MMP13 is a critical target gene during the progression of osteoarthritis
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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.
To investigate the role of the endogenous Mmp13 gene in OA development, tamoxifen was administered to two-week-old Col2CreER;Mmp13 fx/fx (Mmp13 Col2ER ) 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.
The OA progression was decelerated in Mmp13 Col2ER mice 8, 12, and 16 weeks post-surgery. Cartilage grading by blinded observers confirmed decreased articular cartilage degeneration in Mmp13 Col2ER mice at 8, 12 and 16 weeks compared to Cre-negative mice. Histomorphometric analysis demonstrated that Mmp13 Col2ER 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 Mmp13 Col2ER mice. Chondrocyte apoptosis, as determined by TUNEL staining, was higher in control mice compared to Mmp13 Col2ER 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.
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.
KeywordsArticular Cartilage MMP13 Activity Medial Collateral Ligament Chondrocyte Apoptosis Terminal Deoxynucleotidyl Transferase dUTP Nick
Alcian Blue/Hematoxylin/Orange G
a disintegrin and metalloproteinase with thrombospondin motifs
analysis of variance
bone morphogenetic protein 2
type II collagen
type X collagen
enzyme-linked immunosorbent assay
- H & E
hematoxylin and eosin
Safranin O/Fast Green
terminal deoxynucleotidyl transferase dUTP nick end labelling
Osteoarthritis (OA) is a degenerative joint disease and is a major cause of disability. Currently, there is no treatment capable of altering its progression. The major pathological characteristics of OA include progressive loss of articular cartilage, osteophyte formation, and changes in peri-articular and subchondral bone [1, 2]. The articular cartilage receives most of the attention in OA studies because the primary pathologic feature seen in OA is gross articular cartilage damage.
Matrix metalloproteinase (MMP) 13 is a major enzyme that targets cartilage for degradation. Compared to other MMPs, the expression of MMP13 is more restricted to connective tissue . It not only targets type II collagen in cartilage for degradation, but also degrades proteoglycan, types IV and type IX collagen, osteonectin and perlecan in cartilage . Clinical investigation revealed that patients with articular cartilage destruction have high MMP13 expression , suggesting that increased MMP13 may be associated with cartilage degradation. Studies have also shown that Mmp13-overexpressing transgenic mice develop a spontaneous OA-like articular cartilage destruction phenotype . The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of aggrecanases also contributes to proteoglycan/aggrecan depletion and are associated with cartilage degradation during OA. ADAMTS4 and 5 were identified as the major aggrecanases during OA development. Deletion of the Adamts5 gene or double knockout of Adamts4 and Adamts5 prevented cartilage degradation in a surgically induced murine knee OA model [7, 8]. These findings indicate that catabolic enzymes play a significant role in OA progression and support the development of therapies targeting these enzymes as a strategy to decelerate articular cartilage degradation.
Meniscal injuries are among the most common causes of post-traumatic OA in humans. The meniscus is a C-shaped cartilage that functions as a shock-absorbing, load bearing, stability enhancing, and lubricating cushion in the knee joint. Studies show that loss of meniscus integrity and function leads to OA in humans [9, 10]. The meniscal-ligamentous injury (MLI)-induced murine OA model was initially developed by Clements et al.  and this injury model has been further modified and developed in recent studies . This mouse model also permits study of the development of trauma-induced OA in defined genetic backgrounds. In this model, the ligation of the medial collateral ligament coupled with disruption of the meniscus from its anterior-medial attachment induces reproducible OA development over a three-month period . In the present studies, we will determine the role of MMP13 in MLI-induced OA progression. We will use Col2CreER;Mmp13 fx/fx (Mmp13 Col2ER ) mice to block Mmp13 expression in chondrocytes and use MMP13 inhibitors to inhibit MMP13 activity.
Meniscal/ligamentous injury-induced osteoarthritis model and treatment of MMP13 inhibitor
Breeding of Col2CreER;Mmp13 fx/fx mice (Mmp13 Col2ER ).
a) Col2CreER x Mmp13 fx/fx
a) Col2CreER;Mmp13 fx/wt
b) Col2CreER;Mmp13 fx/wt x Mmp13 fx/fx
b) Col2CreER;Mmp13 fx/fx
c) Col2CreER;Mmp13 fx/fx x Mmp13 fx/fx
c) Col2CreER;Mmp13 fx/fx and Mmp13 fx/fx
Histology and histomorphometry
Knee joints from each group were harvested and prepared for sectioning and analysis. Samples were fixed in 10% neutral buffered formalin (VWR, Radnor, PA, USA) for three days, then decalcified with formic acid (Decal Chemical Corp., Suffern, NY, USA) for seven days. After neutralizing with Cal-arrest (Decal Chemical Corp.), samples were processed and embedded in paraffin. Three μm thick mid-saggital sections at three different levels (50 μm apart) were cut from the medial compartment of the joints. The sections were stained with Alcian blue/H&E (AB/H&E) and safranin O/Fast Green (SO/FG). Histomorphometric measurements were performed using OsteoMeasure software (OsteoMetrics, Inc., Atlanta, GA, USA). AB/H&E- or SO/FG-stained areas were outlined on projected images of each histologic section to determine articular cartilage area and thickness.
Immunohistochemistry and TUNEL staining
Three μm thick paraffin sections were baked at 60°C overnight. Slides were then deparaffinized, rehydrated, and digested with pepsin. DAKO endogenous blocking reagent (Dako S2003, Carpinteria, CA, USA) was then used to quench endogenous peroxidase for 10 minutes. Normal horse serum or normal goat serum (Vector S-2000, Burlingame, CA, USA) was used to block non-specific binding sites for 20 minutes. Collagen II or collagen X primary antibodies (Thermo Scientific, Rockford, IL, USA) were added and the slides were incubated at 4°C overnight. Secondary biotinylated horse anti-mouse antibody (Vector BA-2000) or goat anti-mouse antibody (Vector BA-9200) was added for 30 minutes on the second day, followed by incubation with streptavidin (Pierce 21130, Rockford, IL, USA) for 30 minutes. Positive staining was detected by Romulin AEC Chromagen (Biocare Medical RAEC810L, Concord, CA, USA). To detect chondrocyte apoptosis after meniscal surgery, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a kit based on the manufacturer's instruction (Promega, Fitchburg, WI, USA).
Grading of cartilage structure
Tissue sections were stained with Alcian blue/Orange G and graded by two blinded observers based on the scoring system developed by Chambers et al. . In brief, each section was assigned a grade as follows: 0 = normal cartilage, that is, lack of superficial zone fibrillation or clefting; 1 = mild superficial fibrillation; 2 = fibrillation and/or clefting extending below the superficial zone; 3 = mild (< 20%) loss of articular cartilage; 4 = moderate (20% to 80%) loss of articular cartilage; 5 = focal loss of cartilage to the subchondral bone (eburnation); 6 = severe (> 80%) loss of articular cartilage. The progression of OA in different groups was evaluated using this method. Three slides of each sample were analyzed and five mice were used in each group.
MMP13 activity assay
The ability of CL82198 to inhibit MMP13 activity in vitro was determined using the SensoLyte 520 MMP13 Assay Kit (AnaSpec, Fremont, CA, USA). Five ng of MMP13 and 10 μg/mL of CL82198, or control substrate were added into the MMP13 Assay Kit. MMP13 activity was detected following the manufacturer's instructions. Primary sternal chondrocytes were isolated from three-day-old wild-type (WT) pups as previously described . Cells were plated in 12-well-plates and treated with bone morphogenetic protein 2 (BMP-2) (100 ng/ml) alone or BMP-2 with the MMP-13 inhibitor CL82198 (1, 5 and 10 μM) for 60 hours. The culture media were collected and MMP13 activity was determined using the SensoLyte 520 MMP13 Assay Kit (AnaSpec) following the manufacturer's instructions.
Results of all quantitative assays involving multiple doses and time points were analyzed using analysis of variance (ANOVA) followed by Dunnett's test. For experiments comparing two groups, unpaired student's t-test was applied. P < 0.05 was considered to be a significant difference.
OA progression was decelerated in Mmp13Col2ER mice
Col2 and proteoglycan loss and chondrocyte apoptosis are reduced in Mmp13Col2ER mice
CL82198 decelerates MLI-induced OA progression
To investigate the efficacy of CL82198 in the inhibition of OA progression, we performed MLI surgery on 10-week-old WT mice, followed by i.p.injection of saline, 1, 5, or 10 mg/kg of CL82198  every other day, beginning one day after surgery, for 12 weeks. Knee joints were harvested 12 weeks post-surgery. Histological results revealed that OA progression, most notably cartilage loss down to and even below the tidemark in saline control mice, was decelerated with 1, 5, or 10 mg/kg of CL82198 injection, particularly with the 10 mg/kg dose (Figure 3C). Cartilage grading results were consistent and treatment with 5 and 10 mg/kg doses of CL82198 had significantly lower scores than the saline control (Figure 3D). Histomorphometric analysis revealed that the articular cartilage area at the proximal tibiae was increased 9%, 15% and 43% (Figure 3E); total cartilage area was increased 21%, 19% and 38% (Figure 3F); articular cartilage thickness at the proximal tibiae was increased 11%, 37% and 70% (Figure 3G); and total cartilage thickness was increased 23%, 27% and 50% (Figure 3H) after injection of 1, 5, and 10 mg/kg of CL82198, respectively.
CL82198 has protective effects on MLI-induced Col2 and proteoglycan loss and chondrocyte apoptosis
OA is a degenerative joint disease and is the most prevalent form of arthritis. The major symptom of this disease is progressive cartilage break down and eventual complete loss of articular cartilage [1, 19]. The pathogenesis of OA is not well understood, and there is currently no treatment to alter the progression of OA. In this study, we demonstrated that cartilage-specific deletion of Mmp13 decelerated the progression of OA. We also demonstrated that the MMP13 inhibitor CL82198 prevents OA progression in the MLI-induced murine knee OA model and may, therefore, represent a novel treatment option for OA patients to preserve articular cartilage and joint function instead of simply alleviating OA symptoms as existing treatments do.
Recently, Little et al. showed that global knockout of Mmp13 could prevent articular cartilage erosion . However, there are several limitations related to this study. 1) The Mmp13 global knockout mice have a few abnormalities including focal regions of bony union in growth plates, tendency of metaphyseal flaring and increased trabecular bone mass that may affect OA study . 2) OA is a chronic, progressive disease, and their study only assessed articular cartilage degradation four- and eight-weeks post-surgery-induced OA. 3) Their study relied on semi-quantitative histologic methods to characterize OA progression.
To understand better the role of MMP13 in OA development and progression, we generated inducible cartilage-specific Mmp13 Col2ER mice. Tamoxifen was administered when the mice were two-weeks-old, followed by MLI knee joint surgery to induce OA in Mmp13 Col2ER and Cre-negative control mice [22, 12]. Our results demonstrate that while articular cartilage is nearly normal four weeks post-surgery in both groups, injured mice progressively develop an OA-like phenotype at the later 8-, 12-, and 16-week time points. The histomorphometric data showed that cartilage area and thickness at the proximal tibiae was significantly different at 12 and 16 weeks post-surgery in the Mmp13 Col2ER group compared to the control group. However, the total cartilage area and thickness only had a significant difference at 16 weeks post-surgery. This is likely because there was little evidence of cartilage degeneration or loss on the femoral condyle in either experimental group at 4, 8, 12, or 16 weeks post-surgery, which is consistent with previous studies using this model [20, 12].
In addition to morphologic changes in articular cartilage structure, OA is also characterized by changes in matrix composition as well as changes in articular chondrocyte activity, including inappropriate levels of apoptosis [23, 24, 25]. Col2 and proteoglycan content were decreased in both Mmp13 Col2ER and control groups following MLI surgery. However, cartilage specific deletion of the Mmp13 gene partially prevented Col2 and proteoglycan loss. Deletion of the Mmp13 gene inhibited MLI-induced ColX expression as well. This finding is not unexpected as the cartilage is more intact in Mmp13 Col2ER mice compared to control mice at each time point. TUNEL staining revealed that articular chondrocytes underwent apoptosis in both the control and Mmp13 Col2ER group. However, deletion of the Mmp13 gene dramatically reduced articular chondrocyte apoptosis following MLI, potentially because the more intact cartilage in Mmp13 Col2ER mice may provide chondrocytes with environmental survival cues. Little et al. also showed increased articular chondrocyte apoptosis after surgery, but they did not identify a difference between the control and the Mmp13 KO groups. In their surgery model, they transected the medial meniscotibial ligament to destabilize the medial meniscus. In our surgery model, we transected the medial collateral ligament, detached the anterior horn of the medial meniscus from the tibial plateau, and created a tear. Thus, their surgical procedure may elicit a milder OA phenotype than our procedure, and this milder OA phenotype may be the reason for the apparent disparity in the apoptosis results in our and their studies.
Over the past 30 years, several MMP inhibitors have been developed as candidates for the treatment of arthritis, cancer and cardiovascular diseases . However, most of these compounds have failed for a variety of reasons, including non-specificity and toxicity. Currently, no MMP inhibitor has been used in clinics . Recently, Baragi et al. developed an MMP13 inhibitor, ALS 1-0635, and evaluated the efficacy of this compound in a rat OA model . They gave ALS 1-0635 to rats twice-daily beginning one day before surgically-induced OA for three weeks, and found that ALS 1-0635 has chondro-protective effects. However, ALS 1-0635 only had an effect at a dose of 60 mg/kg. The large dose and frequent administrations of this compound suggest a relatively low specificity for ALS 1-0635 compound.
To explore the therapeutic potential of MMP13 inhibition for OA treatment, we investigated the ability of CL82198, a specific MMP13 inhibitor, to inhibit MMP13 activity in vitro. CL82198 is a chemical compound. Unlike ALS 1-0635 and other MMP13 inhibitors which exert their effects via metal chelation, CL82198 binds to the S1' pocket of MMP13 and showed no effects on MMP-1, -9, or TACE . We found that CL82198 can block more than 90% of MMP13 activity when it reacts with active MMP13 directly. Since cartilage degeneration and articular chondrocyte dysfunction are hallmarks of OA, we isolated primary sternal chondrocytes from three-day-old WT pups to determine the effect of CL82198 on the activity of MMP13 secreted by chondrocytes undergoing hypertrophy. We treated sternal chondrocytes with BMP2 (100 ng/ml) for 60 hours to induce hypertrophy and MMP13 production/secretion. Meanwhile, the cells were also treated with CL82198 with BMP2 to inhibit BMP2-induced MMP13 activity. We found that CL82198 inhibited > 90% of MMP13 activity produced by BMP2-treated primary chondrocytes.
Next, we determined the efficacy of CL82198 in vivo. We performed MLI surgery on 10-week-old WT mice, followed by i.p. injection of saline or 1, 5, or 10 mg/kg of CL82198  every other day, beginning one day after surgery. Histological data revealed that OA progression was decelerated following CL82198 administration, with the most pronounced effect at 10 mg/kg. Results of cartilage grading and histomorphometric analysis were consistent with histological data and treatment with CL82198 at 5 and 10 mg/kg doses resulted in significantly lower scores than the saline control.
Consistent with histological and histomorphometric findings, results from SO/FG staining and Col2 and ColX IHC revealed less proteoglycan and Col2 loss and a decreased ColX level in the CL82198 treatment group. Results of TUNEL staining indicate that about 20% fewer chondrocytes underwent apoptosis with treatment of CL82198 (10 mg/kg) following MLI compared to saline control mice. Taken together, these results suggest that OA progression is decelerated by the application of a MMP13 inhibitor.
Genetic manipulation is a powerful method to study the genetic causes of diseases and chemical compound delivery in vivo is a practical approach to test the efficacy of drug treatment. Here, we tested the role of MMP13 using Mmp13 cKO mice and using a MMP13 inhibitor in a surgically induced OA mouse model. Our studies suggest that inhibition of MMP13 expression or activity could efficiently prevent and decelerate OA progression. Our study provides initial evidence showing that a MMP13 inhibitor may have potential for the treatment of OA progression although future investigation is still required.
Our study reveals that cartilage-specific deletion of the Mmp13 gene or inhibition of MMP13 activity decelerates the OA-like phenotype in a surgically induced OA model. Our studies also indicate that MMP13 inhibition could be used as a potential therapeutic strategy for prevention and treatment of OA.
This work was supported by grants R01 AR051189, R01 AR054465 and R01 AR055915 to D.C. from the National Institutes of Health (NIH). H.J. was partially supported by grant No. 81202710 from the National Natural Science Foundation of China and D.C. was partially supported by grant No. Z2111261 from Zhejiang Provincial Natural Science Foundation of China. We gratefully acknowledge the technical expertise of Ryan Tierney and Sarah Mack within the Center for Musculoskeletal Research Histology, IHC, and ISH Core for the processing of all tissue samples.
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