вход по аккаунту


Fibroblast growth factor 2 is an intrinsic chondroprotective agent that suppresses ADAMTS-5 and delays cartilage degradation in murine osteoarthritis.

код для вставкиСкачать
Vol. 60, No. 7, July 2009, pp 2019–2027
DOI 10.1002/art.24654
© 2009, American College of Rheumatology
Fibroblast Growth Factor 2 Is an
Intrinsic Chondroprotective Agent That
Suppresses ADAMTS-5 and Delays
Cartilage Degradation in Murine Osteoarthritis
Shi-Lu Chia, Yasunobu Sawaji, Annika Burleigh, Celia McLean, Julia Inglis,
Jeremy Saklatvala, and Tonia Vincent
Objective. We have previously identified in articular cartilage an abundant pool of the heparin-binding
growth factor, fibroblast growth factor 2 (FGF-2), which
is bound to the pericellular matrix heparan sulfate
proteoglycan, perlecan. This pool of FGF-2 activates
chondrocytes upon tissue loading and is released following mechanical injury. In vitro, FGF-2 suppresses
interleukin-1–driven aggrecanase activity in human
cartilage explants, suggesting a chondroprotective role
in vivo. We undertook this study to investigate the in
vivo role of FGF-2 in murine cartilage.
Methods. Basal characteristics of the articular
cartilage of Fgf2–/– and Fgf2ⴙ/ⴙ mice were determined by
histomorphometry, nanoindentation, and quantitative
reverse transcriptase–polymerase chain reaction. The
articular cartilage was graded histologically in aged
mice as well as in mice in which osteoarthritis (OA) had
been induced by surgical destabilization of the medial
meniscus. RNA was extracted from the joints of Fgf2–/–
and Fgf2ⴙ/ⴙ mice following surgery and quantitatively
assessed for key regulatory molecules. The effect of
subcutaneous administration of recombinant FGF-2 on
OA progression was assessed in Fgf2–/– mice.
Results. Fgf2–/– mice were morphologically indis-
tinguishable from wild-type (WT) animals up to age 12
weeks; the cartilage thickness and proteoglycan staining
were equivalent, as was the mechanical integrity of the
matrix. However, Fgf2–/– mice exhibited accelerated
spontaneous and surgically induced OA. Surgically induced OA in Fgf2–/– mice was suppressed to levels in WT
mice by subcutaneous administration of recombinant
FGF-2. Increased disease in Fgf2–/– mice was associated
with increased expression of messenger RNA of Adamts5, the key murine aggrecanase.
Conclusion. These data identify FGF-2 as a novel
endogenous chondroprotective agent in articular cartilage.
The structural integrity of articular cartilage is
determined principally by homeostasis of the 2 major
macromolecules of the extracellular matrix, type II
collagen and the chondroitin sulfate–rich proteoglycan
aggrecan. In healthy tissue, there is a balance between
anabolic (synthetic) and catabolic (degradative) processes that allows matrix turnover. Excessive catabolic
activity results in matrix breakdown, a hallmark of
osteoarthritis (OA). One key early event in matrix
breakdown is loss of aggrecan, which is caused by
aggrecanase enzymes, members of the ADAMTS family.
In humans, ADAMTS-4 and ADAMTS-5 are thought to
be the major aggrecanases in cartilage (1,2). In the
mouse, deletion of ADAMTS-5, but not ADAMTS-4,
was shown to protect against the development of OA
and inflammatory arthritis, suggesting that ADAMTS-5
is the main murine aggrecanase (3,4).
We have identified fibroblast growth factor 2
(FGF-2) as a potential regulatory molecule in articular
cartilage. It is bound to the heparan sulfate chains of the
proteoglycan perlecan in the pericellular matrix of hu-
Supported by the Wellcome Trust, the Medical Research
Council of the UK, and the Arthritis Research Campaign. Dr. Chia’s
work was supported by the Lee Kuan Yew Scholarship Fund.
Shi-Lu Chia, MD, PhD, Yasunobu Sawaji, PhD, Annika
Burleigh, BSc, Celia McLean, BSc, Julia Inglis, BSc (Hons), PhD,
Jeremy Saklatvala, MD, PhD, Tonia Vincent, MD, PhD: Imperial
College London, London, UK.
Address correspondence and reprint requests to Tonia Vincent, MD, PhD, Kennedy Institute of Rheumatology, Imperial College
London, Charing Cross Campus, 65 Aspenlea Road, London W6 8LH,
UK. E-mail:
Submitted for publication November 5, 2008; accepted in
revised form April 7, 2009.
man and porcine cartilage, where it acts as a mechanotransducer (5,6). Upon loading, FGF-2 is made available
to cell surface tyrosine kinase receptors and activates
intracellular signaling pathways including ERK, one of
the MAPKs (7). FGF-2 is also released from the pericellular pool upon physical injury to the tissue (8).
In order to determine the role of FGF-2 in
articular cartilage, we recently investigated the influence
of FGF-2 on the breakdown of aggrecan in human
articular cartilage explants. We found that FGF-2 suppressed interleukin-1 (IL-1)– or tumor necrosis factor–
stimulated aggrecanase activity in explants of normal
knee cartilage in a dose-dependent manner (9). Because
of these findings, we investigated whether FGF-2 was
chondroprotective in vivo. Fgf2–/– mice are viable, fertile,
and morphologically indistinguishable from their wildtype (WT) littermates under normal conditions (10). We
examined the knee articular cartilage of naive Fgf2–/–
and Fgf2⫹/⫹ mice, and then we compared both agerelated cartilage degeneration in Fgf2–/– and Fgf2⫹/⫹
mice and cartilage degeneration following surgical destabilization of the medial meniscus (DMM), a wellestablished model of OA.
Mice. The generation of Fgf2-knockout mice has been
described elsewhere (10). A fragment of the Fgf2 gene was
replaced with a 3.2-kb hypoxanthine guanine phosphoribosyltransferase (Hprt) minigene, effectively removing the first 59
amino acids, which are important for heparin and receptor
binding and for mitogenic activity. Heterozygous (Fgf2⫹/–)
breeding pairs were maintained on a 50% 129Sv/50% black
Swiss background and were provided by The Jackson Laboratory (Bar Harbor, ME). Genotyping was performed using
reverse transcriptase–polymerase chain reaction (RT-PCR)
with the following primer pairs: 5⬘-CGA-GAA-GAG-CGACCC-ACA-C-3⬘ (forward) and 5⬘-CCA-GTT-CGG-GGACCC-TAT-T-3⬘ (reverse) for the WT allele, and 5⬘-AGGAGG-CAA-GTG-GAA-AAC-GAA-3⬘ (forward) and 5⬘-CCCAGA-AAG-CGA-AGG-AAC-AAA-3⬘ (reverse) for the target
(deleted) allele. Full-cousin mice were used in all experiments,
and, where possible, experiments were performed with littermate controls.
Surgical induction of OA. Surgical induction of OA
was performed in 10–12-week-old mice by microsurgical release of the anterior horn of the medial meniscus from its tibial
attachment (3). Sham surgery consisted of medial capsulotomy
only. All invasive procedures were approved by the UK Home
Office and followed institutional ethical and procedural guidelines.
Histologic assessment. For FGF-2 and perlecan immunostaining, hips from 6-week-old mice were snap-frozen and
sectioned (6-␮m sections) using a CV1900 cryostat (Leica
Microsystems, Milton Keynes, UK) equipped with a tungsten
blade. Tissue was fixed for 15 minutes with methanol, then
immunostained and viewed using Ultraview confocal microscopy (60⫻ oil immersion lens; PerkinElmer, Waltham, MA) as
described previously (6). For tissues used for scoring of OA,
dissected knee specimens were fixed in 10% formalin, decalcified in dilute formic acid, and embedded in paraffin. Coronal
sections (8-␮m thick) were cut and stained with Safranin O.
Sections were evaluated at 80-␮m intervals.
Severity of cartilage destruction was assessed histologically using a modification of a previously described 7-point
scale (0 ⫽ normal; 1 ⫽ surface fibrillations; 2 ⫽ loss of
superficial cartilage and surface delamination, with shallow
fissures but no frank ulceration; 3 ⫽ ulceration of noncalcified
cartilage only; 4 ⫽ vertical clefts extending into subchondral
bone; 5 ⫽ ulceration extending into calcified cartilage but not
into subchondral bone, ⬍80% cartilage loss; and 6 ⫽ ulceration extending into subchondral bone and/or ⬎80% cartilage
loss) (11). Histologic scoring was performed by 2 blinded
observers. All articular surfaces within each section (medial
femur, medial tibia, lateral femur, lateral tibia) were graded
separately, and the scores were added to give a score for the
section (range 0–24). The histologic summed score (maximum
72) was calculated from the sum of the 3 highest section scores
to provide a composite indicator of both the severity and the
extent of cartilage damage. Intra- and interobserver agreement
were very good, as reflected by intraclass correlation coefficients (ICCs) of ⬎0.8.
Cartilage histomorphometry was performed on uncompressed digital images using MCID Analysis 7.0 software
(Imaging Research, St. Catherines, Ontario, Canada). For
neoepitope immunostaining, embedded sections were deparaffinized and treated for 30 minutes with 0.1% hyaluronidase
in phosphate buffered saline (PBS). Sections were blocked
with normal goat serum and then incubated for 1 hour with
anti-NITEGE antibodies (1:850 dilution; kindly provided by
Dr. John Mort, Shriner’s Hospital, Montreal, Quebec, Canada). Antigen was visualized using streptavidin-conjugated
peroxidase (ABC kit; Vector, Burlingame, CA) according to
the manufacturer’s recommendations.
Cartilage histomorphometry. Safranin O–stained paraffin sections were digitally photographed in uncompressed
TIFF format with specialized software (Spot v4.5; Diagnostic
Instruments, Sterling Heights, MI) using a Spot Insight Color
Mosaic camera (Diagnostic Instruments) attached to a Leitz
Wetzlar Dialux 22EB light microscope (Leitz, Wetzlar, Germany). Histomorphometric analysis was performed with
MCID Analysis 7.0 software. The derivation of the morphometric indices measured is as described below.
Thickness. The general region of interest (e.g., the area
around the tibial plateau or femoral condyle) was outlined. A
preset function based on chromatic thresholding was then used
to precisely select only the cartilaginous component, following
which an algorithm was applied that measured multiple vertical distances across the selected region of cartilage, before
returning the maximum and mean values. These values were
taken as the maximum and mean “thickness” measurements,
Proteoglycan staining. The TIFF image was first converted to a 16-bit grey-scale image in the red channel. The
cartilaginous component was selected as described above. The
averaged optical density (OD) was then read—this corresponded to the OD calculated on a grey scale obtained in the
Figure 1. Pericellular fibroblast growth factor 2 (FGF-2) and perlecan in articular cartilage from
Fgf2⫹/⫹ and Fgf2–/– mice. Hip articular cartilage explants were snap-frozen and then cryosectioned
(6 ␮m). Sections were pretreated with hyaluronidase for 1 hour and then stained with propidium
iodide (PI) and antibodies to perlecan (1:1,000) and FGF-2 (1:1,000). Fluorescence signals were
detected using confocal microscopy. No intensity or background adjustments were made between
sections. Bar ⫽ 20 ␮m.
red component of the light crossing the section. The OD was a
function of the intensity of Safranin O staining. Although this
technique is not fully quantitative, it has been validated
previously and provides an approximate measure of proteoglycan content (12). Each OD reading was normalized to the OD
of the epiphyseal plate in the same tissue section (which acted
as a staining control), and the resulting value was expressed as
a ratio. This normalized OD reading was used as an indicator
of the degree of proteoglycan staining. The repeatability of
measurements made with the MCID software was excellent, with
a vanishingly low variance. The intraobserver and interobserver
ICCs were also ⬎0.9, indicating excellent rater agreement.
Real-time RT-PCR. RNA was extracted from whole
joints following removal of the skin and muscle bulk. Tissue
was snap-frozen and then extracted using TRIzol reagent
(Invitrogen, Paisley, UK) and purified using a spin column kit
(Qiagen, Hilden, Germany). RNA was reverse-transcribed to
complementary DNA and quantified with the TaqMan realtime PCR system (Applied Biosystems, Foster City, CA), using
prevalidated primers/probe mixes obtained from the same
company, at a concentration of 900 nM for each primer and
200 nM for the probe. Real-time PCR was performed using a
RotorGene 6000 thermocycler (Corbett Research, Mortlake,
New South Wales, Australia). Data capture and primary
analysis were carried out with RotorGene 6000 software
(version 1.7) from the same company. The thermocycling
conditions were as follows: denaturation at 95°C for 10 minutes, followed by 45 cycles of a denaturation step at 95°C for 2
seconds, followed by an annealing/extension step at 60°C for 30
seconds. All samples were measured in triplicate and compared with expression levels of the housekeeping gene
Accession codes. RefSeq accession codes were as follows: for Fgf2, NM_008006.1; for Adamts4, NM_172845.1; for
Adamts5, NM_011782.1; for Mmp13, NM_008607.1; for Timp1,
NM_011593.2; and for Timp3, NM_011595.2.
Pharmacologic rescue of Fgf2–/– mice. Recombinant
FGF-2 (R&D Systems, Minneapolis, MN) was administered
subcutaneously at a dosage of 1 ␮g every other day, beginning
10 days before surgical OA induction and continuing until the
time when mice were killed (2 or 4 weeks after surgery).
Control animals were injected with an equal volume of PBS.
Statistical analysis. Nonparametric comparisons were
made using the Mann-Whitney 2-tailed U test unless specified
otherwise. Multiple comparisons were made using one-way
analysis of variance with post hoc analysis as indicated. P
values less than 0.05 were considered significant.
Colocalization of FGF-2 with perlecan in the
pericellular matrix of murine articular cartilage. We
previously demonstrated colocalization of FGF-2 and
perlecan in the pericellular matrix of porcine and human
articular cartilage, and verified this binding through in
vitro studies using surface plasmon resonance (6). Pericellular staining of FGF-2 was confirmed in articular
cartilage of Fgf2⫹/⫹ mice, where it colocalized with
perlecan as determined using confocal microscopy.
Staining for FGF-2 was absent in Fgf2–/– animals, although some nonspecific staining was apparent on the
superficial articular surface. Pericellular distribution of
perlecan was maintained in Fgf2–/– mice (Figure 1).
Increased OA in aged Fgf2–/– mice. We first
examined the appearance and characteristics of the
type II collagen between Fgf2–/– and Fgf2⫹/⫹ mice as
judged by messenger RNA (mRNA) level (Figure 2e).
Spontaneous degeneration of the articular cartilage was assessed histologically at 3, 6, and 12 months.
The articular cartilage in all 4 quadrants of the joint,
from at least 8 sections across the joint, was scored (see
Materials and Methods). To measure the severity as well
as the extent of chondral damage, the scores from the 4
quadrants were added together, and the highest 3 scores
from an individual joint were summed (summed score).
While there was no apparent spontaneous degradation
of the cartilage at age 3 months, at age 6 months both
Fgf2–/– and Fgf2⫹/⫹ mice showed incipient articular
cartilage degradation, which was significantly greater in
the Fgf2–/– mice (Figures 3a and b). By 9 months further
significant increase in disease was apparent in Fgf2–/–
mice (Figure 3a). When the summed scores of the
medial and lateral compartments were considered separately, the Fgf2–/– mouse cartilage showed severe de-
Figure 2. Basal cartilage characteristics in Fgf2⫹/⫹ and Fgf2–/– mice. a,
Converted grey-scale image of a Safranin O–stained section through
the medial compartment of a wild-type (WT) murine knee, showing
articular cartilage and growth plate cartilage delineated (original
magnification ⫻ 20). b, Proteoglycan staining in Fgf2⫹/⫹ mice (solid
bars) and Fgf2–/– mice (open bars). c, Maximum cartilage thickness in
Fgf2⫹/⫹ and Fgf2–/– mice. d, Mean cartilage thickness in Fgf2⫹/⫹ and
Fgf2–/– mice. Values in b–d are the mean and SEM (n ⫽ 4–5 joints per
group). e, Quantitative gene expression of cartilage matrix components
from the joints of 12-week-old Fgf2⫹/⫹ and Fgf2–/– mice. Horizontal
bars indicate the mean. Each point represents a single joint. NS ⫽ not
significant; KO ⫽ knockout; Col II␣(1) ⫽ type II collagen; TIMP-1 ⫽
tissue inhibitor of metalloproteinases 1; MMP-13 ⫽ matrix metalloproteinase 13.
articular cartilage of 3-month-old mice (the age at which
OA was to be induced). No differences in cartilage
thickness or intensity of Safranin O staining were observed between Fgf2–/– and Fgf2⫹/⫹ mice by histomorphometric analysis (Figures 2a–d). We also tested the
integrity of the matrix by nanoindentation, using atomic
force microscopy. No difference in mechanical properties of the matrix was detected between either genotype
(data not shown). There was also no difference in gene
expression of the principal matrix proteins aggrecan and
Figure 3. Accelerated osteoarthritis in Fgf2–/– mice with age. a,
Summed histologic scores of knee cartilage from Fgf2⫹/⫹ mice (solid
bars) and Fgf2–/– mice (open bars) at ages 3, 6, and 9 months. b,
Representative Safranin O–stained histologic sections of knee cartilage from Fgf2⫹/⫹ and Fgf2–/– mice at ages 3, 6, and 9 months. M ⫽
medial compartment; L ⫽ lateral compartment; GP ⫽ growth plate.
Bar ⫽ 200 ␮m. c, Summed histologic scores for the medial and lateral
compartments of the joints of 9-month-old Fgf2⫹/⫹ and Fgf2–/– mice.
Values in a and c are the mean and SEM (n ⫽ 5 joints per group).
Figure 4. Accelerated osteoarthritis in Fgf2–/– mice following destabilization of the medial
meniscus (DMM). a, Summed histologic scores at 2, 4, and 8 weeks following DMM surgery in
Fgf2⫹/⫹ mice (solid bars) and Fgf2–/– mice (open bars) (n ⫽ 12–15 joints per group). b,
Representative histologic appearance of Safranin O–stained whole joint sections from Fgf2⫹/⫹ and
Fgf2–/– mice that had undergone DMM surgery and from Fgf2⫹/⫹ mice following sham operation.
Boxed areas at left are shown at higher magnification at right. GP ⫽ growth plate; L ⫽ lateral
compartment; M ⫽ medial compartment. Bar ⫽ 100 ␮m. c, Summed histologic scores 2, 4, and 8
weeks following DMM surgery in the contralateral, unoperated knees of Fgf2⫹/⫹ mice (solid bars)
and Fgf2⫺/⫺ mice (open bars) (n ⫽ 6 joints per group). d, Summed histologic scores 2 weeks
following DMM surgery in Fgf2⫹/⫹ and Fgf2⫺/⫺ mice following subcutaneous injection with vehicle
(phosphate buffered saline [PBS]) or with recombinant human fibroblast growth factor 2 (rFGF2)
(n ⫽ 6 joints per group). Values in a, c, and d are the mean and SEM. NS ⫽ not significant.
generation in both compartments, while the Fgf2⫹/⫹
mouse cartilage showed severe degeneration only in the
medial compartment (Figures 3b and c). Histologic
analysis also showed that at 9 months, the medial
compartment erosion in Fgf2–/– mice, as compared with
that in WT mice, extended more deeply into the subchondral bone, at times beyond the growth plate (Figure
Increased OA in Fgf2–/– mice following surgical
destabilization of the knee. We next examined the
susceptibility of 12-week-old Fgf2⫹/⫹ and Fgf2–/– mice to
surgically induced OA, using DMM as described previously (3). DMM results in a progressive OA-like disease,
in which the articular cartilage degenerates with little or
no synovitis. We compared the summed scores from
Fgf2⫹/⫹ and Fgf2–/– mice at 2, 4, and 8 weeks following
surgery. Compared with Fgf2⫹/⫹ littermate controls,
acceleration of disease was seen in Fgf2–/– mice at all
time points examined following DMM surgery (Figure
4a). Representative histologic sections are shown from
sham-operated Fgf2⫹/⫹ mice and from DMM-operated
Fgf2⫹/⫹ and Fgf2–/– mice (Figure 4b). Sham-operated
knees (in which the joint capsule was opened but the
meniscotibial ligament was spared) from mice of either
genotype showed no cartilage damage (data not shown).
Slowly progressive cartilage damage was seen, however,
in the contralateral, unoperated knees of Fgf2–/– mice 4
and 8 weeks after DMM surgery. This damage was
significantly greater than that in Fgf2⫹/⫹ mice at 8 weeks
(Figure 4c). This was likely due to increased joint
loading through the unoperated side, because of progressive arthritis in the operated knee. These findings
indicate that the absence of FGF-2 in articular cartilage
hastens matrix breakdown and the development of OA,
under conditions of both increased physiologic (i.e., with
age, and on the unoperated contralateral side) and
pathologic (i.e., the operated side) joint loading.
Subcutaneous delivery of FGF-2 reverses the
accelerated OA phenotype of Fgf2–/– mice following
DMM surgery. Even though we had determined by
nanoindentation and histomorphometry that the cartilage of Fgf2–/– mice was similar to that of WT mice, it
was possible that susceptibility to OA in Fgf2–/– mice was
due to an intrinsic weakness in the tissue that had arisen
during development. In order to address this, we treated
Fgf2–/– mice with subcutaneous recombinant human
FGF-2. Others have achieved therapeutic dosing of
FGF-2 by subcutaneous injection (13) following detailed
kinetic studies in the mouse (14). Although no data are
available on the extent to which FGF-2 is able to
penetrate cartilage via subcutaneous administration, the
presence of detectable FGF-2 in the plasma suggests
that access to the joint is feasible. Mice were pretreated
with either subcutaneous FGF-2 or PBS from 10 days
prior to DMM surgery to 2 or 4 weeks after surgery.
Fgf2–/– mice treated with FGF-2, but not those treated
with PBS, were protected from accelerated OA 2 weeks
following surgery and showed histologic scores comparable with those of Fgf2⫹/⫹ mice treated with PBS
(Figure 4d). A trend toward protection was also seen in
mice treated for 4 weeks following surgery, but this did
not reach significance (data not shown). The ability to
reverse the susceptibility of the strain by FGF-2 treatment suggested that FGF-2 had an active chondroprotective role in mature tissue.
Increased ADAMTS-5 expression and activity in
Fgf2–/– mouse cartilage in vivo. ADAMTS-5 is the major
aggrecan-degrading enzyme in murine cartilage; mice
deficient in ADAMTS-5 are protected from surgically
induced OA as well as from inflammatory arthritis (3,4).
We extracted mRNA from joints of mice 2 weeks
following DMM or sham surgery. Adamts5 mRNA was
increased in joints of both Fgf2⫹/⫹ and Fgf2–/– mice 2
weeks after DMM surgery compared with that in shamoperated and contralateral, unoperated control joints
(Figure 5a). This increase was significantly greater in
Fgf2–/– mice than in Fgf2⫹/⫹ mice. Two other proteinase
Figure 5. Greater induction of Adamts5 mRNA and activity in Fgf2–/–
mice than in Fgf2⫹/⫹ mice following destabilization of the medial
meniscus (DMM). a, Extraction of mRNA from the knees of Fgf2⫹/⫹
and Fgf2–/– mice 2 weeks following DMM or sham surgery (Shm) or
from the unoperated contralateral joint (NO) and quantification by
real-time reverse transcriptase–polymerase chain reaction. ⴱ ⫽ P ⬍
0.05 versus sham-operated knees, by analysis of variance with Bonferroni post hoc test. Each point represents a single joint (n ⫽ 4–6 joints
per group). Horizontal bars indicate the mean. b, Immunohistochemical localization of the aggrecanase-generated neoepitope NVTEGE
stained with immunoperoxidase in the articular cartilage of Fgf2⫹/⫹
and Fgf2–/– mice either 2 weeks following DMM surgery or 2 weeks
following sham operation (Fgf2⫹/⫹ mice only). A nonimmune antibody
control is also shown for DMM-operated Fgf2⫹/⫹ mouse cartilage
(DMM ⫹/⫹ [NI]). Men ⫽ meniscus. Bar ⫽ 100 ␮m.
genes, Adamts4 and Mmp13 (a collagenase), were also
induced in knees following DMM surgery, but there was
no further increase in the Fgf2–/– mouse joints, indicating
that the expression of these proteinases during the
chondral response to joint destabilization was not influenced by endogenous FGF-2 (Figure 5a). We also
looked at regulation of the intrinsic tissue inhibitors of
metalloproteinases (TIMPs) 1 and 3. Timp1, but not
Timp3, was induced following DMM surgery, but no
difference in expression was detected between Fgf2⫹/⫹
and Fgf2–/– mouse knees (Figure 5a). In WT mice,
Table 1. Ct values for genes of interest*
Gene of
* Threshold cycle (Ct) values are listed for Adamts4, Adamts5, Mmp13,
Timp3, and GAPDH from joints subjected to destabilization of the
medial meniscus (DMM) or sham operation (Sham) as well as from
unoperated (NO) joints. Each value is an average of 3–5 samples from
experiments performed in triplicate. The Ct value denotes the cycle
number at which it is possible to measure the amplified polymerase
chain reaction product. The Ct value is inversely proportional to the
copy number of the gene of interest template; a low Ct value represents
a higher initial copy number of the gene of interest in the starting
up-regulation of Fgf2 mRNA was detected in joints
following DMM surgery (Figure 5a).
To confirm that ADAMTS-5–mediated aggrecanolysis was occurring in the articular cartilage following DMM surgery, immunohistochemical evaluation was
performed using an antibody against the aggrecanasegenerated neoepitope (NITEGE). This antibody crossreacts with the cleaved epitope of murine aggrecan
(NVTEGE) that is retained in cartilage following
ADAMTS-mediated cleavage (15). Staining of the
NVTEGE epitope was apparent in the superficial layer
of articular cartilage in both Fgf2⫹/⫹ and Fgf2–/– mouse
cartilage 2 weeks following DMM surgery, but was
significantly reduced in tissue from sham-operated animals. No staining was seen when a nonimmune antibody
was used (Figure 5b). Table 1 shows the threshold cycle
values for the ADAMTS enzymes, matrix metalloproteinase 13 (MMP-13) and TIMP-3. Interestingly, despite
a clear role for ADAMTS-5 in the course of murine OA,
the abundance of ADAMTS-5 (and ADAMTS-4)
mRNA in the joint was low compared with that of
MMP-13 and TIMP-3 mRNA.
This is the first description of an endogenous
matrix-bound growth factor acting as a chondroprotective agent in vivo; mice deficient in FGF-2 developed
accelerated OA with age or following surgical destabilization of the knee. Our earlier observation that FGF-2
inhibits IL-1–driven aggrecanolysis in human explants
(9) suggests that the chondroprotective effect of FGF-2
might be due, at least in part, to suppression of aggrecanolysis mediated by ADAMTS-5. Indeed, Adamts5
mRNA was superinduced in Fgf2–/– mice compared with
Fgf2⫹/⫹ mice 2 weeks following DMM surgery. We were
unable to visualize ADAMTS-5 protein due to lack of
suitable antibodies and the likely low abundance of the
enzyme, although we were able to demonstrate
ADAMTS-mediated aggrecanolysis in the cartilage of
both Fgf2–/– and Fgf2⫹/⫹ mice.
Regulation of ADAMTS-5 gene expression has
not been extensively studied. It was initially described as
a constitutively expressed aggrecanase in bovine (16,17)
and human (18) chondrocytes, although we and others
have demonstrated regulation by inflammatory cytokines in murine (4), bovine (19), and human (9) tissue.
The putative promoter region of the gene has predicted
binding sites for a number of transcription factors,
including NF-␬B, which are likely to be involved in gene
expression following activation of inflammatory signaling (20). It is not clear how FGF-2 influences
ADAMTS-5 gene expression in cytokine-treated cartilage explants. The mechanism by which this occurs in
vivo is likely to be yet more complicated because of the
expression and effects of FGF-2 in tissues other than
articular cartilage.
It is possible that FGF-2 was in part inhibiting
aggrecanase activity through induction of TIMP-3, one
of the 4 TIMP family members, which is a high-affinity
inhibitor of ADAMTS-4 and ADAMTS-5 (21). However, we found no evidence that FGF-2 regulated the
expression of TIMP-3 mRNA. Indeed, Timp3 mRNA
was not differentially expressed in Fgf2–/– and Fgf2⫹/⫹
mice following DMM surgery or in vitro following
stimulation of human cartilage with exogenous FGF-2 or
IL-1 (9).
It is not known what signals and which cells are
responsible for aggrecanase expression following surgi-
cal destabilization of the knee. Although there is little or
no synovitis seen in this model when cartilage degeneration is occurring (after 4 weeks), it is possible that
cytokine production within the synovium or joint cavity
drives proteinase production. It is also possible that
proteinase expression is directly induced in the chondrocyte by altered joint biomechanics. Destabilization of the
meniscus is likely to have 2 main consequences following
the initial cutting injury. The first is increased load
transmitted through the weight-bearing region of the
joint. The second is joint instability resulting in increased shear stress over the articular surfaces. It is not
known whether chondrocytes are able to sense and
respond to such changes, but our previous finding that
simple cutting of cartilage is sufficient to activate JNK
and p38 MAPK pathways and to induce inflammatory
response genes such as IL-1 in the chondrocyte (22)
lends support to such a theory.
The role of FGF-2 in cartilage has been most
studied in the growth plate, where it has an inhibitory
effect on chondrocyte proliferation. Gain-of-function
mutations in human FGF receptor 3 (FGFR-3) (23–27)
and overexpression of FGF-2 (28) or intravenous treatment of mice with recombinant FGF-2 (29) all result in
a reduction in the proliferating zone of the growth plate
and in subsequent shortening of the long bones (achondroplastic dwarfism). The absence of a developmental
phenotype in Fgf2–/– mice is thought to be due to
compensation by other FGF family members such as
FGF-18 which are present in the growth plate (30). It is
possible that Fgf2–/– mice exhibited a mild subclinical
chondrodysplasia that rendered the cartilage more susceptible to OA in adult life. However, subcutaneous
delivery of FGF-2 was able to slow arthritis in Fgf2–/–
mice to levels seen in Fgf2⫹/⫹ animals, suggesting that
accelerated OA in Fgf2–/– mice was not due to an
intrinsic matrix weakness that had arisen during development, but rather that it was due to the loss of
FGF-2–mediated suppression of matrix breakdown in
postnatal tissue.
The disparate functions of FGF-2 in pre- and
postnatal cartilage may be due to differences in receptor
expression; FGFR-3 expression is high in the growth
plate (31), but FGFR-1 and FGFR-2 are the predominant receptors in healthy mature articular cartilage
(Vincent T, Chia S-L: unpublished observations), and it
is probably through these that the anticatabolic effects of
FGF-2 are occurring. Our results suggest that other
FGFs do not compensate for the loss of FGF-2 in adult
cartilage. This may be because of the high relative
abundance of FGF-2 in articular cartilage compared
with other FGFs, and also because of the mechanical
control of its bioavailability, which ensures that FGF-2–
mediated signaling occurs during weight bearing as well
as following chondral injury (7,8).
The role of FGF-2 in tissue injury responses in
other organs has also emerged in recent years; Fgf2–/–
mice have delayed healing following excisional skin
wounding (32), and FGF-2 up-regulates neurogenesis
and protects neurons from degeneration in the adult
hippocampus after traumatic brain injury (33). These
observations identify FGF-2 as a tissue remodeling
cytokine and highlight its importance in tissue homeostasis and repair.
Finally, could loss of the chondroprotective role
of FGF-2 contribute to the development of human OA?
We have observed strong pericellular staining for perlecan and FGF-2 in OA cartilage, suggesting that their
expression is maintained in disease. It is not clear at
present whether mechanical signals are unable to activate FGF-2–mediated chondroprotection in damaged
tissue or whether anticatabolic processes are simply
overwhelmed by degradative ones in established OA.
Nonetheless, understanding the molecular basis for
these observations should enable us to harness the
properties of such anticatabolic cytokines in the future
management of OA.
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. Vincent 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. Chia, Sawaji, Inglis, Saklatvala, Vincent.
Acquisition of data. Chia, Burleigh, McLean, Inglis.
Analysis and interpretation of data. Chia, Sawaji, Saklatvala, Vincent.
1. Malfait AM, Liu RQ, Ijiri K, Komiya S, Tortorella MD. Inhibition
of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in
osteoarthritic cartilage. J Biol Chem 2002;277:22201–8.
2. Song RH, Tortorella MD, Malfait AM, Alston JT, Yang Z, Arner
EC, et al. Aggrecan degradation in human articular cartilage
explants is mediated by both ADAMTS-4 and ADAMTS-5. Arthritis Rheum 2007;56:575–85.
3. Glasson SS, Askew R, Sheppard B, Carito B, Blanchet T, Ma HL,
et al. Deletion of active ADAMTS5 prevents cartilage degradation
in a murine model of osteoarthritis. Nature 2005;434:644–8.
4. Stanton H, Rogerson FM, East CJ, Golub SB, Lawlor KE, Meeker
CT, et al. ADAMTS5 is the major aggrecanase in mouse cartilage
in vivo and in vitro. Nature 2005;434:648–52.
5. Vincent T, Saklatvala J. Basic fibroblast growth factor: an extracellular mechanotransducer in articular cartilage? Biochem Soc
Trans 2006;34:456–7.
6. Vincent TL, McLean CJ, Full LE, Peston D, Saklatvala J. FGF-2
is bound to perlecan in the pericellular matrix of articular cartilage, where it acts as a chondrocyte mechanotransducer. Osteoarthritis Cartilage 2007;15:752–63.
Vincent TL, Hermansson MA, Hansen UN, Amis AA, Saklatvala
J. Basic fibroblast growth factor mediates transduction of mechanical signals when articular cartilage is loaded. Arthritis Rheum
Vincent T, Hermansson M, Bolton M, Wait R, Saklatvala J. Basic
FGF mediates an immediate response of articular cartilage to
mechanical injury. Proc Natl Acad Sci U S A 2002;99:8259–64.
Sawaji Y, Hynes J, Vincent T, Saklatvala J. Fibroblast growth
factor 2 inhibits induction of aggrecanase activity in human
articular cartilage. Arthritis Rheum 2008;58:3498–509.
Zhou M, Sutliff RL, Paul RJ, Lorenz JN, Hoying JB, Haudenschild CC, et al. Fibroblast growth factor 2 control of vascular tone.
Nat Med 1998;4:201–7.
Clements KM, Price JS, Chambers MG, Visco DM, Poole AR,
Mason RM. Gene deletion of either interleukin-1␤, interleukin1␤–converting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee osteoarthritis in mice
after surgical transection of the medial collateral ligament and
partial medial meniscectomy. Arthritis Rheum 2003;48:3452–63.
Pastoureau P, Leduc S, Chomel A, De Ceuninck F. Quantitative
assessment of articular cartilage and subchondral bone histology in
the meniscectomized guinea pig model of osteoarthritis. Osteoarthritis Cartilage 2003;11:412–23.
Jin K, LaFevre-Bernt M, Sun Y, Chen S, Gafni J, Crippen D, et al.
FGF-2 promotes neurogenesis and neuroprotection and prolongs
survival in a transgenic mouse model of Huntington’s disease. Proc
Natl Acad Sci U S A 2005;102:18189–94.
Wagner JP, Black IB, DiCicco-Bloom E. Stimulation of neonatal
and adult brain neurogenesis by subcutaneous injection of basic
fibroblast growth factor. J Neurosci 1999;19:6006–16.
Lark MW, Bayne EK, Flanagan J, Harper CF, Hoerrner LA,
Hutchinson NI, et al. Aggrecan degradation in human cartilage:
evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J Clin Invest
Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR,
et al. Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol Chem 1999;274:
Tortorella MD, Malfait AM, Deccico C, Arner E. The role of
ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in
a model of cartilage degradation. Osteoarthritis Cartilage 2001;9:
Koshy PJ, Lundy CJ, Rowan AD, Porter S, Edwards DR, Hogan
A, et al. The modulation of matrix metalloproteinase and ADAM
gene expression in human chondrocytes by interleukin-1 and
oncostatin M: a time-course study using real-time quantitative
reverse transcription–polymerase chain reaction. Arthritis Rheum
19. Little CB, Hughes CE, Curtis CL, Jones SA, Caterson B, Flannery
CR. Cyclosporin A inhibition of aggrecanase-mediated proteoglycan catabolism in articular cartilage. Arthritis Rheum 2002;46:
20. Thirunavukkarasu K, Pei Y, Wei T. Characterization of the human
ADAMTS-5 (aggrecanase-2) gene promoter. Mol Biol Rep 2007;
21. Kashiwagi M, Tortorella M, Nagase H, Brew K. TIMP-3 is a
potent inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase
2 (ADAM-TS5). J Biol Chem 2001;276:12501–4.
22. Gruber J, Vincent TL, Hermansson M, Bolton M, Wait R,
Saklatvala J. Induction of interleukin-1 in articular cartilage by
explantation and cutting. Arthritis Rheum 2004;50:2539–46.
23. Chen L, Adar R, Yang X, Monsonego EO, Li C, Hauschka PV, et
al. Gly369Cys mutation in mouse FGFR3 causes achondroplasia
by affecting both chondrogenesis and osteogenesis. J Clin Invest
24. Rousseau F, Bonaventure J, Legeai-Mallet L, Pelet A, Rozet JM,
Maroteaux P, et al. Mutations in the gene encoding fibroblast
growth factor receptor-3 in achondroplasia. Nature 1994;371:
25. Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ,
Bocian M, et al. Mutations in the transmembrane domain of
FGFR3 cause the most common genetic form of dwarfism,
achondroplasia. Cell 1994;78:335–42.
26. Wang Y, Spatz MK, Kannan K, Hayk H, Avivi A, Gorivodsky M,
et al. A mouse model for achondroplasia produced by targeting
fibroblast growth factor receptor 3. Proc Natl Acad Sci U S A
27. Webster MK, Donoghue DJ. Constitutive activation of fibroblast
growth factor receptor 3 by the transmembrane domain point
mutation found in achondroplasia. EMBO J 1996;15:520–7.
28. Coffin JD, Florkiewicz RZ, Neumann J, Mort-Hopkins T, Dorn
GW II, Lightfoot P, et al. Abnormal bone growth and selective
translational regulation in basic fibroblast growth factor (FGF-2)
transgenic mice. Mol Biol Cell 1995;6:1861–73.
29. Nagai H, Tsukuda R, Mayahara H. Effects of basic fibroblast
growth factor (bFGF) on bone formation in growing rats. Bone
30. Liu Z, Lavine KJ, Hung IH, Ornitz DM. FGF18 is required for
early chondrocyte proliferation, hypertrophy and vascular invasion
of the growth plate. Dev Biol 2007;302:80–91.
31. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. Fibroblast
growth factor receptor 3 is a negative regulator of bone growth.
Cell 1996;84:911–21.
32. Ortega S, Ittmann M, Tsang SH, Ehrlich M, Basilico C. Neuronal
defects and delayed wound healing in mice lacking fibroblast
growth factor 2. Proc Natl Acad Sci U S A 1998;95:5672–7.
33. Yoshimura S, Teramoto T, Whalen MJ, Irizarry MC, Takagi Y,
Qiu J, et al. FGF-2 regulates neurogenesis and degeneration in the
dentate gyrus after traumatic brain injury in mice. J Clin Invest
Без категории
Размер файла
1 108 Кб
degradation, suppressor, growth, osteoarthritis, intrinsic, adamts, chondroprotective, factors, murine, agenti, cartilage, delayu, fibroblasts
Пожаловаться на содержимое документа