Osteopontin and skeletal muscle myoblasts: association with muscle regeneration and regulation of myoblast function in vitro

Osteopontin is a secreted glycoprotein expressed by many cell types including osteoblasts and lymphocytes; it is a constituent of the extracellular matrix (ECM) in bone, and a mitogen for lymphocytes. To investigate the role of osteopontin in muscle repair and development, expression of osteopontin by muscle cells in vivo and in vitro, and the effects of osteopontin on myoblast function in vitro were investigated. Osteopontin staining was weak in sections of muscle from normal mice, but associated with desmin-positive cells in areas of regeneration in muscles from mdx mice. In immunocytochemical, PCR and ELISA studies, cultured myoblasts were found to express osteopontin and secrete it into medium. Treatment of myoblast cultures with fibroblast growth factor-2, transforming growth factor beta1, interleukin-1beta or thrombin significantly increased osteopontin expression. Osteopontin-coated substrata promoted adhesion and fusion, but not proliferation or migration, of myoblasts. The effect of osteopontin on myoblast adhesion was RGD-dependent. In solution, osteopontin significantly increased proliferation and decreased fusion and migration of myoblasts. These results suggest that myoblasts are an important source of osteopontin in damaged muscle and that osteopontin released by myoblasts may assist in controlling both the myogenic and inflammatory processes during the early stages of muscle regeneration.     

The Role of Catalase in Pulmonary Fibrosis

Background

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

Methods

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

Results

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

Conclusions

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

Osteopontin,inflammation and myogenesis: influencing regeneration,fibrosis and size of skeletal muscle

Osteopontin is a multifunctional matricellular protein that is expressed by many cell types. Through cell-matrix and cell-cell interactions the molecule elicits a number of responses from a broad range of target cells via its interaction with integrins and the hyaluronan receptor CD44. In many tissues osteopontin has been found to be involved in important physiological and pathological processes, including tissue repair, inflammation and fibrosis. Post-natal skeletal muscle is a highly differentiated and specialised tissue that retains a remarkable capacity for regeneration following injury. Regeneration of skeletal muscle requires the co-ordinated activity of inflammatory cells that infiltrate injured muscle and are responsible for initiating muscle fibre degeneration and phagocytosis of necrotic tissue, and muscle precursor cells that regenerate the injured muscle fibres. This review focuses on the current evidence that osteopontin plays multiple roles in skeletal muscle, with particular emphasis on its role in regeneration and fibrosis following injury, and in determining the severity of myopathic diseases such as Duchenne muscular dystrophy.     

Osteopontin: a versatile regulator of inflammation and biomineralization.

Osteopontin is a secreted glycoprotein with a multidomain structure and functions characteristic of a matricellular protein. Osteopontin interacts with cell surface receptors via arginine-glycine-aspartate (RGD)- and non-RGD containing adhesive domains, in addition to binding to components of the structural extracellular matrix. While normally expressed in bone and kidney, osteopontin levels are elevated during wound healing and inflammation in most tissues studied to date. Since 1986, over one thousand studies have been published on osteopontin, including recent experiments in osteopontin-deficient mice. These studies reveal osteopontin as a cell adhesive, signaling, migratory, and survival stimulus for various mesenchymal, epithelial, and inflammatory cells, in addition to being a potent regulator of osseous and ectopic calcification. Based on these reports, a general picture of osteopontin as an important regulator of inflammation and biomineralization is emerging. A common denominator in osteopontin function in these situations is its ability to regulate the function of macrophage and macrophage-derived cells (i.e. osteoclasts). While we have learned much about osteopontin and the processes it appears to regulate over the past decade, many questions regarding this important multifunctional protein remain unanswered and provide important directions for future studies.     

Functional role and species-specific contribution of arginases in pulmonary fibrosis

Lung fibrosis is characterized by increased deposition of ECM, especially collagens, and enhanced proliferation of fibroblasts. l-arginine is a key precursor of nitric oxide, asymmetric dimethylarginine, and proline, an amino acid enriched in collagen. We hypothesized that l-arginine metabolism is altered in pulmonary fibrosis, ultimately affecting collagen synthesis. Expression analysis of key enzymes in the arginine pathway, protein arginine methyltransferases (Prmt), arginine transporters, and arginases by quantitative (q) RT-PCR and Western blot revealed significant upregulation of arginase-1 and -2, but not Prmt or arginine transporters, during bleomycin-induced pulmonary fibrosis in mice. HPLC revealed a concomitant, time-dependent decrease in pulmonary l-arginine levels. Arginase-1 and -2 mRNA and protein expression was increased in primary fibroblasts isolated from bleomycin-treated mice, compared with controls, and assessed by qRT-PCR and Western blot analysis. TGF-beta1, a key profibrotic mediator, induced arginase-1 and -2 mRNA expression in primary and NIH/3T3 fibroblasts. Treatment of fibroblasts with the arginase inhibitor, NG-hydroxy-l-arginine, attenuated TGF-beta1-stimulated collagen deposition, but not collagen mRNA expression or Smad signaling, in fibroblasts. In human lungs derived from patients with idiopathic pulmonary fibrosis, arginase activity was unchanged, but arginase-1 expression significantly decreased when compared with donor lungs. Our results thus demonstrate that arginase-1 is expressed and functionally important for collagen deposition in lung fibroblasts. TGF-beta1-induced upregulation of arginase-1 suggests an interplay between profibrotic agents and l-arginine metabolism during the course of lung fibrosis in the mouse, whereas species-specific regulatory mechanisms may account for the differences observed in mouse and human.     

Eosinophils and T lymphocytes possess distinct roles in bleomycin-induced lung injury and fibrosis

Leukocyte infiltration is characteristic of lung injury and fibrosis, and its role during tissue repair and fibrosis is incompletely understood. We found that overexpression of IL-5 in transgenic mice (IL-5(TG)) or by adenoviral gene transfer increased bleomycin (blm)-induced lung injury, fibrosis, and eosinophilia. Surprisingly, blm-treated IL-5-deficient (IL-5(-/-)) mice also developed pronounced pulmonary fibrosis but characterized by marked T lymphocyte infiltration and absence of eosinophilia. In both murine strains however, induction of lung TGF-beta expression was evident. Purified lung eosinophils from blm-treated IL-5(TG) mice stimulated alpha-smooth muscle actin and collagen expression in mouse lung fibroblasts, without affecting proliferation. Furthermore instillation of purified eosinophils into murine lungs resulted in extension of blm-induced lung fibrosis, thus confirming a role for eosinophils. However, lung T lymphocytes from blm-treated IL-5(-/-) mice were able to stimulate fibroblast proliferation but not alpha-smooth muscle actin or collagen expression. Blocking T cell influx by anti-CD3 Abs abrogated lung fibrosis, thus also implicating T lymphocytes as a key participant in fibrosis. Pulmonary fibrosis in IL-5(TG) mice was preferentially associated with type 2 cytokines (IL-4 and IL-13), whereas fibrotic lesions in IL-5(-/-) animals were accompanied by proinflammatory cytokine (TNF-alpha, IL-1beta, and IFN-gamma) expression. We suggest that eosinophils and T cells contribute distinctly to the development of blm-induced lung fibrosis potentially via their production of different cytokine components, which ultimately induce TGF-beta expression that is intimately involved with the fibrosis.     

Vascular dysfunction in aged mice contributes to persistent lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.     

Loss of PTEN expression by mouse fibroblasts results in lung fibrosis through a CCN2-dependent mechanism

Elevated adhesive signaling promotes fibrosis. Protein phosphatase and tensin homologue (PTEN) dephosphorylates focal adhesion kinase and suppresses the activation of Akt and hence suppresses adhesive signaling. Loss of PTEN expression is associated with lung fibrosis, but whether PTEN expression by type I collagen-expressing cells controls lung fibrosis is unclear. Here, we use mice expressing tamoxifen-dependent cre recombinase expressed under the control of a COL1A2 promoter/enhancer and mice harboring floxed-PTEN and/or floxed-CCN2 alleles to assess whether loss of PTEN expression by type I collagen producing cells results in lung fibrosis in a CCN2-dependent fashion. In vivo, loss of PTEN expression resulted in the overexpression of both collagen type I and the pro-adhesive matricellular protein connective tissue growth factor (CTGF/CCN2). However, α-smooth muscle actin expression was unaffected. Loss of CCN2 expression by lung fibroblasts rescues this phenotype; i.e.., mice deficient in both PTEN and CCN2 in collagen type I-expressing cells do not develop significant collagen deposition in the lung. PTEN expression by collagen type I-expressing cells controls collagen deposition; therapeutic strategies blocking CCN2 may be of benefit in blocking excessive collagen deposition in fibrosis.     

Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model

The pathogenesis of nonalcoholic steatohepatitis (NASH) is poorly defined. Feeding mice a diet deficient in methionine and choline (MCD diet) induces experimental NASH. Osteopontin (OPN) is a Th1 cytokine that plays an important role in several fibroinflammatory diseases. We examined the role of OPN in the development of experimental NASH. A/J mice were fed MCD or control diet for up to 12 wk, and serum alanine aminotransferase (ALT), liver histology, oxidative stress, and the expressions of OPN, TNF-alpha, and collagen I were assessed at various time points. MCD diet-fed mice developed hepatic steatosis starting after 1 wk and inflammation by 2 wk; serum ALT increased from day 3. Hepatic collagen I mRNA expression increased during 1-4 wk, and fibrosis appeared at 8 wk. OPN protein expression was markedly increased on day 1 of MCD diet and persisted up to 8 wk, whereas OPN mRNA expression was increased at week 4. TNF-alpha expression was increased from day 3 to 2 wk, and evidence of oxidative stress did not appear until 8 wk. Increased expression of OPN was predominantly localized in hepatocytes. Hepatocytes in culture also produced OPN, which was stimulated by transforming growth factor-beta and TNF-alpha. Moreover, MCD diet-induced increases in serum ALT levels, hepatic inflammation, and fibrosis were markedly reduced in OPN(-/-) mice when compared with OPN(+/+) mice. In conclusion, our results demonstrate an upregulation of OPN expression early in the development of steatohepatitis and suggest an important role for OPN in signaling the onset of liver injury and fibrosis in experimental NASH.     

Pulmonary vaccination as a novel treatment for lung fibrosis

Pulmonary fibrosis is an untreatable, uniformly fatal disease of unclear etiology that is the result of unremitting chronic inflammation. Recent studies have implicated bone marrow derived fibrocytes and M2 macrophages as playing key roles in propagating fibrosis. While the disease process is characterized by the accumulation of lymphocytes in the lung parenchyma and alveolar space, their role remains unclear. In this report we definitively demonstrate the ability of T cells to regulate lung inflammation leading to fibrosis. Specifically we demonstrate the ability of intranasal vaccinia vaccination to inhibit M2 macrophage generation and fibrocyte recruitment and hence the accumulation of collagen and death due to pulmonary failure. Mechanistically, we demonstrate the ability of lung Th1 cells to prevent fibrosis as vaccinia failed to prevent disease in Rag-/- mice or in mice in which the T cells lacked IFN-γ. Furthermore, vaccination 3 months prior to the initiation of fibrosis was able to mitigate the disease. Our findings clearly demonstrate the role of T cells in regulating pulmonary fibrosis as well as suggest that vaccinia-induced immunotherapy in the lung may prove to be a novel treatment approach to this otherwise fatal disease.     

Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype

In the degenerative disease Duchenne muscular dystrophy, inflammatory cells enter muscles in response to repetitive muscle damage. Immune factors are required for muscle regeneration, but chronic inflammation creates a profibrotic milieu that exacerbates disease progression. Osteopontin (OPN) is an immunomodulator highly expressed in dystrophic muscles. Ablation of OPN correlates with reduced fibrosis and improved muscle strength as well as reduced natural killer T (NKT) cell counts. Here, we demonstrate that the improved dystrophic phenotype observed with OPN ablation does not result from reductions in NKT cells. OPN ablation skews macrophage polarization toward a pro-regenerative phenotype by reducing M1 and M2a and increasing M2c subsets. These changes are associated with increased expression of pro-regenerative factors insulin-like growth factor 1, leukemia inhibitory factor, and urokinase-type plasminogen activator. Furthermore, altered macrophage polarization correlated with increases in muscle weight and muscle fiber diameter, resulting in long-term improvements in muscle strength and function in mdx mice. These findings suggest that OPN ablation promotes muscle repair via macrophage secretion of pro-myogenic growth factors.     

Therapeutic effect of a peptide inhibitor of TGF-β on pulmonary fibrosis

Pulmonary fibrosis encompasses several respiratory diseases characterized by epithelial cell injury, inflammation and fibrosis. Transforming growth factor (TGF)-β1 is one of the main profibrogenic cytokines involved in the pathogenesis of lung fibrosis. It induces fibroblast differentiation into myofibroblasts, which produce high levels of collagen and concomitantly loss of lung elasticity and reduction of the respiratory function. In the present study, we have investigated the effects of P17 (a TGF-β inhibitor peptide) on IMR-90 lung fibroblast differentiation in vitro, as well as on the inhibition of the development of bleomycin-induced pulmonary fibrosis in mice. It was found that in IMR-90 cells, P17 inhibited TGF-β1-induced expression of connective tissue growth factor and α-smooth muscle actin. In vivo, treatment of mice with P17 2days after bleomycin administration decreased lung fibrosis, areas of myofibroblast-like cells and lymphocyte infiltrate. P17 also reduced mRNA expression of collagen type I, fibronectin and the fibronectin splice isoform EDA in the lung, and increased the expression of IFN-γ mRNA. Finally, therapeutic treatment with P17 in mice with already established fibrosis was able to significantly attenuate the progression of lung fibrosis. These results suggest that P17 may be useful in the treatment of pulmonary fibrosis.     

Mechanisms of oncostatin M-induced pulmonary inflammation and fibrosis

Oncostatin M (OSM), an IL-6 family cytokine, has been implicated in a number of biological processes including the induction of inflammation and the modulation of extracellular matrix. In this study, we demonstrate that OSM is up-regulated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and scleroderma, and investigate the pathological consequences of excess OSM in the lungs. Delivery of OSM to the lungs of mice results in a significant recruitment of inflammatory cells, as well as a dose-dependent increase in collagen deposition in the lungs, with pathological correlates to characteristic human interstitial lung disease. To better understand the relationship between OSM-induced inflammation and OSM-induced fibrosis, we used genetically modified mice and show that the fibrotic response is largely independent of B and T lymphocytes, eosinophils, and mast cells. We further explored the mechanisms of OSM-induced inflammation and fibrosis using both protein and genomic array approaches, generating a "fibrotic footprint" for OSM that shows modulation of various matrix metalloproteinases, extracellular matrix components, and cytokines previously implicated in fibrosis. In particular, although the IL-4/IL-13 and TGF-beta pathways have been shown to be important and intertwined of fibrosis, we show that OSM is capable of inducing lung fibrosis independently of these pathways. The demonstration that OSM is a potent mediator of lung inflammation and extracellular matrix accumulation, combined with the up-regulation observed in patients with pulmonary fibrosis, may provide a rationale for therapeutically targeting OSM in human disease.     

Poly(ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage

Oxygen-based therapies expose lung to elevated levels of ROS and induce lung cell damage and inflammation. Injured cells are replaced through increased proliferation and differentiation of epithelial cells and fibroblasts. Failure to modulate these processes leads to excessive cell proliferation, collagen deposition, fibrosis, and chronic lung disease. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death. In this study, we evaluated the role of PARP-1 in lung repair during recovery after acute hyperoxia exposure. We exposed PARP-1 -/- and wild-type mice for 64 h to 100% hyperoxia and let them recover in air for 5-21 days. PARP-1-deficient mice exhibited significantly higher lung cell hyperplasia and proliferation than PARP-1 +/+ animals after 5 and 10 days of recovery. This was accompanied by an increased inflammatory response in PARP-1 -/- compared with wild-type animals, characterized by neutrophil infiltration and increased IL-6 levels in bronchoalveolar lavages. These lesions were reversible, since the extent of the hyperplastic regions was reduced after 21 days of recovery and did not result in fibrosis. In vitro, lung primary fibroblasts derived from PARP-1 -/- mice showed a higher proliferative response than PARP-1 +/+ cells during air recovery after hyperoxia-induced growth arrest. Altogether, these results reveal an essential role of PARP-1 in the control of cell repair and tissue remodeling after hyperoxia-induced lung injury.     

Adenosine-dependent pulmonary fibrosis in adenosine deaminase-deficient mice

Pulmonary fibrosis is a common feature of numerous lung disorders, including interstitial lung diseases, asthma, and chronic obstructive pulmonary disease. Despite the prevalence of pulmonary fibrosis, the molecular mechanisms governing inflammatory and fibroproliferative aspects of the disorder are not clear. Adenosine is a purine-signaling nucleoside that is generated in excess during cellular stress and damage. This signaling molecule has been implicated in the regulation of features of chronic lung disease; however, the impact of adenosine on pulmonary fibrosis is not well understood. The goal of this study was to explore the impact of endogenous adenosine elevations on pulmonary fibrosis. To accomplish this, adenosine deaminase (ADA)-deficient mice were treated with various levels of ADA enzyme replacement therapy to regulate endogenous adenosine levels in the lung. Maintaining ADA-deficient mice on low dosages of ADA enzyme therapy led to chronic elevations in lung adenosine levels that were associated with pulmonary inflammation, expression of profibrotic molecules, collagen deposition, and extreme alteration in airway structure. These features could be blocked by preventing elevations in lung adenosine. Furthermore, lowering lung adenosine levels after the establishment of pulmonary fibrosis resulted in a resolution of fibrosis. These findings demonstrate that chronic adenosine elevations are associated with pulmonary fibrosis in ADA-deficient mice and suggest that the adenosine functions as a profibrotic signal in the lung.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-alpha

Transgenic mice overexpressing human transforming growth factor-alpha (TGF-alpha) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-alpha levels ranged from 388 +/- 40 pg/ml in the lowest expressing line to 1,247 +/- 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-alpha lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line (line 28), and increasing terminal airspace area was associated with increasing TGF-alpha expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-alpha lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.