close

Вход

Забыли?

вход по аккаунту

?

art.30422

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 63, No. 9, September 2011, pp 2700–2710
DOI 10.1002/art.30422
© 2011, American College of Rheumatology
Contributions of Angiogenesis to Inflammation, Joint Damage,
and Pain in a Rat Model of Osteoarthritis
Sadaf Ashraf, Paul I. Mapp, and David A. Walsh
Objective. To determine the contributions of angiogenesis to inflammation, joint damage, and pain
behavior in a rat meniscal transection model of osteoarthritis (OA).
Methods. OA was induced in male Lewis rats (n ⴝ
8 per group) by meniscal transection. Animals were
orally dosed with dexamethasone (0.1 mg/kg/day), indomethacin (2 mg/kg/day), or the specific angiogenesis
inhibitor PPI-2458 (5 mg/kg every other day). Controls
consisted of naive and vehicle-treated rats. Synovial
inflammation was measured as the macrophage fractional area (expressed as the percentage), thickness of
the synovial lining, and joint swelling. Synovial angiogenesis was measured using the endothelial cell proliferation index and vascular density. Channels positive
for vessels at the osteochondral junction were assessed
(osteochondral angiogenesis). Medial tibial plateaus
were assessed for chondropathy, osteophytosis, and
channels crossing the osteochondral junction. Pain behavior was measured as weight-bearing asymmetry.
Results. Dexamethasone and indomethacin each
reduced pain behavior, synovial inflammation, and synovial angiogenesis 35 days after meniscal transection.
Dexamethasone reduced, but indomethacin had no significant effect on, the total joint damage score. PPI-2458
treatment reduced synovial and osteochondral angiogenesis, synovial inflammation, joint damage, and pain
behavior.
Conclusion. Our findings indicate that synovial
inflammation and joint damage are closely associated
with pain behavior in the meniscal transection model of
OA. Inhibition of angiogenesis may reduce pain behavior both by reducing synovitis and by preventing structural change. Targeting angiogenesis could therefore
prove useful in reducing pain and structural damage in
OA.
Osteoarthritis (OA), a common chronic disorder
of the joints, is a major cause of pain and disability in
aging populations. Treatments for OA focus on symptomatic relief. OA is associated with chondropathy,
synovitis, subchondral bone remodeling, and osteophytosis (1). The widely accepted notion that OA is
primarily a disease of the cartilage with associated
subchondral and synovial changes (2) is challenged by
the hypothesis that primary disorder may arise in the
subchondral bone and synovium, with loss of articular
cartilage and osteophyte growth being secondary phenomena (3).
Normal adult human articular cartilage is avascular and aneural. In OA, osteochondral angiogenesis
accompanied by the generation of sympathetic and
sensory nerves (4,5) is observed. Blood vessels and
nerves also penetrate newly formed cartilage at the joint
margins during osteophyte formation (5).
Synovitis may contribute both to symptoms and
to joint damage in OA (1). Increased angiogenesis in the
synovium is associated with chronic synovitis in OA (6).
Synovial angiogenesis may impair chondrocyte function
and homeostasis of the articular cartilage as well as
contributing to articular hypoxia (4,7). Angiogenesis is
not in itself painful, but it may exacerbate pain by
enabling the innervation of tissues and by facilitating
inflammation (8). Therefore, inhibitors of angiogenesis
and/or inflammation have the potential to reduce structural damage and pain in OA.
Intraarticular injection of corticosteroids and oral
or topical application of nonsteroidal antiinflammatory
drugs (NSAIDs) are recommended treatments for knee
Dr. Mapp’s work was supported by the Arthritis Research UK
Pain Centre (grant 18769).
Sadaf Ashraf, PhD, Paul I. Mapp, PhD, David A. Walsh,
FRCP, PhD: University of Nottingham, Nottingham, UK.
Address correspondence to Sadaf Ashraf, PhD, Arthritis
Research UK Pain Centre, Department of Academic Rheumatology,
University of Nottingham, City Hospital, Clinical Sciences Building,
Hucknall Road, Nottingham NG5 1PB, UK. E-mail: [email protected]
nottingham.ac.uk.
Submitted for publication August 2, 2010; accepted in revised
form April 19, 2011.
2700
JOINT PATHOLOGY AND PAIN IN A RAT MODEL OF OA
2701
OA (9). In addition to their antiinflammatory actions,
corticosteroids (e.g., dexamethasone) and NSAIDs (e.g.,
indomethacin) can reduce pain and may also be antiangiogenic (10,11). Antiinflammatory drugs have the potential to slow the progression of OA structural damage
by inhibiting synovitis but are not considered to be
disease modifying in human OA (12). We hypothesized
that angiogenesis may not only facilitate inflammation,
but may also be a mechanism by which inflammation
leads to pain and joint damage in OA. We therefore
proposed that inhibiting angiogenesis might in turn
reduce inflammation and reduce pain and joint damage
in OA.
PPI-2458, an antiangiogenic fumagillin analog,
reduces synovitis and bone and cartilage damage in
animal models of arthritis (13,14). It exerts its effects by
inhibiting methionine aminopeptidase type 2 (MetAP2), triggering growth arrest of endothelial cells (ECs) in
the late G1 phase of the cell cycle, inhibiting EC
proliferation and angiogenesis without affecting inflammatory cytokine release (13,15). Transection of the
medial meniscus in the rat results in joint pathology
similar to that observed in human OA (7,16,17), indicating that this model is useful for the development of
pharmacologic interventions for treatment of, and to
explore mechanisms underlying pain in, OA.
We used the rat meniscal transection model to
evaluate the potential contributions of inflammation and
angiogenesis to joint damage and pain in OA. We first
used the meniscal transection model of OA to show that
antiinflammatory drugs (indomethacin and dexamethasone) inhibited synovitis, pain, and joint damage. PPI2458 was used to specifically inhibit angiogenesis without directly affecting inflammation (13,15) in order to
explore the contribution of angiogenesis to inflammation, joint damage, and pain in OA.
Induction of OA. The medial meniscal transection
model of OA was used to elucidate possible effects of antiinflammatory and antiangiogenic agents in OA (7,17). On day
0, immediately prior to surgery, animals were given a subcutaneous injection of cephalexin (0.005 ml/100 gm) (Ceporex;
Schering-Plough Animal Health), an antibiotic, followed by a
single dose of carprofen (0.01 ml/100 gm) (Rimadyl; Pfizer) to
reduce postoperative pain. The left leg was shaved and surgically prepared. The medial collateral ligament was exposed by
cautery of the connective tissue and muscle layers, and part of
it was removed to uncover the meniscus. The joint space was
visualized, and the meniscus cut through the full thickness at its
narrowest point. To ensure that the underlying articular cartilage did not suffer any trauma while the meniscus was being
cut, the ankle of the animal was twisted to create a space
between the meniscus and the two bone ends. This enabled the
free meniscus to be grasped and retracted before it was cut.
The tibial and femoral articular surfaces were also inspected
under the microscope to ensure no damage was sustained. The
connective tissue layer was closed first, followed by the skin,
with coated Vicryl 8-0 and 4-0 sutures, respectively (Ethicon).
Pharmacologic interventions. Indomethacin and PPI2458. Animals were given a single 500-␮l oral dose of either
indomethacin (2 mg/kg) dissolved in sterile 0.9% normal saline
1 hour prior to intraarticular injection, as previously described
(20) or vehicle control (11% 2-hydroxy-propyl-␤-cyclodextrin
[HP␤CD] buffer in phosphate buffered saline [PBS]), or they
were given vehicle containing the angiogenesis inhibitor PPI2458 (5 mg/kg) 24 hours prior to injection.
Dexamethasone and indomethacin. Animals that had
meniscal transection surgery were dosed daily, starting on day
11 and continuing until day 35 (when they were killed), by oral
gavage with either 500 ␮l of sterile 0.9% normal saline
(vehicle) control (pH 7.4) or vehicle control containing either
dexamethasone (0.1 mg/kg) or indomethacin. These doses
were shown in previous studies to inhibit inflammation in the
joint (21–24).
PPI-2458. Animals that had meniscal transection surgery were dosed every other day, starting on day 11 and
continuing until day 35, by oral gavage with either 500 ␮l of
vehicle (HP␤CD buffer in PBS) or vehicle containing the
angiogenesis inhibitor PPI-2458. This dose of PPI-2458 was
shown in previous studies to suppress angiogenesis and inflammation and to improve bone structure (13,14,19).
Naive animals, rather than sham-operated animals,
were used as additional controls because the previously published literature shows that hind limb weight distribution and
tibiofemoral pathology do not differ between sham-operated
and naive animals beyond the first 7 days after surgery (7,25).
The weights of the animals were monitored throughout the
experiment. Day 11 was chosen as the beginning of the
intervention studies in order to allow adequate time for the
animals to recover from meniscal transection surgery. By day
11, the sutures had also fallen out, and the visible skin wound
over the knees had sufficiently healed.
Joint swelling and pain behavior. Prior to interventions and on days 12, 14, 19, 26, 29, 32, and 35 after surgery, or
at 1, 3, 6, 10, and 24 hours after intraarticular injection, pain
behavior was measured as weight-bearing asymmetry between
contralateral and ipsilateral knees, using an Incapacitance
Meter (Linton Instruments) (25,26). Animals were placed in a
MATERIALS AND METHODS
Animals. In vivo experiments performed on male
Lewis rats (n ⫽ 8 per group, weighing 250–270 gm; Charles
River) were carried out in accordance with UK Home Office
regulations. Animals were housed under standard conditions
with unlimited access to food and water. Prior to receiving
intraarticular injections or meniscal transection surgery, all
rats were anesthetized with isoflurane (2% in O2).
Induction of acute inflammation. A previously established model of acute synovitis (18,19) was used to elucidate
possible direct effects of PPI-2458 on synovitis and pain
behavior. A single 50-␮l intraarticular injection of either 0.03%
carrageenan dissolved in sterile 0.9% normal saline (pH 7.4) or
sterile 0.9% normal saline (pH 7.4) was administered to the
left knee joint cavity on day 0 (18,19).
2702
Perspex tube, such that each paw rested on a separate transducer pad that recorded the animal’s weight distribution over
a period of 3 seconds. Each data point is the average of 5
readings. Joint swelling was also measured at these time points
with digital electronic calipers (Mitutoyo UK), representing
asymmetry of knee diameters (millimeters) between the ipsilateral and contralateral knee joints. Both the incapacitance
and knee diameter measurements were taken by an observer
(SA) who was blinded with regard to the experimental group.
Previous experiments have shown that sham-operated controls
do not display weight-bearing asymmetry or joint swelling by
day 12, and do not display synovitis or OA structural changes
on day 35 (7,25). Naive animals, rather than sham-operated
controls, were therefore used in this study in order to evaluate
how completely the interventions reversed the changes of OA.
Tissue collection and preparation. Thirty-five days
after meniscal transection surgery or 24 hours after intraarticular injection, animals were killed by asphyxiation in carbon
dioxide, and synovia with patellae from the right and left knees
were immediately harvested and snap frozen. Tibiofemoral
joints were isolated by cutting the midfemur and midtibia and
were preserved for 48 hours at room temperature in neutral
buffered formalin (containing 4% formaldehyde). Joints were
subsequently decalcified in 10% formic acid in neutral buffered formalin (containing 4% formaldehyde) for 10 days.
Coronal sections of trimmed joint tissues were then processed
by standard histologic techniques and mounted in wax blocks.
Assessment of synovial morphology. Synovial sections
(5 ␮m thick) were cut using a motorized cryostat. CD31positive cells and proliferating cell nuclear antigen (PCNA)–
immunoreactive CD31-positive cells were used to identify ECs
and proliferating ECs, respectively, in the synovia as two
separate measures of the extent of angiogenesis, as previously
described (27,28). Macrophage infiltration was identified by
immunoreactivity for the monoclonal antibody clone ED1
(29). Nuclei were counterstained with the fluorescent DNA
ligand DAPI (30). PCNA and ED1 were developed with
diaminobenzidine using the glucose oxidase/nickel–enhanced
method. Endothelium markers were developed using SigmaAldrich Fast Red. Staining procedures were similar to those
previously described (18). Synovial lining thickness and cellularity were assessed on hematoxylin and eosin–stained sections. Coronal tissue sections (5 ␮m thick) from wax blocks cut
in a Reichert-Jung rotary microtome were taken from the
midpoint of the joint, as identified by the presence of cruciate
ligament insertions, and stained with Safranin O. Lectin immunohistochemistry was performed to identify ECs, using
Griffonia simplicifolia lectin 1 (GS-1), as previously described
(31,32). Preparations were mounted in DePeX except those to
detect ECs, which were mounted in AquaMount.
Image analysis and quantification. Image analysis and
quantification were performed by an observer (SA) who was
blinded to the experimental details, as previously described
(33). Synovial lining thickness and cellularity were evaluated as
previously described and graded on a scale of 0 (lining cell
layer 1–2 cells thick) to 3 (lining cell layer ⬎9 cells thick and/or
severe increase in cellularity) (7). One whole synovial section
per rat knee examined with a 20⫻ objective lens was used, and
an overall grade best representing the section was given. The
entire medial tibial plateau of the midcoronal sections was
stained with Safranin O and used to assess chondropathy and
ASHRAF ET AL
osteophytosis through a 4⫻ objective lens. Chondropathy and
osteophytosis were evaluated using the system described by
Janusz et al (17), as previously described (25), and 1 tissue
section per rat knee was used to assess chondropathy and
osteophytosis. The total joint damage score (range 0–18) was
calculated as the sum of the chondropathy score and the
osteophyte score. The integrity of the osteochondral junction
was measured as the number of channels present in the
articular cartilage of a Safranin O–stained section of the
medial tibial plateaus, using a 20⫻ objective lens. Osteochondral vascular density was determined by counting the number
of channels containing GS-1 lectin–labeled blood vessels that
crossed the osteochondral junction in a section across the
medial tibial plateau.
Reagents. Antibody to PCNA (clone PC10) was obtained from Dako. Biotinylated rat-adsorbed horse anti-mouse
antibody, lectin GS-1 (L1100), goat anti-lectin (AS 2104),
biotinylated rabbit anti-goat (BA 5000), and avidin–biotin
complexes (ABC kits) were obtained from Vector. Monoclonal
antibodies to rat CD31 (clone TLD-3A12) and to macrophages
Figure 1. Effects of indomethacin and PPI-2458 on synovitis and pain
behavior in a rat model of acute synovitis. Rat knees were injected
intraarticularly with either 0.03% carrageenan or saline on day 0. A
single oral dose of either vehicle control (䊐), the angiogenesis
inhibitor PPI-2458 (5 mg/kg 1 day before carrageenan injection) (Œ),
or indomethacin (2 mg/kg 1 hour prior to carrageenan injection) (E)
was given. Following administration of PPI-2458 and 24 hours after the
injection of carrageenan, measures of synovial inflammation, as represented by macrophage infiltration (A), the synovial lining grade
(lining layer thickness/cellularity) (B), and joint swelling (difference in
knee diameter) (C), were not reduced and remained similar to those in
vehicle-treated controls, whereas the antiinflammatory drug indomethacin reduced these measures of inflammation to the levels in
saline-injected controls (■). Pain behavior, which was measured as the
difference in hind limb weight bearing (D), was increased 6 hours after
carrageenan injection. Weight-bearing asymmetry at 6 hours was
prevented by indomethacin treatment, but not PPI-2458 treatment.
⫹⫹ ⫽ P ⬍ 0.01; ⫹⫹⫹ ⫽ P ⬍ 0.001 versus vehicle controls. ⴱⴱ ⫽ P ⬍
0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus saline controls.
JOINT PATHOLOGY AND PAIN IN A RAT MODEL OF OA
2703
Figure 2. Inhibition of measures of inflammation (synovial macrophage infiltration, synovial lining layer thickness/cellularity grade, and joint
swelling [difference in knee diameter]) after meniscal transection in Lewis rats treated with indomethacin, dexamethasone, or PPI-2458. From day
11 to day 35, rats subjected to meniscal transection were given oral doses of indomethacin (2 mg/kg/day) (blue symbols), dexamethasone (0.1
mg/kg/day) (purple symbols), PPI-2458 (5 mg/kg every other day) (pink symbols), or vehicle control (red symbols). Following administration of either
indomethacin or dexamethasone, macrophage infiltration (A), synovial lining layer thickness/cellularity (E), and joint swelling (I) were significantly
reduced 35 days after meniscal transection as compared with vehicle-treated rats. The levels were not different from those in naive controls (green
symbols). Similarly, following PPI-2458 administration, macrophage infiltration (B), synovial lining layer thickness/cellularity (F), and joint swelling
(J) were also significantly reduced 35 days following meniscal transection as compared with vehicle-treated rats. The levels were not different from
those in naive controls. Joint swelling (measured as the difference in knee diameter) remained higher in vehicle-treated rats subjected to meniscal
transection as compared to naive controls on day 35. All 3 drugs inhibited joint swelling to levels that were not different from those in naive controls
by day 35 (I and J). Vertical broken line in I and J indicates initiation of treatment. Photomicrographs show macrophages (black), as delineated by
immunoreactivity for ED1 in sections from vehicle-treated (C) and naive (D) controls, as well as synovial lining layer thickness (arrowheads) and
synovial cellularity, as indicated by hematoxylin and eosin staining of sections from vehicle-treated (G) and naive (H) controls. Bars ⫽ 100 ␮m.
ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus arthritic, vehicle controls. ⫹ ⫽ P ⬍ 0.05; ⫹⫹ ⫽ P ⬍ 0.01; ⫹⫹⫹ ⫽ P ⬍ 0.001 versus naive
controls.
(clone ED1) were from Serotec. All other chemicals were
obtained from Sigma-Aldrich. PPI-2458 was a kind donation
from GlaxoSmithKline.
Statistical analysis. Four fields per section and 1
section per case were measured for synovial macrophage
infiltration and endothelial cell proliferation data. For the
endothelial fractional area (a measure of vascular density), 4
consecutive sections per case with 4 fields of view per section
were used. These numbers were determined in previous experiments (18,19) to minimize the coefficient of variation and to
assure that the observed mean lies within ⫾12.5% of the true
mean. Data were analyzed using the Statistical package for the
Social Sciences version 16 software (SPSS) and presented
graphically using Prism version 4 software (GraphPad). Parametric data were analyzed using one-way analysis of variance.
Univariate comparisons were made using Student’s t-test, with
2704
ASHRAF ET AL
Figure 3. Synovial angiogenesis (synovial endothelial cell [EC] proliferation and vascular density) and vascularization of channels crossing the
osteochondral junction in the medial tibial plateaus of Lewis rats. From day 11 to day 35, rats subjected to meniscal transection were given oral doses
of either indomethacin (2 mg/kg/day), dexamethasone (0.1 mg/kg/day), PPI-2458 (5 mg/kg every other day), or vehicle control. Following
administration of either indomethacin or dexamethasone, the EC proliferation index (the EC proliferating cell nuclear antigen [PCNA] index) (A)
and vascular density (E) were significantly reduced compared with those in vehicle-treated animals, but were similar to those in naive controls.
Similarly, following PPI-2458 administration, the EC PCNA index (B) and vascular density (F) were also significantly reduced compared with those
in vehicle-treated animals, but were similar to those in naive controls. Photomicrographs show ECs (red), as delineated by immunoreactivity for
CD31 (blue arrows), proliferating nuclei (black), as delineated by immunoreactivity for PCNA (green arrows), and proliferating ECs (black arrows),
which contain PCNA-immunoreactivity nuclei, in sections from vehicle-treated (C) and naive (D) controls, as well as the extent of ECs (red), as
delineated by immunoreactivity for CD31 in sections from vehicle-treated (G) and naive (H) controls. A coronal section of the medial tibial plateau
from a rat subjected to meniscal transection and treated with vehicle (J) shows positive staining of vascular channels crossing (blue asterisks) or
approaching (black asterisks) the osteochondral junction (dotted line separating the underlying bone from the cartilage) with Griffonia simplicifolia
lectin 1 (alkaline phosphatase reaction product). Following PPI-2458 administration, vascular channels crossing the medial osteochondral junction
were reduced (I). Bars ⫽ 100 ␮m. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus vehicle controls.
Bonferroni correction for multiple comparisons. Nonparametric data were analyzed using the Kruskal-Wallis test followed
by the Mann-Whitney test to compare 2 groups, with Bonferroni correction for multiple comparisons.
Interobserver variability for the image analysis, using 8
synovial and medial tibial plateau sections, was determined by
2 observers (SA and PIM) and is expressed as the limits of
agreement (34). The limits of agreement were defined as the
mean ⫾ 1.96 SD, within which 95% of the data are expected to
fall. Scoring systems displayed good agreement between the 2
observers (synovial lining grade limits of agreement –0.4, 0.2;
total joint damage score limits of agreement –5, 3). All other
presented data are from a single observer (SA). Numerical
data are reported as the mean (95% confidence interval [95%
CI]) or as the median (interquartile range [IQR]) in the text,
and for clarity, they are presented graphically as the mean ⫾
SEM, except where indicated otherwise. P values less than 0.05
(2-tailed) were considered statistically significant.
RESULTS
Effects of indomethacin or PPI-2458 on inflammation and pain behavior in carrageenan-induced synovitis. Synovial inflammation, as evidenced by macrophage infiltration, the synovial lining grade, and joint
swelling, was increased 24 hours after intraarticular
injection of carrageenan (Figures 1A–C) and was re-
JOINT PATHOLOGY AND PAIN IN A RAT MODEL OF OA
Figure 4. Histologic changes (chondropathy, osteophytosis, and channels at the osteochondral junction) of the medial tibial plateaus of
Lewis rats on day 35 following meniscal transection. A, Coronal section
from a vehicle-treated arthritic control rat, showing severe, fullthickness cartilage loss (arrows), with a large osteophyte formed at the
joint margin (encircled area), and several channels crossing into (black
asterisks) or approaching (blue asterisks) the cartilage (dotted line
separating the underlying bone from the cartilage). B, Coronal section
from a naive control rat, showing normal smooth cartilage and joint
margins. The chondrocytes are homogeneously distributed throughout
the cartilage, and there is evidence of neither proteoglycan loss nor
channels entering the avascular cartilage (blue asterisk). C, Coronal
section from a dexamethasone-treated rat, showing moderate cartilage
loss, with fibrillation, reduced proteoglycan staining, and reduced
chondrocyte density (arrows). A large osteophyte is evident at the joint
margin (encircled area), with channels crossing into the cartilage
(black asterisk). D, Coronal section from a PPI-2458-treated rat,
showing mild cartilage fibrillation, loss of proteoglycan staining, and
loss of chondrocyte density (arrows). A developing osteophyte can be
seen at the joint margin (encircled area), with some channels crossing
into the cartilage from the underlying subchondral bone (black
asterisk). Safranin O stained; bars ⫽ 100 ␮m.
duced to control levels following treatment with indomethacin, but was unaltered by PPI-2458 treatment. At
6 hours after carrageenan injection, vehicle-treated animals showed a greater weight-bearing asymmetry
(mean 33 gm [95% CI 26, 39]) compared with saline
injected controls (mean 2 gm [95% CI –6, 9]; P ⬍ 0.001),
which returned to control levels after this time point
(Figure 1D). Indomethacin reduced weight-bearing
asymmetry to the levels in saline-injected controls 6
hours after carrageenan injection (Figure 1D). Weightbearing asymmetry was unaffected by PPI-2458 treatment (mean 26 gm [95% CI 12, 40] at 6 hours after
carrageenan injection; P ⫽ 1.00 versus carrageenaninjected, vehicle-treated animals). The synovial EC proliferation index was increased 24 hours after intraartic-
2705
ular injection of carrageenan (mean 8.4% [95% CI 6.7,
10]) and was reduced after treatment with PPI-2458
(mean 1.5% [95% CI 0.8, 2.2]; P ⬍ 0.001) to levels
observed in saline-injected controls (mean 0.8% [95%
CI 0.2, 1.3]; P ⫽ 1.00).
Meniscal transection model of OA. Measures of
inflammation, synovial angiogenesis (EC proliferation
index and vascular density), and vascular channels crossing the osteochondral junction were significantly increased 35 days after meniscal transection surgery (Figures 2 and 3). Macrophages were localized to the
synovial lining and dispersed throughout the synovial
sublining (Figures 2C and D). The synovial lining layer
was thicker, with increased cellularity seen throughout
the synovia (Figure 2G). Blood vessels were distributed
throughout the depth of the rat synovia, occasionally
containing PCNA-immunoreactive nuclei (Figures 3C,
D, G, and H). The meniscal transection model was
associated with loss of articular cartilage, reduced chondrocyte density and reduced proteoglycan staining, with
subchondral bone remodeling, including increased numbers of channels breaching the osteochondral junction
and the formation of osteophytes (Figure 4A and Table
1). Increased pain behavior was also observed (Figure 5).
Effects of inhibiting inflammation on angiogenesis, joint damage, and pain in the meniscal transection
model of OA. Following treatment with antiinflammatory compounds, measures of inflammation were reduced to levels observed in naive controls (Figure 2).
Vehicle-treated animals had a greater difference in knee
diameter on day 12 as compared with naive controls
(mean 0.5 mm [95% CI 0.2, 0.8] versus 0.03 mm [95% CI
–0.03, 0.09]; P ⬍ 0.05), and this persisted to day 35.
Treatment with antiinflammatory drugs reduced knee
joint diameter to that in naive controls by day 35 (Figure
2I). Indomethacin or dexamethasone treatment reduced
indices of synovial angiogenesis (Figure 3).
Indomethacin did not significantly reduce joint
damage scores (chondropathy and/or osteophytosis)
(Table 1), whereas dexamethasone reduced total joint
damage (median score 7 [IQR 7–9]) compared with
vehicle-treated controls (median score 12 [IQR 11–13];
P ⫽ 0.015). This effect was primarily due to a reduction
in the chondropathy score (median score 10 [IQR
10–10] in the vehicle-treated group versus 5 [IQR 5–7] in
the dexamethasone-treated group; P ⫽ 0.03) (Figure 4C
and Table 1). Neither dexamethasone nor indomethacin
affected the numbers of channels crossing the osteochondral junction (Table 1).
Vehicle-treated animals that had undergone meniscal transection surgery showed greater weightbearing asymmetry on day 12 (mean 32 gm [95% CI 25,
2706
ASHRAF ET AL
Table 1. Reduction in OA structural changes in the medial tibial plateaus following antiinflammatory/antiangiogenic treatment in the rat meniscal
transection model of OA*
Antiinflammatory treatment
Antiangiogenic treatment
Structural change
Vehicle
Indo.
DEX
Naive
Vehicle
PPI-2458
Naive
Total damage score (range 0–18)
Osteophyte score (range 0–3)
Cartilage damage score (range 0–15)
No. of channels crossing the medial
osteochondral junction
12 (11–13)
2 (2–3)
10 (10–10)
3 (3–5)
10 (8–12)
2 (2–3)
9 (5–10)
3 (2–4)
7 (7–9)†
2 (1–2)
5 (5–7)†
3 (2–3)
1 (0–2)‡
0 (0–0)§
1 (0–2)‡
1 (0–1)‡
13 (10–13)
3 (3–3)
10 (8–10)
5 (4–6)
8 (4–10)†
1 (0–3)†
6 (4–8)
2 (1–5)
1 (0–1)‡
0 (0–0)§
1 (0–1)‡
1 (0–1)‡
* Following dexamethasone (DEX; 0.1 mg/kg/day) treatment, the total joint damage score (the combined chondropathy and osteophyte scores) was
reduced in rats subjected to meniscal transection as compared to that in vehicle-treated controls. This was mainly due to a reduction in cartilage
damage. Indomethacin (Indo.; 2 mg/kg/day) treatment did not significantly reduce joint damage. Channels crossing the osteochondral junction were
not significantly reduced following treatment with either indomethacin or dexamethasone and remained more abundant in those treatment groups
than in naive control rats. Following PPI-2458 (5 mg/kg every other day) treatment, total joint damage in rats subjected to meniscal transection was
reduced compared with that in vehicle-treated controls. This reduction was mainly due to the attenuation of osteophyte growth. Channels growing
into the cartilage from underlying subchondral bone were not significantly different after PPI-2458 as compared with either naive or vehicle-treated
arthritic controls. Values are the median (interquartile range).
† P ⬍ 0.05 versus vehicle-treated arthritic animals.
‡ P ⬍ 0.01 versus vehicle-treated arthritic animals.
§ P ⬍ 0.001 versus vehicle-treated arthritic animals.
40]) as compared with naive controls (mean 7 gm [95%
CI ⫺2, 16]; P ⬍ 0.001). This asymmetry persisted to day
35 (mean 33 gm [95% CI 25, 42] in those with meniscal
transection versus 8 gm [95% CI 2, 13] in naive controls;
P ⬍ 0.001). Treatment with indomethacin reduced
weight-bearing asymmetry on day 14 (mean 13 gm [95%
CI ⫺9, 27]) as compared with that in vehicle-treated
controls (mean 40 gm [95% CI 23, 56]; P ⬍ 0.05), and by
day 35, completely abolished it to the levels in naive
controls (Figure 5A). Dexamethasone reduced weightbearing asymmetry on day 26 (mean 4 gm [95% CI ⫺9,
17]) as compared to vehicle-treated animals (mean 35
Figure 5. Time course of the inhibition of meniscal transection–induced knee joint pain behavior
following the administration of antiinflammatory or antiangiogenic treatment. A, Pain behavior in
arthritic animals treated with indomethacin (), dexamethasone (Œ), or vehicle control (䊐). B,
Pain behavior in arthritic animals treated with either PPI-2458 (F) or vehicle control. On day 0, the
rats underwent meniscal transection. From day 11 (vertical broken line) to day 35, the arthritic
animals were given oral doses of indomethacin (2 mg/kg/day), dexamethasone (0.1 mg/kg/day),
PPI-2458 (5 mg/kg every other day), or vehicle control. Pain behavior was measured as the
difference in hind limb weight bearing between day 0 and day 35 after meniscal transection.
Vehicle-treated arthritic animals showed increased pain behavior compared to naive controls ({)
12 days following meniscal transection, and this increase was maintained to day 35. Indomethacin
significantly inhibited pain behavior in the arthritic animals on day 14 and completely abolished it
to levels in naive controls by day 35. In dexamethasone-treated animals, pain behavior was inhibited
to levels in naive controls on day 26, and this inhibition was maintained to day 35. PPI-2458–treated
animals showed a reduction in pain behavior on day 19, which was completely inhibited to the levels
in naive controls by day 35. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus naive controls.
JOINT PATHOLOGY AND PAIN IN A RAT MODEL OF OA
2707
gm [95% CI 25, 44]; P ⬍ 0.01), and by day 35, completely
abolished it to the levels in naive controls (Figure 5A).
Effects of inhibiting angiogenesis on inflammation, joint damage, and pain in the meniscal transection
model of OA. By day 35 following treatment with PPI2458, measures of inflammation were reduced to levels
in naive rats (Figure 2). PPI-2458 treatment reduced
indices of synovial angiogenesis and the number of
vascularized channels at the osteochondral junction
(median 1 vascularized channel per medial tibial plateau
section [IQR 0–2]) compared with vehicle-treated controls (median 4 vascularized channels per medial tibial
plateau section [IQR 2–5]; P ⫽ 0.026) (Figure 3).
PPI-2458 reduced the total joint damage score
(median score 8 [IQR 4–10]) as compared with vehicletreated controls (median score 13 [IQR 10–13]; P ⫽
0.016), but these were not completely abolished to levels
in naive rats (Figure 4D and Table 1); this reduction was
primarily due to the attenuation of osteophyte growth
(median score 3 [IQR 3–3] in vehicle-treated controls
versus 1 [IQR 0–3] in PPI-2458–treated rats; P ⫽ 0.044).
PPI-2458 had no significant effect on the integrity of the
osteochondral junction (Table 1).
PPI-2458 treatment reduced weight-bearing
asymmetry (mean 17 gm [95% CI 6, 27]) as compared
with vehicle treatment (mean 39 gm [95% CI 29, 49];
P ⬍ 0.01) in the meniscal transection model of OA on
day 19, and completely abolished it to levels in naive
controls by day 35 (Figure 5B).
each of these respects, and with the increasing recognition that meniscal injury both contributes to OA structural damage and is a consequence of OA, this rat model
of OA effectively mimics aspects of human OA and may
provide important insights into the mechanisms underlying structural changes, pain behavior, and responses to
pharmacologic therapies.
Pain of OA is multifactorial in origin (35). In the
absence of disease-modifying drugs for the treatment of
OA, pharmacologic interventions often target central
pain mechanisms in order to relieve symptoms (36).
Current treatments are often limited by potential adverse events. A better understanding of how joint pathology leads to pain is required in order to develop new
therapies. Synovitis contributes to pain in OA (37). Our
data confirm that antiinflammatory treatments can inhibit pain behavior in an animal model of OA. Dexamethasone reduces joint damage (osteophyte formation
and cartilage loss) in experimental models of OA
(38,39). Our data also suggests that dexamethasone may
be chondroprotective in the meniscal transection model
of OA. However, corticosteroids can also inhibit chondrocyte synthetic functions and may therefore exacerbate joint damage (40). Further research would be
required to better understand what determines the
balance between potentially protective and damaging
effects of corticosteroids.
Angiogenesis is a feature of OA in rats as well as
in humans, both in the synovium and at the osteochondral junction (4,7), and it is associated with synovitis
(2,8). We have shown here that inhibition of angiogenesis by administration of PPI-2454 is associated with
reductions in synovitis, structural damage, and pain
behavior in the meniscal transection model of OA.
Inhibition of angiogenesis therefore potentially offers a
novel therapeutic strategy for OA.
The reduction in synovitis that we observed after
targeting angiogenesis using PPI-2458 was similar in
extent to that observed after systemic treatment with
indomethacin or dexamethasone. PPI-2458 has been
shown to prevent the persistence of synovitis after
combined intraarticular injection of carrageenan and
fibroblast growth factor 2 in rats (19). In that study,
inhibition of synovitis and pain behavior occurred later
after treatment with PPI-2458 than did inhibition of
angiogenesis, and were not observed at 24 hours after
intraarticular injection of carrageenan. This contrasts
with our findings after administration of indomethacin,
and suggests that the reduction in synovitis and pain
behavior at later time points after PPI-2458 administration to rats with OA results from inhibition of angiogen-
DISCUSSION
Using the rat meniscal transection model of knee
OA, we examined whether inflammation may contribute
to joint damage and pain through the stimulation of
angiogenesis. We found that inhibiting inflammation
reduced joint damage and pain, that angiogenesis may
contribute to inflammation and joint damage, and that
inhibiting blood vessel growth may be a useful strategy
for reducing OA pain.
Surgical models of mechanical instability, as induced by meniscal transection, represent posttraumatic
OA, with loss of joint congruity and stability. Pathologic
changes in the joint resemble, but develop more rapidly
than, those seen in human OA, and the translational
validity of animal models of OA requires careful appraisal.
Our findings support previous evidence that the
meniscal transection model results in OA changes in the
medial tibial plateaus (7,17,25,35), loss of integrity of the
osteochondral junction, synovitis, and pain behavior. In
2708
esis, rather than from any direct effect on inflammation
or pain processing. However, inflammation and pain
processing involve complex, heterogeneous pathways,
and it is not possible to completely exclude the possibility that previously unsuspected actions of PPI-2458 may
supplement its antiangiogenic effects. We used weightbearing asymmetry as a measure of pain behavior in this
study because it has clinical validity (patients with knee
OA complain of pain on standing), is reproducible, and
is sensitive to change. Further work will be required to
explore possible effects of interventions on other pain
behaviors, such as withdrawal thresholds following punctuate stimulation (allodynia) or gait disturbance.
Synovitis persists throughout the development of
knee OA after meniscal injury in rats (7), and our
current demonstration of the antiinflammatory effects
of PPI-2458 beginning 12 days after meniscal injury
indicates that angiogenesis inhibition can reduce inflammation even after synovitis has become established.
Synovitis is associated with greater chondropathy
in human OA (41) and predicts the progression of joint
damage in longitudinal studies (41,42). Inhibition of
synovitis is therefore an attractive target for disease
modification in OA, although coincidental and detrimental effects of current antiinflammatory agents on
chondrocyte function (26,40) may limit their efficacy as
disease-modifying agents. In this study, treatment with
either dexamethasone or PPI-2458 reduced the total
joint damage score. Vascular endothelial growth factor
blockade has also been shown to reduce new bone
formation around arthritic joints, an effect that may be
partly mediated by inhibition of endochondral ossification (43). Reduction of synovitis may contribute to the
structural protection afforded by dexamethasone or
PPI-2458 seen in our study. However, dexamethasone
predominantly affected chondropathy in our meniscal
transection model, whereas angiogenesis inhibition reduced osteophyte size. This leads us to suggest that the
structural benefits of PPI-2458 and dexamethasone are
mediated through different mechanisms, reflecting differential effects on inflammatory and angiogenic pathways.
Similar to previous findings (44,45), our data also
suggest that indomethacin reduces synovial inflammation and angiogenesis. We did not observe any significant effect on the total joint damage score with indomethacin, whereas some clinical studies have suggested
that indomethacin may increase the rate of radiologic
deterioration of the joint space in patients with OA of
the knee and hip (46,47).
ASHRAF ET AL
Osteochondral angiogenesis and pain behavior in
the meniscal transection model of OA are reduced by
inhibition of matrix metalloproteinases (25). Our current finding that PPI-2458 treatment inhibited vascularization of channels at the osteochondral junction is
consistent with its antiangiogenic activity. However, the
significant increase in total channel numbers in animals
that had undergone meniscal transection surgery and
were then treated with PPI-2458 as compared with the
nonarthritic control animals indicates that angiogenesis
is not a prerequisite for channel formation. Reductions
in pain and structural damage observed in the osteoarthritic animals following treatment with PPI-2458 may
be partly due to inhibition of osteophyte growth and
osteochondral angiogenesis, as well as inhibition of
synovitis.
The current study has limitations that are common to investigations of complex biologic processes in
intact animals. Although PPI-2458 is well characterized
as a specific inhibitor of angiogenesis, we cannot be
certain which consequences of angiogenesis inhibition
are responsible for its analgesic activity. Similarly, indomethacin and dexamethasone may have effects additional to the inhibition of inflammation, including direct
antiangiogenic actions. Consistent with previous studies
using dexamethasone (48,49), corticosteroid-treated animals failed to gain weight at the same rate as animals in
other groups. This reduction in growth may be due to
negative effects on glucose metabolism, although growth
curves paralleled those of the other treatment groups by
day 35, and blood glucose levels did not differ significantly between the groups at the time they were euthanized (data not shown). Despite these limitations, however, our data support the view that antiinflammatory
and antiangiogenic strategies may have potential not
only to relieve symptoms, but also to modify structural
progression in OA.
In conclusion, these findings indicate that synovitis and total joint damage are closely associated with
pain behavior in OA, and this may be partly explained by
angiogenesis in the synovium and at the osteochondral
junction. Targeting angiogenesis could therefore prove
useful in reducing pain and structural damage in OA,
either alone or in combination with other drugs.
ACKNOWLEDGMENTS
The authors thank Dr. Inma Rioja (GlaxoSmithKline,
London, UK) for providing the angiogenesis inhibitor PPI2458. Research using the antiinflammatory drugs was con-
JOINT PATHOLOGY AND PAIN IN A RAT MODEL OF OA
2709
ducted in collaboration with Mr. Phillipe Miska and Dr.
Caroline Baudouin (Laboratoires Expanscience, Courbevoie,
France).
Heitmeyer SA. Induction of osteoarthritis in the rat by surgical
tear of the meniscus: inhibition of joint damage by a matrix
metalloproteinase inhibitor. Osteoarthritis Cartilage 2002;10:
785–91.
Walsh DA, Rodway HA, Claxson A. Vascular turnover during
carrageenan synovitis in the rat. Lab Invest 1998;78:1513–21.
Ashraf S, Mapp PI, Walsh DA. Angiogenesis and the persistence
of inflammation in a rat model of proliferative synovitis. Arthritis
Rheum 2010;62:1890–8.
Pinheiro RM, Calixto JB. Effect of the selective COX-2 inhibitors,
celecoxib and rofecoxib in rat acute models of inflammation.
Inflamm Res 2002;51:603–10.
Cuzzocrea S, Mazzon E, Di Paola R, Genovese T, Muia C, Caputi
AP, et al. Effects of combination M40403 and dexamethasone
therapy on joint disease in a rat model of collagen-induced
arthritis. Arthritis Rheum 2005;52:1929–40.
Takagi T, Tsao PW, Totsuka R, Suzuki T, Murata T, Takata I.
Dexamethasone prevents the decrease of bone mineral density in
type II collagen-induced rat arthritis model. Jpn J Pharmacol
1998;78:225–8.
Sharma JN. Modifications in tissue kallikrein activity with indomethacin and prednisolone treatment in arthritic rats. Inflammopharmacology 2010;18:113–7.
Ku G, Faust T, Lauffer LL, Livingston DJ, Harding MW. Interleukin-1␤ converting enzyme inhibition blocks progression of type
II collagen-induced arthritis in mice. Cytokine 1996;8:377–86.
Mapp PI, Walsh DA, Bowyer J, Maciewicz RA. Effects of a
metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis.
Osteoarthritis Cartilage 2010;18:593–600.
Bove SE, Calcaterra SL, Brooker RM, Huber CM, Guzman RE,
Juneau PL, et al. Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of
monosodium iodoacetate-induced osteoarthritis. Osteoarthritis
Cartilage 2003;11:821–30.
Waseem NH, Lane DP. Monoclonal antibody analysis of the
proliferating cell nuclear antigen (PCNA): structural conservation
and the detection of a nucleolar form. J Cell Sci 1990;96:121–9.
Male D, Rahman J, Linke A, Zhao W, Hickey W. An interferoninducible molecule on brain endothelium which controls lymphocyte adhesion mediated by integrins. Immunology 1995;84:453–60.
Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of
mononuclear phagocytes in lymphoid organs: distinct macrophage
subpopulations in the rat recognized by monoclonal antibodies
ED1, ED2 and ED3. Immunology 1985;54:589–99.
Sanna PP, Jirikowski GF, Lewandowski GA, Bloom FE. Applications of DAPI cytochemistry to neurobiology. Biotech Histochem
1992;67:346–50.
Orgad U, Alroy J, Ucci A, Merk FB. Histochemical studies of
epithelial cell glycoconjugates in atrophic, metaplastic, hyperplastic, and neoplastic canine prostate. Lab Invest 1984;50:294–302.
Alroy J, Goyal V, Skutelsky E. Lectin histochemistry of mammalian endothelium. Histochemistry 1987;86:603–7.
Seegers HC, Hood VC, Kidd BL, Cruwys SC, Walsh DA. Enhancement of angiogenesis by endogenous substance P release and
neurokinin-1 receptors during neurogenic inflammation. J Pharmacol Exp Ther 2003;306:8–12.
Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet
1986;1:307–10.
Bove SE, Laemont KD, Brooker RM, Osborn MN, Sanchez BM,
Guzman RE, et al. Surgically induced osteoarthritis in the rat
results in the development of both osteoarthritis-like joint pain
and secondary hyperalgesia. Osteoarthritis Cartilage 2006;14:
1041–8.
McColl GJ. Pharmacological therapies for the treatment of osteoarthritis. Med J Aust 2001;175 Suppl: S108–11.
18.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Ashraf had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Ashraf, Mapp, Walsh.
Acquisition of data. Ashraf, Mapp, Walsh.
Analysis and interpretation of data. Ashraf, Mapp, Walsh.
19.
20.
21.
22.
REFERENCES
1. Ashraf S, Walsh DA. Angiogenesis in osteoarthritis. Curr Opin
Rheumatol 2008;20:573–80.
2. Walsh DA. Angiogenesis and arthritis. Rheumatology (Oxford)
1999;38:103–12.
3. Imhof H, Breitenseher M, Kainberger F, Trattnig S. Degenerative
joint disease: cartilage or vascular disease? Skeletal Radiol 1997;
26:398–403.
4. Walsh DA, Bonnet CS, Turner EL, Wilson D, Situ M, McWilliams
DF. Angiogenesis in the synovium and at the osteochondral
junction in osteoarthritis. Osteoarthritis Cartilage 2007;15:743–51.
5. Suri S, Gill SE, Massena de Camin S, Wilson D, McWilliams DF,
Walsh DA. Neurovascular invasion at the osteochondral junction
and in osteophytes in osteoarthritis. Ann Rheum Dis 2007;66:
1423–8.
6. Haywood L, McWilliams DF, Pearson CI, Gill SE, Ganesan A,
Wilson D, et al. Inflammation and angiogenesis in osteoarthritis.
Arthritis Rheum 2003;48:2173–7.
7. Mapp PI, Avery PS, McWilliams DF, Bowyer J, Day C, Moores S,
et al. Angiogenesis in two animal models of osteoarthritis. Osteoarthritis Cartilage 2008;16:61–9.
8. Bonnet CS, Walsh DA. Osteoarthritis, angiogenesis and inflammation. Rheumatology (Oxford) 2005;44:7–16.
9. Conaghan PG, Dickson J, Grant RL, on behalf of the Guideline
Development Group. Care and management of osteoarthritis in
adults: summary of NICE guidance. BMJ 2008;336:502–3.
10. Folkman J, Ingber DE. Angiostatic steroids: method of discovery
and mechanism of action. Ann Surg 1987;206:374–83.
11. Madhok R, Wijelath E, Smith J, Watson J, Sturrock RD, Capell
HA. Is the beneficial effect of sulfasalazine due to inhibition of
synovial neovascularization? J Rheumatol 1991;18:199–202.
12. Ding C. Do NSAIDs affect the progression of osteoarthritis?
Inflammation 2002;26:139–42.
13. Bainbridge J, Madden L, Essex D, Binks M, Malhotra R, Paleolog
EM. Methionine aminopeptidase-2 blockade reduces chronic
collagen-induced arthritis: potential role for angiogenesis inhibition. Arthritis Res Ther 2007;9:R127.
14. Lazarus DD, Doyle EG, Bernier SG, Rogers AB, Labenski MT,
Wakefield JD, et al. An inhibitor of methionine aminopeptidase
type-2, PPI-2458, ameliorates the pathophysiological disease processes of rheumatoid arthritis. Inflamm Res 2008;57:18–27.
15. Griffith EC, Su Z, Turk BE, Chen S, Chang YH, Wu Z, et al.
Methionine aminopeptidase (type 2) is the common target for
angiogenesis inhibitors AGM-1470 and ovalicin. Chem Biol 1997;
4:461–71.
16. Bendele AM. Animal models of osteoarthritis. J Musculoskelet
Neuronal Interact 2001;1:363–76.
17. Janusz MJ, Bendele AM, Brown KK, Taiwo YO, Hsieh L,
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
2710
37. Grunke M, Schulze-Koops H. Successful treatment of inflammatory knee osteoarthritis with tumour necrosis factor blockade. Ann
Rheum Dis 2006;65:555–6.
38. Williams JM, Brandt KD. Triamcinolone hexacetonide protects
against fibrillation and osteophyte formation following chemically
induced articular cartilage damage. Arthritis Rheum 1985;28:
1267–74.
39. Pelletier JP, Di Battista JA, Raynauld JP, Wilhelm S, MartelPelletier J. The in vivo effects of intraarticular corticosteroid
injections on cartilage lesions, stromelysin, interleukin-1, and
oncogene protein synthesis in experimental osteoarthritis. Lab
Invest 1995;72:578–86.
40. Annefeld M, Erne B, Rasser Y. Ultrastructural analysis of rat
articular cartilage following treatment with dexamethasone and
glycosaminoglycan-peptide complex. Clin Exp Rheumatol 1990;8:
151–7.
41. Walsh DA, Yousef A, McWilliams DF, Hill R, Hargin E, Wilson
D. Evaluation of a Photographic Chondropathy Score (PCS) for
pathological samples in a study of inflammation in tibiofemoral
osteoarthritis. Osteoarthritis Cartilage 2009;17:304–12.
42. Ledingham J, Regan M, Jones A, Doherty M. Factors affecting
radiographic progression of knee osteoarthritis. Ann Rheum Dis
1995;54:53–8.
43. Miotla J, Maciewicz R, Kendrew J, Feldmann M, Paleolog E.
ASHRAF ET AL
44.
45.
46.
47.
48.
49.
Treatment with soluble VEGF receptor reduces disease severity in
murine collagen-induced arthritis. Lab Invest 2000:80:1195–205.
Bernardi A, Zilberstein AC, Jager E, Campos MM, Morrone FB,
Calixto JB, et al. Effects of indomethacin-loaded nanocapsules in
experimental models of inflammation in rats. Br J Pharmacol
2009;158:1104–11.
Wang HM, Zhang GY. Indomethacin suppresses growth of colon
cancer via inhibition of angiogenesis in vivo. World J Gastroenterol 2005;11:340–3.
Rashad S, Revell P, Hemingway A, Low F, Rainsford K, Walker F.
Effect of non-steroidal antiinflammatory drugs on the course of
osteoarthritis. Lancet 1989;2:519–22.
Huskisson EC, Berry H, Gishen P, Jubb RW, Whitehead J, for the
LINK Study Group, Longitudinal Investigation of Nonsteroidal
Antiinflammatory Drugs in Knee Osteoarthritis. Effects of antiinflammatory drugs on the progression of osteoarthritis of the knee.
J Rheumatol 1995;22:1941–6.
Caparroz-Assef SM, Bersani-Amado CA, Kelmer-Bracht AM,
Bracht A, Ishii-Iwamoto EL. The metabolic changes caused by
dexamethasone in the adjuvant-induced arthritic rat. Mol Cell
Biochem 2007;302:87–98.
Roussel D, Dumas JF, Augeraud A, Douay O, Foussard F,
Malthiery Y, et al. Dexamethasone treatment specifically increases
the basal proton conductance of rat liver mitochondria. FEBS Lett
2003;541:75–9.
Документ
Категория
Без категории
Просмотров
4
Размер файла
842 Кб
Теги
art, 30422
1/--страниц
Пожаловаться на содержимое документа