Treatment with PI3K inhibitors significantly attenuated the TGF‐β1‐ induced EMT, CTGF expression and collagen I synthesis in lung epithelial cells. The interference of CTGF expression impaired the basal and TGF‐β1‐stimulated collagen I deposition, but did not affect the process of EMT. Our data indicate that the signal pathway of TGF‐β1/PI3K/CTGF plays an important role in the fibrogenesis of human lung epithelial cells, which may be a novel therapeutic approach to prevent and treat pulmonary fibrosis.     

Prostaglandins but not leukotrienes alter extracellular matrix protein deposition and cytokine release in primary human airway smooth muscle cells and fibroblasts

Eicosanoids are lipid-signaling mediators released by many cells in response to various stimuli. Increasing evidence suggests that eicosanoids such as leukotrienes and prostaglandins (PGs) may directly mediate remodeling. In this study, we assessed whether these substances could alter extracellular matrix (ECM) proteins and the inflammatory profiles of primary human airway smooth muscle cells (ASM) and fibroblasts. PGE(2) decreased both fibronectin and tenascin C in fibroblasts but only fibronectin in ASM. PGD(2) decreased both fibronectin and tenascin C in both ASM and fibroblasts, whereas PGF(2α) had no effect on ECM deposition. The selective PGI(2) analog, MRE-269, decreased fibronectin but not tenascin C in both cell types. All the PGs increased IL-6 and IL-8 release in a dose-dependent manner in ASM and fibroblasts. Changes in ECM deposition and cytokine release induced by prostaglandins in both ASM and fibroblasts were independent of an effect on cell number. Neither the acute nor repeated stimulation with leukotrienes had an effect on the deposition of ECM proteins or cytokine release from ASM or fibroblasts. We concluded that, collectively, these results provide evidence that PGs may contribute to ECM remodeling to a greater extent than leukotrienes in airway cells.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.