Plasminogen activator inhibitor type-1 deficiency attenuates murine antigen-induced arthritis

K. Van Ness, V. Chobaz-Péclat, M. Castellucci1, A. So and N. Busso

Centre Hospitalier Universitaire Vaudois, Laboratoire de Rhumatologie, Nestlé 05-5029, 1011 Lausanne, Switzerland and
1 Istituto di Morfologia, Universita di Ancona, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To examine the role of plasminogen activator inhibitor type-1 (PAI-1), the major fibrinolytic inhibitor, in vivo during murine antigen-induced arthritis (AIA).

Methods. AIA was induced in PAI-1-deficient mice and control wild-type mice. Arthritis severity was evaluated by technetium 99m (99mTc) uptake in the knee joints and by histological scoring. Intra-articular fibrin deposition was examined by immunohistochemistry and synovial fibrinolysis quantitated by tissue D-dimer measurements and zymograms.

Results. Joint inflammation, quantitated by 99mTc uptake, was significantly reduced in PAI-1-/- mice on day 7 after arthritis onset (P<0.01). Likewise, synovial inflammation, evaluated by histological scoring, was significantly decreased in PAI-1-deficient mice on day 10 after arthritis onset (P<0.001). Articular cartilage damage was significantly decreased in PAI-1-/- mice, as shown by histological grading of safranin-O staining on day 10 after arthritis onset (P<0.005). Significantly decreased synovial accumulation of fibrin was observed by day 10 in arthritic joints of PAI-1-/- mice (P<0.005). Accordingly, the synovial tissue content of D-dimers, the specific fibrin degradation products generated by plasmin, were increased in PAI-1-/- mice (P<0.02). Finally, as expected, PA activity was increased in synovial tissues from PAI-1-/- mice, as shown by zymographic analysis.

Conclusions. These results indicate that deficiency of PAI-1 results in increased synovial fibrinolysis, leading to reduced fibrin accumulation in arthritic joints and reduced severity of AIA.

KEY WORDS: PAI-1, Rheumatoid arthritis, Mice, Fibrin, Fibrinolysis, Inflammation.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The extravascular formation and deposition of fibrin is a hallmark of inflammation. This process helps to promote tissue repair, but fibrin, if it is not removed, may also perpetuate local inflammation. Intra-articular fibrin deposition represents one of the most striking features of rheumatoid arthritis (RA), a chronic inflammatory joint disease characterized by synovial inflammation and, in its later stages, cartilage and bone destruction [1, 2]. Although a T-cell-driven process is thought to initiate the disease, other mechanisms, such as ongoing coagulation and fibrin persistence within the joint, may be important in sustaining inflammation in RA [3, 4].

Accumulation of intra-articular fibrin is the result of the balance between coagulation and fibrinolysis [4]. Degradation of the fibrin matrix is primarily mediated by plasmin [5], though other proteases can also play a role. The generation of plasmin from plasminogen is tightly regulated by the concerted actions of the two plasminogen activators (PA), the urokinase-type (uPA) and tissue-type PA (tPA), and their specific inhibitors (PAIs) [6]. PAI-1 is the major circulating PAI and controls the rate of plasmin generation by forming irreversible inhibitory complexes with uPA and tPA [7]. In synovia from RA patients and in experimental murine arthritis, PAI-1 antigen and mRNA were increased significantly compared with normal tissues [8, 9]. Concomitantly, increased levels of uPA and fibrin D-dimer, a plasmin-specific fibrin degradation product, were detected in synovial tissues and biological fluids from RA patients [8, 10, 11]. Taken together, these results indicate that, despite increased PAI-1 levels, there is an ongoing plasmin-mediated fibrinolysis in RA.

There is accumulating evidence that PAI-1 is a multifunctional protein that is involved not only in extracellular matrix proteolysis, but also in cellular adhesion and migration through its binding site for vitronectin [12]. Recently, a role of PAI-1 in tumour angiogenesis has been demonstrated [13], although it remains to be established whether this activity of PAI-1 is mediated by regulation of proteolytic activity and/or by vitronectin-dependent effects.

Within the joint, increased PAI-1 expression may have a beneficial effect on arthritis, by inhibiting plasmin-mediated matrix degradation of bone and cartilage [14, 15], or a deleterious effect by favouring fibrin accumulation or by promoting angiogenesis. Our previous observations in mice deficient in uPA indicate that fibrin accumulation may by itself perpetuate synovial inflammation [16]. In order to elucidate the possible in vivo functions of PAI-1 in arthritis and to evaluate if it has a protective or deleterious role in joint inflammation, we studied the effects of PAI-1 deficiency on the course of murine antigen-induced arthritis (AIA), an experimental model of arthritis which shows many of the histological features of RA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
PAI-1-deficient mice (PAI-1-/-) and wild-type littermate mice (PAI-1+/+) on a mixed Ola129/C57BL/6 background [17] were originally provided by Dr Carmeliet (University of Leuven, Belgium) and bred in our animal housing facility.

Induction of arthritis
AIA in mice was established as described previously [18]. Briefly, animals were immunized on days 0 and 7 with 100 µg methylated bovine serum albumin (mBSA; Sigma Chemical Company, Buchs, Switzerland) emulsified in 0.1 ml complete Freund's adjuvant containing 200 µg mycobacterial strain H37RA (Difco, Basel, Switzerland) by intradermal injection at the base of the tail. On day 0, animals received, as an additional adjuvant, 2x109 heat-killed Bordetella pertussis organisms (Berna, Bern, Switzerland) injected intraperitoneally. Arthritis was induced in the right knee on day 21 by intra-articular injection of 100 µg of mBSA in 10 µl sterile phosphate-buffered saline (PBS), the left knee being injected with sterile PBS alone. Approval was obtained from the Service Vétérinaire cantonal, Lausanne, Switzerland, for these experiments.

Isotopic quantification of joint inflammation
Joint inflammation was measured by 99mTc uptake in the knee joint as described [19]. Briefly, mice were first sedated by intraperitoneal administration of sodium pentobarbital (50 mg/kg) and then injected subcutaneously in the neck region with 10 µCi 99mTc. The accumulation of the isotope in the knee was determined by external gamma-counting after 15 min. The ratio of 99mTc uptake in the inflamed arthritic knee to 99mTc uptake in the contralateral control knee was calculated. A ratio higher than 1.1 indicated joint inflammation.

Histological grading of arthritis
Mice were killed and the knees were dissected and fixed in 10% buffered formalin for 4 days. Fixed tissues were decalcified for 3 weeks in 15% ethylenediamine tetraacetic acid (EDTA), dehydrated and embedded in paraffin. Sagittal sections (6 µm) of the whole knee joint were stained with safranin-O and counterstained with fast green/iron haematoxylin. Histological sections were graded by two observers unaware of animal genotype or treatment. Synovial cell infiltrate and exudate were scored from 0 to 6 (0=no cells; 6=maximum number of inflammatory cells). Cartilage proteoglycan depletion (damage), reflected by loss of safranin-O staining intensity, was scored on a scale from 0 (fully stained cartilage) to 6 (totally unstained cartilage) in proportion to severity.

Fibrin immunohistochemistry
Paraffin-embedded sections were processed for fibrin immunohistochemistry exactly as described before [15]. Fibrin immunostaining in the synovial membrane was graded independently by two observers unaware of animal treatment on a scale from 0 (no fibrin at all) to 6 (maximum of fibrin staining).

Cryostat section preparation
Dissected knees were embedded in Tissue-Tek OCT, then immediately frozen in pre-cooled hexane and stored at -70°C until use. Sections were cut on a motor-driven Leica cryostat with a retraction microtome and a tungsten carbide knife at a cabinet temperature of -25°C.

Tissue protein extract preparation
Cryostat sections of joint tissue were homogenized in 50 mM Tris–HCl pH 7.5, containing 110 mM NaCl, 10 mM EDTA and 0.1% NP-40. The homogenate was centrifuged at 4000 g for 10 min at 4°C and the supernatant stored at -20°C. Protein content of the tissue extracts was measured by the method of Bradford using BSA as a standard.

D-dimer measurements
The D-dimer concentration in tissue extracts was measured by a commercially available enzyme-linked immunosorbent assay (ELISA) kit designed for human D-dimer (Asserachrom D-Di; Diagnostica Stago, Asnières, France), which cross-reacts with murine D-dimer. The content of murine D-dimer was calculated according to the human D-dimer standard curve, normalized per mg of protein and expressed as the percentage of D-dimer in control mice.

Plasminogen activators zymographies
Tissue protein extracts were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) zymography as described [15]. Briefly, after SDS–PAGE of the samples, the gel was washed in Triton X-100 and layered over a casein underlay containing 2% non-fat dry milk, 0.9% agar and 40 µg/ml of purified human plasminogen in PBS (with 0.9 mM Ca2+ and 1 mM Mg2+). Underlays were incubated in a humidity chamber of 37°C for 3–4 h, during which PAs diffused from the gel into the underlay, converting plasminogen into plasmin, which in turn lysed the insoluble casein. Zones of plasminogen-dependent caseinolysis appeared as black areas when visualized under dark-ground illumination. Photographs were taken using dark-ground illumination.

Statistical analysis
The Wilcoxon rank sum test for unpaired variables (two-tailed) was used to compare differences between groups. P values <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PAI-1 deficiency reduces technetium uptake in affected knee joints
To explore whether the deficiency of PAI-1 had an effect on the course of AIA, we measured knee joint inflammation in control and PAI-1-deficient mice by the ratio of 99mTc uptake in the inflamed arthritic joint over that of the non-arthritic contralateral knee joint at different time points up to day 10 (Fig. 1Go). In PAI-1-/- mice, we observed an attenuation of the inflammatory response throughout the duration of the experiment, although only the decrease observed on day 7 reached a statistically significant value (day 7, 25% decrease, P<0.01).



View larger version (31K):
[in this window]
[in a new window]
 
FIG. 1.  Time course of knee joint inflammation in PAI-1-/- mice with AIA. Joint inflammation was measured by external gamma-counting of 99mTc uptake on days 3, 7 and 10 after antigen injection into the right knee. Results are expressed as the ratio of 99mTc uptake in the right (R) arthritic knee joint to that in the left (L) non-inflamed contralateral knee joint, a value higher than 1.1 indicating joint inflammation. For each time point, mean+S.E.M. of the ratio is shown. WT, wild-type mice (n=11–12); KO, PAI-1-/- mice (n=9–12). *P<0.05 was considered significant.

 

PAI-1 deficiency ameliorates histological features of AIA
To determine whether the observed decrease in knee joint inflammation in PAI-1-deficient mice was associated with specific histopathological changes, we compared the histological features of arthritic knee joints from control and PAI-1-deficient mice (Fig. 2Go). In both groups of animals, arthritis was histologically present in all knees which had been injected with mBSA. In wild-type mice, on day 10 of AIA, the synovial membrane was thickened (Fig. 2AGo), mainly as a result of invasion by inflammatory cells. In PAI-1-deficient mice, synovial infiltrate was significantly decreased in comparison with control mice [4.33±0.3 in control mice (n=20) vs 2.13±0.4 in PAI-1-/- mice (n=15) on day 10 after arthritis onset; P<0.001] by day 10 of AIA (compare Fig. 2AGo and B and see histological scoring in Fig. 3Go).



View larger version (129K):
[in this window]
[in a new window]
 
FIG. 2.  Histology and immunohistology of whole knee-joint sections of control mice and PAI-1-/- mice with AIA. (A, C) Safranin-O-stained sections of arthritic knee joints on day 10 after arthritis induction. Note the difference in thickness of synovial membrane (sy), which was thicker in the control arthritic mice than in the PAI-1-/- mice. Note also proteoglycan staining of the articular cartilage (ca), which was less in control arthritic mice than in PAI-1-/- mice. (B, C) Fibrin(ogen) immunostaining (fib) of adjacent sections.

 


View larger version (24K):
[in this window]
[in a new window]
 
FIG. 3.  Histological grading of arthritic knee joints. Cell infiltrate, cell exudate and cartilage damage were scored histologically using an arbitrary scale from 0 to 6. Results are expressed as mean+S.E.M. WT, wild-type mice (n=20); KO, PAI-1-/- mice (n=15). *P<0.05 was considered significant.

 
The effect of AIA on articular cartilage was evaluated. Induction of arthritis in control mice led to a decrease in the proteoglycan content, as demonstrated by loss of safranin-O staining (Fig. 2Go). In PAI-1-deficient mice, the decrease in proteoglycan staining was less severe, as shown by histological grading of safranin-O staining [3.03±0.2 in control mice (n=20) vs 1.8±0.3 in PAI-1-/- mice (n=15), on day 10 after arthritis onset; P<0.005] (Fig. 3Go).

Fibrin deposition is decreased in arthritic joints of PAI-1-deficient mice
Because of the established role of PAI-1 in fibrinolysis, we wished to test the hypothesis that the loss of PAI-1 results in decreased fibrin deposition. Fibrin content in knee joints was analysed by fibrin immunohistochemistry (Fig. 2CGo) and by scoring (Fig. 4bGo) on day 10 of AIA. In arthritic PAI-1+/+ mouse knee joints, fibrin was found in the synovium, the synovial fluid and on the surface of the articular cartilage (Fig. 2CGo). Decreased amounts of fibrin were detected in PAI-1-deficient mice. On the basis of immunohistological scoring, PAI-1-/- mice showed about 40% reduction in the level of fibrin [3.3±0.22 in control mice (n=20) vs 2.03±0.26 in PAI-1-/- mice (n=15) on day 10 after arthritis onset; P<0.005].



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 4.  Fibrin scoring and fibrin D-dimer measurements in arthritic knee joints. Intra-articular fibrin deposition was scored in the synovial membrane using an arbitrary scale from 0 to 6. WT, wild-type mice (n=20); KO, PAI-1-/- mice (n=15). D-dimers were measured by ELISA in synovial tissue extracts (wild-type mice, n=10, PAI-1-/- mice, n=10). Results are expressed as percentages of control wild-type mice. *P<0.05 was considered significant.

 

PA activity and fibrinolytic activity are increased in arthritic synovial membrane from PAI-1-deficient mice
On day 10 of AIA, tissue extracts were prepared from arthritic knee joints and analysed by zymography (Fig. 5Go). This allowed clear distinction between uPA and the other plasminogen activator, tPA, which migrated at 48 and 72 kDa respectively. In arthritic synovial tissues of PAI-1-/- mice, both uPA and tPA were detected. In wild-type mice, only uPA activity was detectable in all the tissue extracts analysed. In these extracts, tPA was involved in the high molecular weight tPA-PAI-1 complexes that became visible after longer development of the zymography (not shown), whereas uPA, produced as a single chain proenzyme, remained uncomplexed.



View larger version (72K):
[in this window]
[in a new window]
 
FIG. 5.  PA enzyme assays of knee synovial tissues. Ten micrograms of proteins from synovial tissue of arthritic knee joints from wild-type (WT) and PAI-1-/- (KO) mice were analysed, after SDS-PAGE, by PA zymography. Zones of caseinolysis appear as black areas when visualized under dark-ground illumination.

 
Direct evidence of ongoing plasmin-mediated fibrinolysis can be provided by the generation of specific fibrin degradation products, such as fibrin D-dimers. In a preliminary experiment, to test the specificity of a D-dimer ELISA, which was initially devised for human D-dimers, we tested synovial tissue extracts from fibrinogen-deficient mice, which lacking fibrin, are devoid of D-dimers. No detectable D-dimers were measured in these samples. The degree of fibrinolysis in the compromised joint was thus evaluated by D-dimer measurements in synovial tissue extracts from both control and PAI-1-/- mice (Fig. 4bGo). In accordance with increased PA activity, PAI-1-deficient mice also had significantly increased D-dimer levels [1.94±0.15 ng/mg protein in tissue extracts from control mice (n=10) vs 6.37±1.7 in PAI-1-/- mice (n=10) on day 10 after arthritis onset; P<0.02]. This observation is in agreement with the increased zymographic PA activity, mainly due to tPA, observed in joints during AIA in PAI-1-deficient mice.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Disturbances in the regulation of the balance between fibrin deposition and elimination have been implicated in a number of inflammatory conditions, including certain rheumatic diseases. In this context, we wanted to investigate how alterations in PAI-1 expression would affect the clinical and histological manifestations of AIA, a murine model of RA. Our experiments showed that, in PAI-1-deficient mice, the severity of AIA was reduced compared with wild-type littermates, as assessed by technetium uptake and by histology. Technetium uptake was significantly reduced on day 7, and histological assessment of different parameters of joint inflammation and damage on day 10 showed consistent reductions in the degree of synovial inflammation and cartilage damage. This milder phenotype of AIA in PAI-1-/- animals was associated with histological evidence for a reduction in synovial fibrin deposition and, at the same time, with an increased level of fibrin D-dimers in joint tissues. Taken together, these results strongly suggest that, in the absence of PAI-1, the unopposed PA activities of uPA and tPA result in increased fibrinolysis. We have previously demonstrated that impaired fibrinolysis due to uPA or plasminogen deficiency leads to exacerbation of arthritis [16]. Our current data on the effects of PAI-1 deficiency provide additional evidence to support the importance of efficient fibrinolysis in the resolution of joint inflammation.

Previous experiments in PAI-1 knockout mice have shown that, beside the control of fibrinolysis, there are other PAI-1-mediated biological effects to be considered. One such is the role of PAI-1 in angiogenesis, which may explain why PAI-1-deficient mice were less prone to tumour invasion and the observed correlations between increased serum PAI-1 levels and poor cancer prognosis in man [20]. We cannot rule out this mechanism to explain the reduction in inflammatory changes in AIA, although our microscopic assessment of the synovial vasculature did not reveal any gross differences in vascularity (data not shown). Another mechanism may be the action of PAI-1 on leucocyte adhesion and migration through the uPA/uPAR system [21]. Consistent with this, our histological evaluation showed that synovial inflammatory cell infiltrate was significantly reduced in PAI-1-deficient mice.

Our results suggest that PAI-1 plays an important role in inflammation. Under basal conditions, PAI-1-deficient mice did not have an abnormal phenotype apart from a mild hyperfibrinolytic state. However, on induction of tissue injury, expression of PAI-1 exacerbates hyperoxia-induced pulmonary damage in mice [22]. Furthermore, animals overexpressing PAI-1 developed a more severe lung injury and deposition of fibrin and collagen-rich matrix after bleomycin challenge or hypoxia [22, 23]. The findings are best accounted for by the role of PAI-1 in regulating fibrinolysis during inflammation and tissue repair, though, as mentioned earlier, non-fibrinolytic effects cannot be totally excluded. Taken together with the observations made in mice deficient in uPA, plasminogen and fibrinogen [16, 24, 25], these results underline the key role of a coordinated and regulated fibrinolytic pathway in the resolution of inflammation.

In RA, PAI-1 is significantly increased in both synovial fluid and the synovial membrane. Although the inhibition of PA action by PAI-1 may be beneficial because of its inhibitory effect on plasmin-mediated cartilage breakdown [15], this could be offset by the proinflammatory effects of reduced fibrinolysis. Indeed, an association between more severe disease and reduced fibrinolytic activity, as evidenced by levels of D-dimers and the ratio between PAI-1 and tPA, has been observed in clinical studies [26]. These findings corroborate the hypothesis that changes in fibrinolysis may affect synovial inflammation, and suggest that this pathway may be a target in the development of future therapeutic interventions.


    Acknowledgments
 
We thank Carole Morard for her technical assistance. We are indebted to Dr Eric Kolodziesczyk for his continuous support in microscopy analysis.


    Notes
 
Correspondence to: N. Busso. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Firestein GS. Etiology and pathogenesis of rheumatoid arthritis. In: Kelley WN, Harris ED, Ruddy S, Sledge C, eds. Textbook of rheumatology. Philadelphia: W. B. Saunders, 1997:851–97.
  2. Jasani MK. Fibrin: metabolism, immunopathogenesis and significance in rheumatoid arthritis. In: Panayi GS, Johnson PM, eds. Immunopathogenesis of rheumatoid arthritis. Surrey: Red Books, 1978:137–46.
  3. Cicala C, Cirino G. Linkage between inflammation and coagulation: an update on the molecular basis of the crosstalk. Life Sci1998;62:1817–24.[ISI][Medline]
  4. Weinberg JB, Pippen AM, Greenberg CS. Extravascular fibrin formation and dissolution in synovial tissue of patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum1991;34:996–1005.[ISI][Medline]
  5. Collen D, Lijnen HR. Fibrin-specific fibrinolysis. Ann N Y Acad Sci1992;667:259–71.[ISI][Medline]
  6. Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest1991;88:1067–72.[ISI][Medline]
  7. Eitzman DT, Ginsburg D. Of mice and men. The function of plasminogen activator inhibitors (PAIs) in vivo. Adv Exp Med Biol1997;425:131–41.[ISI][Medline]
  8. Busso N, Péclat V, So A, Sappino AP. Plasminogen activation in synovial tissues: Differences between normal, osteoarthritis, and rheumatoid arthritis joints. Ann Rheum Dis1997;56:550–7.[Abstract/Free Full Text]
  9. Salvi R, Péclat V, So A, Busso N. Enhanced expression of genes involved in coagulation and fibrinolysis in murine arthritis. Arthritis Res2000;2:504–12.[ISI][Medline]
  10. Carmassi F, de Negri F, Morale M, Song KY, Chung SI. Fibrin degradation in the synovial fluid of rheumatoid arthritis patients: a model for extravascular fibrinolysis. Semin Thromb Hemost1996;22:489–96.[Medline]
  11. Wallberg-Jonsson S, Rantapaa-Dahlqvist S, Nordmark L, Ranby M. Mobilization of fibrinolytic enzymes in synovial fluid and plasma of rheumatoid arthritis and spondyloarthropathy and their relation to radiological destruction. J Rheumatol1996;23:1704–9.[ISI][Medline]
  12. Waltz DA, Natkin LR, Fujita RM, Wei Y, Chapman HA. Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin. J Clin Invest1997;100:58–67.[Abstract/Free Full Text]
  13. Bajou K, Noel A, Gerard RD et al. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med1998;4:923–8.[ISI][Medline]
  14. Ronday HK, Smits HH, Quax PH et al. Bone matrix degradation by the plasminogen activation system. Possible mechanism of bone destruction in arthritis. Br J Rheumatol1997;36:9–15.[ISI][Medline]
  15. van der Laan WH, Pap T, Ronday HK et al. Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of a cell surface-targeted plasmin inhibitor. Arthritis Rheum2000;43:1710–8.[ISI][Medline]
  16. Busso N, Péclat V, van Ness K et al. Exacerbation of antigen-induced arthritis in urokinase-deficient mice. J Clin Invest1998;102:41–50.[Abstract/Free Full Text]
  17. Carmeliet P, Kieckens L, Schoonjans L et al. Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization. J Clin Invest1993;92:2746–55.[ISI][Medline]
  18. Brackertz D, Mitchell GF, Mackay IR. Antigen-induced arthritis in mice. I. Induction of arthritis in various strains of mice. Arthritis Rheum1977;20:841–50.[ISI][Medline]
  19. Kruijsen MW, van den Berg WB, Van De Putte LB, van den Broek WJ. Detection and quantification of experimental joint inflammation in mice by measurement of 99mTc-pertechnetate uptake. Agents Actions1981;11:640–2.[ISI][Medline]
  20. Harbeck N, Thomssen C, Berger U et al. Invasion marker PAI-1 remains a strong prognostic factor after long-term follow-up both for primary breast cancer and following first relapse. Breast Cancer Res Treat1999;54:147–57.[ISI][Medline]
  21. Edwards DR, Murphy G. Cancer. Proteases—invasion and more. Nature1998;394:527–8.[ISI][Medline]
  22. Barazzone C, Belin D, Piguet PF, Vassalli JD, Sappino AP. Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury. J Clin Invest1996;98:2666–73.[Abstract/Free Full Text]
  23. Eitzman DT, McCoy RD, Zheng X et al. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest1996;97:232–7.[Abstract/Free Full Text]
  24. Kitching AR, Holdsworth SR, Ploplis VA et al. Plasminogen and plasminogen activators protect against renal injury in crescentic glomerulonephritis. J Exp Med1997;185:963–8.[Abstract/Free Full Text]
  25. Romer J, Bugge TH, Pyke C et al. Impaired wound healing in mice with a disrupted plasminogen gene. Nature Med1996;2:287–92.[ISI][Medline]
  26. Kopeikina LT, Kamper EF, Koutsoukos V, Bassiakos Y, Stavridis I. Imbalance of tissue-type plasminogen activator (t-PA) and its specific inhibitor (PAI-1) in patients with rheumatoid arthritis associated with disease activity. Clin Rheumatol1997;16:254–60.[ISI][Medline]
Submitted 31 January 2001; Accepted 20 July 2001