Abstract
Rationale: Activation of the coagulation cascade has been demonstrated in pulmonary fibrosis. In addition to its procoagulant function, various coagulation proteases exhibit cellular effects that may also contribute to fibrotic processes in the lung.
Objective: To investigate the importance of protease-activated receptor (PAR)-2 and its activators, coagulation factor VIIa (FVIIa)/tissue factor (TF), in the development of idiopathic pulmonary fibrosis (IPF).
Methods: Expression and localization of PAR-2 and its activators were examined in IPF lung tissue. The ability of PAR-2 to mediate various cellular processes was studied in vitro.
Measurements and Main Results: Expression of PAR-2 was strongly elevated in IPF lungs and was attributable to alveolar type II cells and fibroblasts/myofibroblasts. Transforming growth factor-β1, a key profibrotic cytokine, considerably enhanced PAR-2 expression in human lung fibroblasts. FVIIa stimulated proliferation of human lung fibroblasts and extracellular matrix production in a PAR-2–dependent manner, but did not initiate differentiation of fibroblasts into myofibroblasts. PAR-2/FVIIa-driven mitogenic activities were mediated via the p44/42 mitogen-activated protein kinase pathway and were independent of factor Xa and thrombin production. Proproliferative properties of FVIIa were markedly potentiated in the presence of TF and abrogated by TF antisense oligonucleotides. Hyperplastic alveolar type II cells overlying fibroblastic foci were found to be the source of FVII in IPF lungs. Moreover, TF colocalized with PAR-2 on fibroblasts/myofibroblasts in IPF lungs.
Conclusions: The PAR-2/TF/FVIIa axis may contribute to the development of pulmonary fibrosis; thus, interference with this pathway confers novel therapeutic potential for the treatment of IPF.
Receptor-mediated cellular effects of blood coagulation factors play an increasingly important role in the development of idiopathic pulmonary fibrosis (IPF). However, the contribution of protease-activated receptor-2 (PAR-2) to IPF is not known.
Activation of PAR-2 by factor VIIa/tissue factor complex induces fibroblast/myofibroblast proliferation and extracellular matrix production. Thus, up-regulation of PAR-2 and its activators in the lungs of patients with IPF may make a significant contribution to fibrotic process in the lung.
Accumulating evidence suggests that activation of the coagulation cascade may play a role in the pathogenesis of pulmonary fibrosis. Increased procoagulant activity has been observed in the bronchoalveolar lavage fluids (BALFs) of patients with IPF. The procoagulant activity observed under these conditions arises from the imbalance between locally produced pro- and anticoagulant factors, in combination with leakage of plasma proteins into the alveolar space. Tissue factor (TF) in association with factor VIIa (FVIIa) and inhibition of urokinase by plasminogen activator inhibitor-1 are major factors that are responsible for the intraalveolar accumulation of fibrin (7, 8). Fibrin deposits have been found in lung biopsies from patients with pulmonary fibrosis, and they have been associated with the development of fibrotic lesions (9).
Besides their important role in fibrin formation, it is now well recognized that coagulation proteases exert profibrotic cellular effects via activation of protease-activated receptors (PARs). At present, four PARs have been described: PAR-1 to PAR-4. Thrombin activates PAR-1, PAR-3, and PAR-4, whereas factor Xa (FXa) cleaves either PAR-1 or PAR-2, depending on cell type and cofactor expression. PAR-2 is also activated by trypsin, tryptase, neutrophil proteinase-3, TF/FVIIa, and TF/FVII/FXa complexes (10, 11). To date, some studies have investigated the contribution of PAR-1 to the development of pulmonary fibrosis. Activation of PAR-1 by FXa or thrombin was found to promote fibroblast proliferation, procollagen production, cytokine release, and fibroblast-to-myofibroblast differentiation (12–14). A potential role of PAR-1 in fibrotic processes was further underscored by the finding that PAR-1–deficient mice are protected from bleomycin-induced lung fibrosis (15). In line with this observation, direct inhibition of FXa or thrombin attenuated the fibrotic response in the bleomycin model as well (14, 16).
Although the role of PAR-1 in the development of lung fibrosis has been documented, much less is known about the contribution of PAR-2 to this pathological condition. PAR-2 has been suggested to play a role in tissue repair processes in the liver, pancreas, and kidney (17–19). Therefore, it is tempting to hypothesize that PAR-2 may play a role in the fibroproliferative process occurring in the lungs of patients with IPF. To explore this idea, the expression and activity of the PAR-2/TF/FVII axis was investigated in IPF lungs, with particular attention being paid to PAR-2–driven lung fibroblast activation, the major hallmark of fibrotic processes. Some of the results of these studies have been previously reported in the form of an abstract (20).
A detailed description of routine methodologies is provided in the online supplement. Only nonstandard procedures and specialized materials are described in this section.
The investigations have been conducted according to Declaration of Helsinki principles and were approved by the local institutional review board and ethics committee. Informed consent was obtained from either the patients or their next-of-kin. BALF was obtained by flexible fiberoptic bronchoscopy from 20 spontaneously breathing healthy volunteers and from 40 spontaneously breathing patients with IPF. Diagnosis of IPF was settled on the basis of the American–European Consensus Criteria (1). In 20 patients, diagnosis was confirmed by surgical lung biopsy and forwarded an UIP pattern in every case. In addition, lung tissue was obtained from 24 patients with IPF who underwent lung transplantation. IPF diagnosis was based on both clinical criteria as well as proof of an UIP pattern. Unused donor lungs served as a control (n = 10). Table 1 shows the demographic and clinical characteristics of the patient cohort.
Variable | IPF (BALF) | IPF (lung tissue) |
|---|---|---|
| Subjects, n | 40 | 24 |
| Age (yr), mean ± SD | 60.3 ± 12.1 | 56.3 ± 18.7 |
| Sex (male/female), n/n | 30/10 | 19/5 |
| Smoking status (never/former/current), n/n/n | 20/15/5 | 10/14/0 |
| FVC% predicted, mean ± SD | 65.2 ± 25.0 | 57.4 ± 19.5 |
| DlCO% predicted, mean ± SD | 45.6 ± 13.8 | 29.1 ± 18.0 |
| Histological confirmation of a UIP pattern, % | 50 | 100 |
Fibroblasts and alveolar type II cells were isolated from donor and IPF lungs as described (21, 22). Fibroblasts were stimulated for various times with FXa (50 nM), FVIIa (50 nM), thrombin (65 U/L), inactivated FVIIa (FVIIi; 50 nM) (all from American Diagnostica, Greenwich, CT); peptide agonist for PAR-2 (AP; 2-furoyl-LIGRLO-NH2, 100 μM), scrambled peptide control (RP; trans-cinnamoyl-OLIGRL-NH2, 100 μM) (both kindly provided by A. Meinhardt, Department of Anatomy and Cell Biology, Justus Liebig University, Giessen, Germany); or transforming growth factor (TGF)-β1 (10 ng/ml; R&D Systems, Wiesbaden, Germany). In some experiments, cells were transfected with small interfering RNA (siRNA) sequences directed against human PAR-2 (Santa Cruz Biotechnology, Santa Cruz, CA) or TF (Ambion, Austin, TX) or with vectors containing full-length cDNA for human PAR-2 or TF.
Proliferation of human lung fibroblasts (HLFs) was determined by a DNA synthesis assay based on the uptake of [3H]thymidine.
Statistical analyses were performed in R version 2.3.1. Deviations from the normal distribution were tested by Shapiro-Wilk test. All in vitro data were normally distributed and therefore these data are presented as means (± SD). Clinical data are given as medians and interquartile range. The box-and-whisker plots indicate the median and first and third quartiles; the whiskers are extended to the most extreme value inside the 1.5-fold interquartile range. Differences between two groups were tested by Student t test and Wilcoxon rank sum test according to the distribution of the data. All tests were performed with an undirected hypothesis (two-sided). The level of statistical significance was set at 5%.
Initially, we analyzed the expression of PAR-2 in IPF lungs. Elevated PAR-2 mRNA (Figure 1A) and protein (Figures 1B and 1C) levels were observed in the lung homogenates of patients with IPF. Immunohistochemical staining of donor lung sections demonstrated immunoreactivity for PAR-2 in alveolar macrophages and alveolar type II (ATII) cells (Figure 1D), whereas in IPF lungs strong PAR-2 staining was associated with hyperplastic ATII cells and fibroblasts/myofibroblasts (Figures 1D and 1E). Western blot analysis revealed enhanced production of PAR-2 in fibroblasts isolated from IPF lungs as compared with fibroblasts isolated from donor lungs (Figures 1F and 1G). Immunofluorescence analysis confirmed these results (Figure 1H, top). Furthermore, fibroblasts derived from IPF lungs showed a marked increase in the number of α-smooth muscle actin (α-SMA)–positive cells, indicating their myofibroblast phenotype (Figure 1H, bottom). As fibroblasts are key effector cells in the development of fibrosis, in further studies we focused on the role of PAR-2 in the regulation of various processes in this cell population.
Figure 1. Expression of protease-activated receptor (PAR)-2 is elevated in idiopathic pulmonary fibrosis (IPF) lungs. (A) PAR-2 expression in lung homogenates of donors (n = 10) and patients with IPF (n = 24) as assessed by quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR results are expressed as ΔCt (difference in cycle threshold [Ct] between the housekeeping gene [PBGD; porphobilinogen deaminase] and the gene of interest). Significance level is indicated. (B) PAR-2 expression in lung homogenates of donors and patients with IPF as assessed by Western blotting. The specificity of the detected band was proven by preincubating the anti–PAR-2 antibody (αPAR-2) with its blocking peptide (BP). Representative donors (4 of 10) and patients (9 of 24) are shown. (C) Densitometric analysis of (B). Significance level is indicated. (D) Representative serial lung sections from patients with IPF and donors, stained for PAR-2, α-smooth muscle actin (α-SMA), or pro–surfactant protein C (proSP-C). Negative controls, including no anti–PAR-2 antibody, isotype control, and anti–PAR-2 antibody preabsorbed with blocking peptide, are demonstrated. Arrowheads indicate alveolar type II (ATII) cells, asterisks represent alveolar macrophages, and arrows indicate fibroblasts/myofibroblasts. Original magnification, ×20. Scale bar: 100 μm. (E) Double immunostaining of IPF lung sections for PAR-2 and α-SMA. Arrows indicate PAR-2– and α-SMA–positive fibroblasts/myofibroblasts. Insets show the negative control. Original magnification, ×43. Scale bar: 20 μm. (F) PAR-2 expression in human lung fibroblasts (HLFs) isolated from donor (n = 10) and IPF (n = 24) lungs. The specificity of the detected band was proven by preincubating anti–PAR-2 antibody (αPAR-2) with its blocking peptide (BP). Representative donors (4 of 10) and patients (4 of 24) are shown. (G) Densitometric analysis of (F). Significance level is indicated. (H) Donor and IPF lung fibroblasts cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1% penicillin–streptomycin were stained for PAR-2 (top) and α-SMA (bottom). Insets show the negative control. Original magnification, ×40. Scale bar: 10 μm.
[More] [Minimize]To investigate how PAR-2 expression may be regulated in pulmonary fibrosis, donor HLFs were stimulated with the key profibrotic growth factor TGF-β1, and PAR-2 production was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. TGF-β1 increased PAR-2 mRNA and protein expression, with maximal effects observed at 20 and 72 hours of treatment, respectively (Figures 2A–2C). Similar results were obtained when HLFs were treated with other profibrotic cytokines, such as platelet-derived growth factor-BB or insulin-like growth factor-1 (data not shown).
Figure 2. Transforming growth factor (TGF)-β1 up-regulates protease-activated receptor (PAR)-2 expression in human lung fibroblasts (HLFs). (A and B) Time course of PAR-2 expression in donor HLFs after TGF-β1 (10 ng/ml) stimulation as assessed by (A) quantitative real-time polymerase chain reaction (qRT-PCR) and (B) Western blotting. qRT-PCR results are expressed as ΔCt (difference in cycle threshold [Ct] between the housekeeping gene [PBGD; porphobilinogen deaminase] and the gene of interest). Data are shown as means ± SD, n = 4. *P < 0.05. The Western blot illustrated is from one representative experiment of four. (C) Densitometric analysis of (B). Data are presented as means ± SD; n = 4. *P < 0.05; **P < 0.01.
[More] [Minimize]We next investigated the induction of donor HLF proliferation by PAR-2 activators such as FXa and FVIIa. Both FXa and FVIIa increased lung fibroblast proliferation as assessed by cell counting (Figure 3A). Elevated HLF proliferation was also evident by enhanced [3H]thymidine incorporation (Figure 3B) and increased immunostaining for Ki67 (Figures 3C and 3D). In accordance with these data, a time-dependent rise in the expression of cyclin D1 on stimulation of HLFs with FXa or FVIIa was observed (see Figure E1 in the online supplement). Of note, mitogenic effects induced by FXa and FVIIa were as pronounced as those induced by thrombin (Figures 3A−3D). Neither the thrombin inhibitor hirudin nor the FXa inhibitor TENSTOP blocked FVIIa-induced proliferation of HLFs (Figure 3E). This indicates that FVIIa-dependent mitogenic effects are not a result of thrombin and FXa generation, which are the downstream products of FVIIa activation.
Figure 3. Factor Xa (FXa), FVIIa, and thrombin stimulate proliferation of human lung fibroblasts (HLFs). (A) Donor HLFs were stimulated for 24 hours with FXa (50 nM), FVIIa (50 nM), or thrombin (65 U/L). Cell proliferation was assessed by cell counting. Data are presented as means ± SD from four independent experiments. **P < 0.01; ***P < 0.001. (B) [3H]thymidine incorporation in HLFs treated for 24 hours with FXa (50 nM), FVIIa (50 nM), or thrombin (65 U/L). The results are expressed as relative proliferation, compared with unstimulated cells. Data represent mean values ± SD from four independent experiments, each performed in triplicate. **P < 0.01; ***P < 0.001. (C) Proliferation of HLFs stimulated for 24 hours with FXa (50 nM), FVIIa (50 nM), or thrombin (65 U/L) as assessed by immunostaining for the proliferation marker Ki67. Original magnification, ×20. Scale bar: 10 μm. DAPI = 4′,6-diamidino-2-phenylindole. (D) Quantification of Ki67-positive cells. The number of Ki67-positive cells was determined by counting four randomly chosen fields per slide from four independent experiments. Data are shown as means ± SD. **P < 0.01. (E) HLFs were stimulated for 24 hours with FXa (50 nM), FVIIa (50 nM), or thrombin (65 U/L) in the absence or presence of hirudin (1,000 U/L) or TENSTOP (0.5 μM). Cell proliferation was assessed by [3H]thymidine incorporation. The results are expressed as relative proliferation compared with unstimulated vehicle-treated cells. Data represent mean values ± SD from four independent experiments, each performed in triplicate. **P < 0.01; ***P < 0.001; NS = not significant. For all proliferation assays, cells were seeded in 48-well plates, growth arrested in serum-free Dulbecco's modified Eagle's medium, and stimulated for 24 hours in serum-free medium with respective reagents.
[More] [Minimize]To determine the potential role of PAR-2 in FXa- and FVIIa-induced cell proliferation, donor HLFs were transfected with PAR-2– and/or TF-specific siRNAs and then stimulated with the respective protease. Transfection of HLFs with PAR-2 (siPAR-2) or TF (siTF) siRNA caused significant knockdown of these proteins as demonstrated by RT-PCR and Western blotting (Figure 4A). Moreover, PAR-2 siRNA did not affect expression of PAR-1, indicating target gene specificity (Figure E2). As shown in Figure 4B, knockdown of PAR-2 abolished the mitogenic response of HLFs to FVIIa but did not influence the proliferation rate induced by FXa or thrombin. Depletion of TF almost completely inhibited the mitogenic response of HLFs to FVIIa but did not affect HLF proliferation on stimulation with FXa or thrombin (Figure 4B). Similar results were obtained when the cells were simultaneously transfected with siRNAs specific for PAR-2 and TF.
Figure 4. Factor VIIa (FVIIa)–induced proliferation of human lung fibroblasts (HLFs) requires protease-activated receptor (PAR)-2 and tissue factor (TF). (A) Determination of knockdown efficiency in HLFs after transfection with small interfering RNA (siRNA) against PAR-2 (siPAR-2; top) or TF (siTF; bottom) as assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. siR = scrambled siRNA. Data are representative of three independent experiments. (B) Effect of PAR-2 and TF knockdown on donor HLF proliferation. Forty-eight hours after transfection with siR, siRNA against PAR-2 (siPAR-2), and/or siRNA against TF (siTF), HLFs were stimulated for 24 hours in serum-free medium with FXa (50 nM), FVIIa (50 nM), or thrombin (65 U/L) and cell proliferation was assessed by [3H]thymidine incorporation. The results are expressed as relative proliferation compared with unstimulated siR-transfected cells. Data represent mean values ± SD from four independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001; NS = not significant. (C) Overexpression of PAR-2 (left) and TF (right) in HLFs as assessed by Western blotting. Data are representative of four independent experiments. (D) Effect of PAR-2 and TF overexpression on donor HLF proliferation. Forty-eight hours after transfection with empty vector (pcDNA3.1), PAR-2–expressing vector (pPAR-2) and/or TF-expressing vector (pTF) HLFs were stimulated for 24 hours in serum-free medium with FXa (50 nM), FVIIa (50 nM), or thrombin (65 U/L) and cell proliferation was assessed by [3H]thymidine incorporation. The results are expressed as relative proliferation compared with unstimulated pcDNA3.1-transfected cells. Data represent mean values ± SD from four independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001; NS = not significant. (E) Donor HLFs were treated for 24 hours in serum-free medium with FVIIa (50 nM), inactivated FVIIa (FVIIi; 50 nM), peptide agonist for PAR-2 (AP; 100 μM), or scrambled peptide control (RP; 100 μM). Cell proliferation was assessed by [3H]thymidine incorporation. The results are expressed as relative proliferation compared with unstimulated cells. Data represent mean values ± SD from three independent experiments, each performed in triplicate. **P < 0.01; ***P < 0.001; NS = not significant. (F) Donor and idiopathic pulmonary fibrosis (IPF) fibroblasts were seeded in 48-well plates, growth arrested in serum-free Dulbecco's modified Eagle's medium, and then stimulated for 24 hours in serum-free medium with FVIIa (50 nM). Cell proliferation was assessed by [3H]thymidine incorporation. The results are expressed as relative proliferation compared with unstimulated donor cells. Data represent mean values ± SD from four independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01; NS = not significant.
[More] [Minimize]To further confirm a crucial role of PAR-2 and TF in FVIIa-induced HLF proliferation, we evaluated the effect of PAR-2 and/or TF overexpression on this process. Transfection of PAR-2 (pPAR-2) or TF (pTF) vectors into HLFs considerably increased expression of these proteins as assessed by Western blotting (Figure 4C). Overexpression of PAR-2 and TF alone partially enhanced cell proliferation on treatment with FVIIa. This effect was strongly potentiated when PAR-2 and TF were overexpressed simultaneously (Figure 4D). Of note, overexpression of PAR-2 and TF alone or together did not induce any changes in HLF proliferation in response to FXa or thrombin (Figure 4D). Collectively, these data strongly suggest that PAR-2 and TF are required for FVIIa-triggered HLF proliferation but are dispensable for the induction of HLF proliferation by FXa or thrombin.
To determine the role of FVIIa catalytic activity in the induction of donor HLF proliferation, cells were incubated with FVIIi. FVIIi did not exert any proproliferative effects on HLFs, indicating the requirement of FVII proteolytic activity for the induction of HLF proliferation (Figure 4E). Two-furoyl-LIGRLO-NH2, a potent and selective PAR-2 agonist peptide (AP [23]), served as positive control.
As PAR-2 expression was strongly up-regulated in diseased lung fibroblasts, we sought to study the proliferation rate of donor and IPF fibroblasts in response to FVIIa. Under basal conditions no difference in growth rate between donor and IPF fibroblasts was noted; however, on FVII stimulation the magnitude of the mitogenic response was greater in fibroblasts isolated from the lungs of patients with IPF (Figure 4F).
Intracellular mitogen-activated protein kinase (MAPK) signaling pathways play an important role in cell proliferation. Therefore, we investigated the activation of various MAPKs and Akt in response to FVIIa stimulation. FVIIa induced rapid phosphorylation of p44/42 with maximal response within 15–30 minutes. In contrast, no activation of p38, c-Jun N-terminal kinase (JNK), and Akt was observed (Figure 5A). To examine whether FVIIa-stimulated phosphorylation of p44/42 is PAR-2/TF dependent, we transfected donor HLFs with PAR-2– and TF-specific siRNAs before incubation with FVIIa. Knockdown of PAR-2 and TF resulted in significant inhibition of FVIIa-induced p44/42 activation (Figure 5B). No p44/42 phosphorylation was observed when the cells were incubated with FVIIi, indicating the requirement of FVII catalytic activity for induction of the intracellular signaling pathway (Figure 5C). Moreover, FVIIa-stimulated proliferation of donor HLFs was significantly inhibited in the presence of the selective inhibitors of MAPK/ERK kinase-1,2 (MEK1/2), PD98059 and U0126 (Figure 5D). Together, these data indicate that FVIIa stimulates HLF proliferation via the p44/42 signaling pathway in a PAR/2-TF–dependent manner.
Figure 5. Factor VIIa (FVIIa)–driven protease-activated receptor (PAR)-2–dependent proliferation of human lung fibroblasts (HLFs) is mediated by p44/42 kinase. (A) Donor HLFs were treated for the indicated times with FVIIa (50 nM) and the activation of p44/42, c-Jun N-terminal kinase (JNK) (p54/46), p38, and Akt kinases as assessed by phosphorylation was analyzed by Western blotting. Phosphoproteins were detected with phospho-specific antibodies. Equal loading was confirmed with pan-specific antibodies. Data are representative of four independent experiments. (B) Impact of PAR-2 and tissue factor (TF) knockdown on FVIIa-induced p44/42 kinase phosphorylation. Forty-eight hours before FVIIa (50 nM) stimulation cells were transfected with scrambled small interfering RNA (siR) or siRNA against PAR-2 (siPAR-2) and TF (siTF). Activation of p44/42 as assessed by phosphorylation was examined by Western blotting. Data are representative of four independent experiments. (C) Phosphorylation of p44/42 kinase in HLFs stimulated for 15 minutes with FVIIa (50 nM), inactivated FVIIa (FVIIi; 50 nM), peptide agonist for PAR-2 (AP; 100 μM), or scrambled peptide control (RP; 100 μM). Data are representative of four independent experiments. (D) Donor HLFs were seeded in 48-well plates, growth arrested in serum-free Dulbecco's modified Eagle's medium, and then treated in serum-free medium with MAPK/ERK kinase (MEK)-1,2 inhibitor PD98059 (10 μM) or U0126 (20 μM) for 1 hour before incubation with FVIIa (50 nM) for 24 hours. Cell proliferation was assessed by [3H]thymidine incorporation. The results are expressed as relative proliferation compared with unstimulated cells. Data represent mean values ± SD from four independent experiments, each performed in triplicate. *P < 0.05; **P < 0.01; NS = not significant.
[More] [Minimize]Differentiation of fibroblasts into myofibroblasts is central to the pathogenesis of pulmonary fibrosis, and therefore we examined whether FVIIa promotes this phenotypic change. As assessed by qRT-PCR and Western blotting, exposure of donor HLFs to FVIIa did not induce expression of the myofibroblast marker α-SMA (Figures 6A–6C). In contrast, a strong stimulation of α-SMA synthesis was observed in response to TGF-β1 (Figures 6A–6C). Similar results were obtained by immunofluorescence staining (Figure 6D).
Figure 6. Factor VIIa (FVIIa) does not induce differentiation of fibroblasts into myofibroblasts. (A and B) Time course of α-smooth muscle actin (α-SMA) expression in human lung fibroblasts (HLFs) after stimulation with FVIIa (50 nM) or transforming growth factor (TGF)-β1 (10 ng/ml) as assessed by (A) quantitative real-time polymerase chain reaction (qRT-PCR) or (B) Western blotting. qRT-PCR results are expressed as ΔCt (difference in cycle threshold [Ct] between the housekeeping gene [PBGD; porphobilinogen deaminase] and the gene of interest). Data are shown as means ± SD. *P < 0.05; **P < 0.01. The Western blot illustrated is from one representative experiment of three. (C) Densitometric analysis of (B). Data are demonstrated as means ± SD, n = 3. *P < 0.05; **P < 0.01. (D) Immunofluorescence for detection of α-SMA in HLFs treated for 48 hours with FVIIa (50 nM) or TGF-β1 (10 ng/ml). Inset shows the negative control. Original magnification, ×40. Scale bar: 10 μm.
[More] [Minimize]Excessive deposition of ECM proteins in the lung is a hallmark of IPF, and therefore we examined whether FVII stimulates production of ECM proteins, such as fibronectin (FN), osteopontin (OPN), and collagen I. Exposure of donor HLFs to FVIIa stimulated the synthesis of OPN and FN in a time-dependent manner. qRT-PCR analysis demonstrated the strongest induction of OPN and FN mRNA expression at 20 hours of treatment (Figure 7A). Maximal OPN and FN protein levels were achieved within a 72-hour stimulation period (Figures 7B and 7C). Similar results were obtained when donor HLFs were treated for 72 hours with the PAR-2 agonist peptide (AP), 2-furoyl-LIGRLO-NH2 (Figures 7D and 7E). To examine whether FVIIa stimulated OPN and FN synthesis in a PAR-2/TF–dependent manner, we transfected HLFs with PAR-2– and TF-specific siRNAs. As shown in Figures 7F and 7G, knockdown of PAR-2 and TF resulted in significant inhibition of OPN and FN expression after FVIIa stimulation. We further evaluated the extent of OPN and FN synthesis during FVIIa treatment in donor and diseased fibroblasts. IPF fibroblasts exhibited moderately higher OPN and FN expression at baseline in comparison with donor fibroblasts. Interestingly, on FVII stimulation OPN and FN production was greatly augmented in fibroblasts isolated from IPF lungs (Figures 7H and 7I). Of note, stimulation of donor and IPF fibroblasts with FVIIa did not have any effect on collagen I production (data not shown).
Figure 7. Factor VIIa (FVIIa) induces osteopontin (OPN) and fibronectin (FN) expression in human lung fibroblasts (HLFs). (A and B) Time course of OPN and FN expression in donor HLFs after FVIIa (50 nM) stimulation as assessed by (A) quantitative real-time polymerase chain reaction (qRT-PCR) and (B) Western blotting. qRT-PCR results are expressed as ΔCt (difference in cycle threshold [Ct] between the housekeeping gene [PBGD; porphobilinogen deaminase] and the gene of interest). Data are shown as means ± SD, n = 3. *P < 0.05; **P < 0.01. The Western blot illustrated is from one representative experiment of four. (C) Densitometric analysis of (B). Data are presented as means ± SD; n = 4. *P < 0.05; **P < 0.01. (D) Induction of OPN and FN expression in HLFs treated for 72 hours with protease-activated receptor (PAR)-2 agonist peptide (AP; 100 μM) or scrambled peptide control (RP; 100 μM) as assessed by Western blotting. The Western blot illustrated is from one representative experiment of three. (E) Densitometric analysis of (D). Data are presented as means ± SD; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Effect of PAR-2 and tissue factor (TF) knockdown on FVIIa-induced OPN and FN expression. Forty-eight hours after transfection with scrambled small interfering RNA (siR), siRNA against PAR-2 (siPAR-2), and siRNA against TF (siTF), HLFs were stimulated for 48 hours with FVIIa (50 nM) and OPN and FN expression was analyzed by Western blotting. The Western blot illustrated is from one representative experiment of three. (G) Densitometric analysis of (F). Data are presented as means ± SD; n = 3. *P < 0.05; **P < 0.01. (H) Expression of OPN and FN in donor and idiopathic pulmonary fibrosis (IPF) fibroblasts stimulated for 48 hours with FVIIa (50 nM) as assessed by Western blotting. The Western blot illustrated is from one representative experiment of three. (I) Densitometric analysis of (H). Data are presented as means ± SD; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.
[More] [Minimize]As expression of PAR-2 was found to be elevated in the lungs of patients with IPF and as FVII was identified as an important PAR-2 activator stimulating fibroblast proliferation and ECM production, we next sought to examine FVII antigen level and its activity in IPF bronchoalveolar lavage fluid (BALF). Increased FVII antigen level and activity were detected in BALF of patients with IPF (Figures E3A and E3B, and Reference 8). Immunohistochemical analysis revealed no FVII-positive staining in donor lung tissue sections, whereas a strong positive signal for FVII was observed in hyperplastic ATII cells overlying fibroblast foci in IPF lungs (Figure 8A). Moreover, an elevated FVIIa mRNA level was detected by qRT-PCR in ATII cells isolated from the lungs of patients with IPF (Figure 8B). This suggests that FVII, which is produced by hyperplastic ATII cells, may act in an auto- and paracrine fashion to regulate cellular activities in IPF lungs.
Figure 8. Hyperplastic alveolar epithelial cells are the source of factor VII (FVII) in idiopathic pulmonary fibrosis (IPF) lungs. (A) Representative lung tissue sections from donor and patients with IPF, stained for FVII (brown) and pro–surfactant protein C (proSP-C; red). Arrowheads indicate alveolar type II (ATII) cells. Original magnification, ×40. Scale bar: 20 μm. (B) FVII expression in ATII cells isolated from donor (n = 3) and IPF (n = 3) lungs as assessed by quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR results are expressed as ΔCt (difference in cycle threshold [Ct] between the housekeeping gene [PBGD; porphobilinogen deaminase] and the gene of interest). Significance level is indicated.
[More] [Minimize]As our results demonstrated a requirement for TF for FVIIa-induced PAR-2–dependent stimulation of HLF proliferation and ECM production, we sought to analyze TF expression in fibroblasts isolated from donor and IPF lungs. A pronounced increase in TF mRNA (Figure 9A) and protein (Figures 9B and 9C) expression was observed in fibroblasts isolated from IPF lungs. These results were confirmed by immunofluorescence analysis (Figure 9D). Moreover, laser-assisted microdissection in combination with qRT-PCR showed up-regulation of TF expression in fibroblast foci of IPF lungs in comparison with alveolar septae of donor lungs (Figure E4). Immunohistochemical studies revealed colocalization of TF and PAR-2 in fibroblasts/myofibroblasts in IPF lung tissue (Figure 9E). Dual immunofluorescence staining of isolated IPF fibroblasts confirmed colocalization of TF and PAR-2 on the cell membrane (Figure 9F).
Figure 9. Tissue factor (TF) expression is elevated in fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) lungs. (A) TF mRNA expression in human lung fibroblasts (HLFs) isolated from donor (n = 10) and IPF (n = 24) lungs. Quantitative real-time polymerase chain reaction (qRT-PCR) results are expressed as ΔCt (difference in cycle threshold [Ct] between the housekeeping gene [PBGD; porphobilinogen deaminase] and the gene of interest). Significance level is indicated. (B) TF protein expression in HLFs isolated from donor (n = 10) and IPF (n = 24) lungs. Representative donors (4 of 10) and patients (4 of 24) are shown. (C) Densitometric analysis of (B). Significance level is indicated. (D) Human lung fibroblasts isolated from donor and IPF lungs stained for TF. Inset shows the negative control. Original magnification, ×43/1.25–0.75 oil objective. Scale bar: 10 μm. (E) Representative serial lung sections from patients with IPF, stained for α-smooth muscle actin (α-SMA), protease-activated receptor (PAR)-2, or TF. Magnified fields demonstrate TF-positive fibroblasts/myofibroblasts. Inset shows the isotype control. Original magnification, ×20. Scale bar: 100 μm. (F) Immunofluorescence staining of fibroblasts isolated from IPF lungs for PAR-2 and TF. Inset shows the negative control. The magnified field shows colocalization of PAR-2 and TF on the cell membrane, as indicated by arrows. Original magnification, ×60/1.25–0.75 oil objective. Scale bar: 5 μm.
[More] [Minimize]There are several potential mechanisms by which activation of coagulation proteinases may contribute to fibrotic processes in acutely and chronically injured lungs. Excessive intraalveolar deposition of fibrin is thought to inhibit surfactant function and to provide a provisional matrix on which fibroblasts can proliferate and produce collagen (24). Furthermore, fibrin may serve as a reservoir of profibrotic growth factors (25). However, because lung injury and pulmonary fibrosis occur in fibrinogen-null mice after bleomycin administration (26), fibrin deposition does not seem to be essential for the development of lung fibrosis. This observation indicates that the cellular rather than the procoagulant effects of coagulation proteinases may play a critical role for inflammatory and fibrotic processes in the lung. Some of these processes appear to be mediated via proteolytic activation of PARs. It has already been demonstrated that thrombin and FXa stimulate proliferation of fibroblasts, their differentiation into myofibroblasts, accelerate procollagen production, and increase expression of profibrotic cytokines via proteolytic activation of PAR-1 (12–14, 27, 28). Moreover, the absence of PAR-1 affords protection from bleomycin-induced lung fibrosis (15).
Much less is known about a possible contribution of PAR-2 to fibrotic processes in various organs and in the lung in particular. Increased PAR-2 expression was detected in pancreatic and experimental rat liver fibrosis and was shown to correlate with the extent of interstitial fibrosis in IgA nephropathy (17–19). In pulmonary diseases, high expression of PAR-2 has been observed in bronchopulmonary dysplasia and infant respiratory distress syndrome (29). In the present study, we demonstrate increased PAR-2 expression in the lung tissue of well-characterized patients with IPF whose diagnosis was, in accordance with current guideline recommendations, established in a multidisciplinary approach featuring experienced clinicians, radiologists, and pathologists in this field. PAR-2 was localized to hyperplastic ATII cells and fibroblasts/myofibroblasts in fibrotic lungs. In addition, fibroblasts isolated from IPF lungs showed significantly higher PAR-2 production than did fibroblasts extracted from donor lungs. In line with this observation TGF-β1, a cytokine known to be crucially involved in the pathogenesis of IPF, strongly induced PAR-2 synthesis in donor lung fibroblasts. Thus, it seems plausible that although quiescent tissue fibroblasts constitutively express a low level of PAR-2, conditions that promote fibroblast activation might considerably influence PAR-2 expression. This is further supported by the results demonstrating that transformation of PAR-2–negative to PAR-2–positive fibroblasts occurs in a wound scratch model as well as in normal and hypertrophic scars of humans (30, 31). Taken together, tissue injury/damage may relay a signal for PAR-2 induction that drives physiological tissue repair to a pathological tissue response culminating in fibrosis.
As a consequence, we investigated the role of PAR-2 in FVIIa- and FXa-induced HLF proliferation. Our experiments demonstrated that PAR-2 plays a pivotal role in FVII-induced proliferation of HLFs whereas mitogenic activities exerted by FXa are PAR-2 independent. In addition, our studies revealed that induction of HLF proliferation requires FVII catalytic activity, but does not involve FXa or thrombin, downstream components of the coagulation system. Thus, mitogenic effects observed in response to FVIIa are not the result of the initiation of the coagulation cascade but are triggered directly by FVIIa, which activates PAR-2, eliciting effects comparable to PAR-2 agonist peptide. We further demonstrated the requirement for TF in FVIIa-induced PAR-2–dependent HLF proliferation. This observation agrees with previous studies showing that FVIIa signals via PAR-2 only in the presence of TF (32, 33). In contrast, depletion or overexpression of TF did not influence the responsiveness of lung fibroblasts to FXa, suggesting that other FXa-binding sites might exist that help to localize FXa to the cell surface and promote its interaction with PAR-1. In this regard, effector cell protease receptor-1 (EPR-1) has been identified as an FXa receptor; however, its importance as such is not clear and needs further investigation (34, 35). In addition, we found that TF/FVIIa-induced PAR-2 activation resulted in phosphorylation of p44/42. Activation of p44/42 was necessary for FVIIa-driven mitogenic activities, as preincubation of lung fibroblasts with MEK1/2-specific inhibitors completely abolished these cellular effects. The central role of the p44/42 signaling pathway in TF/FVIIa/PAR-2–mediated cellular activities has already been demonstrated and was found to be essential not only for the stimulation of cell proliferation but also for the induction of gene expression (36, 37).
As our data indicate that activation of PAR-2 requires assembly of TF and FVIIa, we next investigated the expression and localization of these two molecules in the lungs of patients with IPF. Immunohistochemical analysis of IPF lung tissue sections revealed that hyperplastic epithelial cells overlying fibroblastic foci are prominent sites of FVII immunoreactivity. Furthermore, ATII cells isolated from IPF lungs expressed higher levels of FVII as compared with cells extracted from donor lungs. These data provide compelling evidence that FVII may be locally generated in the injured lung. This is in line with our previous studies demonstrating that in acute and chronic interstitial lung diseases the alveolar compartment represents an important source of hemostatic factors (38, 39). Moreover, we report that PAR-2 colocalizes with TF on hyperplastic ATII cells and fibroblasts/myofibroblasts in IPF lungs, thereby providing a cell surface–binding site for FVIIa, which subsequently enables cleavage of the receptor. The positive staining for TF in IPF fibroblasts/myofibroblasts contrasts with previous data documenting TF immunostaining in IPF lungs confined to the hyperplastic alveolar epithelium and occasional macrophages (9, 14, 40). Various reasons could underlie the differing findings. First, technical/methodological aspects could play a role. Different immunohistochemistry protocols were applied in our study versus the previous studies. Importantly, fibroblast staining for TF in IPF lung tissue was clearly weaker compared with the epithelium, and not all fibroblasts stained positive for TF, suggesting that different subpopulations of fibroblasts with unique phenotypes and functions may exist under the given conditions. This is in line with previous studies highlighting evidence of fibroblast heterogeneity, for example, with respect to expression of surface markers, ability to synthesize collagen, and cytokine production (41). However, in addition to immunohistochemistry, we used several other assays to confirm TF expression in lung fibroblasts from patients with IPF. We detected significant amounts of TF at the mRNA and protein levels in lung fibroblasts isolated from patients with IPF. TF was also expressed in fibroblasts isolated from donor lungs, albeit to a significantly lower degree. This is in line with previous studies showing that normal human fibroblasts from different tissues constitutively express TF (37, 42, 43). Moreover, our unpublished data indicate that TGF-β1 increases TF expression in donor HLFs, which is in accordance with a previous report (44). Analogous to the observations in isolated fibroblasts, we also observed significantly increased TF expression in microdissected fibroblast foci of IPF lungs. In addition to technical/methodological aspects there are other possible, patient-related, explanations for the different findings in our study versus the other studies with respect to TF localization to fibroblasts/myofibroblasts in IPF lungs. Differences in medication at the time of specimen sampling could play a role, for example, regarding prednisolone treatment that was previously shown to impact on TF expression (45). Furthermore, in contrast to the previous studies, which examined specimens obtained from diagnostic surgical lung biopsies (9, 14, 40), we investigated lung explant specimens obtained at the time of transplantation, thus likely representing different stages of the disease. It is possible that alterations in the lung microenvironment over time may lead to the induction or enhancement of TF expression in fibroblasts. In fact, TF antigen levels in BALF were previously found to be significantly higher in advanced compared with nonadvanced patients with IPF (40), and previous studies have demonstrated that different stimuli (e.g., growth factors or inflammatory mediators) may induce or enhance TF expression in different cell populations including fibroblasts (43, 44, 46). Here, pulmonary infections could play a role. Infections are a common cause of rapid deterioration requiring hospitalization in patients with IPF (47). Those events typically occur in an advanced disease stage and have a negative impact on the long-term prognosis (47). Therefore, they could contribute to changes in the lung microenvironment in the later phase of the disease, leading to alterations in the expression of pathogenetically relevant factors such as TF and to subsequent disease progression. Altogether, this could indicate that PAR-2–mediated, TF-dependent cellular effects potentiate, rather than initiate, the fibrotic response. However, future studies are required to investigate this aspect in more detail and to support the considerations.
Taken together, we propose a model whereby FVIIa released by hyperplastic epithelial cells acts in an auto- and paracrine fashion on PAR-2/TF–positive ATII cells and fibroblasts, respectively. Although FVII-induced PAR-2-TF–mediated fibroblast activities included enhanced grow rate and elevated production of FN and OPN, the impact of the PAR-2/TF/FVIIa axis on ATII behavior needs further investigation.
Myofibroblasts are important effector cells in pulmonary fibrosis, responsible for production of cytokines and ECM proteins within fibrotic foci. In contrast to FXa, a potent inducer of fibroblast/myofibroblast differentiation and collagen production (6), stimulation of lung fibroblasts with FVIIa did not promote any of the aforementioned processes (3, 5, 48). This indicates that FXa and FVIIa not only activate different receptors, but also exert different biological effects.
Interestingly, one study demonstrated no effect of PAR-2 deficiency on the development of bleomycin-induced lung fibrosis in mice (49). This is supported by unpublished data from our group showing that PAR-2−/− mice are not protected from pulmonary fibrosis, using the same model. However, the apparent discrepancy between our clinical and in vitro data on the one hand and the in vivo results on the other hand may well be explained by the fact that mouse lung fibroblasts, in contrast to human lung fibroblasts, do not express PAR-2 (Reference 32, and our unpublished observation). Thus, mouse models are hardly suitable for drawing conclusions with respect to a possible contribution of PAR-2 to the pathogenesis of human lung fibrosis.
In conclusion, our study provides compelling evidence that activation of PAR-2 by the FVIIa/TF complex has a capacity to drive the fibrotic response to lung injury by inducing fibroblast proliferation and ECM production, thereby linking activation of the coagulation cascade to the induction of fibroproliferative processes. Thus, we present a rationale for strategies aimed at blocking PAR-2 in IPF, a progressive and fatal disorder without effective treatment thus far.
The authors thank Gisela Mueller and Horst Thiele for excellent technical assistance. The authors also thank Walter Klepetko, Director of the Department of Cardiothoracic Surgery, University of Vienna, for kindly providing human lung tissue.
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