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Effects of tenidap on the progression of osteoarthritic lesions in a canine experimental model. Suppression of metalloprotease and interleukin-1 activity

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ARTHRITIS & RHEUMATISM
Val. 40, No. 2, February 1997, pp 284-294
0 1997, American College of Rheumatology
284
EFFECTS OF TENIDAP ON THE PROGRESSION OF
OSTEOARTHRITIC LESIONS IN A CANINE EXPERIMENTAL MODEL
Suppression of Metalloprotease and Interleukin-1 Activity
JULIO C. FERNANDES, JOHN P. CARON, JOHANNE MARTEL-PELLETIER, DRAGAN JOVANOVIC,
FRANCOIS MINEAU, GINETTE TARDIF, IVAN G. OTTERNESS, and JEAN-PIERRE PELLETIER
Objective. To study, in vivo, the therapeutic effectiveness of tenidap, an antirheumatic drug, on the
progression of lesions in an experimental osteoarthritis
(OA) dog model. The action of tenidap on the activity
and expression of metalloproteases in cartilage, as well
as on the bioactivity of interleukin-1 (IL-1) in synovial
fluid, was determined.
Methods. The anterior cruciate ligament of the
right stifle joint of 20 mongrel dogs was sectioned
through a stab wound. Dogs were divided into 3 groups:
group I (n = 7) received no treatment, group I1 (n = 6)
was treated with oral omeprazole (20 mg/day), and
group I11 (n = 7) received oral omeprazole (20 mg/day)
and a therapeutic dosage of oral tenidap (3 mg/kg twice
daily). Four weeks following surgery, the untreated dogs
(group I) were killed, and drug treatments were begun
for the other dogs (groups I1 and 111). These dogs
received medication for 8 weeks (weeks 4-12) and then
were killed. Evaluations were made of the incidence and
size of osteophytes as well as of the size and grade of
cartilage erosions on both the condyles and plateaus.
____
Supported in part by grants from Pfizer International, New
York, and the Medical Research Council of Canada.
Julio C. Fernandes, MD, Dragan Jovanovic, MD, PhD,
Fraqois Mineau, BSc: Louis-Charles Simard Research Center, Osteoarthritis Research Unit, Notre-Dame Hospital, Montreal, Quebec,
Canada; John P. Caron, DVM, MVSc: Louis-Charles Simard Research
Center, Osteoarthritis Research Unit, Notre-Dame Hospital, Montreal, Quebec, Canada, and Michigan State University, East Lansing;
Johanne Martel-Pelletier, PhD, Ginette Tardif, PhD, Jean-Pierre
Pelletier, MD: University of Montreal, and Louis-Charles Simard
Research Center, Osteoarthritis Research Unit, Notre-Dame Hospital, Montreal, Quebec, Canada; Ivan G. Otterness, PhD: Pfizer Inc.,
Groton, Connecticut.
Address reprint requests to Jean-Pierre Pelletier, MD, Rheumatic Disease Unit, Notre-Dame Hospital, 1560 Sherbrooke Street
East, Montreal, Quebec H2L 4M1, Canada.
Submitted for publication March 13, 1996; accepted in revised
form August 21, 1996.
Histologic examination of the severity of the cartilage
lesions and synovial inflammation was also performed. Activity levels of collagenase, stromelysin,
and gelatinase as well as collagenase-1, collagenase-3,
and stromelysin-1 messenger RNA were determined in
the cartilage. The level of IL-1 activity in the synovial
fluid was also measured.
Results. Among the dogs with OA, lesions were
more severe a t 12 weeks than a t 4 weeks. Group 111
(tenidap-treated) dogs had a slightly reduced incidence
of osteophytes compared with the group I1 (12-week OA)
dogs (71% versus loo%), and the size of the osteophytes
was significantly diminished (mean f SEM 1.75 f 0.69
mm versus 4.38 f 0.64 mm). Macroscopically, tenidap
decreased the size (condyles 6.00 +- 2.18 mm’ versus
21.08 f 6.70 mm’, plateaus 15.50 f 4.77 mm’ versus
35.0 f 3.64 mm’) and the grade (condyles 0.57 & 0.20
versus 1.17 -C 0.21, plateaus 1.07 f 0.22 versus 2.00 2
0.25) of the cartilage lesions compared with the 12-week
OA dogs. At the histologic level, the severity of cartilage
lesions was also decreased in the tenidap-treated dogs
versus the 12-week OA dogs, both on the condyles
(3.43 +- 0.54 versus 5.55 f 0.38) and on the plateaus
(3.39 f 0.35 versus 5.54 f 0.60). All 3 OA groups
showed a significant and similar level of synovial inflammation. Tenidap markedly decreased collagenase,
stromelysin, and gelatinase activity, as well as the level
of expression of collagenase-3 in the cartilage. Interestingly, the activity level of IL-1 in synovial fluid was also
significantly reduced in the tenidap-treated dogs.
Conclusion. Tenidap markedly reduced the severity of OA lesions, indicating the effect of this drug in
decreasing the progression of disease. It appears that
the drug acts by reducing the activity and/or expression
of metalloproteases in cartilage, a process known to play
a major role in the pathophysiology of OA lesions. This
EFFECTS OF TENIDAP ON OA PROGRESSION
effect could be mediated by the suppressive effect of
tenidap on IL-1 activity.
Osteoarthritis (OA) is a degenerative disease of
the articular cartilage that is associated with a variable
degree of synovitis (1).Advances in the understanding of
chondrocyte and cartilage matrix biology, in conjunction
with refined methods of evaluating the progression of
OA, have substantially increased the means of therapeutic intervention in this disease. Although all the pathogenetic mechanisms of joint destruction in inflammatory
and articular degenerative diseases are not yet fully
understood, chondrocytes are thought to play a key role
in the alteration of cartilage matrix macromolecule
homeostasis (1). Therefore, there is growing interest in
OA therapies that target the altered chondrocytic metabolism (2). In addition, since synovial inflammation
plays a significant role in the clinical stage of this disease
(3,4), therapy might well be aimed at the proinflammatory cytokines (4,5).
In OA, the degradation of the cartilage extracellular matrix, with subsequent erosion of the tissue, is
mediated in part by proteolytic enzymes (1,4). Among
these enzymes, matrix metalloproteinases (MMP) such
as collagenase-1, collagenase-3, stromelysin, and gelatinase are likely to be among those playing a predominant
role (1,4,6).
MMP are secreted as proenzymes and activated
extracellularly through an enzymatic cascade (1,4,7).
The paracrine and autocrine secretions of proinflammatory cytokines in OA cartilage and synovium, such as
interleukin-1 (IL-1) and tumor necrosis factor a
(TNFa), are likely to be involved in up-regulating the
expression and synthesis of these proteolytic enzymes
(4,7). Moreover, enhancement of MMP biologic activity,
which is related to an imbalance in the level of their
physiologic activators and inhibitors (8-1 1), appears to
be a key factor in limiting OA cartilage degradation
(1,477).
Transection of the anterior cruciate ligament
(ACL) of dog knees has proven to be an interesting and
reliable experimental model of O A (12,13). In this
model, MMP similar to those found in human OA have
been demonstrated to contribute to the degradation of
OA cartilage (14-16). This model is a useful tool to test
a drug’s effects on the progression of the disease, as well
as its action on the main pathophysiologic mechanisms
involved in cartilage degradation. Using this model, it
has been shown that intraarticular corticosteroid injections can reduce the progression of OA, which is associated with a reduction in the synthesis of stromelysin by
285
chondrocytes (14,15). Doxycycline, administered orally,
has also been shown to reduce the severity of OA lesions
and MMP activity (16) in the dog ACL model. These
studies give strong evidence that control of MMP
synthesisiactivity should be a therapeutic target in OA
treatment.
Tenidap ([Z]-5-chloro-2,3 dihydro-3-[hydroxy-2thienyl methylene]-oxo-1H-indole-1-carboxamide,
sodium salt) is a novel antirheumatic drug of the oxindole
class (17). This drug has a dual action: inhibition of
cyclooxygenases (18) and production of proinflammatory cytokines such as IL-1, IL-6, and TNFa, in human
cell (19) and synovial membrane (20) explants from
patients with rheumatoid arthritis. In this experimental
OA dog model, in vivo administration of tenidap under
prophylactic conditions markedly reduced progression
of cartilage lesions and osteophyte formation as well as
intensity of synovial inflammation (21). At the same
time, tenidap was also found to decrease stromelysin and
collagenase activity in both cartilage and synovial
membrane, which, along with marked inhibition of
collagenase-1 expression, suggests that this drug acts by
modulating MMP synthesis. Whether this effect is direct
or indirect, i.e., through cytokine regulation (19,20,22),
remains to be determined. Therefore, the properties of
this drug and its clinical effectiveness in O A patients
(23) make it a potential and novel alternative to existing
treatments.
Drugs used in the treatment of human OA are
prescribed at a time when lesions already exist, often in
association with synovial inflammation. Exploration of a
drug’s beneficial effect on the OA process should be
carried out under conditions as near as possible to
clinical reality. Since tenidap may be able to intervene in
the disease’s adverse effects, we investigated the in vivo
results of orally administered tenidap on the progression
of lesions, using the experimental OA dog model. It
must, however, be noted that the treatment was begun at
a time when the O A lesions were only at a relatively
early stage of the disease. Macroscopic and histologic
evaluations of cartilage and synovial membranes were
carried out, as well as determination of the activity and
expression of 3 MMP in OA cartilage and of IL-1
bioactivity in the synovial fluid.
MATERIALS AND METHODS
Experimental groups. Twenty adult crossbred dogs
weighing 20-25 kg each were used in this study. Surgical
sectioning of the ACL of the right knee, through a stab wound,
was performed on all the dogs (14,15,21). The surgery was
286
carried out on animals that were anesthetized with intravenous
pentobarbital sodium (25 m a g ) and intubated. Following
surgery, the dogs were kept in animal care facilities within
our hospital for 1 week and then sent to a housing farm
where they were left free to exercise in a large field for 4-6
hours every day.
The animals were randomly divided into 3 treatment
groups: group I (4-week O A , n = 7) received no treatment,
group I1 (12-week O A , n = 6) received oral omeprazole (20
mdday; Astra Pharma, Mississauga, Ontario, Canada), and
group I11 (12-week tenidap-treated; n = 7) received oral
omeprazole (20 mgiday) and oral tenidap (3 mg/kg twice daily;
Pfizer Central Research, Groton, CT). Drug treatment was
initiated 4 weeks after surgery and lasted 8 weeks. The animals
were killed 4 weeks (group I) or 12 weeks (groups I1 and 111)
following surgery. Because of their gastrointestinal sensitivity
to antirheumatic drugs, the tenidap-treated dogs were given
omeprazole, which proved to fully protect them from drugrelated gastrointestinal side effects, but did not interfere with
the progression of experimental OA lesions (21).
Drug administration and circulating drug level. Omeprazole's effect on tenidap pharmacokinetics in dogs was
determined in a previous study (21), and those results indicated that it does not alter tenidap plasma concentrations.
Moreover, when tenidap was administered orally (3 mg/kg
twice daily) in combination with omeprazole (20 mg/day), the
therapeutic blood concentrations were similar to those observed in tenidap-treated human OA patients (24).
Macroscopic grading of lesions. Immediately after the
dogs were killed, their right knees were dissected, placed on
ice, and examined for gross morphologic changes, including
the presence of osteophyte formation and cartilage lesions, as
previously described (14,1521). Examinations were performed
by 2 independent, blinded observers. The degree of osteophyte
formation was graded by measuring the maximal width (mm)
of the spur on each femoral condyle. The cartilage changes on
the medial and lateral femoral condyles and tibial plateaus
were graded separately under a dissecting microscope (Stereozoom; Bausch & Lomb, Rochester, NY).
The depth of the erosion was graded on a scale of 0-4,
as follows: 0 = surface appears normal, 1 = minimal fibrillation or a slight yellowish discoloration of the surface, 2 =
erosion extending into superficial or middle layers only, 3 =
erosion extending into deep layers, and 4 = erosion extending
to subchondral bone. The surface area of the articular surface
changes was measured and expressed in mm2.
Histologic grading. Histologic evaluation was performed on sagittal sections of cartilage from the lesional areas
of each femoral condyle and tibial plateau, as previously
described (14,21). Specimens were dissected and fixed in 10%
buffered formalin and embedded in paraffin for histologic
study. Serial sections (5 pm) were prepared and stained with
Safranin 0. The severity of the OA lesions was graded on a
scale of 0-14 by 2 independent observers using the histologic1
histochemical scale of Mankin et a1 (25). This scale evaluates
the severity of OA lesions based on the loss of staining with
Safranin 0 (scale 0-4), cellular changes (scale 0-3), invasion
of the tidemark by blood vessels (scale 0-l), and structural
changes (scale 0-6). On the latter scale, 0 indicates normal
cartilage structure and 6 signifies erosion of the cartilage down
FERNANDES ET AL
to the subchondral bone. This scoring system was based on the
most severe histologic changes within each cartilage section.
Representative specimens of synovial membrane from
the medial and lateral knee compartments were dissected from
underlying tissues. The specimens were fixed in 10% buffered
formalin, embedded in paraffin, sectioned (5 pm), and stained
with hematoxylin and eosin. For each compartment, 2 synovial
membrane specimens were examined for scoring purposes and
the highest score from each compartment was retained. The
average was calculated and considered as a unit for the whole
knee. The severity of synovitis was graded on a scale of 0-10
(14) by 2 independent observers, by adding the scores for 3
histologic criteria: synovial lining cell hyperplasia (scale 0-2),
villous hyperplasia (scale 0-3), and degree of cellular infiltration by mononuclear and polymorphonuclear cells (scale 0-5).
Metalloprotease activity assay. After macroscopic examination was performed and sections were obtained for
histologic evaluation, the remaining cartilage from the femoral
condyles and tibial plateaus was carefully dissected from the
underlying bone. For each dog, the cartilage was pooled and
divided into 2 equal parts: one was used to measure MMP
activity and the other for RNA extraction (see below).
Extraction of MMP from the cartilage was carried out
as previously described (21). The tissue was sliced and homogenized in the extraction buffer (50 mM Tris HCI, 10 mMCaCI,,
2M guanidine HCl, 0.05% Brij-35, pH 7.5). The mixture was
stirred overnight at 4°C and then centrifuged (18,OOOg for 30
minutes at 4°C). The supernatant was extensively dialyzed (48
hours at 4°C) against an assay buffer (50 mMTris HCl, 10 mM
CaCI,, 0 . N NaC1, 0.05% Brij-35, pH 7.5) using Spectrapor 4
dialysis tubing with a 12,000-Da cutoff (Spectrum Medical
Industries, Los Angeles, CA).
The collagenase activity in tissue extract was measured
using telopeptide-free type I collagen from rat tail tendon
acetylated with I4C-acetic anhydride (26). One hundred
microliters of 14C-collagensuspension (12,000 disintegrations
per minute) was incubated under the following conditions: 1)
with a 100-pl aliquot of tissue extract in the presence of 1 mM
aminophenylmercuric acid (APMA), and 2) with a 100-pl
aliquot of tissue extract containing 1mM APMA and 25 mM
EDTA, to serve as a blank. Each solution was incubated for 48
hours at 30"C, after which each was centrifuged at 12,OOOg for
15 minutes at 4°C. The radioactivity contained in the supernatant was determined using a beta scintillation counter (Beta
Rack, Model 1218; LKB, Stockholm, Sweden). The total
enzymatic activity was expressed in units per gram of tissue wet
weight, in which 1 unit corresponded to the hydrolysis of 1 pg
of substrate/hour at 30°C. More than 90% of the collagenase
activity was inhibited by EDTA.
The stromelysin activity in the cartilage extract was
measured by the method of Chavira et a1 (27), using azocoll
(Calbiochem-Behring, San Diego, CA) as substrate. One hundred microliters of the azocoll suspension was incubated in a
manner similar to the collagenase assay, except that 1 mM
1,lO-phenanthroline served as the blank. After incubation (48
hours at 37"C), this solution was centrifuged (12,0008 for 15
minutes at 4°C) and the supernatant's optical density was
determined by spectrophotometric analysis. The stromelysin
activity was expressed in units per gram of tissue wet weight, in
which 1 unit corresponded to the hydrolysis of 1 pg of
EFFECTS OF TENIDAP ON OA PROGRESSION
substrateihour at 37°C. The azocoll-digesting activity in the
extracts was inhibited >90% by 1,lO-phenanthroline.
The gelatinase activity in the tissue extracts was measured as previously described (28), after the collagenase substrate (see above) was denatured by heating at 60°C for 30
minutes. Experimental conditions were the same as for the
collagenase activity assay, but incubation was done for 24
hours at 37°C. Enzymatic activity was expressed in units per
gram of tissue wet weight, in which 1 unit corresponded to the
hydrolysis of 1 pg of substrateihour at 37°C.
IL-1 assay. IL-1 activity was measured in the synovial
fluid. Samples were collected using a sterile syringe, transferred into sterile tubes, centrifuged, and stored at -80°C. The
IL-1 activity was assessed according to the ability of the
synovial fluid to promote incorporation of 3H-thymidine by
D10.G4.1 (D10) murine T cells in the presence of concanavalin
A (Con A, type IVS; Sigma, St. Louis, MO), as described
previously (29) but with slight modifications. The test fluid
(100 pl) was incubated in 96-well tissue culture plates (#3072
Falcon; Becton-Dickinson, Mississauga, Ontario, Canada) with
4 X 10' D10 cells and 2.5 pg/ml of Con A in Click's medium
(Sigma) containing 10% fetal calf serum, 2 mM L-glutamine,
50 p M 2-mercaptoethanol, 20 mgiml a-methyl-D manoside,
100 unitsiml penicillin, and 100 pg/ml streptomycin. Cultures
were made in triplicate and incubated for 72 hours at 37°C in
a 5% C0,/95% air incubator.
At 6 hours before the end of the incubation period, the
rate of DNA synthesis was determined by measuring the
incorporation of 1 pCi/well of 3H-thymidine (specific activity
11.7 Ciimmole; Amersham, Oakville, Ontario, Canada). Recombinant human IL-1/3 (rHuIL-lP; Genzyme, Cambridge,
MA) served as the control. Units were determined using a
standard curve calculated against the levels of rHuIL-lp.
Results were expressed in arbitrary unitsiml.
RNA extraction. Total RNA from cartilage was isolated as previously described (21). Each cartilage specimen was
homogenized in 6M guanidine hydrochloride containing 25
mM sodium citrate, pH 7, 25 mM EDTA, 0.5% sarkosyl, and
100 mM 2-mercaptoethanol, followed by the addition of 3M
sodium acetate buffer, pH 5, 0.25 volume of saturated phenol,
and isoamyl alcohol/chloroform (1:49). This solution was vigorously shaken, cooled at 4°C for 1 hour, and centrifuged
(12,000g for 30 minutes at 4°C). The aqueous phase was mixed
with isopropanol and allowed to stand for 18 hours at -20°C.
The solution was centrifuged at 12,OOOg (for 20 minutes at 4°C)
and the pellet was resuspended in a 4M guanidine isothiocyanate buffer containing cesium trifluoroacetate (2.01 gmiml;
Pharmacia Biotech, Baie d'Urf6, Quebec, Canada) and centrifuged again at 4°C for 24 hours at 100,OOOg in an SW 40 Ti
rotor (Beckman Instruments, Mississauga, Ontario, Canada).
The RNA pellet was dissolved in 20 mM sodium acetate buffer,
pH 5, containing 0.5% sodium dodecyl sulfate, 1 mM EDTA
and extracted once with preheated (60°C) saturated phenol,
followed by precipitation with absolute ethanol. After centrifugation (13,00Og, 20 minutes at 4"C), the RNA pellet was
solubilized in diethyl pyrocarbonate-treated sterile water, and
the RNA was quantitated spectrophotometrically.
Northern blotting. Total RNA was resolved on 1.2%
agarose-formaldehyde gels, as previously described (21).
Three micrograms of RNA was used for each cartilage specimen. Following transfer to nylon membranes (Hybond N;
287
Figure 1. Femoral condyles from A, the 4-week osteoarthritis (OA)
dogs, B, the 12-week OA dogs, and C, the tenidap-treated OA dogs.
Note the large osteophyte formation along the condylar ridge in the
12-week OA dogs. Only small osteophytes were present in the dogs
treated with tenidap and in the 4-week OA dogs. Dogs were killed
either at 4 weeks (A) or at 12 weeks (B and C) after surgery.
Amersham) overnight at 4°C in 10 mM sodium acetate buffer,
pH 7.8, containing 20 mM Tris and 0.5 mM EDTA, the RNA
was cross-linked to the membranes by exposure to ultraviolet
light. Specific probes for dog collagenase-1 and GAPDH,
described previously (21), were developed in our laboratory.
Primers for collagenase-3 and stromelysin-1 were synthesized
FERNANDES ET AL
288
Table 1. Femoral condyle and tibial plateau macroscopic grading*
Femoral condyles
Group
No. of
anima1s
OA, 4 weeks?
OA, 12 weeks$
Tenidap, 12 weeks (P)§
7
6
7
* Values are the mean
Tibia1 plateaus
Size, mmz
Grade,
0-4 scale
Size, mm2
Grade,
0-4 scale
7.68 t 2.50
21.08 F 6.70
6.00 2 2.18
(0.03)
0.64 i 0.17
1.17 t 0.21
0.57 Z 0.20
(0.06)
26.86 t 5.26
35.00 t 3.64
15.50 5 4.77
(0.01)
1.07 t 0.20
2.00 2 0.25
1.07 Z 0.22
(0.02)
5 SEM.
t Animals were killed and tissue was examined 4 weeks after surgery.
$Animals were killed and tissue was examined at 12 weeks. Omeprazole (20 mgiday) was given orally for
8 weeks, beginning 4 weeks after surgery.
5 Animals were killed and tissue was examined at 12 weeks. Tenidap (3 mgikg twice daily) and omeprazole
(20 mgiday) were given orally for 8 weeks, beginning 4 weeks after surgery. Statistical analysis was done
by Mann-Whitney U test. P values are in comparison with the omeprazole-treated 12-week osteoarthritis
(OA) group. Significance was also obtained when the grade of the tibial plateaus of the omeprazoletreated group was compared with the 4-week OA group ( P < 0.02).
and used to amplify fragments specific for those genes from
dog chondrocyte RNA. These fragments were ligated to the
vector polymerase chain reaction I1 (Invitrogen, San Diego,
CA) and subsequently sequenced in order to verify the genes'
identity. The sequences for collagenase-3 were 5 ' CITAGAGGTGACTGGCAAAC-3' (sense primer) and 5'CTGGTAATGGCATCAAGGGA-3' (antisense primer), corresponding to positions 232-251 basepairs and 869-888 bp,
respectively, from the published sequence (30). The amplified
fragment was 658 bp. The sequences for stromelysin-1 primers
were 5'-GAAAGTCTGGGAAGAGGTGACTCCAC3'
(sense
primer) and 5'-CAGTGTTGGCTGAGTGAAAGAGACCC-3'
(antisense primer), corresponding to positions 414-440 bp and
671-697 bp, respectively, from the published sequence (31). The
amplified fragment was 274 bp.
Detection was done by a luminescent method using
DIG-11-UTP (Boehringer Mannheim Biochemica, Mannheim, Germany) with Lumigen PPD (4-methoxy-4-[3phosphatephenyl]Spiro-[l,2-dioxetane-3,2'-adamantane]
disodium salt) as substrate for alkaline phosphatase conjugated to
anti-DIG antibody Fab fragments, as previously described
(21). The detection was carried out following the Boehringer
Mannheim User's Guide (1993).
The membranes were then subjected to autoradiography using Kodak XAR-5 film (Eastman Kodak, Rochester,
NY) at room temperature. All autoradiographies were subjected to laser scanning densitometry (GS-300; Hoefer Scientific Instruments, San Francisco, CA) to determine the levels
of messenger RNA (mRNA). Results were expressed as the
relative amount of mRNA normalized to the level of GAPDH
mRNA.
Statistical analysis. Mean values and standard errors
of the mean were calculated. Statistical analysis was done using
the Mann-Whitney U test. P values less than 0.05 were
considered significant.
RESULTS
Macroscopic findings. Osteophytes. Osteophytes
were present on 43% of the condyles from the 4-week
OA dogs (group I), whereas the 12-week OA dogs
(group 11) showed osteophytes on all condyles (100%).
The incidence of osteophytes in the tenidap-treated dogs
(71%) was slightly lower than that found in the 12-week
OA dogs. The mean f SEM width of the osteophytes in
the 4-week OA dogs (0.86 t 0.39 mm) was -5 times
lower than that in the 1Zweek OA dogs (4.38 t 0.64)
(Figures 1A and B). In the tenidap-treated dogs, the
osteophytes were much smaller (1.75 2 0.69 mm) (Figure 1C) compared with those in the 12-week OA dogs
(P < 0.005).
Cartilage lesions. In the 4-week OA dogs, small
fibrillated lesions of a mild grade were present on both
condyles. Larger lesions of a slightly more severe grade
were present on the tibial plateaus (Table 1 and Figures
2A and B). Lesions from both the medial and lateral
plateaus were of approximately similar size, with a
slightly more severe grade found in the lateral compartment. In the 12-week O A dogs, the lesions on both the
condyles and plateaus were larger and had a more severe
grade compared with those in the 4-week OA dogs
(Table 1 and Figures 2C and D).
The tenidap-treated dogs showed a marked and
significant reduction in severity of cartilage lesions on
both the condyles and plateaus (Figures 2E and F).
Compared with the 12-week OA group, this reduction
was significant in both the size and the grade of the
lesions on the tibial plateaus and in the size of the lesions
on the femoral condyles (Table 1). A definite trend
toward a reduction in the grading score (P < 0.06)
(Table 1) was also noted for the femoral condyles. The
severity of the lesions in the tenidap-treated dogs was
similar to that in the 4-week dogs, except for the lesion
EFFECTS OF TENIDAP ON OA PROGRESSION
Figure 2. Macroscopic appearance of cartilage from the femoral
condyles (A, C, and E) and tibial plateaus (B, D, and F) of dogs with
osteoarthritis (OA). In the 4-week dogs with OA, pitting of the central,
weight-bearing region of a condyle and a plateau are evident (A and
B). In the 12-week dogs with OA, erosions and pitting of the condyle
and tibial plateau are shown (C and D). In the tenidap-treated dogs,
pitted areas of cartilage can be seen on the condyles and plateaus (E
and F).
size on the tibial plateaus, which was smaller in the
tenidap-treated group.
Synovial membrane. Synovia from all 3 groups
showed similar changes and exhibited definite signs of
inflammation. The synovium was hypertrophic and demonstrated a red-yellowish discoloration, with a large
number of blood vessels.
Microscopic findings. Cartilage. Specimens from
both the 4-week OA (Figures 3A and B) and the
12-week OA (Figures 3C and D) groups showed morphologic changes characteristic of OA, while none of the
specimens indicated an invasion of the tidemark by
blood vessels. The histologic score (Figure 4) for the
lesions on the condyles and plateaus was higher in the
12-week OA dogs (mean ? SEM 5.55 5 0.38 and 5.54 t0.60, respectively) than in the 4-week OA dogs (2.11 2
0.32 and 3.43 t- 0.50, respectively). In the tenidaptreated dogs, the lesions on the condyles and plateaus
(3.43 ? 0.54 and 3.39 t- 0.35, respectively) (Figures 3E
and F and Figure 4) were significantly less severe
compared with those in the 12-week OA dogs. The
289
Figure 3. Representative sections of articular cartilage from the femoral condyles (A, C, and E) and tibial plateaus (B, D, and F) of the
4-week dogs with osteoarthritis (OA) (A and B), the 12-week dogs with
OA (C and D), and the tenidap-treated dogs (E and F). (Safranin 0
stained, original magnification X 10.)
tenidap-treated dogs had only mild histologic lesions,
and the histologic scores on the condyles and plateaus
were similar to those for the 4-week OA group.
Cloning and/or hypercellularity was consistently
present in specimens from the 12-week OA group
Femoral Condyles
Tibia1 Plateaus
**
d
a4
4 weeks
Q4
Teni@
12 weeks
Q4
4 weeks
Tenidap
12 weeks
Q4
Figure 4. Histologic grading of cartilage from the femoral condyles
and tibial plateaus of dogs with osteoarthritis (OA) (see Table 1 for
description of the treatment groups). Bars show the mean and SEM
histologic score (see ref. 24 for description of scoring system) for
lesions from the 4-week OA dogs, the 12-week OA dogs, and the
tenidap-treated dogs. P values were calculated using the MannWhitney U test. * = P < 0.0008 versus 4-week OA dogs, P < 0.02
versus tenidap-treated dogs; ** = P < 0.01 versus 4-week OA dogs,
P < 0.005 versus tenidap-treated dogs.
FERNANDES ET AL
290
(Figures 3C and D). The mean SEM cell score (scale
0-3) in the condyles was higher in the 12-week OA
group (2.00 ? 0.00) than in the tenidap-treated animals
(1.33 -+ 0.18; P < 0.03), but the score in the plateaus was
not significantly different (12-week OA group 1.67 ?
0.18 versus tenidap-treated group 1.29 ? 0.16). This
smaller difference was largely due to a decrease in the
number of specimens showing cell cloning in the
plateaus.
The structural changes (scale 0-6) on the condyles and plateaus were less severe in the 4-week OA
group (mean 5 SEM 0.54 % 0.25 and 1.00 2 0.26,
respectively) than in the 12-week OA group (1.73 -I 0.19
and 2.17 2 0.39, respectively). The tenidap-treated dogs
showed less severe structural changes on the femoral
condyles (0.96 t 0.32; P 5 0.07) and on the tibia1
plateaus (1.00 5 0.31; P < 0.02) than did the 12-week
OA dogs.
Similar scores for Safranin 0 staining (scale 0-4)
were found in both the condyles and the plateaus when
the scores for the 4-week OA group (mean t SEM
1.11 % 0.08 and 1.36 ? 0.16, respectively) were compared with those for the tenidap-treated group (1.07 %
0.12 and 1.18 2 0.11, respectively). These scores, in turn,
were lower than those in the 12-week OA group (1.50 ?
0.18 and 2.17 f 0.39, respectively).
Synovial membrane. The synovium from all 3
groups was thick, had numerous villosities, and showed
synovial lining cell hyperplasia (2-5 layers) and marked
infiltration mononuclear cells. The histologic scores
(scale 0-10) were nearly equal among the 3 groups, with
no significant differences between the 4-week OA dogs
(mean % SEM 4.57 % 0.57), the 12-week OA dogs
(4.67 ? 0.44), and the tenidap-treated dogs (5.50 t
0.71).
Metalloprotease activity. Collagenase. The mean
collagenase activity in the cartilage of the 4-week OA
dogs was much higher (-2-fold) than that found in the
12-week OA dogs (P < 0.06) (Figure 5). In the tenidaptreated dogs, the cartilage enzyme level was lower than
1.28
in the 12-week OA dogs (mean t SEM 3.78
versus 6.34 ? 0.79).
Stuomelysin. The stromelysin activity in the cartilage from each group is illustrated in Figure 5. The
enzymatic activity was slightly lower for the 12-week OA
dogs than for the 4-week OA dogs. Enzymatic activity in
the tenidap-treated dogs was significantly lower ( P <
0.05) compared with the 12-week OA group.
Gelatinase. The gelatinase activity (Figure 5 ) in
the cartilage of dogs from the 12-week OA group was
about twice as high as that found in the 4-week OA
Collagenase
+_
f_
T
OA
4 weeks
Stromelysin
Gelatinase
*Wl T
TeNdap
12 weeks
OA
OA
4 weeks
TeNdap
12 weeks
OA
Q4
4WeekS
Tenidap
l2WeekS
Q4
Figure 5. Enzymatic activity levels of collagenase, stromelysin, and
gelatinase in cartilage of dogs with osteoarthritis (OA) (see Table 1for
description of the treatment groups). The enzymatic activity is expressed as units/gm of tissue wet weight (w.w.). Bars show the mean
and SEM unitsigm. P values were calculated using the Mann-Whitney
U test. * = P < 0.06 versus 4-week OA dogs; ** = P < 0.05 versus
tenidap-treated dogs; *** = P < 0.06 versus 4-week OA dogs, P < 0.03
versus tenidap-treated dogs.
group (P < 0.06), and was significantly higher than the
activity found in the tenidap-treated dogs ( P < 0.03).
Metalloprotease gene expression level. The level
of expression of collagenase-1 and stromelysin-1 in the
cartilage was similar among all 3 groups (data not
shown). However, the level of expression of collagenase-3
was found to be significantly lower (P < 0.02) in the
tenidap-treated dogs compared with that observed in the
12-week OA dogs (Figure 6).
IL-1 activity in synovial fluid. As illustrated in
Figure 7, IL-1 activity was low in the 4-week OA dogs
and rose -5-fold in the 12-week OA dogs. Interestingly,
the tenidap-treated dogs exhibited a statistically significant decrease (P < 0.004) in activity compared with the
12-week OA dogs.
DISCUSSION
This study showed that tenidap decreased the
progression of existing cartilage lesions in the experimental dog model of OA. This preventive action included a reduction in the articular cartilage breakdown
and osteophyte growth, along with inhibition of MMP
activity in the early experimental OA cartilage.
In previous studies using this dog model of OA,
we demonstrated that tenidap, given prophylactically at
the time of surgery, markedly reduced several parameters of the pathologic process, including osteophyte
formation, cartilage lesions, and synovial inflammation
(21). Under the present experimental conditions, with
tenidap treatment given when lesions were already
EFFECTS OF TENIDAP ON OA PROGRESSION
Treatment
4 weeks
1
291
12 weeks
I
I
1
Tenidap
Collagenase-3
c 18s
GAPDH
4-
Relative expression of mRNA
0,ss f 0.09
(Normalized lo GAPDH)
0.59 f 0.14
18s
0.22 t 0.05
fP4W
Figure 6. Levels of collagenase-3 messenger RNA (mRNA) in cartilage of dogs with osteoarthritis (OA). Total RNA was analyzed
by Northern hybridization using collagenase-3 and GAPDH RNA probes (see Table 1 for description of the treatment groups).
Bottom lane shows the mean ? SEM ratio of collagenase-3 mRNA to GAPDH mRNA in each group. P values were determined by
Mann-Whitney U test. Levels in the tenidap-treated group were significantly lower compared with those in the 12-week OA group.
present (albeit at a very early stage), the drug was also
found to markedly reduce the size of osteophytes. In
addition, the incidence of osteophytes was reduced, but,
not surprisingly, this reduction was smaller than that
seen under the previous, prophylactic protocol, in which
some spurs were present or in formation when treatment
began. Although the exact mechanism for the reduction
of osteophyte outgrowth remains to be determined, it
may be related to factors associated with the inflamed
synovium. Along with mechanical factors, it was suggested that growth factors and cytokines are involved in
the formation and growth of osteophytes, since these
molecules can induce growth and differentiation of
mesenchymal cells (32,33). Intraarticular injections of
0.05
E
.A
z-1
&?
'2 c
e2
z
5
0.04
0.03
om
E
0.01
OaO
a4
4 weeks
M
Tenidap
12 weeks
Figure 7. Levels of interleukin-I (IL-I) activity in synovial fluid of
dogs with osteoarthritis (OA) (see Table 1 for description of the
treatment groups). The activity was expressed as arbitrary units/ml,
calculated using a standard curve against levels of recombinant human
IL-lP. Bars show the mean and SEM unitsirnl. * = P < 0.001 versus
4-week OA dogs, P < 0.004 versus tenidap-treated dogs, by MannWhitney U test.
rHuIL-1 receptor antagonist markedly decreased the
incidence and size of osteophytes in the ACL dog model
(34). This finding clearly points to a role for IL-1,
directly or indirectly, in the genesis of osteophytes.
In the present study, tenidap was found to significantly reduce IL-1 activity in the synovial fluid. This
finding makes it tempting to speculate that tenidap's
effect on osteophytes may be mediated in some way
through inhibition of IL-1 synthesis. This reduction in
the level of IL-1 could also have induced a decrease in
the rate of mitosis, producing a down-modulation in the
action of transforming growth factor p (35-37) or of
other growth factors that influence bone and cartilage
metabolism (38,39).
The major effect of tenidap, by gross and microscopic observation, was preservation of the cartilage
structure. Modest fibrillation was observed at 4 weeks,
and this did not progress during tenidap treatment over
the 8-week period. In contrast, the untreated animals
showed substantial fibrillation. This implies that tenidap
provided protection for the cartilage collagen network
underlying the cartilage structure. Tenidap treatment
resulted in no change in the intensity of Safranin 0
staining of the cartilage between the 4-week OA dogs
and the 12-week tenidap-treated dogs. In our previous
study under prophylactic conditions (21), tenidap was
found to protect against loss of Safranin 0 staining only
in the femoral condyles. In the present study, no protective effect was observed in either the femoral condyles or
the tibia1 plateaus. In both studies, tenidap inhibited
stromelysin activity in the cartilage. These results suggest that the loss of proteoglycan staining may have been
due to enzymes other than stromelysin.
292
It is of interest to note that the level of expression
of collagenase-1, collagenase-3, and stromelysin in control groups did not vary over time, while the collagenase
activity decreased in the presence of an increasing level
of IL-1. These findings may be related to the level of
IL-1 present at 4 weeks, which may have been sufficient
to induce a maximum stimulation of MMP expression in
chondrocytes. The inverse correlation between the level
of IL-1 in synovial fluid and the collagenase activity
could possibly be due to the role played by other factors,
such as protease activators and inhibitors, which regulate the activity of MMP over the course of the disease.
The progression of cartilage lesions over time, in the
presence of a decreasing level of collagenase activity,
may indicate that the damage created in the collagen
network by proteolytic enzymes in the early stages of OA
may have already become irreversible. The marked
decrease in 3 MMP activities (stromelysin, gelatinase,
and, to a lesser extent, collagenase) in the cartilage of
the tenidap-treated dogs provides evidence that these
enzymes could play a major role in the pathophysiology
of OA. The absence of statistical significance in the level
of reduction of collagenase activity by tenidap is likely
due to a Type I1 error.
There are several possible explanations for
tenidap’s action which are not mutually exclusive.
Tenidap interfered directly with the activity and/or
synthesis of the MMP in the cartilage. However, only 1
MMP, collagenase-3, was shown to be inhibited by this
drug at the expression level. This finding is not surprising, since the synthesis of MMP has been shown to be
discoordinately regulated (40). Tenidap could have
acted indirectly on MMP synthesis through an inhibition
of IL-1 synthesis (20). The significant decrease in IL-1
activity in the synovial fluid of the tenidap-treated dogs
is consistent with this possibility.
Detailed analysis of the histologic findings
showed that structural damage in the cartilage, indicative of collagen network breakdown, was marked at 12
weeks after surgery, when the score was approximately
twice as high as at 4 weeks. Tenidap appeared to stop the
progression of these structural changes, since the score
in the tenidap-treated group was similar to that noted at
4 weeks. The protective effect of tenidap on cartilage
collagen could be related to the decreased MMP levels.
It was believed that the collagen network changes in OA
cartilage were related to the action of collagenase-1,
because this enzyme was the only one known to induce
primary cleavage of type I1 collagen. In addition to
collagenase-1, a new MMP, collagenase-3, has recently
been identified in human articular cartilage and is likely
FERNANDES ET AL
involved in OA cartilage degradation (6). It has been
shown that collagenase-3 can cleave type I1 collagen at a
higher velocity rate than collagenase-1 (6,41). Collagenolytic activity measured in the articular cartilage is,
therefore, likely to be a combination effect of these 2
collagenases.
The absence, in this study, of an effect of tenidap
on collagenase-1 expression is surprising and contrasts
with our earlier study findings made under prophylactic
conditions (21). The discovery that another collagenase
is present in human OA cartilage leads us to suspect that
these enzymes play different roles in the pathologic
process, along with having different levels of regulation.
Under the present protocol, we showed that tenidap
inhibited expression of collagenase-3. Thus, the decreased collagenolytic activity could be related, at least
in part, to reduced production of collagenase-3. The
decrease in stromelysin and gelatinase activity by
tenidap, likely due to an action of the drug at the
posttranscriptional level in cartilage, is also of major
importance, since these enzymes may act not only on
proteoglycan or other matrix macromolecules, but also
on different collagen types. Stromelysin, for example,
has the ability to cleave the native type IX collagen in all
3 chains, as well as type I1 collagen telopeptides. Moreover, stromelysin is important for full activation of
collagenase (42).
The presence of chondrocyte cloning was seen
frequently in the 12-week OA cartilage. It has been
suggested that these pathologic changes in the cartilage
could have occurred in response to growth factors and/or
to cytokines (43). The significant reduction in chondrocyte cloning in the tenidap-treated dogs may indicate
that this drug can reduce the synthesis or action of
factors responsible for the proliferation of chondrocytes.
In conclusion, the present findings demonstrate
that tenidap, when used therapeutically, is able to reduce
the progression of experimental OA lesions. This may be
due to a reduction in MMP activity/synthesis within the
cartilage, perhaps through a cytokine-modulating activity of the drug.
ACKNOWLEDGMENTS
The authors thank the laboratory technicians for their
assistance, Ms Santa Fiori and Ms Mary McCutcheon for their
secretarial support, and Dr. Jean-Pierre Raynauld for his
statistical expertise.
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Clinical Images: Steroid-induced subcutaneous tissue atrophy
This 44-year-old woman with rheumatoid arthritis was given triamcinolone acetonide (120 mg; 40 mg/ml), injected intramuscularly
into her right upper thigh by her general practitioner, for a flare of her disease. This injection was helpful in suppressing the active
disease, but left her with an indentation at the site of the injection, secondary to subcutaneous tissue atrophy. Intramuscular
methylprednisolone and triamcinolone are sometimes used in the treatment of inflammatory rheumatic diseases. They should be
administered via the deep intramuscular route into the upper, outer quadrant of the buttock areas to avoid this complication.
D. W. T. Ching, MBChB, MRCP (UK)
P. M. Dellow, RGON, BN
Timaru Hospital
Timaru, New Zealand
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