Plasminogen activator inhibitor-1 deficiency enhances subchondral osteopenia after induction of osteoarthritis in mice

Abstract

Background: Subchondral osteopenia is important for the pathophysiology of osteoarthritis (OA). Although previous studies suggest that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is related to bone metabolism, its role in OA remains unknown. We therefore investigated the roles of PAI-1 in the subchondral bone in OA model mice.

Methods: Wild type (WT) and PAI-1-deficient (KO) mice were ovariectomized (OVX), and then destabilization of the medial meniscus (DMM) surgery was performed.

Results: DMM and OVX significantly decreased the trabecular bone mineral density of the subchondral bone evaluated by quantitative computed tomography in PAI-1 KO mice. The effects of OVX and/or PAI-1 deficiency on the OARSI score for the evaluation of the progression of knee degeneration were not significant. PAI-1 deficiency significantly augmented receptor activator nuclear factor κB ligand mRNA levels enhanced by IL-1β in mouse primary osteoblasts, although it did not affect osteoblast differentiation. Moreover, PAI-1 deficiency significantly increased osteoclast formation from mouse bone marrow cells.

Conclusion: We showed that PAI-1 deficiency accelerates the subchondral osteopenia after induction of OA in mice. PAI-1 might suppress an enhancement of bone resorption and subsequent subchondral osteopenia after induction of OA in mice.

Keywords: Osteoarthritis; Osteoclast; Ovariectomy; Plasminogen activator inhibitor-1; Subchondral bone.

Fig. 1

Effects of DMM, OVX and PAI-1 deficiency on the BMD of the tibial subchondral bone in mice. a qCT images of the subchondral bone in WT and PAI-1 KO mice 8 weeks after DMM or sham surgery with or without OVX. b The trabecular BMD of the subchondral bone was assessed by qCT in WT and PAI-1 KO mice 8 weeks after DMM or sham surgery with or without OVX. Regions of interest were defined as 240-μm (10-slice) segments from 72 μm proximal to the end of the proximal growth plate towards the joint. Data represent the mean ± SEM of 10–11 mice. **p < 0.01 and *p < 0.05. c Hematoxylin and Eosin-stained sections from the subchondral bone in WT and PAI-1 KO mice 8 weeks after DMM or sham surgery with or without OVX. Scale bars = 200 μm

Fig. 2

Effects of DMM, OVX and PAI-1 deficiency on articular cartilage degeneration in mice. Cartilage degeneration was assessed using the OARSI histological scoring system in WT and PAI-1 KO mice 8 weeks after DMM or sham surgery with or without OVX. Each knee was evaluated using 6 grades for the progression of arthropathy. Data represent the mean ± SEM of 10–11 mice. **p < 0.01

Fig. 3

Effects of IL-1β on the expressions of PAI-1 and TGF-β₁ in primary mouse osteoblasts. a Primary osteoblasts were obtained from 3-day-old female WT mice. Total RNA was extracted from primary osteoblasts cultured with or without 10 ng/mL IL-1β or 1 ng/ml TGF-β₁ for 24 h. The levels of PAI-1 or GAPDH mRNA were assessed by real-time PCR. Data represent the mean ± SEM. n = 6 for each group. **p < 0.01. b Primary osteoblasts were obtained from 3-day-old female WT and PAI-1 KO mice. Total RNA was extracted from primary osteoblasts cultured with or without 10 ng/mL IL-1β for 24 h. Then, the levels of TGF-β₁ or GAPDH mRNA were assessed by real-time PCR. Data represent the mean ± SEM. n = 6 for each group

Fig. 4

Effects of PAI-1 deficiency on osteoblast differentiation in mouse osteoblasts and bone marrow cells. a Primary osteoblasts were obtained from 3-day-old female WT and PAI-1 KO mice. Total RNA was extracted from primary osteoblasts cultured with or without 10 ng/mL IL-1β or 1 ng/ml TGF-β₁ for 24 h. The levels of Runx2, Osterix, ALP, Col-1, OCN or GAPDH mRNA were assessed by real-time PCR. Data represent the mean ± SEM. n = 6 for each group. *p < 0.05. b Primary osteoblasts were obtained from 3-day-old female WT mice. Total RNA was extracted from primary osteoblasts cultured with or without 10 ng/mL IL-1β or 1 ng/ml TGF-β₁ in the presence or absence of 1 μM SB421542 for 24 h. The levels of Osterix or GAPDH mRNA were assessed by real-time PCR. Data represent the mean ± SEM. n = 6 for each group. **p < 0.01. c Bone marrow cells were obtained from 8-week-old female WT and PAI-1 KO mice. Total RNA was extracted from the bone marrow cells cultured with or without 200 ng/ml BMP-2 or 10 ng/mL IL-1β for 48 h. The levels of Osterix, ALP, OCN or GAPDH mRNA were assessed by real-time PCR. Data represent the mean ± SEM. n = 5 for each group. **p < 0.01 and *p < 0.05

Fig. 5

Effects of PAI-1 deficiency on the expression of RANKL and OPG in mouse osteoblasts. Primary osteoblasts were obtained from 3-day-old female WT and PAI-1 KO mice. Total RNA was extracted from primary osteoblasts cultured with or without 10 ng/mL IL-1β or 1 ng/ml TGF-β₁ in the presence or absence of 1 μM SB421542 for 24 h. The levels of RANKL, OPG or GAPDH mRNA were assessed by real-time PCR. Data represent the mean ± SEM. n = 6 for each group. **p < 0.01 and *p < 0.05

Fig. 6

Effects of PAI-1 deficiency on osteoclast formation from mouse bone marrow cells. After bone marrow cells obtained from WT and PAI-1 KO mice cultured with M-CSF (50 ng/ml) for 3 days, cells were treated with or without IL-1β in the presence of M-CSF (50 ng/ml) and RANKL (50 ng/ml) for 3 days. Then, TRAP-positive MNCs were counted. Data represent the mean ± SEM. n = 6 for each group. **p < 0.01