Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis

Abstract

Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3'-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.

Keywords: Inflammation; Osteoarthritis.

Figure 1. Key molecules in the fibrinolysis pathway are dysregulated in human OA joints.

(A) Unsupervised hierarchical clustering of uPA and uPAR expression in microarray data set on synovial membranes from healthy individuals (n = 7) or those with early- (n = 10) or end-stage OA (n = 9). Scale bar indicates z score. (B) ELISA analysis of plasmin levels in knee joint synovial fluids from individuals with OA (n = 6), ACL tear (n = 8), or DMT (n = 3) and in the plasma from healthy individuals (n = 8). (C and D) ELISA analysis of activated uPA (C) or uPAR (D) levels in synovial fluid of knee joints and serum from individuals with confirmed OA (n = 10). (E) Representative images from immunohistochemical staining of tPA, uPA, uPAR, and isotype control in damaged knee cartilage (left) and synovium (right) of OA from individuals who underwent total knee replacement. The arrowhead indicates positive staining for tPA, uPA, and uPAR. For panels B–D, data are the mean ± SEM of duplicates or triplicates and are representative of at least 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by 2-tailed t test or 1-way ANOVA. The test in panel B is 1-way ANOVA and in panels C and D is Mann-Whitney U test. For panel E, scale bar is 200 μm; cartilage and synovial tissues from n = 5 individuals were analyzed; and the representative images were shown.

Figure 2. Genetic deficiency or pharmacological blockade of plasmin attenuates OA in mice.

(A) Representative cartilage degeneration in Safranin-O–stained sections of the medial region of stifle joints from Plg^+/+ (n = 10) and Plg^–/– (n = 5) male mice 20 weeks after DMM and quantification of the cartilage degeneration. (B) Representative cartilage degeneration in Safranin-O–stained sections of the medial region of stifle joints from C57BL/6J mice treated for 12 weeks with intra-articular injected plasmin (n = 10) or PBS (n = 10) and quantification of the cartilage degeneration. (C and D) Representative cartilage degeneration in Safranin-O–stained sections of the medial region of stifle joints from C57BL/6J mice subjected to DMM and treated with intra-articular injection of antiplasmin (n = 10), α2-macroglobulin (n = 10), or PBS (n = 10) (C) or with i.p. injection of tranexamic acid (n = 6) or PBS (n = 6) (D) for 12 weeks and quantification of the cartilage degeneration. Arrowheads indicate areas of cartilage degeneration. Scale bars, 200 μm. All data are the mean ± SEM of triplicates and are representative of 3 independent experiments. *P < 0.05, **P < 0.01. The test in panels A, B, and D is Mann-Whitney U test. Panel C uses 1-way ANOVA.

Figure 3. Genetic deficiency of PAI-1 accelerates OA while deficiency of tPA attenuates OA in DMM mice.

(A and B) Representative cartilage degeneration in Safranin-O–stained sections of the medial region of stifle joints from Serpine1^+/+ (n = 7) and Serpine1^–/– (n = 8) mice (A) or Plat^+/+ (n = 6) and Plat^–/– (n = 7) mice (B) 20 weeks after DMM and quantification of the cartilage degeneration. Arrowheads indicate areas of cartilage degeneration. Scale bar, 200 μm. All data are the mean ± SEM of duplicates or triplicates and are representative of at least 2 independent experiments. **P < 0.01 by Mann-Whitney U test.

Figure 4. Plasmin contributes to the initiation and progression of OA through multiple mechanisms: the degradation of lubricin and cartilage proteoglycan, activation of pro-MMPs, and induction of inflammatory and degradative mediators from synovial cells.

(A) Representative images from immunofluorescence staining of plasmin (green, left), staining of nuclei (blue, middle), and merging (right) in the undamaged articular cartilage area from individuals with knee OA who underwent total knee replacement. (B) Representative images from immunohistochemical staining of plasmin in the damaged articular cartilage area (upper left), the synovium (upper right), and the isotype controls (bottom, respectively) from individuals with OA. The arrowhead indicates binding of plasmin on the surface of chondrocytes (upper left) and cells of the synovial lining (upper right). (A and B) Scale bar, 200 μm; cartilage and synovial tissues from n = 5 individuals were analyzed. (C) Degradation of recombinant lubricin, shown on SDS-PAGE gel stained with Coomassie blue, by plasmin, but not activated tPA or uPA after 4 hours’ 37°C incubation. Red arrowhead shows the lubricin stained with Coomassie blue in different conditions: vehicle, plasmin, tPA, and uPA. (D) ELISA quantification of soluble sGAG released from cartilage explants from individuals with OA, treated with vehicle, pro–MMP-13, plasmin, or plasmin + pro–MMP-13. (E) Quantitative PCR (qPCR) analysis of OA-related inflammatory and degradative mediators as well as VEGFα in human primary synoviocytes, derived from the knee joints of individuals with OA, with or without plasmin stimulation. (F and G) qPCR analysis of relative gene expression levels of OA-related inflammatory and degradative mediators in synovial tissue (F) or articular cartilage from Plg^+/+ (n = 5) and Plg^–/– (n = 5) mice 20 weeks after DMM. All data are the mean ± SEM of triplicates and are representative of 3 independent experiments. **P < 0.01, and ***P < 0.001. The test in panel D is 1-way ANOVA. The test in panels E–G is 2-tailed t test.

Figure 5. Another key fibrinolysis molecule, uPA, and its receptor, uPAR, also play critical roles in the pathogenesis of OA.

(A and B) Representative cartilage degeneration in Safranin-O–stained sections of the medial region of stifle joints from Plau^+/+ (n = 10) and Plau^–/– (n = 9) (A) and Plaur^+/+ (n = 9) and Plaur^–/– (n = 9) mice (B) 20 weeks after DMM and quantification of the cartilage degeneration. Arrowheads indicate areas of cartilage degeneration. Scale bar, 200 μm. (C and D) MicroPET/CT imaging of mouse knee joints 20 weeks after DMM or sham surgery and quantification of relative ⁶⁸Ga uptake levels in these joints (n = 7). Mice were i.v. injected with ⁶⁸Ga-NODAGA-AE105 (C) or ⁶⁸Ga-NODAGA-AE105 plus unlabeled AE105 (D). (E and F) qPCR analysis of relative mRNA expression levels of OA-related inflammatory, degradative mediators as well as VEGFα in synovial tissues from Plau^+/+ (n = 5) and Plau^–/– (n = 5) mice (E) or Plaur^+/+ (n = 5) and Plaur^–/– (n = 5) mice (F) 20 weeks after DMM. Data are the mean ± SEM of duplicates or triplicates and are representative of at least 2 independent experiments. *P < 0.05, ** P < 0.01, and *** P < 0.001. The test in panels A and B is Mann-Whitney U test. The test in panels C–F is 2-tailed t test.

Figure 6. uPA induces inflammatory and degradative mediators via PI3K and AKT downstream signaling pathways to promote OA.

(A–C) qPCR analysis of OA-related inflammatory and degradative mediators as well as VEGFα in human monocyte-derived macrophages (A), primary synoviocytes (B), and cartilage chondrocytes (C); all cells except macrophages are derived from the knee joints of individuals with OA who underwent total knee replacement, stimulated with or without uPA. (D–H) ELISA validation of protein levels of IL-1β (D), IL-6 (E), IL-8 (F), and MMP-13 (G) in human macrophages stimulated with or without uPA. All data are the mean ± SEM of triplicates and are representative of 3 independent experiments. (H) Western blot analysis of phosphorylation of signaling molecules in human primary synoviocytes, derived from the knee joints of individuals with OA, that were either unstimulated or stimulated with uPA (15 minutes and 30 minutes). Red arrows denote changes in phosphorylation levels over time. Data are representative of at least 3 independent experiments. (I) ELISA quantification of soluble sGAG released from cartilage explants from individuals with OA, treated with PBS, pro–MMP-13 alone, uPA alone, or uPA and pro–MMP-13 together. All data are the mean ± SEM of triplicates and are representative of 3 independent experiments. **P < 0.01, and ***P < 0.001. The test in panels A–G is 2-tailed t test. The test in panel I is 1-way ANOVA.

Figure 7. Targeted therapy for knee articular cartilage homeostasis.

Overview of plasminogen effectors, enzymes, and proteins that can be targeted by pharmacological inhibitors to restore cartilage homeostasis and treat osteoarthritis.