The peroxisome proliferator–activated receptor γ agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: In vivo protective effects mediated through the inhibition of key signaling and catabolic pathways

Authors


Abstract

Objective

Emerging evidence indicates that peroxisome proliferator–activated receptor γ (PPARγ) may have protective effects in osteoarthritis (OA). The aim of this study was to evaluate the in vivo effect of a PPARγ agonist, pioglitazone, on the development of lesions in a canine model of OA, and to explore the influence of pioglitazone on the major signaling and metabolic pathways involved in OA pathophysiologic changes.

Methods

OA was surgically induced in dogs by sectioning of the anterior cruciate ligament. The dogs were then randomly divided into 3 treatment groups in which they were administered either placebo, 15 mg/day pioglitazone, or 30 mg/day pioglitazone orally for 8 weeks. Following treatment, the severity of cartilage lesions was scored. Cartilage specimens were processed for histologic and immunohistochemical evaluations; specific antibodies were used to study the levels of matrix metalloproteinase 1 (MMP-1), ADAMTS-5, and inducible nitric oxide synthase (iNOS), as well as phosphorylated MAPKs ERK-1/2, p38, JNK, and NF-κB p65.

Results

Pioglitazone reduced the development of cartilage lesions in a dose-dependent manner, with the highest dosage producing a statistically significant change (P < 0.05). This decrease in lesions correlated with lower cartilage histologic scores. In addition, pioglitazone significantly reduced the synthesis of the key OA mediators MMP-1, ADAMTS-5, and iNOS and, at the same time, inhibited the activation of the signaling pathways for MAPKs ERK-1/2, p38, and NF-κB.

Conclusion

These results indicate the efficacy of pioglitazone in reducing cartilage lesions in vivo. The results also provide new and interesting insights into a therapeutic intervention for OA in which PPARγ activation can inhibit major signaling pathways of inflammation and reduce the synthesis of cartilage catabolic factors responsible for articular cartilage degradation.

Osteoarthritis (OA), one of the most common arthritic conditions, is primarily related to the progressive erosion of articular cartilage associated with synovial inflammation. The changes in cartilage are linked to a combination of mechanical and biochemical factors (1). The subchondral bone remodeling that takes place during the evolution of OA is believed to be an important factor in cartilage degradation (2–4). Its contribution seems to be associated with both the abnormal biophysical properties of the tissue and the excess synthesis of many catabolic factors, including growth hormones and cytokines, that can modulate the metabolism of OA cartilage (5–8).

Synovial inflammation, a common finding in patients with symptomatic OA, has also been demonstrated to contribute to the evolution of structural changes in the disease (9, 10). This latter phenomenon is probably related to the synthesis and release, by the synovial membrane, of factors that are capable of inducing cartilage catabolism and other structural changes (1). Synovial inflammation would seem to play a determinant role in the progression of cartilage degradation, by stimulating the synthesis of proteases and catabolic factors that can degrade the extracellular matrix, for example, the matrix metalloproteinases (MMPs), proteases of the ADAMTS family, and other oxidative products such as nitric oxide (NO) (1, 11–13).

The synthesis of the catabolic factors involved in the pathophysiologic mechanisms of OA is controlled by numerous pathways that can regulate their expression or activity. For instance, the synthesis/action of proinflammatory cytokines can be down-regulated by either antiinflammatory cytokines, the release of soluble receptors, or natural antagonists (14). In recent years, the family of nuclear receptors, including peroxisome proliferator–activated receptor γ (PPARγ), has generated particular interest because of its potential role in regulating the synthesis of a large number of catabolic factors involved in arthritis. Several studies have demonstrated that the activation of PPARγ by natural ligands can reduce the expression and synthesis of those catabolic and inflammatory factors that have the most relevant role in diseases such as OA (15–17). These factors include inflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and IL-6 (18), MMPs such as MMP-1 and MMP-13 (15, 16), and NO (17). This action of the PPARγ agonists is mediated through the inhibition of a number of signaling pathways, including the NF-κB and MAPK pathways (17, 18). A recent study has provided preliminary evidence that pioglitazone, a PPARγ agonist, can reduce the progression of experimental OA in a guinea pig model of meniscectomy-induced arthritis (19).

The aim of the present study was to explore the effects of pioglitazone in a dog model of anterior cruciate ligament transection (ACLT)–induced OA, to gain more insight into how this receptor agonist may reduce the progression of the structural changes that occur in OA. We explored, in depth, several major mechanisms through which in situ activation of the PPARγ system could modulate the progression of the disease.

MATERIALS AND METHODS

Experimental groups.

Twenty-three adult crossbred dogs (2–3 years old, each weighing 20–25 kg) were used in this study. Surgical sectioning of the ACL of the right knee, through a stab wound, was performed on all dogs as previously described (20). Following surgery, the dogs were housed on a farm, where they had free access to exercise in a large enclosure.

The dogs were randomly distributed into 3 treatment groups. Group 1 (n = 8) consisted of dogs that were given placebo treatment (encapsulated methylcellulose), while groups 2 and 3 (n = 8 and n = 7 dogs per group, respectively) were given pioglitazone orally once per day, every day including weekends, at a dosage of 15 mg/day or 30 mg/day, respectively. Drug treatment was initiated immediately after surgery, and the dogs were killed 8 weeks later. All animal care personnel were blinded to the treatment groups. The study protocol was approved by the institutional ethics committee.

Macroscopic grading.

Immediately after the dogs were killed, the right knee of each dog was placed on ice and dissected. Each knee was examined for gross morphologic changes, as previously described, by 2 independent observers who were blinded to the treatment groups (21).

The degree of osteophyte formation was graded by measuring the maximal width (in mm) of the spur on both the medial and the lateral femoral condyles, using a digital caliper (Digimatic Caliper; Mitutoyo, Kawasaki, Japan). These 2 values, recorded for each dog, were considered separately for the purposes of statistical analysis.

Cartilage changes on the medial and the lateral femoral condyles and the tibial plateaus were graded separately. Macroscopic changes to the cartilage were graded as follows. Changes to the articular surface area were measured, with results expressed in mm2. In addition, the depth of erosions was graded on a scale of 0–4, where 0 = normal appearance of the surface, 1 = minimal fibrillation or a slight yellowish discoloration of the surface, 2 = erosion extending into the superficial or middle layers, 3 = erosion extending into the deep layers, and 4 = erosion extending to the subchondral bone. The cartilage macroscopic score was then calculated using the formula, surface × depth, as previously described (21). This method allowed evaluation of the OA lesions on a continuous scale, where a grade of 0 indicates the absence of lesions, and the higher the grade, the more severe the lesion. The final macroscopic score consisted of the sum of the scores (surface × depth) of each of the lesions present on the medial and the lateral femoral condyles or the tibial plateaus. The total joint score consisted of the sum of the final scores obtained for both condyles and the tibial plateaus (21).

Histologic grading.

Cartilage was removed from the areas of the lesions identified on macroscopic evaluation. Histologic evaluation was then performed on sagittal sections of cartilage from the lesional areas of each femoral condyle and each tibial plateau, as previously described (20, 21). Specimens were dissected, fixed in TissuFix #2 (Laboratoires Gilles Chaput, Montreal, Quebec, Canada), and embedded in paraffin for histologic evaluation. Serial sections (5 μm) were stained with Safranin O. The severity of the OA lesions was graded on a scale of 0–13, by 2 independent observers who were blinded to the treatment, using the histologic scale modified from the method of Mankin et al (22). This scale was used to evaluate the severity of OA lesions based on the loss of Safranin O staining (scale 0–4), cellular changes (scale 0–3), and structural changes (scale 0–6, in which a grade of 0 indicates normal cartilage structure while a grade of 6 indicates erosion of the cartilage down to the subchondral bone). The final histologic score, expressed as the mean, represented the most severe histologic changes within the cartilage lesions on the femoral condyles or the tibial plateaus.

Immunohistochemistry.

Cartilage specimens from the femoral condyles and the tibial plateaus were processed for immunohistochemical analysis, as previously described (21, 23). Cartilage was fixed in TissuFix #2 for 24 hours, and then embedded in paraffin. Serial sections (5 μm) of paraffin-embedded specimens were placed on Superfrost Plus slides (Fisher Scientific, Nepean, Ontario, Canada), deparaffinized in toluene, rehydrated in a reverse-graded series of ethanol, and either preincubated with 0.25 units/ml chondroitinase ABC (Sigma-Aldrich Canada, Oakville, Ontario, Canada) in phosphate buffered saline (PBS), pH 8.0, for 60 minutes at 37°C or heated in 10 mM citrate buffer, pH 6.0, at 68°C for 20 minutes (for ERK-1/2 and MMP-1).

The specimens were subsequently washed in PBS, incubated in 0.3% Triton X-100 for 20 minutes, and then placed in 3% hydrogen peroxide/PBS for 15 minutes. Slides were further incubated with a blocking serum (Vectastain ABC kit; Vector, Burlingame, CA) for 60 minutes, after which they were blotted and then overlaid with a primary mouse monoclonal antibody against MMP-1 (dilution 1/40, Calbiochem ref. no. 444209; EMD Biosciences, Darmstadt, Germany), rabbit polyclonal antibody against ADAMTS-5 (dilution 1/50, Cedarlane ref. no. CL1-ADAMTS5; Cedarlane, Hornby, Ontario, Canada), rabbit polyclonal antibody against inducible NO synthase (iNOS) (dilution 1/200, Santa Cruz ref. no. SC-650; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal antibody against phospho–ERK-1/2 (dilution 1/25, Cell Signaling ref. no. 9106; Cell Signaling Technology, Beverly, MA), rabbit polyclonal antibody against phospho-p38 (dilution 1/25, Biosource ref. no. 44-684G; Biosource, Camarillo, CA), rabbit polyclonal antibody against phospho–NF-κB p65 (dilution 1/15, Santa Cruz ref. no. SC-372), and rabbit polyclonal antibody against phospho-JNK (dilution 1/15, Santa Cruz ref. no. SC-12882), for 18 hours at 4°C in a humidified chamber.

Each slide was washed 3 times in PBS (pH 7.4) and stained using the avidin–biotin–peroxidase complex method (Vectastain ABC kit), which entails incubation in the presence of a biotin-conjugated secondary antibody for 45 minutes at room temperature, followed by the addition of the avidin–biotin–peroxidase complex for 45 minutes. All incubations were carried out in a humidified chamber at room temperature, and the color was developed with 3,3′-diaminobenzidine (Dako Diagnostics Canada, Mississauga, Ontario, Canada) containing hydrogen peroxide. The slides were counterstained with eosin.

To determine the specificity of staining, 3 control procedures were carried out in accordance with the same experimental protocol as described above. The first procedure involved omission of the primary antibody, the second involved substitution of the primary antibody with an autologous preimmune serum, and the third involved immunoadsorption with the corresponding recombinant proteins (for 1 hour at 37°C) for MMP-1 (R&D Systems, Minneapolis, MN) or immunizing peptide for ADAMTS-5 (Cedarlane) and iNOS (Santa Cruz Biotechnology). All control experiments revealed background staining only, in the superficial or deep zones of cartilage (results not shown), as has been demonstrated previously (21, 24). For the experiments with phosphorylated antibodies, 2 control procedures were carried out, involving, first, omission of the primary antibody, and second, substitution of the primary antibody with the same concentration of autologous preimmune serum. These 2 control experiments also revealed background staining only, and the results have been published previously (25). Since there is, as yet, no recombinant protein or control peptide available for the phosphorylated antibodies, the third control experiment was not done.

Serial sections were prepared from each block of cartilage, and slides from each specimen were processed for immunohistochemical analysis. Each section was examined under a light microscope (Leitz Orthoplan; Leica, St. Laurent, Quebec, Canada) and photographed with a CoolSNAP cf Photometrics camera (Roper Scientific, Rochester, NY).

Morphometric analysis.

The presence of the different antigens in the cartilage was quantified using our previously reported method (20, 21, 23), with estimates obtained by determining the number of chondrocytes that stained positive in the entire thickness of cartilage. Three sections from each femoral condyle and each tibial plateau were examined. Each set of specimens was scored separately, with the results expressed as the mean for each specimen. In addition, the cartilage was divided into 6 microscopic fields (3 in the superficial zone and 3 in the deep zone) (40× magnification; Leica) to determine the mean results for each zone. Prior to evaluation, it was ensured that an intact cartilage surface for each OA specimen could be detected and used as a marker for validation of the morphometric analysis. The superficial zone of cartilage corresponds to the superficial and upper intermediate layer, and the deep zone corresponds to the lower intermediate and deep layer.

The total number of chondrocytes and the number of chondrocytes staining positive for the specific antigen were determined. The final results were expressed as the percentage of chondrocytes staining positive for the antigen, which yielded the cell score, with the maximum score being 100%. Each slide was read by 2 independent readers (CB and MB) who were blinded to the treatment groups. The final score was determined from consensus of the 2 readers. For the purposes of statistical analysis, the staining results obtained from the femoral condyles and the tibial plateaus were considered separately. Because the staining in the deep zone of cartilage was negligible for each of the studied antibodies, the data presented are from the superficial zone of cartilage only.

Statistical analysis.

Results for each group are primarily expressed as the median (range). Statistical analysis was performed using the Mann-Whitney U test. P values less than or equal to 0.05 were considered significant.

RESULTS

Characteristics of the experimental animals.

No clinical signs of drug toxicity were noted in any of the treatment groups. There was no significant change in the body weight of the dogs in any of the 3 treatment groups during the study period.

Macroscopic findings.

The degree of osteophyte formation was similar among the OA joints from all 3 experimental groups. The median size of the osteophytes was 9.3 mm (range 5.1–15.8 mm) in the placebo-treated group, 8.8 mm (range 6.1–12.7 mm) in the 15 mg/day pioglitazone group, and 9.1 mm (range 4.8–13.1 mm) in the 30 mg/day pioglitazone group.

In the placebo-treated group, the size of the lesions found on the cartilage surface was moderately severe and was of a similar grade between the femoral condyles and the tibial plateaus, with a trend toward lesions being more pronounced on the condyles and plateaus from the medial side (Figure 1). Lesions on the tibial plateaus and on the femoral condyles, although not always found at the same topographic locations, were preferentially localized in the weight-bearing zones of the joint. However, as seen in Figure 1, some of the lesions were localized in the joint periphery.

Figure 1.

Macroscopic appearance of osteoarthritic (OA) cartilage from the femoral condyles and tibial plateaus of placebo-treated and pioglitazone-treated dogs with OA at 8 weeks after surgery. Erosion and pitting (areas indicated by red circles) of the condyles and plateaus were evident in the placebo-treated dogs with OA. In the pioglitazone-treated dogs, a decrease in the severity of lesions on the condyles was seen, particularly after a dosage of 30 mg/day. A = anterior; P = posterior.

Assessment of the macroscopic score of joint lesions (Table 1) demonstrated that treatment with pioglitazone significantly reduced (P < 0.05) the total joint score of lesions, indicative of a decreased severity of the lesions both on the condyles and on the plateaus. The most pronounced effect was observed on the femoral condyles after treatment with 30 mg/day pioglitazone, the highest dosage of the drug tested, which resulted in a significant reduction in the macroscopic score (P < 0.05). This decrease in the severity of the lesions was mainly related to a reduction in the size of the lesions on the femoral condyles. On the medial femoral condyles, the median surface size of the lesions was 88.2 mm2 (range 30.2–137.5 mm2) in the placebo group, compared with 57.0 mm2 (range 9.3–108.4 mm2) in the dogs treated with pioglitazone at 15 mg/day (P < 0.02 versus placebo) and 23.7 mm2 (range 3.8–92.3 mm2) in the dogs treated with pioglitazone at 30 mg/day (P < 0.02 versus placebo); on the lateral femoral condyles, these values were 56.4 mm2 (range 14.9–96.7 mm2), 56.2 mm2 (range 32.0–112.4 mm2), and 28.1 mm2 (range 2.9–106.2 mm2), respectively. In contrast, on the tibial plateaus, the size of the lesions remained similar among all 3 treatment groups (results not shown).

Table 1. Cartilage macroscopic score of lesions on the femoral condyles and tibial plateaus of osteoarthritic (OA) canine joints*
GroupNo. of dogsMacroscopic score
Femoral condylesTibial plateausTotal joint
  • *

    Values are the median (range) macroscopic score of joint lesions per group. Scores were calculated as the sum of the surface × depth of cartilage lesions, and the total joint score is the sum of the final scores obtained for the condyles and the plateaus.

  • P < 0.05 versus the placebo group, by Mann-Whitney U test.

OA placebo control8200.3 (69.9–273.2)186.6 (85.1–277.0)399.4 (155.0–502.3)
Pioglitazone-treated OA, 15 mg/day8167.2 (74.1–255.5)206.5 (161.6–343.9)369.9 (248.2–519.9)
Pioglitazone-treated OA, 30 mg/day782.6 (34.4–242.2)156.0 (63.0–236.4)270.8 (145.6–416.5)

Histologic findings.

Lesional joint cartilage in the placebo-treated group exhibited morphologic changes, including cartilage fibrillation and fissures, changes in cellularity, such as cloning and hypocellularity, and loss of Safranin O staining, as previously described (11, 20, 21) (Figure 2). A decrease in the histologic score of the medial femoral condyles, indicative of a reduced severity of the lesions, was observed in dogs with OA treated with pioglitazone (median histologic score 6.25 [range 3–12] in the placebo group versus 4.25 [range 3–7] in the 15 mg/day pioglitazone group and 5.5 [range 5–6] in the 30 mg/day pioglitazone group); this difference was significant following a dosage of 15 mg/day pioglitazone as compared with the placebo group (P < 0.05). This reduction in histologic scores was mainly related to a protective effect of pioglitazone treatment on the severity of structural changes and the loss of Safranin O staining.

Figure 2.

Histologic analysis of representative sections of osteoarthritic (OA) cartilage from the medial femoral condyles of placebo-treated and pioglitazone-treated dogs with OA (Safranin O stained; original magnification × 100).

Immunohistochemical and morphometric findings.

The immunohistochemical study was carried out on serial sections of cartilage. Analysis of cartilage catabolic factors showed that the specimens from the placebo-treated dogs contained a large number of chondrocytes, particularly in the superficial layers of cartilage, that stained positive for MMP-1, ADAMTS-5, and iNOS (Figure 3A). There was a clear and significant decrease in the number of chondrocytes staining positive for the 3 catabolic factors in the 2 groups of dogs that were treated with either dosage (15 or 30 mg/day) of pioglitazone (Figure 3B).

Figure 3.

A, Expression of matrix metalloproteinase 1 (MMP-1), ADAMTS-5, and inducible nitric oxide synthase (iNOS) in representative sections of cartilage from placebo-treated dogs with osteoarthritis (OA) and pioglitazone (15 mg/day and 30 mg/day)–treated dogs with OA. Sections were immunostained with antibodies against MMP-1, ADAMTS-5, or iNOS. Positive cells are indicated by dark brown staining (original magnification × 100). B, Levels of MMP-1, ADAMTS-5, and iNOS in OA cartilage, as determined by morphometric analysis of immunostaining. Box plots represent the first and third quartiles, the line within each box represents the median, and the bars outside the box represent the spread, or range, of values. P values were calculated by Mann-Whitney U test.

The exploration of the different cell signaling pathways revealed that the specimens from the placebo-treated dogs contained a large number of chondrocytes, localized in the superficial layers of cartilage, that stained positive for the phosphorylated forms of ERK-1/2, p38, and NF-κB (Figure 4A). Pioglitazone inhibited, in a dose-dependent manner, activation (phosphorylation) of ERK-1/2 as well as p38 and NF-κB, with significant inhibition at the highest dosage tested (30 mg/day) (Figure 4B). Several chondrocytes in the superficial zone of cartilage stained positive for phospho-JNK in the placebo-treated group (median phospho-JNK–positive cells 19.44%, range 14.80–24.34%). Treatment with pioglitazone, at either dosage tested, did not affect activation of JNK (median phospho-JNK–positive cells 17.88%, range 12.50–27.27% in the 15 mg/day–treated group and 18.07%, range 14.13–26.26% in the 30 mg/day–treated group).

Figure 4.

A, Expression of phospho–ERK-1/2, p38, and NF-κB p65 in representative sections of cartilage from placebo-treated dogs with osteoarthritis (OA) and pioglitazone (15 mg/day and 30 mg/day)–treated dogs with OA. Sections were immunostained with antibodies against phospho–ERK-1/2, p38, and NF-κB p65. Positive cells are indicated by dark brown staining (original magnification × 100). B, Levels of phospho–ERK-1/2, p38, and NF-κB p65 in OA cartilage, as determined by morphometric analysis of immunostaining. Box plots represent the first and third quartiles, the line within each box represents the median, and the bars outside the box represent the spread, or range, of values. P values were calculated by Mann-Whitney U test.

DISCUSSION

This study provides new and interesting findings on the potential for PPARγ agonists to have chondroprotective effects on OA structural changes. Our results offer new insights into the innate mode of action of this therapeutic intervention on the disease process. We demonstrated that treatment with pioglitazone, a PPARγ agonist, was able to reduce the development of cartilage erosions. This effect was correlated with the action of the drug in reducing the synthesis of major catabolic factors that are capable of degrading cartilage macromolecules and that have a negative impact on the survival of chondrocytes. The effects of pioglitazone were found to be clearly related to its marked inhibitory activity with regard to several major cell signaling pathways, in particular, the MAPKs ERK-1/2, p38, and NF-κB.

Experimental models of OA are often used to test therapeutic efficacy in the context of developing new disease-modifying OA drugs (DMOADs). These models allow for the evaluation of the effects of drug treatment on the progression of structural changes and also on the innate mechanisms through which these drugs can exert their mode of action. At this time, it is not known whether the data obtained from animal model experimentation can be extrapolated to clinical observations. The experimental dog model of ACLT-induced OA has been used extensively for such purposes (20, 21, 23, 25–27) and could thus be an exception and serve as a useful tool in the study of human OA. Studies have recently shown that the positive effects of doxycycline on OA structural changes in an experimental dog model of OA (28) could also be seen in patients with knee OA treated with this drug (29). These findings are interesting, but before any final judgment can be made, the results need to be further validated by confirmatory studies.

Pioglitazone was more effective at reducing the severity of the lesions on the femoral condyles than on the tibial plateaus. It is important to note that we have already observed similar differences when testing the effects of other drugs in this model (20, 21, 26, 27). Although the exact reason for these differences remains unknown, it is likely that the effectiveness of the drugs on the different pathophysiologic mechanisms of OA may vary between the condyles and the plateaus. In the present study, pioglitazone was found to preferentially reduce the development of cartilage lesions on the femoral condyles. These findings are very much in accordance with those previously reported in a study in which pioglitazone was used as treatment in a guinea pig model of medial meniscectomy–induced OA (19). In the latter model, the protective effects of the drug were noted on the medial tibial plateaus, which is to be expected, since the lesions are preferentially distributed at that anatomic site in the guinea pig model. However, and as previously reported in the study using the guinea pig model, pioglitazone treatment had no effect on osteophyte formation. This drug seems to have very little, if any, effect on bone metabolism, since pioglitazone treatment had no effect on subchondral bone remodeling in an experimental dog model of OA (Pelletier JP, et al: personal communication).

Pioglitazone also induced a reduction in the severity of lesions according to the cartilage histologic score. This effect was mainly related to a reduction in the severity of structural changes and loss of matrix aggrecans, as shown by the protective effect of the drug on the loss of Safranin O staining. These findings are again consistent with those by Kobayashi et al (19) in the guinea pig model, and suggest that pioglitazone treatment has significant positive effects on major OA cartilage catabolic processes.

The PPARγ agonists have been shown to exert pleiotropic effects on mechanisms of inflammation and cell signaling (18). More specifically, with regard to the pathophysiologic mechanisms of arthritis, studies have demonstrated that the activation of PPARγ could inhibit a number of important pathways responsible for the structural changes that occur during OA. For instance, PPARγ agonists were found to inhibit production of IL-1β, NO, and MMP-13 in vitro in human OA chondrocytes (15, 17), and to inhibit MMP-1 and IL-1β in human arthritis synovial fibroblasts (16, 30). The inhibition of MMP-1 production was found to be associated with a reduction in activator protein 1 (AP-1) binding activity (16). In vivo, in the guinea pig OA model, treatment with pioglitazone was found to reduce the levels of IL-1β and MMP-13 in cartilage chondrocytes (19).

The erosion of OA cartilage is a complex process associated with a combination of metabolic and mechanical factors. The excess production of proteases, mainly by OA chondrocytes, is believed to be among the most important contributing metabolic factors involved in the degradation of the major cartilage matrix macromolecules, such as type II collagen and the aggrecans (1). Major collagenolytic enzymes found in OA are mostly members of the MMP family and include MMP-1 and MMP-13. The present study results show that pioglitazone was able to reduce MMP-1 levels in OA cartilage. These findings, as well as those by Kobayashi et al (19) with regard to the effects of pioglitazone on MMP-13 in the guinea pig model, can provide an explanation for the protective effects of pioglitazone on cartilage structure.

The present study findings also show that pioglitazone treatment was able to reduce the levels of ADAMTS-5, an enzyme involved in the degradation of aggrecan, a major cartilage matrix component responsible for the ability of cartilage to resist compressive loads. Many enzymes, including MMPs, have been linked to the degradation of aggrecan and proteoglycan monomers in OA cartilage (31–35). ADAMTS-5 is 1 of 2 aggrecanase proteases that are members of the ADAMTS family (31). ADAMTS-5 has been found at increased levels in OA cartilage (36). Moreover, a recent study of genetically modified mice in which the catalytic domain of ADAMTS-5 was deleted showed that after surgically induced joint instability, the course of cartilage destruction was abrogated (37). These results clearly demonstrate that ADAMTS-5 is the primary aggrecanase responsible for aggrecan degradation in this OA model. Accordingly, the findings of the present study show that pioglitazone could reduce the levels of ADAMTS-5 in OA cartilage, which provides another possible explanation as to how pioglitazone may exert its chondroprotective effects. Moreover, these findings complement the previous findings regarding the inhibition of MMP-1 and MMP-13.

The excess production of NO in OA tissue is believed to be an important contributing factor to cartilage catabolism (1). Moreover, the increased level of NO has been linked to chondrocyte apoptosis (38, 39). The role of NO in the pathophysiologic mechanisms of OA is also supported by a recent study showing that selective inhibition of iNOS could reduce the progression of experimental OA in dogs (11). This reduction of iNOS levels in OA chondrocytes by pioglitazone should also lead to reduced production of NO, and therefore this could be another factor that contributes to the positive effect of pioglitazone on the OA process.

The effects of pioglitazone on the synthesis of OA catabolic factors were found to be likely related to the inhibition of major intracellular signaling pathways. Noticeable effects on 2 MAPKs (ERK-1/2 and p38) and on NF-κB were observed. Our findings are consistent with those of a number of studies exploring the function of the PPARγ system. Although, so far, very little is known about the molecular mechanisms by which PPARγ modulates kinase activities, observations from several previous studies seem most relevant to the present results. One recent study demonstrated that another PPARγ agonist, resiglitazone, was capable of inhibiting the in vitro activity of p38 and NF-κB, with a subsequent reduction in proinflammatory gene expression (40). Similar findings with regard to the effects of troglitazone on ERK-1/2 were also reported (41). The level of phosphorylation of JNK and p38 have also been shown to be diminished in response to specific stimuli in PPARγ-deficient mice (40).

The effects of PPARγ on the synthesis of inflammatory mediators and catabolic factors have been studied extensively. PPARγ agonists have been shown to be potent inhibitors of the synthesis of inflammatory cytokines, such as IL-1β, TNFα, and IL-6, by monocyte/macrophage cells and other cell types (18, 40). The effects of pioglitazone in terms of reducing the synthesis of the MMPs, including MMP-1, could be attributed to multiple mechanisms (15, 16). The reduction in the activity of both the p38 and the ERK-1/2 pathways is likely to be a very important mechanism, since these pathways are predominant in the regulation of expression of MMP-1 in chondrocytes (17, 21, 42). Moreover, and as previously reported, the inhibition of the AP-1 DNA binding activity (16), as well as the inhibition of the expression of the c-Fos subunit (43), could also contribute to the inhibitory effect of pioglitazone. In the context of the results of the present study, it is not surprising that previous studies have demonstrated inhibition of MMP-13 synthesis by pioglitazone (19), since the expression of MMP-13 seems to be preferentially mediated through the p38 pathway (44, 45), which was herein shown to be inhibited by treatment with pioglitazone.

The inhibition of NF-κB activation is an interesting finding and could explain, at least in part, the reduction in the level of iNOS in OA chondrocytes (15). However, again, and as was previously seen with MMP-1, other pathways, such as ERK-1/2, have also been shown to be involved in the regulation of iNOS gene expression in chondrocytes (21). Therefore, the effect of pioglitazone on iNOS synthesis could very well result from a combined inhibition of both pathways. This finding with regard to the inhibition of the activation of NF-κB by pioglitazone could also explain the action of the drug in inhibiting ADAMTS-5, since the synthesis of this enzyme was reported to be blocked by an IκB kinase inhibitor, BMS-345541 (46). The inhibition of the activation of NF-κB by pioglitazone could be one of several possible mechanisms of action, as previously reported (18). One attractive hypothesis lies in the possible blockage of IκB degradation or phosphorylation by pioglitazone (18). Finally, and as has been previously reported (47), the effect of pioglitazone may also be possibly explained by the inhibition of DNA binding of the p65 NF-κB subunits.

In summary, this study provides new information on the potential DMOAD effects of PPARγ agonists. It also presents interesting findings about the mechanisms through which treatment with pioglitazone could reduce the development of cartilage lesions. Insights into the in vivo inhibitory activity of the drug on major signaling pathways that regulate the synthesis of OA cartilage catabolic factors were also gained. These findings are particularly important in the context of establishing new targeted therapies for the treatment of patients with OA.

AUTHOR CONTRIBUTIONS

Dr. Pelletier had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Martel-Pelletier, Fahmi, Pelletier.

Acquisition of data. Boileau, Mineau, Boily,

Analysis and interpretation of data. Boileau, Martel-Pelletier, Fahmi, Pelletier.

Manuscript preparation. Boileau, Martel-Pelletier, Mineau, Boily.

Statistical analysis. Boileau, Boily, Pelletier.

ROLE OF THE STUDY SPONSOR

This study was supported by a grant provided by Takeda Pharmaceuticals. The study sponsor was not involved in the study set-up, data collection, or analysis and interpretation of the data, and had no influence on the publishing of the data.

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