Key molecules in the fibrinolysis pathway are dysregulated in human OA joints. To gain insight into the role of fibrinolysis in OA, we investigated whether genes involved in the fibrinolysis pathway were dysregulated in OA joints. Unsupervised hierarchical clustering analysis of gene expression microarray data sets on synovial membranes from individuals with early- or end-stage OA and healthy individuals revealed an upregulation of uPA and uPAR in the early- or end-stage OA joints (Figure 1A). No mRNA expression of plasminogen was detected in the synovial tissues from knee joints of either OA or other joint diseases, suggesting that plasminogen translocates into the synovium from circulation. Bulk RNA-sequencing data comparing gene expression in degenerative meniscus tear (DMT), rheumatoid arthritis (RA), and OA showed that PLAU expression was significantly upregulated in OA compared with DMT, with minor or no changes in PLAU receptor (PLAUR) (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.173603DS1).

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.

Next, we determined if fibrinolysis protein levels were also dysregulated in OA joints. Higher levels of plasmin were detected in the synovial fluids (SFs) of individuals with OA when compared with healthy individuals by ELISA analysis. Plasmin levels were elevated in the SFs of individuals with anterior cruciate ligament (ACL) tear, who are at high risk for OA, and those with DMT, reflective of early OA, as compared with healthy individuals (Figure 1B). Plasmin levels from SFs of patients with ACL tear and DMT were significantly lower compared with OA. Our results showed that there was an increase in plasmin levels following traumatic joint injury (ACL), during early-stage OA (DMT), and during end-stage OA (Figure 1B). We also found higher levels of uPA and uPAR in the SFs of individuals with OA, when compared with plasma (Figure 1, C and D).

Corroborating these results, immunohistochemical analysis of damaged cartilage and synovium from individuals with OA showed positive staining of tPA, uPA, and uPAR compared with isotype controls (Figure 1E). Our data indicate that fibrinolysis is aberrant in OA joints.

Genetic deficiency or pharmacological blockade of plasmin attenuates OA in mice. To understand the role of plasminogen and plasmin in the pathogenesis of posttraumatic OA, we examined the effects of genetic deficiency in the plasminogen gene Plg on the development of OA in the DMM mouse model. We surgically induced OA through DMM in both B6.129P2-Plgtm1Jld/J (Plg–/–) and Plg+/+ mice. Twenty weeks after DMM, Plg–/– mice showed significantly less cartilage degeneration than Plg+/+ mice (Figure 2A). Consistent with the protection seen in Plg–/– mice, intra-articular injection of plasmin for 12 weeks alone was sufficient to promote cartilage degeneration in C57BL/6J mice (Figure 2B). Additionally, intra-articular treatment with antiplasmin or α2-macroglobulin, a plasmin inhibitor (30), for 12 weeks following DMM, attenuated the progression of OA in C57BL/6J mice (Figure 2C). Furthermore, treatment with tranexamic acid, an antifibrinolytic (31), for 12 weeks mitigated cartilage degeneration in C57BL/6J DMM mice (Figure 2D). Taken together, these results show that plasmin plays a central role in the pathogenesis of OA.

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.

Genetic deficiency of PAI-1 accelerates OA while deficiency of tPA attenuates OA in DMM mice. Next, we determined whether regulators of plasmin activation contribute to OA development. We tested whether genetic deficiency in either the plasmin inhibitor, PAI-1, or in the tissue plasminogen activator, tPA, affected the development of OA in a DMM mouse model. Mice that were genetically deficient for PAI-1 (B6.129S2-Serpine1tm1Mlg/J; Serpine1–/–) displayed more cartilage degeneration when compared with their wild-type (Serpine1+/+) littermates, 20 weeks after DMM (Figure 3A). In contrast, mice deficient in tPA (C.129S2-Plattm1Mlg/J; Plat–/–) had less cartilage degeneration than their wild-type (Plat+/+) littermates when subjected to DMM (Figure 3B). Our data further indicate that the dysregulation of plasmin activation functions in the development of OA.

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.

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. Given that aberrant activation of plasmin played a role in the pathogenesis of OA, we investigated how OA development was affected. We used immunohistochemistry to visualize the localization of plasminogen/plasmin in articular cartilage from individuals with OA. We found that plasminogen/plasmin was localized on the surfaces of cartilage in undamaged areas, while plasminogen/plasmin was undetected on chondrocytes located inside this area (Figure 4A). In contrast, we observed positive staining of plasminogen/plasmin on chondrocytes within the damaged cartilage area and synovium of individuals with OA (Figure 4B). These findings suggest that the superficial articular cartilage is resistant to damage caused by plasmin, and once the superficial cartilage is degraded, plasmin will continue to damage chondrocytes. Plasmin on synovial cells influenced synovium hemostasis.

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.

We then investigated potential mechanisms by which plasmin contributes to synovial hemostasis imbalance and cartilage degradation. Lubricin, produced by synovial cells and chondrocytes, is a surface-active mucinous glycoprotein that imparts boundary lubrication between joint surfaces. It plays a role in reducing friction and in maintaining a wear-resistant property in articulating joints (32). We tested plasmin’s ability to degrade recombinant lubricin in vitro. Upon incubation with plasmin, lubricin was undetected by Coomassie stain on an SDS-PAGE gel, whereas lubricin was present after incubation with PBS, activated tPA, and uPA (Figure 4C). This indicates that plasmin, but not activated tPA or uPA, degrades lubricin.

Sulfated glycosaminoglycans (sGAGs) are important molecular indicators of healthy cartilage and are present at lower concentrations in OA (33, 34). sGAG, released from the cartilage by degradation, is an indicator of proteoglycan damage. We found that treatment with plasmin of cartilage explants from individuals with OA resulted in increased sGAG release when compared with cartilage treated with PBS (Figure 4D). Additionally, proteinases, such as MMPs (e.g., MMP-13) produced by chondrocytes and synovial cells in the joint, play a significant role in the pathological destruction and eventual loss of cartilage in OA (35). Incubation of cartilage explants with zymogen pro–MMP-13 alone did not lead to a higher release of sGAG. However, coincubation of plasmin and pro–MMP-13 led to a marked increase in sGAG release when compared with plasmin alone (Figure 4D). Further, no changes in sGAG levels were detected when cartilage explants were treated with activated tPA, in either the presence or absence of pro–MMP-13 (Supplemental Figure 2). These results are consistent with the notion that plasmin can degrade proteoglycans and other connective tissue components (36) and that plasmin can activate the latent form of MMPs, which can specifically degrade the ECM (37, 38).

In addition to the effect of plasmin on lubricin degradation and cartilage proteoglycan degradation, we examined whether it could directly induce production of inflammatory and degradative mediators in the joint. When stimulated with plasmin, human primary synoviocytes derived from the knee joints of individuals with OA expressed OA-related inflammatory CCL2 and degradative MMP14, ADAMTS4, and ADAMTS5 mediators as well as VEGFA (Figure 4E). Expression levels of inflammatory Cxcl15 and Ccl2 and degradative Mmp3 and Adamts5 mediators were significantly reduced in synovial tissue (Figure 4F) and Il6, Cxcl15, Ccl2, Mmp3, Mmp13, and Vegfα in articular cartilage (Figure 4G) from Plg–/– mice as compared with Plg+/+ control mice 20 weeks after DMM. Together, our results indicate that plasmin promotes inflammatory and degradative mediators in OA joints, which may contribute to the pathogenesis of OA.

Fibrinolysis molecule, uPA, and its receptor, uPAR, also play a critical role in the pathogenesis of OA. After detecting elevated levels of uPA and uPAR in OA joints, we determined if genetic loss of function impacted the progression of OA in mice following DMM. We found that genetic deficiency of uPA (B6.129S2-Plautm1Mlg/J; Plau–/–) and uPAR (B6.129P2-Plaurtm1Jld/J; Plaur–/–) decreased cartilage degeneration as compared with their wild-type littermates, 20 weeks after DMM (Figure 5, A and B), suggesting the involvement of both uPA and uPAR in the pathogenesis of OA.

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.

Next, we studied uPA and uPAR binding in OA joints by quantifying the uptake of AE105, a core uPA/uPAR binding peptide, in the articular joints of C57BL/6J DMM mice. Radiolabeled AE105, 68Ga-NODAGA-AE105 (39), was injected i.v. 20 weeks after DMM or sham surgery. Deposition of AE105 was determined by microPET/CT imaging of mouse knee joints. Mice subjected to DMM took up a higher concentration of 68Ga-NODAGA-AE105 compared with the sham-operated side of joints (Figure 5C). Coinjection of competing, nonradioactive labeled AE105 diminished the difference in radiolabeled uPA uptake, verifying the binding specificity of AE105 to uPAR (Figure 5D). Our data show that uPA/uPAR binding increased in a mouse model of DMM-induced OA.

We also examined if genetic deficiencies in uPA and uPAR mitigated DMM-induced OA pathologies by affecting the production of inflammatory and degradative mediators by cells in the joint. Indeed, reduced gene expression levels of OA-related inflammatory I1b, Il6, Cxcl15, and Ccl2 and degradative Mmp3, Mmp13, and Adamts5 mediators, but not Adamts4, were detected in synovial tissues from Plau–/– DMM mice (Figure 5E). Reduced gene expression levels of OA-related inflammatory Il1b, Il6, Cxcl15, and Ccl2 and degradative Mmp13 and Adamts5 mediators were also detected in synovial tissues from Plaur–/– mouse knee joints subjected to DMM (Figure 5F).

uPA induces inflammatory and degradative mediators via PI3K and AKT downstream signaling pathways to promote OA. After finding that mouse synovial tissue deficient for uPA and uPAR had reduced expression of inflammatory and degradative mediators, we tested whether uPA was able to induce production of these mediators in different cell types within the joint. We observed an increase in expression of IL1B, IL6, CXCL8 CCL2, CCL5, PTGS2, and VEGFA when human monocyte-derived macrophages were stimulated with uPA (Figure 6A). A similar transcriptomic signature was detected in synoviocytes with a marked upregulation of ILB, IL6, CXCL8, CCL2, CCL5, PTGS2, and MMP13 but not TNFA or VEGFA (Figure 6B). In cartilage chondrocytes from OA knee joints, we noted a significant increase in IL6, CXCL8, CCL2, CCL5, PTGS2, MMP13, and VEGFA (Figure 6C). In some cases, elevated transcription led to an increase in protein translation as measured by ELISA (Figure 6, D–G). Higher IL-1β protein concentration was detected in uPA-stimulated chondrocytes, while higher IL-6 and IL-8 were seen in uPA-stimulated macrophages, synoviocytes, and chondrocytes, as well as increased pro–MMP-13 protein levels in uPA-stimulated synoviocytes. The induction of inflammatory and degradative mediators by uPA is similar to the effect of plasmin that we observed before (Figure 4E).

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.

In light of the above findings, we investigated the intracellular pathways through which uPA induces changes in the expression of inflammatory and degradative genes in OA. Upon stimulation with uPA, human primary synoviocytes, derived from the knee joint of individuals with OA, showed transient increases in PI3K phosphorylation and PDK1, 15 minutes after stimulation. AKT and ERK remained phosphorylated at 15 and 30 minutes after stimulation (Figure 6H). Our data suggest that, analogous to the mechanism in other diseases such as cancer (40), uPA activates a cascade of known target molecules and thereby induces inflammation and tissue degradation in the OA joint.

Finally, we also investigated the role of uPA in cartilage degradation in OA (Figure 6I). We found that treatment of cartilage explants from individuals with OA with uPA resulted in increased release of sGAG into the supernatant of cultures, while incubation of cartilage explants with pro–MMP-13 and uPA did not lead to an increase in sGAG release when compared with cartilage treated with PBS and pro–MMP-13 (Figure 4D). Our data suggest that similar to plasmin, uPA can directly degrade certain proteoglycans (41), despite its inability to degrade lubricin (Figure 4C), and cannot activate MMP zymogens to further degrade proteoglycan.