Centre Hospitalier Universitaire Vaudois, Laboratoire de Rhumatologie, Nestlé 05-5029, 1011 Lausanne, Switzerland and
1 Istituto di Morfologia, Universita di Ancona, Italy
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Abstract |
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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.
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Introduction |
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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.
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Materials and methods |
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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 TrisHCl 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 sulphatepolyacrylamide gel electrophoresis (SDSPAGE) zymography as described [15]. Briefly, after SDSPAGE 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 34 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.
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Results |
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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. 2). 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. 2A
), 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. 2A
and B and see histological scoring in Fig. 3
).
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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. 2C) and by scoring (Fig. 4b
) 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. 2C
). 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].
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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. 5). 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.
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Discussion |
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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.
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Acknowledgments |
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Notes |
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References |
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