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Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor–1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1–/– mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1–/– mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.
In patients with acute and chronic fibrotic lung diseases, fibrin forms within the lung due to a leakage of plasma from damaged vasculature and activation of the coagulation cascade. The extravascular fibrin is not cleared in a timely fashion because of a marked increase in the expression of plasminogen activation inhibitor–1 (PAI-1) relative to urokinase-type plasminogen activator (uPA) (1–3). Transgenic animal experiments have firmly established a causal link between the level of PAI-1 and the severity of fibrosis. Specifically, mice with impaired systemic plasminogen activation to plasmin as the result of overexpression of a PAI-1 transgene develop a more exuberant fibrotic response following bleomycin injury than do littermate controls (4). Similarly, poor outcomes are noted in mice with a targeted deletion of the plasminogen gene (5). Conversely, mice with a targeted deletion of their Pai1 gene are profoundly resistant to lung fibrosis in the bleomycin model, and they also have significantly improved survival (4, 6). Transgenic overexpression (7), adenoviral delivery (8), and aerosolization (9) of uPA all limit lung fibrosis and improve survival following lung injury. Thus, a robust body of evidence indicates that plasminogen activation protects from lung fibrosis. However, an explanatory mechanism for this protective effect is unclear. Although it was originally attributed to plasmin-mediated breakdown of fibrin, it has been shown that fibrosis is not substantially inhibited in mice genetically lacking fibrinogen (6, 10). More recently, the antifibrotic actions of plasminogen activation to plasmin have been ascribed to other mechanisms including the proteolytic release of HGF (6, 11).
Another well-known antifibrotic factor in the lung is PGE2. PGs, including PGE2, are generated via conversion of arachidonic acid to PGH2 via COX-1 or COX-2 enzymes. Via E prostanoid receptor 2–mediated (EP2-mediated) increases in intracellular cyclic AMP, PGE2 directly inhibits major pathobiologic functions of effector fibroblasts including chemotaxis, proliferation, collagen synthesis, and differentiation to myofibroblasts (12–15). Derangements of PG synthesis are present in fibrotic diseases in humans and animal models of pulmonary fibrosis. Reduced PGE2 levels have been reported in bronchoalveolar lavage fluid and conditioned medium from alveolar macrophages of patients with idiopathic pulmonary fibrosis (IPF) (16, 17). Fibroblasts from IPF patients are unable to upregulate the COX-2 enzyme and are thereby deficient in PGE2 production (18–20); it has recently been suggested that this defect may be the consequence of histone deacetylation of the COX2 gene promoter (21). The relevance of such an impairment is suggested by the facts that pharmacologic (administration of indomethacin) (22) or genetic (gene deletion of Cox2) (23) reduction in PGE2 synthesis in the lung as well as gene deletion of EP2 (24) augment bleomycin-induced fibrosis in mice. By contrast, protection against experimental fibrosis has been observed when endogenous PGE2 is overproduced (21, 25, 26) or when exogenous PGE is administered (27). Interestingly, a reduction in PGE2 responsiveness mediated by a loss of the EP2 receptor has also been described in fibroblasts derived from bleomycin-treated mice (24) and IPF patients (28). Thus, diminished PGE2 production and/or signaling characterize lung fibrosis and are likely to be pathophysiologically significant.
Here we hypothesized that the antifibrotic actions of plasminogen activation are mediated by the induction of PGE2 synthesis. To date, there are no reports of cross-regulation between plasminogen activation and PGE2 in the lung. Only a few examples of crosstalk between these two systems (in colon cancer, gastric fibroblasts, and osteoblasts) are noted in the literature (29–31), and no study has reported that plasminogen activation promotes PGE2 synthesis in any organ or cell. Herein we demonstrate that (a) the plasminogen activation pathway enhances PGE2 production in vivo and in relevant cell types in vitro; (b) PGE2 synthesis accounts for direct inhibitory effects on fibroblasts of plasminogen activation to plasmin; (c) PGE2 elaboration proceeds via a plasmin/HGF/COX-2 pathway; and (d) the HGF/COX-2/PGE2 axis accounts for the protection against pulmonary fibrosis observed in Pai1–/– mice.
We have previously reported that Pai1–/– mice are protected from bleomycin-induced lung fibrosis (4, 32). To determine whether this protected phenotype is associated with increased PGE2 production, we measured PGE2 levels in lung homogenates of WT or Pai1–/– mice during the fibrotic phase at day 21 after bleomycin treatment (33). Figure Figure11 demonstrates that the protected Pai1–/– mice produced significantly greater levels of PGE2 in the lung than did WT mice. In order to determine the cellular source of increased PGE2 production in the lung, we next analyzed the effects of plasminogen activation on 3 cell types known to be critical to the development of pulmonary fibrosis: fibroblasts, fibrocytes, and alveolar epithelial cells (AECs).
Fibroblasts were isolated from lung explants harvested at day 14 from saline- or bleomycin-treated murine lungs and serum starved for 24 hours in serum-free medium (SFM) consisting of 0.1% BSA-DMEM. Thereafter, cells were exposed to 10 U/ml uPA, 45 mU/ml plasminogen, or the two in combination. After 24 hours, supernatants were collected, and PGE2 levels were measured by ELISA. The addition of either uPA alone or plasminogen alone had no influence on the levels of PGE2 produced by fibroblasts purified from either saline- (Figure (Figure2A)2A) or bleomycin-treated mice (data not shown). However, treatment with uPA plus plasminogen led to a significant increase in PGE2 secretion in fibroblasts from both saline- and bleomycin-treated (Figure (Figure2B)2B) mice when compared with cells cultured in SFM alone.
PAI-1 inhibits uPA-mediated activation of plasminogen to plasmin. To determine whether endogenous capacity for plasminogen activation also regulates PGE2 synthesis, we purified fibroblasts as above from WT or Pai1–/– mice and cultured the cells for 24 hours in the presence of SFM alone or SFM containing 10 U/ml uPA and 45 mU/ml plasminogen. Basal production of PGE2 was higher in fibroblasts from Pai1–/– mice. In addition, the induction of PGE2 synthesis in Pai1–/– fibroblasts by plasminogen activation was significantly greater than that in fibroblasts purified from WT mice (Figure (Figure2C).2C). These results suggest that the proteolytic function of uPA is important for the induction of PGE2 generation.
We next wished to extend these findings to fibrocytes, mesenchymal cells of bone marrow origin that contribute to pulmonary fibrogenesis (34, 35). The ability of these cells to elaborate PGE2 has not previously been reported in any context. Fibrocytes were purified from the lungs of saline- or bleomycin-treated mice. As in fibroblasts, the addition of uPA alone or plasminogen alone had no effect on PGE2 secretion (Figure (Figure3A).3A). Culture of fibrocytes with the combination of 10 U/ml uPA and 45 mU/ml plasminogen led to an increase in PGE2 production in fibrocytes from both saline- and bleomycin-treated mice (Figure (Figure3B).3B). Fibrocytes purified from Pai1–/– mice showed enhanced secretion of PGE2 compared with WT fibrocytes in the presence of uPA plus plasminogen (Figure (Figure3C).3C). It is also evident from these data that the capacity of fibrocytes to produce PGE2 is substantially lower than that of fibroblasts.
The fact that uPA alone was incapable of stimulating PGE2 synthesis suggests that this effect is not the result of uPA ligation of its receptor, uPAR, but rather is the result of its ability to convert plasminogen to plasmin. To confirm this, we cultured murine fibrocytes and fibroblasts with 50 mU/ml murine plasmin. Plasmin was indeed able to enhance PGE2 secretion in both mesenchymal cell types isolated from saline- (Figure (Figure4A)4A) and bleomycin-treated (data not shown) lungs. To test whether the enzymatic activity of plasmin was necessary for PGE2 generation, we examined the effects of its inhibitor, α2-antiplasmin. As shown in Figure Figure4B,4B, addition of α2-antiplasmin significantly attenuated the ability of plasminogen activation to stimulate PGE2 secretion. Similar results were noted in fibrocytes (data not shown).
We next investigated whether COX-2 enzyme induction accounts for the ability of plasminogen activation to increase PGE2 synthesis. Fibroblasts and fibrocytes were isolated from saline- or bleomycin-treated mice and cultured as above. After 24 hours, cell lysates were collected, and COX-2 protein expression was evaluated by Western blot and its mRNA examined by real-time RT-PCR (Figure (Figure5).5). Provision of uPA plus plasminogen or of plasmin alone indeed led to increased COX-2 mRNA and protein expression as compared with cells cultured in SFM in both fibroblasts (Figure (Figure5,5, A and B) and fibrocytes (Figure (Figure5,5, C and D). Since autocrine expression of COX-2 and production of PGE2 are well known to inhibit fibroblast expression of the important matrix protein collagen I (15, 36), we also examined levels of collagen I protein and the α1 chain of procollagen I mRNA in these cell lysates. A simultaneous reduction in (pro)collagen I mRNA and protein accompanied the induction of COX-2 elicited by plasminogen activation to plasmin in fibroblasts (Figure (Figure5,5, A and B) as well as fibrocytes (Figure (Figure5,5, C and D).
On a per-cell basis, AECs are the most prodigious producers of PGE2 within the lung (37). AECs were isolated from saline- and bleomycin-treated mice and adhered to fibronectin-coated plates. After 3 days of adherence, cells were washed with PBS and serum starved for 24 hours in SFM. Then, medium was changed, and 10 U/ml uPA, 45 mU/ml plasminogen, or the combination of both was added to the wells. After 24 hours, the supernatants were collected, and PGE2 levels were measured by ELISA. Similar to the results seen with mesenchymal cells, neither uPA nor plasminogen alone had a measurable effect, but the two together elicited a significant increase in PGE2 production in both normal (Figure (Figure6A)6A) and fibrotic (Figure (Figure6B)6B) AECs as compared with SFM controls. The greater capacity for PGE2 synthesis of AECs compared with mesenchymal cells is apparent. When cells were treated with 50 mU/ml plasmin alone, PGE2 secretion increased to levels similar to those observed with addition of uPA plus plasminogen (data not shown). Both basal and plasminogen activation–stimulated PGE2 production were significantly greater in Pai1–/– than WT cells (Figure (Figure6C).6C). We next treated AECs with SFM, uPA plus plasminogen, or plasmin as above and measured Cox2 mRNA induction by real-time RT-PCR. Both reagent and enzymatically generated plasmin resulted in 5- to 7-fold increases in Cox2 mRNA (Figure (Figure6D). 6D).
In conjunction with the results obtained in murine cells, we used a primary human fetal lung fibroblast cell line, IMR-90, to test the effect of plasminogen system components on human fibroblasts. Although uPA had no effect, plasminogen alone or plasmin enhanced PGE2 synthesis in IMR-90 cells (Figure (Figure7A).7A). The efficacy of plasminogen alone in IMR-90 cells, which was not observed in murine cells, reflects the fact that these human cells endogenously produce and secrete more uPA than do murine fibroblasts (Figure (Figure7B).7B). Thus, the endogenous uPA can activate exogenously supplied plasminogen to plasmin. The effects of plasmin on PGE2 paralleled its effects on COX-2 protein expression and were inversely related to expression of collagen I protein (Figure (Figure7C).7C). Importantly, treatment of IMR-90 cells with the COX-1/2 inhibitor indomethacin prevented inhibition of collagen I synthesis by plasminogen (Figure (Figure7D),7D), implying that plasmin’s suppressive effect in fibroblasts was indeed dependent on its ability to enhance prostanoid generation.
Plasmin’s effects could likewise be mediated via its activation of the G protein–coupled protease activated receptor PAR-1. Involvement of this receptor was explored using the specific PAR-1 agonist peptide TFLLRN and the antagonist peptide FLLRN (38, 39) (both at 100 μM). As shown in Figure Figure8,8, the agonist was unable to significantly enhance COX-2 expression or PGE2 synthesis above control levels (Figure (Figure8A).8A). Similar results were obtained in murine lung fibroblasts and fibrocytes (data not shown). Importantly, the antagonist was unable to attenuate the effect of plasmin on induction of COX-2 or PGE2 levels (Figure (Figure8B).8B). These results argue against the involvement of PAR-1 in both murine and human mesenchymal cells.
Since plasmin’s effects on COX-2/PGE2 were independent of PAR-1, we utilized IMR-90 cells to test the alternative possibility that they were mediated by activation or release of a growth factor that was itself capable of inducing COX-2. HGF is produced as a biologically inactive precursor, pro-HGF, and can be sequestered either in the extracellular matrix or on the cell membrane (40). Plasmin can cleave pro-HGF and release it from matrix (11, 41, 42), whereupon active HGF can interact with its receptor, c-Met, to oppose fibrogenesis. In renal (43) and bronchial (44) epithelial cells, HGF has been shown to induce COX-2 and increase PGE2 generation, but such an action has not been reported in fibroblasts. Addition of plasmin to IMR-90 cell cultures caused a time-dependent increase in total HGF protein (Figure (Figure9A);9A); the rapid kinetics of this response (peak at 2 hours, with rapid decline thereafter) argue against an underlying transcriptional mechanism and instead suggest proteolytic activation and/or release from cells/matrix. We next incubated cells with HGF at 1 or 10 ng/ml (concentrations spanning the peak concentration of approximately 6 ng/ml measured in Figure Figure9A)9A) and observed a dose-dependent induction of COX-2 protein (Figure (Figure9B).9B). A blocking antibody against the HGF receptor largely abrogated the ability of plasmin to increase PGE2 production (Figure (Figure9C),9C), while a nonspecific IgG did not, implicating HGF in this action of plasmin. When IMR-90 cells were incubated with plasminogen and levels of HGF, PGE2, and COX-2 were measured over a 24-hour period (Figure (Figure9D),9D), kinetic analysis showed that the rise in HGF preceded the increase in COX-2 expression, which in turn preceded the production of PGE2. These results demonstrate that HGF, released from cells/matrix by plasmin, is instrumental in upregulating COX-2 and PGE2, thereby contributing to the antifibrotic effect of plasminogen activation.
To test the in vivo relevance of the HGF/COX-2/PGE2 axis in mediating the antifibrotic actions of plasminogen activation, we evaluated the effects of a selective c-Met inhibitor, PHA-665752, on lung hydroxyproline accumulation in Pai1–/– mice. This inhibitor has previously been reported to exert optimal antitumor actions in mice in vivo at the dosage that we employed (45). Because prominent local irritant effects limit its repeated administration at a given site, we alternated its daily administration between i.v. and s.c. routes from days 10 to 20 after bleomycin treatment and harvested lungs for analysis at day 21 (see protocol in Figure Figure10A);10A); control animals received vehicle alone. Administration of the c-Met inhibitor significantly increased collagen content in Pai1–/– mice (Figure (Figure10B).10B). Similar results were observed at day 14 (data not shown). To determine the impact of c-Met inhibition on the prostanoid synthetic pathway in the protected Pai1–/– mice, we quantified COX-2 protein (Figure (Figure10C)10C) and PGE2 (Figure (Figure10D)10D) in lung homogenates. PHA-665752 significantly reduced COX-2 and PGE2 in parallel with its enhancement of lung collagen. Together, these data indicate that the HGF/c-Met/COX-2/PGE2 axis mediates the in vivo protection against experimental pulmonary fibrosis associated with exaggerated plasminogen activation.
Previous studies have documented that lung fibroblasts from IPF patients manifest an inability to upregulate COX-2 in response to a variety of inducing agents (18, 23). We therefore wished to examine plasmin- and HGF-induced PGE2 synthesis in fibroblasts from IPF patients exhibiting the characteristic histopathologic pattern of usual interstitial pneumonia (UIP) and utilized cells obtained from nonfibrotic lung resected from adult patients without IPF of a similar age for comparison. Normal adult lung fibroblasts exhibited an increase in PGE2 production over baseline in response to both plasmin (Figure (Figure11A)11A) and HGF (Figure (Figure11B),11B), while fibroblasts from UIP lung were unable to augment PGE2 synthesis in response to either stimulus. HGF release into the medium increased in response to plasmin in both normal and UIP fibroblasts, and no significant difference between them was evident (Figure (Figure11C). 11C).
Although research in the pathogenesis of pulmonary fibrosis has been dominated by studies investigating fibroblast activation signals, evidence indicates that tissue remodeling is also characterized by a relative deficiency in counterregulatory antifibrotic signals. Two such antifibrotic signals are PGE2 and plasminogen activator activity. Each of these has been shown to be deficient in patients with IPF, and deficiency of each has been established to be pathogenically important in animal models of pulmonary fibrosis. Although PGE2 has previously been shown to modulate expression of plasminogen activation system components such as PAI-1 (29–31), the influence of the plasminogen activation system on PGE2 production has not been investigated previously. We found that plasminogen activation augmented PGE2 secretion in 3 cell types known to be important players in the development of pulmonary fibrosis — fibroblasts, fibrocytes, and AECs. This action was seen in both normal and fibrotic cells in mice as well as in human adult and fetal lung fibroblasts. Increased PGE2 generation was independent of uPA interaction with uPAR and was instead attributable to the enzymatic activities of uPA and of plasmin; however, PAR-1 was not the proteolytic target of plasmin. Rather, the operative mechanism involved the enzymatic release of HGF by plasmin and subsequent HGF-induced upregulation of COX-2. In vitro and in vivo experiments using Pai1–/– mice established that augmentation of PGE2 biosynthesis is also a consequence of exaggerated endogenous plasmin activity. Moreover, our data suggest that the HGF/COX-2/PGE2 axis is critical for the antifibrotic actions of plasmin in vitro and in vivo. Finally, plasmin- and HGF-mediated PGE2 synthesis was impaired in fibroblasts from IPF patients, indicating a defect in this newly identified form of antifibrotic crosstalk.
In murine fibroblasts and fibrocytes, stimulation with uPA plus plasminogen led to increased PGE2 synthesis. Treatment with uPA or plasminogen alone was insufficient to produce this effect. In human cells, which as we demonstrate secrete higher levels of uPA constitutively, the addition of plasminogen alone was able to induce PGE2 generation. Several lines of evidence argue that the ability of plasminogen activation to accomplish this was mediated by the enzymatic activity of plasmin rather than via ligation of the uPA receptor uPAR. First, treatment with uPA alone failed to promote PGE2 production. Second, the ability of plasminogen activation to induce PGE2 production was inhibited by the addition of α2-antiplasmin. Third, the genetic absence of PAI-1, which removes the major inhibitor of plasminogen activation, augmented PGE2 synthesis in vitro and in vivo. Although PAR-1 is a potential proteolytic target for plasmin and its activation has been reported to be capable of COX-2 induction (39, 46), experiments with PAR-1 agonist and antagonist peptides failed to support a role for this G protein–coupled receptor in the enhancement of PGE2 synthesis. This may be explained by the fact that although plasmin can activate PAR-1, thrombin is the principal protease for PAR-1 (47).
PGE2 opposes fibroproliferative responses to lung injury by mechanisms that include inhibiting inflammatory and immune responses (48), promoting epithelial cell integrity (43, 49), and downregulating activation of a number of fibroblast functions including proliferation (15), migration (50, 51), myofibroblast differentiation (52), and collagen I accumulation (15). Although plasminogen activation shares with PGE2 the ability to restrain fibrogenesis, its actions on fibroblast phenotypes are poorly characterized, but likely complex. Plasmin has been shown to promote fibroblast proliferation via its ability to activate the PAR-1 receptor (53), but also to promote lung fibroblast apoptosis (54). In this report, we demonstrate that generation of plasmin can limit collagen I synthesis. Inhibition of collagen expression was independent of PAR-1 but was prostanoid dependent, as judged by the ability of the COX-1/2 inhibitor indomethacin to abrogate this effect. While the contribution of other prostanoids to collagen inhibition cannot be excluded, PGE2 is the most likely mediator of this effect, as it is the predominant prostanoid produced by both AECs and lung fibroblasts (26, 55). The net result of these actions of plasmin would be to limit fibrosis in vivo. In fact, our results demonstrating that Pai1–/– mice, which are protected from bleomycin-induced pulmonary fibrosis, have elevated levels of PGE2 in the lung support this contention. Similarly, the fact that plasmin cannot induce the secretion of PGE2 in fibroblasts from patients with IPF is also consistent with the known global defect in COX-2 upregulation in these cells (18, 23), a defect that may be explained by epigenetic silencing (21).
Plasmin can cleave a number of matrix proteins, including fibronectin, laminin, proteoglycans, and basement membrane (type IV) collagen, and is also known to activate a number of growth factors including TGF-β and HGF. Active HGF is an important effector of antifibrotic actions in vivo (56–58) and has been shown to specifically contribute to the antifibrotic influence of plasminogen activation (32). Exogenous administration of HGF (59) or HGF gene transfer (60, 61) has been shown to reduce the development of bleomycin-induced pulmonary fibrosis in vivo. HGF has been shown previously to suppress collagen I synthesis in fibroblasts (62), to augment collagenolytic activity in epithelial cells (57), and to induce COX-2 and PGE2 synthesis in epithelial cells (44). Our results provide evidence that plasmin increases HGF (via some combination of proteolytic cleavage of pro-HGF and release from matrix sequestration; ref. 11) and this induces COX-2 synthesis. Importantly, the ability of an HGF receptor–blocking antibody to abrogate the effects of plasmin on COX-2/PGE2 synthesis demonstrates that HGF is responsible for plasmin’s effects on human lung fibroblast collagen synthesis. The inability of uPA to produce this same effect argues that plasmin-mediated proteolytic release of HGF from the matrix is critical in this process. Finally, the fact that a c-Met inhibitor abolished the protection against bleomycin-induced fibrosis observed in Pai1–/– mice in parallel with decreases in COX-2 protein and PGE2 serves to implicate the HGF/COX-2/PGE2 axis in the resistance of these plasmin-rich animals in vivo.
Transcription of the HGF gene is well known to be stimulated by substances that increase cyclic AMP, including PGE2 (63). It is therefore likely that this ability of HGF to promote PGE2 synthesis is part of a positive feedback loop that results in amplification of fibroblast HGF production. This, in turn, further contributes to an antifibrotic milieu by virtue of the ability of HGF to promote epithelial cell survival (64) and to inhibit epithelial-mesenchymal transdifferentiation (43, 65). It has long been known that plasminogen activation is antifibrotic but that its mechanisms do not necessarily involve fibrin degradation (6, 10). Our results provide new insight into potential mechanisms whereby plasminogen activation exerts its antifibrotic effects in vivo.
C57BL/6 mice were purchased from The Jackson Laboratory. Animal protocols were approved by the University of Michigan Committee on the Use and Care of Animals. Pai1–/– mice on a C57BL/6 background as described previously (66) were bred in-house.
Murine uPA, plasmin, and plasminogen were purchased from Molecular Innovations. Human uPA was purchased from American Diagnostica, and human plasminogen, human plasmin, HGF (containing both active and pro-forms), and the nonselective COX inhibitor indomethacin were purchased from Sigma-Aldrich. Primary antibodies for immunoblot analysis were obtained from the following suppliers: anti–COX-2 from Cayman Chemical; anti-human and anti-mouse antibodies against type 1 collagen both from CedarLane Laboratories; anti–α-tubulin from Sigma-Aldrich; anti-uPA from Oxford Biomedical. Secondary anti-murine antibodies for Western blot analysis were purchased from Pierce Biotechnology and Cell Signaling Technology. Secondary anti-rabbit antibodies for Western blot were purchased from Cell Signaling Technology. Bleomycin was purchased from Sigma-Aldrich. PGE2 was obtained from Cayman Chemicals and dissolved in DMSO. Blocking antibody against the HGF receptor c-Met was obtained from R&D Systems. PGE2 ELISA kits were purchased from Assay Designs or Cayman Chemicals, and HGF ELISA kit was purchased from R&D Systems. The PAR-1 agonist TFLLRN and the PAR-1–blocking peptide FLLRN were purchased from AnaSpec. The His-tagged α2-antiplasmin was purchased from Molecular Innovations. The c-Met inhibitor PHA-665752 was purchased from Tocris Bioscience.
Mice were anesthetized with intraperitoneal ketamine/xylazine, and the trachea was exposed by incision. Bleomycin was dissolved in PBS and instilled intratracheally (i.t.) using a 27-gauge needle at a dose of 0.00135 U/g mouse in a volume of 50 μl. In some experiments, WT and Pai1–/– mice were treated with the selective c-Met inhibitor PHA-665752 (45) (25 mg/kg) daily from days 10 to 20 after bleomycin treatment via i.v. or s.c. routes on alternate days; this dosage was selected because it was determined from dose-response experiments to exhibit optimal antitumor effects in mice in vivo (45). Lungs were harvested on day 21 after bleomycin treatment and analyzed for COX-2 protein by immunoblot analysis, PGE2 by ELISA, and hydroxyproline content as previously described (24).
Fibroblasts and fibrocytes were isolated from lungs of mice on day 14 after saline or bleomycin treatment, as described (35). Murine lungs were perfused via the right ventricle with 5 ml 0.9% NaCl and removed under aseptic conditions. Lungs were minced with scissors in DMEM containing 10% fetal calf serum. Lungs from a single animal were placed in 15 ml medium in tissue culture flasks. Mesenchymal cells were allowed to grow out of the minced tissue, and when cells reached 70% confluence, they were passaged following trypsinization. Cells were grown for 14 days (2–3 passages) before being used. To separate fibrocytes from fibroblasts, cells were incubated with anti-CD45 Abs coupled to magnetic beads (Miltenyi Biotec). Labeled cells were then sorted by binding the cell population to positive selection columns using a SuperMACS apparatus (Miltenyi Biotec) according to the manufacturer’s instructions. Immunohistochemical staining or flow cytometry staining on this population confirmed that these fibrocytes were CD45+collagen I+. In contrast, the fibroblast population was CD45–collagen I+.
Type II AECs were isolated from mice using the method of Corti and coworkers (67). Following anesthesia and heparinization, the mouse was exsanguinated and the pulmonary vasculature perfused. The trachea was cannulated, and the lungs were filled with 1–2 ml Dispase (BD Biosciences), followed by 1 ml of low-melting-point agarose. Lungs were then removed and placed in iced PBS to harden the agarose. The lungs were placed in 2 ml Dispase and incubated for 45 minutes at 24°C. Subsequently the lung tissue was teased from the airways and minced in DMEM with 0.01% DNase. The lung mince was gently passed successively through 100-, 40-, and 25-μm nylon mesh filters. Bone marrow–derived cells were removed by magnetic depletion using anti-CD32 and anti-CD45 Abs. Mesenchymal cells were removed by overnight adherence in a petri dish. The nonadherent AECs were then plated at 500,000 cells/well on fibronectin-coated 24-well plates. Cells were maintained in DMEM with penicillin/streptomycin/amphotericin B and 10% fetal calf serum at 37°C in 5% CO2. The final adherent population included only 4% nonepithelial cells at day 2 in culture by intermediate filament staining.
IMR-90 fetal human lung fibroblasts were obtained from the Coriell Institute for Medical Research at passage 4. They were cultured in DMEM plus 10% FBS at 37°C in 5% CO2 and used for experimentation at passage 6–9. As previously described (68), primary adult “normal” human lung fibroblasts were isolated from the margins of lung tissue resected from patients with suspected lung cancer that displayed normal lung histology, whereas IPF fibroblasts were cultured from lung biopsy specimens of patients diagnosed with IPF whose tissue histopathology showed UIP. Patients in the two groups were of similar age, and specimens from both groups were obtained with written informed consent under a protocol approved by the Institutional Review Board of the University of Michigan. Primary fibroblasts were cultured in the above medium and used at passage 7. For experimentation, cells were trypsinized, counted, and plated in Falcon 6-well plates (BD Biosciences) at 500,000–800,000 cells per well. They were allowed to adhere for 6–8 hours and then cultured in serum-free DMEM for 18–24 hours. At the time of experimental treatment, the cells were 60%–80% confluent.
ELISA was used to quantify PGE2 and HGF per the manufacturers’ instructions in cell-free supernatants. In lung homogenates, lipids were extracted using C18 Sep-Pak cartridges (Waters) as previously described (22), and PGE2 was then measured by ELISA in reconstituted lipid extracts.
HGF receptor blocking antibody or nonspecific IgG (both at 20 μg/ml) were added to the medium 10 minutes prior to the addition of plasmin and allowed to incubate with the cells for 18 hours. The medium was then removed and assayed for levels of PGE2.
Murine cells were washed with ice-cold PBS and 200 μl cold lysis buffer (1% w/w Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M NaH2PO4, 0.02 M EDTA, 0.05 M NaF, 0.002 M NA3VO4, and 1:100 dilution of Calbiochem Protease Cocktail Set II (Calbiochem-Novabiochem) was added to each sample. For human samples, the lysis buffer contained PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS. 1× Protease inhibitor from Roche was added fresh each time along with a 1:100 dilution of 0.2 M sodium orthovanadate. Lysates were assayed for total protein concentration using the DC Protein Assay (Bio-Rad). For mouse cells, 4 μg of protein from each sample was then analyzed for expression of COX-2 or β-actin using methods that have been described previously (52). For human samples, 20–25 μg of protein was loaded prior to testing for collagen I, COX-2, or tubulin.
Zymography was performed to determine the molecular size of plasminogen activators, using the method of Powell-Jones (69) with modifications. The cell supernatants were subjected to SDS-PAGE using 10% slab gels containing a-casein (7 mg/ml; Sigma-Aldrich) and Glu-plasminogen (20 mg/ml). The gel was washed in 1% Tween 80 for 1 hour at 37°C and incubated in 0.1% Tween 80 in PBS overnight at room temperature. The gel was then stained with Coomassie blue and destained in a solution of 10% acetic acid and 50% methanol. The molecular weights of the lytic bands were calculated by comparison to human and mouse uPA standards.
Data are presented as mean ± SEM of values determined from the indicated number of experiments. Data analysis employed GraphPad Prism software, using Student’s t test or ANOVA with Bonferroni correction, as appropriate, to determine significant differences between group means. In all instances, a P value less than 0.05 was considered significant.
The authors thank Bi Yu, Natalya Subbotina, and Carol Wilke for technical assistance. This work was supported by NIH grants HL087846 and AI065543, a grant from the Pulmonary Fibrosis Foundation, and a Career Investigator Award from the American Lung Association of Michigan (B.B. Moore); NIH grants HL058897 and HL094311 (to M. Peters-Golden) and HL078871 (to T.H. Sisson); and a Parker B. Frances Fellowship, an American Thoracic Society Career Development Award, and NIH grant HL094657 (to S.K. Huang).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2010;120(6):1950–1960. doi:10.1172/JCI38369.