Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.     

TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment?

Fibroblast proliferation and extracellular matrix accumulation characterize idiopathic pulmonary fibrosis (IPF). We evaluated the presence of tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4; collagenase-1, -2, and -3; gelatinases A and B; and membrane type 1 matrix metalloproteinase (MMP) in 12 IPF and 6 control lungs. TIMP-1 was found in interstitial macrophages and TIMP-2 in fibroblast foci. TIMP-3 revealed an intense staining mainly decorating the elastic lamina in vessels. TIMP-4 was expressed in IPF lungs by epithelial and plasma cells. TIMP-2 colocalized with Ki67 in fibroblasts, whereas TIMP-3 colocalized with p27 in inflammatory and epithelial cells. Collagenase-1 was localized in macrophages and alveolar epithelial cells, collagenase-2 was localized in a few neutrophils, and collagenase-3 was not detected. MMP-9 was found in neutrophils and subepithelial myofibroblasts. Myofibroblast expression of MMP-9 was corroborated in vitro by RT-PCR. MMP-2 was noticed in myofibroblasts, some of them close to areas of basement membrane disruption, and membrane type 1 MMP was noticed in interstitial macrophages. These findings suggest that in IPF there is higher expression of TIMPs compared with collagenases, supporting the hypothesis that a nondegrading fibrillar collagen microenvironment is prevailing.     

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.     

Effects of basic fibroblast factor (bFGF) on MMP-2, TIMP-2, and type-I collagen levels in human lung carcinoma fibroblasts

Matrix metalloproteinases (MMP's) and tissue inhibitors of metalloproteinases (TIMP's) possess a preponderant role in the metabolism of the major extracellular matrix protein, collagen, and are thought to be important in the mechanism of tumor invasion. Lung cancer occupies the first position in mortality and the second position in incidence, among all cancers. In the present investigation, we studied the effect of basic fibroblast growth factor (bFGF) on collagen, matrix metalloproteinase-2 (MMP-2), and tissue metalloproteinase inhibitor-2 (TIMP-2) levels in normal and carcinoma lung tissue fibroblast cultures. MMP-2 was selected because of its high specificity in the degradation of type IV collagen, major component of the basal membrane. The effect of bFGF on MMP-2, TIMP-2, total collagen, and type I collagen levels of normal and carcinoma lung fibroblast cultures was investigated at 0, 10, and 100 ng/ml. Statistical analysis was carried out using the Mann-Whitney-U test and possible correlations were searched using the Spearman correlation analysis method. MMP-2, TIMP-2, total collagen, and type-1 collagen levels based on cell counts (10(3) cells) showed no statistically significant differences between the carcinoma and normal fibroblast cultures. However, positive correlations were found between MMP-2 and TIMP-2 in normal (P = 0.016) and carcinoma (P = 0.001) tissue fibroblast cultures. Positive correlations were also found between total collagen and TIMP-2 levels in normal and carcinoma tissue fibroblast cultures (P = 0.002 and P = 0.032). Total collagen and TIMP-2 levels also showed positive and strong correlations in all cultures except in 100 ng/ml bFGF concentrations. In addition, type I collagen and MMP-2 levels showed positive significant correlations only in normal and carcinoma control cultures, while type I collagen and TIMP-2 levels showed positive correlations in all cultures except carcinoma fibroblasts at 100 ng/ml bFGF. It may be concluded that bFGF does not affect MMP-2, TIMP-2, total collagen, and type-1 collagen levels in fibroblast cultures grown from human carcinoma and normal lung tissues. However, bFGF was noted, in vitro, to disturb the equilibrium which normally exists between the four parameters, both in normal and carcinoma tissue fibroblasts.     

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.     

Comparison of SP-A and LPS effects on the THP-1 monocytic cell line

Surfactant protein A (SP-A) increases production of proinflammatory cytokines by monocytic cells, including THP-1 cells, as does lipopolysaccharide (LPS). Herein we report differences in responses to these agents. First, polymyxin B inhibits the LPS response but not the SP-A response. Second, SP-A-induced increases in tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-8 are reduced by >60% if SP-A is preincubated with Survanta (200 microgram/ml) for 15 min before addition to THP-1 cells. However, the LPS effects on TNF-alpha and IL-8 are inhibited by <20% and the effect on IL-1beta by <50%. Third, at Survanta levels of 1 mg/ml, SP-A-induced responses are reduced by >90%, and although the inhibitory effects on LPS action increase, they still do not reach those seen with SP-A. Finally, we tested whether SP-A could induce tolerance as LPS does. Pretreatment of THP-1 cells with LPS inhibits their response to subsequent LPS treatment 24 h later, including TNF-alpha, IL-1beta, and IL-8. Similar treatment with SP-A reduces TNF-alpha, but IL-1beta and IL-8 are further increased by the second treatment with SP-A rather than inhibited as with LPS. Thus, whereas both SP-A and LPS stimulate cytokine production, their mechanisms differ with respect to inhibition by surfactant lipids and in ability to induce tolerance.     

Surfactant protein A regulates surfactant phospholipid clearance after LPS-induced injury in vivo

Previous in vitro studies have suggested that surfactant protein A (SP-A) may play a role in pulmonary surfactant homeostasis by mediating surfactant secretion and clearance. However, mice made deficient in SP-A [SP-A (-/-) animals] have relatively normal levels of surfactant compared with wild-type SP-A (+/+) animals. We hypothesize that SP-A may play a role in surfactant homeostasis after acute lung injury. Bacterial lipopolysaccharide was instilled into the lungs of SP-A (-/-) mice and SP-A (+/+) mice to induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (-/-) animals, although injury did not alter [3H]choline or [14C]palmitate incorporation into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in surfactant synthesis/secretion 12 h after injury. Clearance of [3H]DPPC from the lungs of injured SP-A (-/-) animals was decreased by approximately 40%. Instillation of 50 microg of exogenous SP-A rescued both the clearance defect and the increased phospholipid defect in injured SP-A (-/-) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.     

Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro

We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing.     

Alpha-defensin enhances expression of HSP47 and collagen-1 in human lung fibroblasts

Neutrophils and lung fibroblasts are thought to play a role in the pathogenesis of pulmonary fibrosis. We reported previously that heat shock protein 47 (HSP47), a collagen-specific molecular chaperon, and collagen-1 synthesis were involved in pulmonary fibrosis, and that plasma levels of alpha-defensins (HNP; human neutrophil peptide), cationic proteins with antimicrobial and cytotoxic activity in neutrophils, were significantly higher in patients with idiopathic pulmonary fibrosis than in control subjects. Here, we investigated the direct effect of HNP-1 in vitro on the expression of HSP47 and collagen-1 in human lung fibroblasts (NHLF). HNP-1 at 5 microg/ml induced fibroblast proliferation but at concentrations >50 microg/ml, HNP-1 reduced cell viability. Incubation of NHLF with 10 to 25 microg/ml of HNP-1 for 24-h increased the expression of HSP47 and collagen-1 mRNAs (p<0.05). The levels of HSP47 protein also increased significantly at 50 microg/ml, and those of collagen-1 protein increased at 10 to 50 microg/ml of HNP-1 (p<0.05). The mitogen-activated protein kinase (MAPK) signaling pathway in NHLF was activated by HNP-1 stimulation, but inhibitor of MEK (PD98059) did not block HNP-1-induced HSP47 protein production. Our results suggest that alpha-defensin is a fibrogenic mediator that promotes collagen synthesis through the upregulation of HSP47 and collagen-1 in lung fibroblasts and participates in the pathogenesis of neutrophil-induced pulmonary fibrosis.     

Evaluation of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 on bovine inner cell mass outgrowth in vitro

Effects of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) on bovine inner cell mass (ICM) outgrowth and proteinase production in vitro were determined. Inner cell masses were isolated immunosurgically from day 7 embryos (day 0 = onset of estrus) and cultured for 96 h. In experiment 1, cellular outgrowth and gelatinase production were evaluated for ICM cultured on collagen IV, fibronectin, or laminin. More (P < 0.05) ICM generated cellular outgrowth on fibronectin (71%). compared with collagen IV (0%) or laminin (15%). Inner cell mass and outgrowth areas were greatest (P < 0.05) on fibronectin after 96 h of culture, compared with laminin. Although the incidence of cellular outgrowth on laminin was limited, numbers of cells in outgrowths supported by laminin were similar (P > 0.10) to fibronectin except at 72 h of culture, where more (P < 0.05) cells were in laminin than in fibronectin outgrowths. Gelatinase activity was not detected in conditioned medium. In experiment 2, cellular outgrowth and plasminogen activator production by ICM cultured on fibronectin in medium containing 0 or 10 microg/ml TIMP-2 were evaluated. Inner cell mass and outgrowth areas, and numbers of cells in outgrowths were greater (P < 0.05) in 10 compared with 0 microg/ml TIMP-2 at 96 h of culture. Mean plasminogen activator activity in conditioned medium from ICM cultured in 10 microg/ml TIMP-2 was greater (P < 0.05) compared with 0 microg/ml TIMP-2 (16.2 +/- 4.8 versus 6.7 +/- 1.4 x 10(-3) IU/ml, respectively). These results demonstrate that cellular outgrowth from bovine ICM is supported by fibronectin and is stimulated by TIMP-2.     

Fibroblasts from the muscles of Duchenne muscular dystrophy patients are resistant to cell detachment apoptosis

Extracellular matrix (ECM) proteins, including collagen and growth factors, are greatly increased in tissue fibrosis and mainly secreted by fibroblasts. We previously demonstrated that muscle-derived fibroblasts from Duchenne muscular dystrophy (DMD) patients have a profibrotic phenotype, that includes significantly reduced expression of tissue inhibitor of metalloprotease 3 (TIMP-3) compared to control. Since TIMP-3 induces apoptosis in various cell types, we hypothesized increased resistance of DMD fibroblasts to apoptosis. To address this, we evaluated apoptotic nuclei, caspase 3, caspase 3 substrate expression, and migration and adhesion properties of muscle-derived fibroblasts, after applying different apoptosis-inducing treatments. We found that DMD fibroblasts were less susceptible to cell death, more adhesive, and had greater tendency to migrate than control fibroblasts — findings further supported by alterations in FAK and ERK/MAPK expression. Resistance to apoptosis and greater adhesion are likely to contribute to muscle fibrosis so a pharmacological treatment that targets dysregulated pathways involved in cell detachment apoptosis (anoikis) may limit the progressive fibrotic remodeling characteristic of DMD.     

TGF-β1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-β1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-β1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-β1 and serum significantly augmented collagen gel contraction. TGF-β1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-β1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-β induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.     

SP-B and SP-C containing new synthetic surfactant for treatment of extremely immature lamb lung

Although superiority of synthetic surfactant over animal-driven surfactant has been known, there is no synthetic surfactant commercially available at present. Many trials have been made to develop synthetic surfactant comparable in function to animal-driven surfactant. The efficacy of treatment with a new synthetic surfactant (CHF5633) containing dipalmitoylphosphatidylcholine, phosphatidylglycerol, SP-B analog, and SP-C analog was evaluated using immature newborn lamb model and compared with animal lung tissue-based surfactant Survanta. Lambs were treated with a clinical dose of 200 mg/kg CHF5633, 100 mg/kg Survanta, or air after 15 min initial ventilation. All the lambs treated with air died of respiratory distress within 90 min of age. During a 5 h study period, Pco(2) was maintained at 55 mmHg with 24 cmH(2)O peak inspiratory pressure for both groups. The preterm newborn lamb lung functions were dramatically improved by CHF5633 treatment. Slight, but significant superiority of CHF5633 over Survanta was demonstrated in tidal volume at 20 min and dynamic lung compliance at 20 and 300 min. The ultrastructure of CHF5633 was large with uniquely aggregated lipid particles. Increased uptake of CHF5633 by alveolar monocytes for catabolism was demonstrated by microphotograph, which might be associated with the higher treatment dose of CHF5633. The higher catabolism of CHF5633 was also suggested by the similar amount of surfactant lipid in bronchoalveolar lavage fluid (BALF) between CHF5633 and Survanta groups, despite the 2-fold higher treatment dose of CHF5633. Under the present ventilation protocol, lung inflammation was minimal for both groups, evaluated by inflammatory cell numbers in BALF and expression of IL-1β, IL-6, IL-8, and TNFα mRNA in the lung tissue. In conclusion, the new synthetic surfactant CHF5633 was effective in treating extremely immature newborn lambs with surfactant deficiency during the 5 h study period.     

Relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts

Relaxin is believed to play a role in connective tissue remodeling during pregnancy (Bell, R.J., Eddie, L. W., Lester, A. R., Wood, E. C., Johnston, P.D., and Niall, H. D. (1987) Obstet. Gynecol. 69, 585-589; MacLennan, A. H. (1983) Clin. Reprod. Fertil. 2, 77-95). In the present study, normal human fibroblasts exposed to concentrations of a synthetic bioactive relaxin peptide from 0.1 to 10 ng/ml synthesized and secreted the metalloproteinase procollagenase, which was immunoprecipitable as a doublet of 52 and 57 kDa by a monoclonal antibody to human collagenase. The stimulation in procollagenase protein expression was reflected in an elevation in procollagenase mRNA levels. Media conditioned for 48 h by relaxin-treated fibroblasts (0.1 ng/ml) contained 1.7 units/ml activatable collagenase compared with 0.2 units/ml by untreated fibroblasts. In addition, relaxin caused a modest decrease in the levels of tissue inhibitor of metalloproteinases, as detected by reverse zymography and Northern analysis. Relaxin was also a potent modulator of the collagen secretory phenotype of these fibroblasts. Relaxin at 100 ng/ml down-regulated collagen secretion by 40%. When fibroblasts were treated simultaneously with cytokines such as transforming growth factor beta or interleukin 1 beta, which stimulated collagen synthesis to at least 9-fold of basal levels, relaxin at 100 ng/ml was able to down-regulate collagen expression by up to 88%. This decrease was reflected by changes at the mRNA level. These results indicate that relaxin can cause significant collagen turnover both by stimulating collagenase expression and by down-modulating collagen synthesis and secretion.     

Tolbutamide alters glucose transport and metabolism in the embryonic mouse heart

BACKGROUND: Tolbutamide is a sulfonylurea oral hypoglycemic agent widely used for the treatment of non insulin-dependent diabetes mellitus. Tolbutamide produces dysmorphogenesis in rodent embryos and becomes concentrated in the embryonic heart after maternal oral dosing. Tolbutamide increases glucose metabolism in extra-pancreatic adult tissues, but this has not previously been examined in embryonic heart. METHODS: CD-1 mouse embryos were exposed on GD 9.5 to tolbutamide (0, 100, 250, or 500 microg/ml) for 6, 12, or 24 hr in whole-embryo culture. Isolated hearts were evaluated for (3)H-2DG uptake and conversion of (14)C-glucose to (14)C-lactate. Glut-1, HKI, and GRP78 protein levels were determined by Western analysis, and Glut-1 mRNA was measured by RT-PCR. RESULTS: Cardiac (3)H-2DG uptake increased after exposure to 500 microg/ml tolbutamide for 6 hr, and 100, 250, or 500 microg/ml tolbutamide for 24 hr, compared to controls. Glycolysis increased after exposure to 500 microg/ml tolbutamide for 6 or 24 hr compared to controls. Glut-1 protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 12 or 24 hr, and Glut-1 mRNA increased in hearts exposed to 500 microg/ml tolbutamide for 24 hr compared to controls. HKI protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 6 hr, but not 12 or 24 hr. There was no effect on GRP78 protein levels in hearts exposed to tolbutamide for 6, 12, or 24 hr. CONCLUSIONS: Tolbutamide stimulates glucose uptake and metabolism in the embryonic heart, as occurs in adult extra-pancreatic tissues. Glut-1 and HKI, but not GRP78, are likely involved in tolbutamide-induced cardiac dysmorphogenesis.     

Nitric oxide in the pathogenesis of diffuse pulmonary fibrosis

By studying the responses of nitric oxide in pulmonary fibrosis, the role of inducible nitric oxide synthase in diffuse pulmonary fibrosis as caused by lipopolysaccharide (LPS) treatment was investigated. When compared to rats treated with LPS only, the rats pretreated with 1400W (an iNOS-specific inhibitor) were found to exhibit a reduced level in: (i) NOx (nitrate/nitrite) production, (ii) collagen type I protein expression, (iv) soluble collagen production, and (iv) the loss of body weight and carotid artery PO2. In the pulmonary fibroblast culture, exogenous NO from LPS-stimulated secretion by macrophages or from a NO donor, such as DETA NONOate, was observed to induce the expression of TIMP-1, HSP47, TGF-beta1, and collagen type I as well as the phosphorylation of SMAD-2. After inhalation of NO for 24 h, an up-regulation of collagen type I protein was also noted to occur in rat pulmonary tissue. The results suggest that the NO signal pathway enhanced the expression of TGF-beta1, TIMP-1, and HSP47 in pulmonary fibroblasts, which collectively demonstrate that the NO signal pathway could activate the SMAD-signal cascade, by initiating a rapid increase in TGF-beta1, thereby increasing the expression of TIMP-1 and HSP47 in pulmonary fibroblasts, and play an important role in pulmonary fibrosis.