Pioglitazone a peroxisome proliferatoractivated receptor ╨Ю╤Ц agonist reduces the progression of experimental osteoarthritis in guinea pigs.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 2, February 2005, pp 479–487 DOI 10.1002/art.20792 © 2005, American College of Rheumatology Pioglitazone, a Peroxisome Proliferator–Activated Receptor ␥ Agonist, Reduces the Progression of Experimental Osteoarthritis in Guinea Pigs Tetsuya Kobayashi,1 Kohei Notoya,1 Takako Naito,1 Satoko Unno,1 Akihiro Nakamura,1 Johanne Martel-Pelletier,2 and Jean-Pierre Pelletier2 Objective. To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator– activated receptor ␥ (PPAR␥) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP-13) and interleukin-1␤ (IL-1␤) in articular cartilage. Methods. The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/ day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP-13 and IL-1␤ in OA cartilage chondrocytes were evaluated by immunohistochemistry. Results. OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP-13 and IL-1␤ in cartilage from placebo-treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP-13 and IL-1␤ in OA cartilage. Conclusion. This is the first in vivo study demonstrating that a PPAR␥ agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP-13 and IL-1␤, which are known to play an important role in the pathophysiology of OA lesions. Osteoarthritis (OA) is a degenerative disease and the major cause of disability in humans. Aging, mechanical stress and traumatic injury, genetic susceptibility, and metabolic predispositions are considered risk factors for this disease. An important feature of OA is the degradation of articular cartilage, composed of abundant extracellular matrix rich in sulfated proteoglycan and type II collagen. This process is likely related to the excess synthesis and release of several catabolic factors such as proinflammatory cytokines, matrix metalloproteinases (MMPs), and nitric oxide (NO) in the tissue (1). OA is characterized by a shift of the balance between production of cartilage matrix and proteolytic degradation toward increased proteolysis. The most influential causative proteases in OA are MMPs, a family of zinc-containing, calcium-dependent proteases. An MMP that is of particular interest in the degradation of cartilage in pathologic conditions is MMP-13 (collagenase 3). MMP-13 has been found to be elevated in both rheumatoid arthritis (RA) and OA joint tissues and, more particularly, in articular cartilage (2,3). This enzyme cleaves the native collagen and is 5–10-fold 1 Tetsuya Kobayashi, MS, Kohei Notoya, PhD, Takako Naito, BS, Satoko Unno, AS, Akihiro Nakamura, PhD: Takeda Pharmaceutical Co. Ltd., Osaka, Japan; 2Johanne Martel-Pelletier, PhD, JeanPierre Pelletier, MD: Hôpital Notre-Dame, Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada. Address correspondence and reprint requests to Kohei Notoya, PhD, Pharmacology Research Laboratories I, Pharmaceutical Research Division, Takeda Pharmaceutical Co. Ltd., 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan. E-mail: email@example.com. Submitted for publication May 16, 2004; accepted in revised form October 21, 2004. 479 480 more active on type II collagen than are MMP-1 and MMP-8, other collagenases also present in this tissue (3,4). Both chondrocytes and synoviocytes express these MMPs, and proinflammatory cytokines such as interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) induce or enhance their production (1). These cytokines are also potent inducers of NO in chondrocytes (5). NO contributes to cartilage degradation by 1) inhibiting the synthesis of cartilage matrix, 2) inhibiting the production of IL-1 receptor antagonist, and 3) enhancing MMP activity and inducing chondrocyte apoptosis (6–9). To date, the accumulated findings show that selective inhibition of IL-1, MMPs, or inducible NO synthase (iNOS) could reduce the progression of structural changes in experimental OA (10). Thus, the modulation of these catabolic factors may lead to the identification of new therapeutic targets for the treatment of OA in humans. A potential candidate for the down-regulation of gene expression of these catabolic factors is the peroxisome proliferator–activated receptor ␥ (PPAR␥), a member of the nuclear receptor family. It was originally characterized as a regulator of adipocyte differentiation and lipid metabolism (11,12). Recently, PPAR␥ was shown to be expressed in other cell types, including macrophages, T lymphocytes, endothelial cells, synoviocytes, and chondrocytes (13–17). Ligands for PPAR␥ include certain polyunsaturated fatty acids, the thiazolidinedione class of antidiabetic drugs, a variety of nonsteroidal antiinflammatory drugs, and the prostaglandin D2 metabolite 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ2) (18,19). Several lines of evidence have demonstrated that PPAR␥ agonists not only regulate lipid and glucose homeostasis, but also may diminish inflammatory processes. PPAR␥ activation results in the inhibition of various inflammatory events, such as the production of IL-1␤, TNF␣, and IL-6 in monocytes/macrophages as well as the proliferation and production of IL-2 by T lymphocytes (13,20,21). Furthermore, an antiinflammatory role for PPAR␥ agonists has been described in joint connective tissue cells. Fahmi et al recently reported that PPAR␥ agonists can suppress the expression of iNOS and MMP-13 in human chondrocytes, as well as the expression of MMP-1 in human synovial fibroblasts (22,23). These actions of PPAR␥ agonists were proven through repression of the activities of many transcription factors, including NF-B, activator protein 1 (AP-1), STATs, and nuclear factors of activated T cells (21–24). On the basis of these findings, we found it highly relevant to investigate the in vivo effect of pioglitazone, a synthetic agonist for PPAR␥, on the progression of OA KOBAYASHI ET AL structural lesions and major pathophysiologic pathways. In this study, we used guinea pigs with a partial medial meniscectomy of the right knee joint. MATERIALS AND METHODS Animal experiments. All animal experiments in this study were carried out in accordance with the ethics guidelines established by the Experimental Animal Care and Use Committee of Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). Male Hartley guinea pigs (330–360 gm; SLC, Shizuoka, Japan) were used. A partial medial meniscectomy was performed according to the method of Meacock et al (25). Prior to surgery, the animals were anesthetized with an intramuscular injection of a mixture of ketamine (50 mg/kg, 0.2 ml; Sankyo, Tokyo, Japan) and xylazine (20 mg/kg, 0.1 ml; Bayer, Leverkusen, Germany). After the skin overlying the right hindknee joint was shaved and sterilized, a small incision was made over the medial side of the joint to expose the medial collateral ligament. Using iris scissors, this ligament was transected, and the distal half of the medial meniscus was released and finally excised. Particular care was taken to avoid any damage to the cartilage surfaces. All animals were permitted to move freely in the cage after surgery. The guinea pigs were placed into 4 treatment groups: group 1 (n ⫽ 12) comprised unoperated guinea pigs that received no treatment (normal); group 2 (n ⫽ 12) consisted of operated OA guinea pigs that received placebo treatment (methylcellulose solution) (control); groups 3 and 4 (n ⫽ 12 per group) comprised OA guinea pigs that received pioglitazone (Takeda Pharmaceutical Co. Ltd.) suspended in 0.5% (weight/volume) methylcellulose at a dosage of 2 mg/kg/day (group 3) or 20 mg/kg/day (group 4). Pioglitazone was administered twice daily as a suspension in 0.5% methylcellulose, by oral gavage into the stomach. Treatment was initiated 1 day following surgery and continued for 4 weeks, including weekends, after which all animals were killed. Macroscopic grading. Immediately thereafter, the right knee was dissected and evaluated for morphologic change of the tibial plateaus, according to the criteria described by Pelletier et al (26). Briefly, the depth of erosion in articular cartilage was graded on a scale of 0–4, in which 0 ⫽ a normal-appearing surface, 1 ⫽ minimal fibrillation or a slight yellowish discoloration of the surface, 2 ⫽ erosion extending into the superficial or middle layers only, 3 ⫽ erosion extending into the deep layers, and 4 ⫽ erosion extending to the subchondral bone. The surface area (size) of lesions was measured using Adobe Photoshop image analysis software (version 6.0; Adobe Systems, San Jose, CA), with results expressed in mm2. In addition, the degree of osteophyte formation was evaluated by measuring the maximum width of the spur on each tibial plateau, with results expressed in mm. Histologic grading. Histologic grading was performed on sagittal sections of cartilage from the damaged area of each tibial plateau. Specimens were dissected, fixed in a 10% formalin neutral buffer solution (Wako, Osaka, Japan) for 2 days, subjected to decalcification in a 5% formic acid–formalin solution for 10 days, and embedded in paraffin for histologic evaluation. Serial sections (6 m) were stained with hematoxylin and eosin or Safranin O–fast green. The severity of the OA lesions was graded on a scale of 0–12 by 2 blinded observers PIOGLITAZONE IN EXPERIMENTAL OA 481 Figure 1. Macroscopic appearance of cartilage from the tibial plateaus of A, normal, B, placebo-treated osteoarthritic (OA), and C, pioglitazone (20 mg/kg/day)–treated OA guinea pigs. Erosion and pitting (areas indicated by circles in B and C) were evident in the placebo-treated OA animals. In the pioglitazone-treated animals, the lesions were less severe compared with those in the placebo-treated controls. (TK and KN), using histologic/histochemical criteria modified from the criteria by Mankin et al (27). This scale evaluates the severity of OA lesions based on the loss of Safranin O staining (score range 0–4), cellular changes (score range 0–3), and structural changes (score range 0–5, in which 0 ⫽ normal cartilage structure and 5 ⫽ erosion of the cartilage down to the subchondral bone). The scoring system was based on the most severe histologic changes within each cartilage section. Immunohistochemistry. Cartilage specimens were processed for immunohistochemical analysis in accordance with the method of Huebner et al (28) with some modifications. Paraffin sections (6 m) were deparaffinized and hydrated using xylene and a graded alcohol series. The sections were washed in distilled water and exposed to 3% H2O2 for 10 minutes at room temperature to quench any endogenous peroxidase activity. Slides were further incubated with a 1.5% normal goat serum solution (Vectastain Elite ABC kit; Vector, Burlingame, CA) for 30 minutes at room temperature to suppress nonspecific binding, and were blotted and then overlaid with a rabbit polyclonal antibody against MMP-13 (1.0 mg/ml, 1:5,000; Triple Point Biologics, Portland, OR) or a rabbit polyclonal antibody against IL-1␤ (85 mg/ml, 1:1,700; Rockland, Gilbertsville, PA) at 4°C for 18 hours in a humidified chamber. The primary polyclonal antibody against MMP-13 recognizes the latent proMMP-13 and the active form of the enzyme. The rabbit polyclonal antibody against IL-1␤ recognizes the mature form of the cytokine. This antiserum does not recognize human IL-1␣. Each slide was washed 3 times in phosphate buffered saline (pH 7.4; Takara Shuzo, Shiga, Japan), and biotinylated goat anti-rabbit IgG secondary antibody was applied to sections for 30 minutes, followed by Vectastain ABC Elite reagent, an avidin–biotin–peroxidase complex (Vectastain ABC Elite kit; Vector), for 30 minutes. The antibody was detected using 0.1% (1 mg/ml) diaminobenzidine tetrahydrochloride and 0.02% H2O2 in a 0.1 mole/liter Tris buffer (Vector). Slides were counterstained with hematoxylin. To determine the specificity of staining, 3 control procedures were carried out using the same experimental protocol: 1) use of adsorbed immune serum (1 hour at 37°C) with a 20-fold molar excess of recombinant MMP-13 or IL-1␤, 2) omission of the primary antibody, and 3) substitution of the primary antibody with an autologous preimmune serum. The purified antigens used were human recombinant MMP-13 (Genzyme, Cambridge, MA) or human recombinant IL-1␤ (Genzyme). Each section was examined with a light microscope (10⫻ magnification) and scored separately. The presence of the antigen was estimated by determining the number of chondrocytes staining positive in the cartilage. The total number of chondrocytes and the number of chondrocytes positive for MMP-13 or IL-1␤ were counted over a defined area using a microscope grid. The results were expressed as the percentage of positive chondrocytes (cell score), with the maximum possible score being 100%. Each slide was evaluated by 2 blinded observers (TK and KN). Statistical analysis. Results are expressed as the mean ⫾ SEM. Statistical significance was assessed with the Shirley-Williams test (29,30). P values of less than 0.025 were considered significant. RESULTS Drug administration and drug-circulating levels. Pioglitazone was administered twice daily in each group, at total daily dosages of 2 mg/kg and 20 mg/kg. The pharmacokinetic properties of pioglitazone were evaluated after a single oral administration to fed guinea pigs at a dose of 10 mg/kg; the maximum plasma concentration (Cmax) was 7.5 g/ml and the time to reach Cmax was 0.83 hours. The value for the area under the curve (0–24 hours) (AUC0–24 hours) was 26.4 g 䡠 hour/ml. Thus, for a dosage of 20 mg/kg/day, the likely total AUC0–24 hours would be 52.8 g 䡠 hour/ml. Of note, the therapeutic dose of pioglitazone in humans is 30 mg/day, with a total AUC0–24 hours of pioglitazone and its active metabolites of 41.6 g 䡠 hour/ml. Moreover, in repeated-dose toxicology studies (up to 13 weeks) in rats, no toxicity was found at AUCs as high as 64–85 g 䡠 hour/ml. 482 KOBAYASHI ET AL Table 1. Size of osteophytes and macroscopic and histologic grading of cartilage from the tibial plateaus of normal, placebo-treated (OA), and pioglitazone-treated OA guinea pigs* Group Normal OA Placebo Pioglitazone, 2 mg/kg/day Pioglitazone, 20 mg/kg/day Macroscopic grading Osteophyte size, mm Size, mm2 Depth, 0–4 scale Histologic grading, 0–12 scale 0 0 0 0.58 ⫾ 0.23 1.3 ⫾ 0.23 0.97 ⫾ 0.12 1.0 ⫾ 0.11 7.0 ⫾ 0.66 6.5 ⫾ 1.2 4.1 ⫾ 0.90† 2.4 ⫾ 0.15 1.7 ⫾ 0.33 1.1 ⫾ 0.23† 6.7 ⫾ 0.53 5.0 ⫾ 0.81 4.2 ⫾ 0.80† * Guinea pigs underwent a partial medial meniscectomy of the right knee joint. The osteoarthritic (OA) groups were treated with placebo or received pioglitazone at 2 mg/kg or 20 mg/kg orally for 4 weeks, beginning the day following surgery. Values are the mean ⫾ SEM (n ⫽ 12). † P ⱕ 0.025 versus OA placebo, by Shirley-Williams test. Characteristics of the experimental animals. No clinical signs of drug toxicity were noted in each treatment group. Treatment with pioglitazone did not alter the level of plasma glucose in the OA guinea pigs (mean ⫾ SEM 145 ⫾ 5.05 mg/dl in the placebo group versus 149 ⫾ 2.53 mg/dl in the 20 mg/kg/day pioglitazone group). There was no significant difference in bodyweight gain among the OA guinea pigs from the 3 treatment groups during the study period (results not shown). Macroscopic findings. Cartilage from unoperated guinea pigs had a normal appearance. In placebotreated OA guinea pigs, macroscopic damage was of a moderate degree on the tibial plateaus. OA guinea pigs treated with pioglitazone at 2 mg/kg/day and 20 mg/kg/ day showed dose-dependent decreases in the grade (depth) and surface area (size) of macroscopic lesions compared with the placebo-treated OA guinea pigs, and the differences were statistically significant for the group treated with the higher dosage of the drug (54% reduction in grade and 41% reduction in surface area among OA guinea pigs receiving 20 mg/kg/day) (Figure 1 and Table 1). The size of osteophytes was similar between the OA guinea pigs that received the pioglitazone treatment and those that received the placebo treatment, indicating that pioglitazone had no effect on osteophyte formation (Table 1). Histologic findings. Cartilage from placebotreated OA guinea pigs exhibited morphologic changes, including fibrillation, hypocellularity, and loss of Safra- Figure 2. Representative hematoxylin and eosin–stained sections of cartilage from the tibial plateaus of A and D, normal, B and E, placebo-treated osteoarthritic (OA), and C and F, pioglitazone (20 mg/kg/day)–treated OA guinea pigs. Original magnification ⫻ 40 in A–C, ⫻ 100 in D–F. PIOGLITAZONE IN EXPERIMENTAL OA Figure 3. Representative Safranin O– and fast green–stained sections of cartilage from the tibial plateaus of A, normal, B, placebo-treated osteoarthritic (OA), and C, pioglitazone (20 mg/kg/day)–treated OA guinea pigs. Original magnification ⫻ 100. Figure 4. Expression of matrix metalloproteinase 13 (MMP-13) (A–C) and interleukin-1␤ (IL-1␤) (D–F) in representative sections of cartilage from the tibial plateaus of normal (A and D), placebo-treated osteoarthritic (OA) (B and E), and pioglitazone (20 mg/kg/day)–treated OA (C and F) guinea pigs. These sections were immunostained with a polyclonal antibody against MMP-13 or IL-1␤. Positive cells showed dark brown staining. No specific staining was detected in cartilage treated with immunoadsorbed serum (G). Original magnification ⫻ 40. 483 484 KOBAYASHI ET AL Table 2. Cartilage cell scores for MMP-13 and IL-1␤ in normal, placebo-treated (OA), and pioglitazone-treated OA guinea pigs* Group Normal OA Placebo Pioglitazone, 2 mg/kg/day Pioglitazone, 20 mg/kg/day MMP-13–positive cells IL-1␤–positive cells 32 ⫾ 3.4 45 ⫾ 4.5 74 ⫾ 3.8 63 ⫾ 4.8 36 ⫾ 4.9† 84 ⫾ 3.6 76 ⫾ 4.9 57 ⫾ 4.5† * Values are the mean ⫾ SEM; percentage of positive chondrocytes (i.e., cell score) of 12 animals per group. MMP-13 ⫽ matrix metalloproteinase 13; IL-1␤ ⫽ interleukin-1␤; OA ⫽ osteoarthritis. † P ⱕ 0.025 versus OA placebo, by Shirley-Williams test. nin O staining. A significant decrease in the severity of lesions was obtained with the administration of pioglitazone at 20 mg/kg/day (37% reduction in histologic severity score compared with the placebo group). The reduction in the histologic score was largely due to a decrease in the severity of structural changes and to attenuation of the loss of Safranin O staining (Figures 2 and 3 and Table 1). Immunohistochemical findings. In specimens of cartilage from the tibial plateaus of normal guinea pigs, only chondrocytes within the superficial layers stained positive for MMP-13. In specimens of OA cartilage from placebo-treated guinea pigs, large numbers of positive chondrocytes were detected in the middle and deep cartilage layers, and the percentage of MMP-13–positive cells was increased compared with that in normal cartilage (Figure 4 and Table 2). Immunohistochemical findings from experiments using the antibody that recognized IL-1␤ were similar among all specimens of OA cartilage (Figure 4 and Table 2). Pioglitazone-treated OA guinea pigs demonstrated dose-dependent decreases in both MMP-13 and IL-1␤ cell scores, and these reached statistical significance in the higher dosage group (90% and 69% reductions, respectively, compared with the placebo-treated OA guinea pigs) (Figure 4 and Table 2). No background staining was observed in the negative control when each primary antibody was omitted (Figure 4G). DISCUSSION This study is the first to provide evidence that pioglitazone, a synthetic agonist for PPAR␥, can reduce the severity of experimental OA in vivo. The Hartley guinea pigs used in this study had normal plasma glucose concentrations and pioglitazone did not alter these levels, nor did it affect body-weight gain. These results are consistent with the findings of Ikeda et al (31). Recently, Dumond et al suggested a minor role for leptin, the product of the ob gene, as a key regulator of chondrocyte metabolism and indicated that leptin may contribute to the pathophysiology of OA (32). Pioglitazone, however, has no effect on the plasma level of leptin in animals and humans (33,34), although treatment with troglitazone was reported to decrease ob gene expression in animals (35). Therefore, the observed chondroprotection by pioglitazone is likely the result of local actions of the thiazolidinedione derivative in the joints, rather than due to systemic metabolic effects. Pioglitazone may act directly on articular chondrocytes and inhibit catabolic responses in experimental OA. Recent studies have shown that PPAR␥ is expressed in rat and human chondrocytes (17,22), and PPAR␥ ligands such as thiazolidinedione rosiglitazone and 15d-PGJ2 inhibit IL-1␤–induced production of NO and MMP-13, probably by repressing the activation of AP-1 and NF-B in human chondrocytes (22). Pioglitazone also inhibits IL-1␤–induced production of MMP-13 in human chondrocytes in vitro (Notoya K and Unno S: unpublished observations). Since MMP-13 is overexpressed by chondrocytes in OA cartilage (3,4), inhibiting this enzyme is of great importance because of its ability to degrade type II collagen (4). The protective effect of pioglitazone observed in this study seems to be due to the reduction of MMP-13 synthesis caused by interference with IL-1 signaling in articular chondrocytes. In fact, our immunohistochemical analysis revealed that treatment with pioglitazone reduced the percentage of chondrocytes in articular cartilage that stained positive for MMP-13, which had been increased in surgically induced OA. Another immunohistochemical experiment performed with the antibody that specifically recognized IL-1␤ suggested that pioglitazone was capable of inhibiting the production of this cytokine in OA chondrocytes. There is evidence that human chondrocytes synthesize IL-1 in response to the pathologic condition (36,37), and that local production of this cytokine represents an alternative or even prime source for MMP induction within OA cartilage (38). Therefore, the local autocrine function for IL-1 is also a possible target for pioglitazone to inhibit cartilage destruction. This is supported in the present study by the similar expression profiles of IL-1␤ and MMP-13 in cartilage specimens from placebo-treated or pioglitazone-treated OA guinea pigs. The molecular mechanisms underlying the inhibition of cytokine production by PPAR␥ agonist pioglitazone are still unclear. In addition to IL-1␤, other PIOGLITAZONE IN EXPERIMENTAL OA cytokines produced in arthritic joints, such as IL-6 and TNF␣, are inhibited by PPAR␥ agonists (20). Treatment with rosiglitazone was demonstrated to reduce plasma levels of IL-1␤, TNF␣, and IL-6 in a mouse model of collagen-induced arthritis (39), and pioglitazone was shown to decrease the TNF␣ level in skeletal muscle in Wister fatty rats (33). These common mechanisms may contribute to the PPAR␥ agonist–induced reduction in the synthesis of these proinflammatory cytokines, especially in muscle cells and chondrocytes, because both cell types are derived from mesenchymal stem cells and may share common cellular machinery to regulate inflammatory responses. Moreover, it has been reported in the literature that some of these ligands could act via both PPAR␥-dependent and -independent pathways. This issue was not examined herein and further studies are warranted. MMP-13– and IL-1␤–immunopositive chondrocytes were observed in cartilage specimens even from normal guinea pigs (10 weeks of age), and the percentages of positively stained chondrocytes for these antigens were relatively higher than those in non-OA cartilage specimens from other species, including humans (26,38). These findings are consistent with those of Huebner et al, who detected MMP-13 protein in cartilage chondrocytes both from 2-month-old guinea pigs without pathologic joint damage and from 12-month-old guinea pigs with loss of cartilage and advanced OA (28). Hartley guinea pigs, used in the present study, have been known to show a spontaneous degeneration of cartilage (40). Therefore, these immunohistochemical observations may represent an early molecular manifestation of the OA process, which would indicate the presence of OA-related changes in the articular cartilage of Hartley guinea pigs. Synovium is also a potential target for chondroprotection by pioglitazone via mechanisms of antiinflammatory activity. Synovial fibroblast cells from patients with RA and OA were shown to express PPAR␥ (16,23). In these cells, PPAR␥ agonists prevented IL-1–induced production of MMP-1, at least in part through a reduction in the binding of AP-1 to DNA (23). In mouse adjuvant arthritis models, administration of pioglitazone or rosiglitazone significantly inhibited the expression of iNOS in both the ankle and temporomandibular joints via inhibition of the NF-B pathway (41). Kawahito et al also reported that PPAR␥ ligands inhibited the growth of synoviocytes in vitro through apoptosis and were potent in suppressing chronic inflammation and pannus formation in adjuvant-induced arthritis in rats (16). Further work is needed to elucidate the effect of piogli- 485 tazone on synovial inflammation in this animal model of OA. In the present study, the doses of pioglitazone were high compared with those used in antidiabetic studies, to show the therapeutic effects on OA cartilage (31,42). The differences between the effective doses of pioglitazone used for OA and type 2 diabetes mellitus likely depend on the level of functional PPAR␥ in each target cell or target tissue. Reverse transcription– polymerase chain reaction (RT-PCR) and/or Western blot analysis showed that the expression of PPAR␥ was weak in cultured rat chondrocytes and human synoviocytes, compared with adipose tissue (16,17). Our preliminary results using quantitative RT-PCR also revealed that the level of PPAR␥ expression was much higher in adipose tissue than in the cartilage of guinea pigs (Naito T and Notoya K: unpublished observations). However, since diabetes mellitus is a systemic risk factor for the development of arthritis (43), the administration of pioglitazone to patients with diabetes in conjunction with OA may lead to the removal of the risk factors for OA via the improvement of insulin resistance and severe obesity, and so may be effective in reducing cartilage destruction through direct chondroprotection, even at the regular clinical doses used. Thus, the PPAR␥ ligand may have interesting potential for the treatment of type 2 diabetes mellitus with OA. In conclusion, the present study demonstrates that pioglitazone, a synthetic agonist for PPAR␥, slows the progression of experimental OA in vivo. This phenomenon appears to be associated with a reduction in the synthesis of MMP-13 and IL-1␤ within the cartilage. ACKNOWLEDGMENTS We are grateful for the expert technical assistance of Nobutaka Tsuruta (Takeda Pharmaceutical Co. Ltd.). We also thank Drs. Hiroyuki Odaka, Haruhiko Makino, Takayuki Doi, Yasuo Sugiyama, and Takashi Sohda (Takeda Pharmaceutical Co. Ltd.) for encouragement throughout this work. REFERENCES 1. Martel-Pelletier J, di Battista J, Lajeunesse D. Biochemical factors in joint articular degradation in osteoarthritis. In: Reginster JY, Pelletier JP, Martel-Pelletier J, Henrotin Y, editors. Osteoarthritis: clinical and experimental aspects. Berlin: Springer-Verlag; 1999. p. 156–87. 2. Stahle-Backdahl M, Sandstedt B, Bruce K, Lindahl A, Jimenez MG, Vega JA, et al. Collagenase-3 (MMP-13) is expressed during human fetal ossification and re-expressed in postnatal bone remodeling and in rheumatoid arthritis. Lab Invest 1997;76:717–28. 3. Reboul P, Pelletier JP, Tardif G, Cloutier JM, Martel-Pelletier J. The new collagenase, collagenase-3, is expressed and synthesized 486 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. KOBAYASHI ET AL by human chondrocytes but not by synoviocytes. J Clin Invest 1996;97:2011–9. Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest 1997;99:1534–45. Stadler J, Stefanovic-Racic M, Billiar TR, Curran RD, McIntyre LA, Georgescu HI, et al. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide. J Immunol 1991;147:3915–20. Clancy RM, Amin AR, Abramson SB. The role of nitric oxide in inflammation and immunity [review]. Arthritis Rheum 1998;41: 1141–51. Pelletier JP, Mineau F, Ranger R, Tardif G, Martel-Pelletier J. The increased synthesis of inducible nitric oxide inhibits IL-1ra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation. Osteoarthritis Cartilage 1996;4: 77–84. Murrell GA, Jang D, Williams RJ. Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun 1995;206:15–21. Notoya K, Jovanovic DV, Reboul P, Martel-Pelletier J, Mineau F, Pelletier JP. The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2. J Immunol 2000;165:3402–10. Pelletier JP, Haraoui B, Fernandes JC. New and future therapies for osteoarthritis. In: Reginster JY, Pelletier JP, Martel-Pelletier J, Henrotin Y, editors. Osteoarthritis: clinical and experimental aspects. Berlin: Springer-Verlag; 1999. p. 387–408. Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in fibroblasts by PPAR ␥2, a lipid-activated transcription factor. Cell 1994;79:1147–56. Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA. Peroxisome proliferator-activated receptor (PPAR) ␥: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology 1994;135:798–800. Ricote M, Li AC, Millson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-␥ is a negative regulator of macrophage activation. Nature 1998;391:79–82. Clark RB, Bishop-Bailey D, Estrada-Hernandez T, Hla T, Puddington L, Padula SJ. The nuclear receptor PPAR ␥ and immunoregulation: PPAR ␥ mediates inhibition of helper T cell responses. J Immunol 2000;164:1364–71. Xin X, Yang S, Kowalski J, Gerritsen ME. Peroxisome proliferator-activated receptor ␥ ligands are potent inhibitors of angiogenesis in vitro and in vivo. J Biol Chem 1999;274:9116–21. Kawahito Y, Kondo M, Tsubouchi Y, Hashiramoto A, BishopBailey D, Inoue K, et al. 15-deoxy-⌬12,14-PGJ2 induces synoviocytes apoptosis and suppresses adjuvant-induced arthritis in rats. J Clin Invest 2000;106:189–97. Bordji K, Grillasca JP, Gouze JN, Magdalou J, Schohn H, Keller JM, et al. Evidence for the presence of peroxisome proliferatoractivated receptor (PPAR) ␣ and ␥ and retinoid z receptor in cartilage. J Biol Chem 2000;275:12243–50. Willson TM, Wahli W. Peroxisome proliferator-activated receptor agonists. Curr Opin Chem Biol 1997;1:235–41. Lehmann JM, Lenhard JM, Oliver BB, Ringold GM, Kliewer SA. Peroxisome proliferator-activated receptors ␣ and ␥ are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J Biol Chem 1997;272:3406–10. Jiang C, Ting AT, Seed B. PPAR-␥ agonists inhibit production of monocyte inflammatory cytokines. Nature 1998;391:82–6. Yang XY, Wang LH, Chen T, Hodge DR, Resau JH, DaSilva L, et al. Activation of human T lymphocytes is inhibited by peroxisome proliferator-activated receptor ␥ (PPAR ␥) agonists PPAR ␥ 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. co-association with transcription factor NFAT. J Biol Chem 2000;275:4541–4. Fahmi H, di Battista JA, Pelletier JP, Mineau F, Ranger P, Martel-Pelletier J. Peroxisome proliferator–activated receptor ␥ activators inhibit interleukin-1␤–induced nitric oxide and matrix metalloproteinase 13 production in human chondrocytes. Arthritis Rheum 2001;44:595–607. Fahmi H, Pelletier JP, di Battista JA, Cheung HS, Fernandes JC, Martel-Pelletier J. Peroxisome proliferator-activated receptor ␥ activators inhibit MMP-1 production in human synovial fibroblasts likely by reducing the binding of the activator protein 1. Osteoarthritis Cartilage 2002;10:100–8. Fahmi H, Pelletier JP, Martel-Pelletier J. PPAR ␥ ligands as modulators of inflammatory and catabolic responses in arthritis: an overview. J Rheumatol 2002;29:3–14. Meacock SC, Bodmer JL, Billingham ME. Experimental osteoarthritis in guinea pigs. J Exp Pathol 1990;71:279–93. Pelletier JP, Fernandes JC, Brunet J, Moldovan F, Schrier D, Flory C, et al. In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes. Arthritis Rheum 2003;48:1582–93. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 1971;53:523–37. Huebner JL, Otterness IG, Freund EM, Caterson B, Kraus VB. Collagenase 1 and collagenase 3 expression in a guinea pig model of osteoarthritis. Arthritis Rheum 1998;41:877–90. Shirley E. A non-parametric equivalent of Williams’ test for contrasting increasing dose levels of a treatment. Biometrics 1977;33:386–9. Williams DA. A note on Shirley’s nonparametric test for comparing several dose levels with a zero-dose control. Biometrics 1986;42:183–6. Ikeda H, Taketomi S, Sugiyama Y, Shimura Y, Sohda T, Meguro K, et al. Effects of pioglitazone on glucose and lipid metabolism in normal and insulin resistant animals. Arzneimittelforschung 1990; 40:156–62. Dumond H, Presle N, Terlain B, Mainard D, Loeuille D, Netter P, et al. Evidence for a key role of leptin in osteoarthritis. Arthritis Rheum 2003;48:3118–29. Murase K, Odaka H, Suzuki N, Tayuki H, Ikeda H. Pioglitazone time-dependently reduces tumour necrosis factor-␣ level in muscle and improves metabolic abnormalities in Wister fatty rats. Diabetologia 1998;41:257–64. Satoh N, Ogawa Y, Usui T, Tagami T, Kono S, Uesugi H, et al. Antiatherogenic effect of pioglitazone in type 2 diabetic patients irrespective of the responsiveness to its antidiabetic effect. Diabetes Care 2003;26:2493–9. Zhang B, Graziano MP, Doebber TW, Leibowitz MD, WhiteCarrington S, Szalkowski DM, et al. Down-regulation of the expression of the obese gene by an antidiabetic thiazolidinedione in Zucker diabetic fatty rats and db/db mice. J Biol Chem 1996;271:9455–9. Fujisawa T, Hattori T, Takahashi K, Kuboki T, Yamashita A, Takigawa M. Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1. J Biochem (Tokyo) 1999;125:966–75. Gemba T, Valbracht J, Alsalameh S, Lotz M. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocytes activation by the 29-kDa amino-terminal fibronectin fragment. J Biol Chem 2002;277:907–11. Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and PIOGLITAZONE IN EXPERIMENTAL OA proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes. Arthritis Rheum 2001;44:585–94. 39. Cuzzocrea S, Mazzon E, Dugo L, Patel NS, Serraino I, di Paola R, et al. Reduction in the evolution of murine type II collagen–induced arthritis by treatment with rosiglitazone, a ligand of the peroxisome proliferator–activated receptor ␥. Arthritis Rheum 2003;48:3544–56. 40. Bendele AM, Hulman JF. Spontaneous cartilage degeneration in guinea pigs. Arthritis Rheum 1988;31:561–5. 41. Shiojiri T, Wada K, Nakajima A, Katayama K, Shibuya A, Kudo C, 487 et al. PPAR␥ ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice. Eur J Pharmacol 2002;448:231–8. 42. Maeshiba Y, Kiyota Y, Yamashita K, Yoshimura Y, Motohashi M, Tanayama S. Disposition of the new antidiabetic agent pioglitazone in rats, dogs, and monkeys. Arzneimittelforschung 1997;47: 29–35. 43. Strumer T, Brenner H, Brenner RE, Gunther KP. Non-insulin dependent diabetes mellitus (NIDDM) and patterns of osteoarthritis: the Ulm osteoarthritis study. Scand J Rheumatol 2001;30: 169–71.