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Pioglitazone a peroxisome proliferatoractivated receptor ╨Ю╤Ц agonist reduces the progression of experimental osteoarthritis in guinea pigs.

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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
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:
Submitted for publication May 16, 2004; accepted in revised
form October 21, 2004.
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
structural lesions and major pathophysiologic pathways.
In this study, we used guinea pigs with a partial medial
meniscectomy of the right knee joint.
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
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
(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␤
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.
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.
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*
Pioglitazone, 2 mg/kg/day
Pioglitazone, 20 mg/kg/day
Macroscopic grading
Osteophyte size,
Size, mm2
Depth, 0–4 scale
Histologic grading,
0–12 scale
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
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.
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.
Table 2. Cartilage cell scores for MMP-13 and IL-1␤ in normal,
placebo-treated (OA), and pioglitazone-treated OA guinea pigs*
Pioglitazone, 2 mg/kg/day
Pioglitazone, 20 mg/kg/day
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).
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
The molecular mechanisms underlying the inhibition of cytokine production by PPAR␥ agonist pioglitazone are still unclear. In addition to IL-1␤, other
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
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-
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.
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.
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
by human chondrocytes but not by synoviocytes. J Clin Invest
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
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
Clancy RM, Amin AR, Abramson SB. The role of nitric oxide in
inflammation and immunity [review]. Arthritis Rheum 1998;41:
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:
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
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 ␥
co-association with transcription factor NFAT. J Biol Chem
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
Williams DA. A note on Shirley’s nonparametric test for comparing several dose levels with a zero-dose control. Biometrics
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;
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
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)
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
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,
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:
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:
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