MMP13 is a critical target gene during the progression of osteoarthritis

Abstract

Introduction: Osteoarthritis (OA) is a degenerative joint disease affecting a large population of people. The mechanism of this highly prevalent disease is not fully understood. Currently there is no effective disease-modifying treatment for OA. The purpose of this study was two-fold: 1) to investigate the role of MMP13 in the development of OA; and 2) to evaluate the efficacy of the MMP13 inhibitor CL82198 as a pharmacologic treatment for preventing OA progression.

Methods: To investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13fx/fx (Mmp13Col2ER) and Cre-negative control mice for five days. OA was induced by meniscal-ligamentous injury (MLI) when the mice were 10 weeks old and MLI or sham-operated joints were harvested 4, 8, 12, or 16 weeks after surgery. To evaluate the efficacy of CL82198, MLI surgery was performed on 10-week-old wild type mice. CL82198 or saline was administered to the mice daily beginning immediately after the surgery for up to 16 weeks. The joint tissues collected from both experiments were evaluated by cartilage grading, histology/histomorphometry, immunohistochemistry (IHC), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The ability of CL82198 to inhibit MMP13 activity in vitro was confirmed by ELISA.

Results: The OA progression was decelerated in Mmp13Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13Col2ER mice had a higher articular cartilage area and thickness at 12 and 16 weeks post-surgery compared to the control mice. Results of IHC revealed greater type II collagen and proteoglycan expression in Mmp13Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13Col2ER mice. CL82198 inhibited MMP13 activity in conditioned media from vehicle (>85%) or bone morphogenetic protein 2 (BMP2)-treated (>90%) primary murine sternal chondrocytes. Intraperitoneal injection of CL82198 decelerated MLI-induced OA progression, increased type II collagen and proteoglycan levels, and inhibited chondrocyte apoptosis compared to saline treatment as determined by OA grading, histology, histomorphometry, IHC, and TUNEL staining, respectively.

Conclusions: Mmp13 is critical for OA progression and pharmacologic inhibition of MMP13 is an effective strategy to decelerate articular cartilage loss in a murine model of injury-induced knee OA.

Figure 1

Decelerated osteoarthritis progression in Mmp13^Col2ER mice. Tamoxifen was administered when matrix metalloproteinase (MMP13) conditional knockout (cKO) mice (Mmp13^Col2ER) and Cre-negative control mice were two weeks old (1 mg/10 g body weight, i.p., daily for five days). Meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) surgery was performed on the right hind-limbs when the mice were 10 weeks old. The left hind-limbs were used as sham controls. (A) Knee joint samples were harvested 4, 8, 12, or 16 weeks post-surgery and Alcian blue/Hematoxylin/Orange G staining was performed. Histological results showed decreased articular cartilage degradation in Mmp13^Col2ER mice 8, 12 and 16 weeks post-surgery. (B) Histological grading by blinded observers confirmed decreased articular cartilage degradation in Mmp13^Col2ER mice at 8, 12, and 16 weeks compared to control Cre-negative mice (*P < 0.05). (C) Tibia articular cartilage area was quantified by tracing the Alcian blue-positive area in the proximal tibia. There was no significant difference 4 and 8 weeks post-surgery in Mmp13^Col2ER MLI mice versus control MLI mice. The tibia cartilage area was increased 21% in Mmp13^Col2ER MLI mice compared to control MLI mice 12 weeks post-surgery (*P < 0.05) and increased 31% 16 weeks post-surgery (*P < 0.05). (D) Tibia thickness was quantified by tracing the Alcian blue-positive thickness in the center of the tibial plateau. There was no significant difference in tibial cartilage thickness at 4 or 8 weeks post-surgery. Tibia cartilage thickness was increased 29% in Mmp13^Col2ER MLI mice compared to control MLI mice 12 weeks post-surgery (*P < 0.05) and increased 50% 16 weeks post-surgery (*P < 0.01). (E) Total articular cartilage area was quantified by tracing the Alcian blue-positive area in both the proximal tibia and distal femur. No significant differences were detected 4, 8, and 12 weeks post-surgery. Total cartilage area increased 18% in Mmp13^Col2ER MLI mice compared to control MLI mice 16 weeks post-surgery (*P < 0.01). (F) Total cartilage thickness was quantified by tracing the Alcian blue-positive thickness in the center of the proximal tibia and distal femur. No significant differences were detected at 4, 8, and 12 weeks post-surgery. Total cartilage thickness increased 39% in Mmp13^Col2ER MLI mice compared to control MLI mice 16 weeks post-surgery (*P < 0.01).

Figure 2

Decelerated osteoarthritis progression in Mmp-13^Col2ER mice. Tamoxifen was administered when matrix metalloproteinase (MMP13) conditional knockout (cKO) mice (Mmp13^Col2ER) and Cre-negative control mice were two-weeks-old (1 mg/10 g body weight, intraperitoneal injection, daily for five days). Meniscal-ligamentous injury (MLI) surgery was performed when the mice were 10-weeks-old. Knee joints were harvested eight weeks post-surgery. (A) Safranin O/Fast Green staining was performed to evaluate proteoglycan content. Proteoglycan loss following MLI was reduced in Mmp13^Col2ER mice. (B) Type II collagen (Col2) loss following MLI was reduced in Mmp13^Col2ER mice. Immunohistochemistry (IHC) of Col2 was performed to assess its protein level. (C) IHC of ColX was performed to assess its protein level. Type X collagen (ColX) induction following MLI was reduced in Mmp13^Col2ER mice. (D) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (in green) and 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstaining (in blue) were performed to assess chondrocyte apoptosis. Chondrocyte apoptosis following MLI was reduced in Mmp13^Col2ER mice. (E) Quantification of TUNEL staining positive cells was performed to assess chondrocyte apoptosis.

Figure 3

CL82198 prevents and decelerates MLI-induced osteoarthritis progression. (A and B) CL82198 inhibits matrix metalloproteinase (MMP13) activity in vitro. (A) Addition of 5 ng of MMP13, 5 ng of MMP13 and 10 μg/mL of CL82198, or substrate control into a 96-well plate. MMP13 enzyme activity was determined using the SensoLyte 520 MMP13 Assay Kit, and showed that CL82198 could block > 90% MMP13 activity in vitro. (B) Primary sternal chondrocytes were isolated from three-day-old wild type pups. Cells were plated in 12-well plates and treated with 100 ng bone morphogenetic protein 2 (BMP-2), 100 ng of BMP-2 and 1 μM of CL82198, 100 ng of BMP-2 and 5 μM of CL82198, 100 ng of BMP-2 and 10 μM of CL82198, or vehicle control for 60 hours. Cell culture media was collected and MMP13 activity was determined and showed that CL82198 could inhibit > 90% MMP13 activity produced by BMP-2-treated primary chondrocytes. (C) Inhibition of MMP13 by CL82198 protects against meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA). MLI and sham surgeries were performed on 10-week-old wild type mice. CL82198 was administered to wild type mice one day after surgery by intraperitoneal injection every other day for 12 weeks at doses of 1, 5, or 10 mg/kg body weight. Normal saline was used as a control. Knee joints were collected, and sectioned 12 weeks post-surgery, and Alcian blue/Hematoxylin/Organge G staining was performed. (D) Histological grading by two blinded observers confirmed decreased articular cartilage degradation in mice treated with 1, 5, and 10 mg/kg of CL82198 compared to saline control mice (*P < 0.001). (E) The articular cartilage area at proximal tibiae was quantified by tracing the Alcian blue-positive area in the proximal tibia. Compared to saline MLI, tibial cartilage area was increased 9, 15, and 43% after treatment with 1, 5, and 10 mg/kg of CL82198, respectively (*P < 0.05). (F) Total articular cartilage area was quantified by tracing the Alcian blue-positive area in both the proximal tibia and the distal femur. Total cartilage area was increased 21%, 19%, and 38% with treatment of 1, 5, and 10 mg/kg of CL82198, respectively (*P < 0.05). (G) Tibia cartilage thickness was quantified by tracing the Alcian blue-positive thickness in the center of the tibial plateau. Compared to saline treatment, tibia cartilage thickness was increased 11%, 37%, and 70% with treatment of 1, 5, and 10 mg/kg of CL82198, respectively (*P < 0.05). (H) Total thickness was quantified by tracing the Alcian blue-positive thickness in both the proximal tibia and the distal femur. Total cartilage thickness was increased 23%, 27%, and 50% after injection of 1, 5, or 10 mg/kg CL82198, respectively (*P < 0.05).

Figure 4

CL82198 prevents and decelerates MLI-induced osteoarthritis progression. Meniscal-ligamentous injury (MLI) and sham surgeries were performed on 10-week-old wild type mice. CL82198 was administered to wild type mice beginning one day after surgery by intraperitoneal injection every other day for 12 weeks at doses of 10 mg/kg body weight. Normal saline was used as a vehicle control. (A) Safranin O/Fast green staining was performed to assess proteoglycan content. Proteoglycan loss was reduced following CL82198 treatment. (B) Immunohistochemistry (IHC) of type II collagen (Col2) was performed to assess its protein level. Col2 loss was reduced following CL82198 treatment. (C) IHC of ColX was performed to assess its protein level. Type X collagen (ColX) induction was reduced following CL82198 treatment. (D) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (in green) and 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstaining (in blue) were performed to assess chondrocyte apoptosis. Chondrocyte apoptosis was reduced following CL82198 treatment. (E) Quantification of TUNEL staining positive cells was performed to assess chondrocyte apoptosis.