The antifibrotic effects of plasminogen activation occur via prostaglandin E₂ synthesis in humans and mice

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.

Pai1^–/– mice exhibit increased production of PGE₂ in vivo. 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 1 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).

Figure 1

Bleomycin-treated Pai1^–/– mice overproduce PGE₂. WT or Pai1^–/– mice were injected with bleomycin on day 0. On day 21, lungs were removed and homogenized. Lipids were extracted using C18 cartridges, and levels of PGE₂ were measured by ELISA; n = 5, *P = 0.03.

Plasminogen activation increases PGE₂ secretion in murine lung fibroblasts. 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 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 2B) mice when compared with cells cultured in SFM alone.

Figure 2

Plasminogen activation stimulates PGE₂ release in fibroblasts. (A) Fibroblasts from saline-treated mice were cultured at 5 × 10⁵/ml and serum starved for 24 hours. Cells were then treated with SFM, 10 U/ml uPA, 45 mU/ml plasminogen (PLG), or uPA plus PLG for 24 hours. PGE₂ was then measured by ELISA in cell supernatants; n = 5, ***P < 0.001. (B) Mice were given i.t. saline or i.t. bleomycin on day 0. On day 14, lungs were harvested, minced, and cultured until day 28. Fibroblasts from normal and bleomycin-treated mice (N-FIB and B-FIB, B) were cultured in SFM or with uPA plus PLG (U+P) for 24 hours, and PGE₂ was measured; n = 5 or more in all groups, ***P < 0.001. (C) Fibroblasts were purified from WT or Pai1^–/– mice and were treated with SFM or uPA plus PLG for 24 hours before culture supernatants were analyzed for PGE₂ production via ELISA; n = 5 or more in each group, *P < 0.05, ***P < 0.001.

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 2C). These results suggest that the proteolytic function of uPA is important for the induction of PGE₂ generation.

Plasminogen activation increases PGE₂ secretion in murine fibrocytes. 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 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 3B). Fibrocytes purified from Pai1^–/– mice showed enhanced secretion of PGE₂ compared with WT fibrocytes in the presence of uPA plus plasminogen (Figure 3C). It is also evident from these data that the capacity of fibrocytes to produce PGE₂ is substantially lower than that of fibroblasts.

Figure 3

Plasminogen activation stimulates PGE₂ release in fibrocytes. (A) Fibrocytes from saline-treated mice were cultured at 5 × 10⁵/ml and serum starved for 24 hours. Cells were then treated with SFM, 10 U/ml uPA, 45 mU/ml PLG, or uPA plus PLG for 24 hours. PGE₂ was then measured by ELISA in cell supernatants; n = 3, **P < 0.01. (B) Mice were given i.t. saline or i.t. bleomycin on day 0. On day 14, lungs were harvested, minced, and cultured until day 28. Fibrocytes were then purified. Fibrocytes (FIBCY) from saline-treated, normal, and bleomycin-treated mice (B) were cultured in SFM or with uPA plus PLG for 24 hours, and PGE₂ was measured; n = 5, ***P < 0.001. (C) Fibrocytes were purified from WT or Pai1^–/– mice and were treated with SFM or uPA plus PLG for 24 hours before culture supernatants were analyzed for PGE₂ production via ELISA; n = 4, ***P < 0.001.

Proteolytic actions of uPA and plasmin are responsible for promoting murine mesenchymal cell synthesis of PGE₂. 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 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 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).

Figure 4

Induction of PGE₂ synthesis in mouse lung mesenchymal cells requires plasmin enzymatic activity. (A) Fibroblasts and fibrocytes were purified from saline-treated lungs, serum starved overnight, and cultured for 24 hours in SFM or with 50 mU/ml plasmin. PGE₂ was then measured in cell-free supernatants; n = 3, ***P < 0.001. (B) Fibroblasts from saline-treated mice were cultured in the presence of SFM, 10 U/ml uPA plus 45 mU/ml PLG, or uPA plus PLG plus 30 μg/ml α2-antiplasmin; n = 7 per group, *P < 0.05, ***P < 0.001.

Plasminogen activation leads to increased COX-2 production and decreased collagen synthesis in murine fibroblasts and fibrocytes. 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 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 5, A and B) and fibrocytes (Figure 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 5, A and B) as well as fibrocytes (Figure 5, C and D).

Figure 5

Plasminogen activation induces COX-2 and limits collagen I production in mouse lung mesenchymal cells. Fibroblasts (A and B) and fibrocytes (C and D) from saline-treated mice were cultured for 24 hours in the presence of SFM alone, 10 U/ml uPA plus 45 mU/ml PLG, or 50 mU/ml plasmin. Cell lysates were prepared and analyzed by Western blot for expression of collagen I and COX-2 (A and C). Each lane represents a unique culture. Data are representative of 2 independent experiments. In B and D, total RNA was made from cells cultured as above and analyzed for expression of Cox2 and the α1 chain of procollagen I (Procol I) by real-time RT-PCR. Values were first normalized to expression of β-actin in each sample. Then, the average of the n = 3 SFM-treated cultures was normalized to 1 for each gene.

Plasminogen activation increases PGE₂ secretion and COX-2 expression in murine AECs. 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 6A) and fibrotic (Figure 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 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 6D).

Figure 6

Plasminogen activation stimulates PGE₂ release in AECs. (A) AECs from saline-treated mice were cultured at 1.5 × 10⁶/ml on fibronectin-coated plates and serum starved for 24 hours. Cells were then treated with SFM, 10 U/ml uPA, 45 mU/ml PLG, or uPA + PLG for 24 hours. PGE₂ was then measured by ELISA in cell supernatants; n = 4, **P < 0.01. (B) Mice were given i.t. saline or i.t. bleomycin on day 0. On day 14, AECs were purified. AECs from saline-treated, normal and bleomycin-treated mice (N-AEC and B-AEC, B) were cultured in SFM or with uPA plus PLG for 24 hours, and PGE₂ was measured; n = 3 or more in all groups, **P < 0.01, ***P < 0.001. (C) AECs were purified from WT or Pai1^–/– mice and were treated with SFM or uPA plus PLG for 24 hours before culture supernatants were analyzed for PGE₂ production via ELISA; n = 4 or more in each group, *P < 0.05, **P < 0.01, ***P < 0.001. (D) AECs were purified from saline-treated mice and cultured at 1.5 × 10⁶/ml on fibronectin-coated plates. Cells were serum starved overnight and then cultured in SFM, 10 U/ml uPA plus 45 mU/ml PLG, or with 50 mU/ml plasmin. Total RNA was prepared and analyzed for Cox2 via real-time RT-PCR. Values for each sample were first normalized to β-actin, then the mean value for the SFM group was normalized to 1. n = 2 per group, representative of 2 experiments.

Plasminogen activation promotes PGE₂ synthesis via COX-2 induction, with resultant inhibition of collagen expression in human IMR-90 fibroblasts. 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 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 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 7C). Importantly, treatment of IMR-90 cells with the COX-1/2 inhibitor indomethacin prevented inhibition of collagen I synthesis by plasminogen (Figure 7D), implying that plasmin’s suppressive effect in fibroblasts was indeed dependent on its ability to enhance prostanoid generation.

Figure 7

IMR-90 cell PGE₂ synthesis is regulated by plasminogen activation. (A) IMR-90 cells in SFM were treated with PLG (200 mU/ml), plasmin (100 mU/ml), or uPA (10 U/ml) for 18 hours. Medium was removed from the cells, and PGE₂ was measured by ELISA and is expressed relative to the control value measured in SFM alone; n ≥ 5, *P < 0.05 versus control. (B) Cell supernatants were collected from either saline-treated murine lung fibroblasts or human IMR-90 cells cultured at equal densities for 24 hours. Supernatants were then analyzed by zymography for the ability to activate PLG. A band of the appropriate size for human uPA (54 kDa) is readily distinguishable in the IMR-90 cell supernatants, but the lower-molecular-weight murine uPA band (45 kDa) is very faint in the murine lung fibroblasts; n = 5 per group. (C) IMR-90 cells in SFM were treated with 100 mU/ml plasmin for 18 hours. Media was harvested for PGE₂ determination (triangles), and lysates were harvested for collagen I protein expression (circles) or COX-2 protein expression (squares) as determined by immunoblot analysis and densitometry using α-tubulin as a loading control; n = 3, *P < 0.05 versus control. Collagen I and COX-2 immunoblots are representative of experiments performed in triplicate. (D) IMR-90 cells were treated with SFM (control), PLG (100 mU/ml), or PLG plus indomethacin (Indo; 10 μM) for 18 hours, and collagen I was determined by immunoblot analysis and densitometry using α-tubulin as a loading control; n = 3, *P < 0.05 versus control. Collagen I immunoblots are representative of experiments performed in triplicate. Blots in both C and D are composed from lanes that were run on the same gel but were noncontiguous.

Induction of COX-2 expression and PGE₂ synthesis is independent of PAR-1 signaling. 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 8, the agonist was unable to significantly enhance COX-2 expression or PGE₂ synthesis above control levels (Figure 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 8B). These results argue against the involvement of PAR-1 in both murine and human mesenchymal cells.

Figure 8

Lack of effect of PAR-1 agonist and antagonist peptides on PGE₂ production and COX-2 expression in IMR-90 cells. (A) IMR-90 cells in SFM were treated with the PAR-1 agonist peptide TFLLRN (100 μM) for 18 hours; n = 3. (B) IMR-90 cells in SFM were treated with plasmin (100 mU/ml) with and without the PAR-1 antagonist peptide FLLRN (antag; 100 μM) for 18 hours; n = 3. Where plasmin and FLLRN were coincubated, the antagonist was added 10 minutes before the plasmin. Lysates were harvested, and COX-2 protein expression was determined by immunoblot analysis and densitometry using α-tubulin as a loading control. Medium was removed from the cells for PGE₂ determination. COX-2 immunoblots in A and B are derived from the same experiments, so that differences in densitometric units accurately reflect differences between plasmin (B) and no-plasmin control (A) conditions. None of the comparisons shown represent statistically significant differences. Blots in both A and B are composed from two lanes that were run on the same gel but were noncontiguous.

HGF activation by plasmin mediates upregulation of PGE₂ biosynthesis. 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 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 9A) and observed a dose-dependent induction of COX-2 protein (Figure 9B). A blocking antibody against the HGF receptor largely abrogated the ability of plasmin to increase PGE₂ production (Figure 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 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.

Figure 9

Plasmin induces IMR-90 cell COX-2 expression and PGE₂ synthesis in an HGF-dependent manner. (A) IMR-90 cells in SFM were treated with plasmin (50 mU/ml) for 2, 4, 9, 18, and 24 hours. Total HGF was determined by ELISA and normalized to levels measured in cells incubated with SFM alone; n = 3, *P < 0.05 versus control. At 2 hours, the mean absolute concentration of HGF was 6.12 ng/ml. (B) IMR-90 cells in SFM were treated with HGF (1 or 10 ng/ml) for 18 hours. Lysates were harvested for COX-2 protein expression as determined by immunoblot analysis and densitometry using α-tubulin as a loading control; n = 3, *P = 0.05 versus control (SFM alone). (C) IMR-90 cells in SFM were treated with plasmin (50 mU/ml), plasmin with an HGF receptor blocking antibody (20 μg/ml), or plasmin with a nonspecific IgG (control IgG; 20 μg/ml) for 18 hours (n = 3). PGE₂ was determined and normalized for cellular protein. *P < 0.05 versus control (SFM alone). (D) IMR-90 cells in SFM were treated with PLG (200 mU/ml) for 2, 4, 9, 18, and 24 hours. Lysates were harvested for COX-2 protein expression (inverted triangles) as determined by immunoblot analysis and densitometry using α-tubulin as a loading control. Media was harvested, and PGE₂ (squares) and HGF (circles) were determined and normalized to cellular protein. Data are expressed relative to SFM control and are from 1 experiment representative of 2.

The HGF/COX-2/PGE₂ axis mediates protection against pulmonary fibrosis in Pai1^–/– mice in vivo. 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 10A); control animals received vehicle alone. Administration of the c-Met inhibitor significantly increased collagen content in Pai1^–/– mice (Figure 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 10C) and PGE₂ (Figure 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.

Figure 10

A c-Met inhibitor increases collagen deposition in the lungs of Pai1^–/– mice in parallel with reductions in COX-2 expression and PGE₂ production. (A) Protocol. Pai1^–/– mice were injected with bleomycin (Bleo) on day 0 and were treated with vehicle (l-lactate and 10% polyethylene glycol) alone (control, n = 6) or with the c-Met inhibitor PHA-665752 (25 mg/kg) (n = 7) in vehicle. On alternate days, administration was i.v. via tail vein or s.c., as shown. On day 21, lungs were harvested. (B) Collagen deposition in the right lung was determined by measuring hydroxyproline content. (C) COX-2 levels in left lung homogenates were determined by immunoblot analysis and densitometry. Data are expressed as percent of vehicle-treated mice. (D) PGE₂ levels in the lipid extracts from left lungs were measured by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. Similar results were obtained in a second experiment.

The ability of plasmin and HGF to induce PGE₂ secretion is defective in fibroblasts from patients with IPF. 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 11A) and HGF (Figure 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 11C).

Figure 11

Plasmin and HGF are unable to upregulate PGE₂ production in fibroblasts from patients with IPF. Fibroblasts from IPF patients diagnosed with UIP or fibroblasts obtained from histologically normal (Nml) lung resections were cultured in SFM (control) with or without plasmin (100 mU/ml) (A) or HGF (10 ng/ml) (B) for 18 hours. Medium was removed, and PGE₂ levels were determined by ELISA. The SFM (control) sample for each cell line was normalized to 100% (dotted lines). Data are from cells derived from n = 3 patients per group. *P < 0.05 versus Nml. (C) Fibroblasts from Nml and UIP lung were treated with SFM alone (control) or plasmin (100 mU/ml) for 2 hours (based on the kinetics of HGF release shown in Figure 9A), and HGF levels in the supernatants were determined by ELISA. Data are from cells derived from n = 3 patients per group; *P < 0.05 versus control Nml without plasmin.

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.

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).

Address correspondence to: Bethany B. Moore, 4053 BSRB, 109 Zina Pitcher Place, Ann Arbor, Michigan 48109, USA. Phone: 734.647.8378; Fax: 734.612.2331; E-mail: Bmoore@umich.edu. Or to: Marc Peters-Golden, 6301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, USA. Phone: 734.763.9077; Fax: 734.764.4556; E-mail: petersm@umich.edu.

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.

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