Download cartilage mechanical injury and co-culture with joint capsule tissue

CARTILAGE MECHANICAL INJURY AND CO-CULTURE WITH JOINT CAPSULE TISSUE INCREASE
ABUNDANCE OF ADAMTS-5 PROTEIN AND AGGRECAN G1-NITEGE PRODUCT
+*Lee, J H; **Bai, Y; §Flannery, CR; **Sandy, JD; **Plaas, A; *Grodzinsky, AJ
*MIT, Cambridge, MA; §Wyeth Research, Cambridge, MA; **USF, Tampa, FL
[email protected]
INTRODUCTION: Joint injury in young adults leads to increased risk
for development of osteoarthritis later in life [1]. Mechanical overload of
cartilage explants [2] and co-culture with joint capsule tissue [3] have
been established as in vitro models for the study of joint injury with the
goal of elucidating processes of matrix degradation relevant to disease
progression. These models of joint injury have been shown previously to
increase gene expression of matrix degrading enzymes without affecting
expression of matrix proteins [4,5]. The objectives of this study were to
(1) determine if protein levels of aggrecanase-1 (ADAMTS-4) and
aggrecanase-2 (ADAMTS-5) increase in response to mechanical injury
of cartilage or co-culture with joint capsule tissue, and (2) determine if
aggrecan is cleaved by aggrecanase activity in these injury models.
METHODS: Tissue Harvest & Injury Models: Cartilage disks (3 mm
diam, 1 mm think) from the middle zone of the femoropatellar groove
and pieces of joint capsule tissue (~5 mm diam) were harvested from
knee joints of 1-2 week old calves and maintained separately in medium
(DMEM + 10% FBS) for two days to allow equilibration. On day 2,
location-matched cartilage disks were either maintained in free swelling
culture, mechanically injured (50% strain compression ramp at 1mm/s
followed by immediate release), co-cultured with joint capsule tissue, or
both injured and co-cultured. Immunohistochemistry: On days 4 and 16,
cartilage and joint capsule tissue samples were fixed in formalin,
paraffin embedded, sectioned, stained with anti-ADAMTS-4 (JSCVMA)
or anti-ADAMTS-5 (JSCKNG) antibodies, and counterstained with
Methylgreen. Proteinase K treatment was used for epitope retrieval.
Aggrecan Western Blotting: On day 16, aggrecan was extracted from
cartilage disks in 4M GuHCl + proteinase inhibitors followed by ethanol
precipitation and digestion of GAGs (chondroitinase ABC, keratanase II,
endo-β-galactosidase). Reduced samples were run on 4-15 % gradient
Tris-HCl gels, transferred to nitrocellulose, and blotted with anti-G1
antibody or anti-NITEGE antibody (detects neo-epitope resulting from
aggrecanase cleaveage at Glu 373 of aggrecan core protein). Antibody
binding was detected by chemiluminescence.
RESULTS: Aggrecanase immunostaining: Increased abundance of
ADAMTS-5 was observed in cartilage tissue explants 4 days after
injurious mechanical compression and following 4 days of co-culture
with joint capsule tissue (Fig. 1). Increased staining for ADAMTS-5
continued through 16 days following injury and following injury + coculture; levels returned to control by day 16 in cartilage co-cultured with
joint capsule but not injured (Fig. 1). Staining predominantly appeared
to be associated with chondrocytes rather than in the extracellular
matrix. Staining for ADAMTS-4 was low or absent at all time points
with no discernible difference seen between experimental conditions
(data not shown). Cleavage of Aggrecan: Accumulation of aggrecanasegenerated cleavage products in cartilage explants was observed
following injury, co-culture with joint capsule, and the combination of
injury + co-culture (Fig. 2). The cleavage site was confirmed using an
antibody specific to the neo-epitope generated by cleavage at the Glu373Ala374 bond in the aggrecan core protein (data not shown). Analysis of
conditioned culture medium for aggrecan fragments by Western blotting
was complicated by release of G1 and NITEGE reactive proteins from
the joint capsule tissue when cultured alone (data not shown). This
release from bovine joint capsule alone, which is consistent with
observations of other investigators [6], made analysis of the co-culture
samples problematic in determining the source (cartilage or joint
capsule) of proteins in the culture medium. No increase was observed in
cleavage of aggrecan at the MMP site (VDIPES) in the IGD following
injury or co-culture through 16 days in culture (data not shown).
DISCUSSION: Mechanical injury and co-culture with joint capsule
tissue resulted in increased abundance of ADAMTS-5 in cartilage
explants and cleavage at the aggrecanase site in the interglobular domain
(IGD) of aggrecan. In addition, we observed previously that gene
expression of ADAMTS-5 increased 40-fold while ADAMTS-4
increased only 2-3-fold following injury alone [4]. During co-culture,
gene expression of ADAMTS-4 and ADAMTS-5 were both upregulated
~6-fold [4,5]. ADAMTS-5 protein is also present in human arthritic
synovial tissue [7] and was recently shown to be the primary
aggrecanase responsible for cartilage degradation in two mouse models
of arthritis [8,9]. The present data supports the idea that ADAMTS-5
(and not ADAMTS-4) is also responsible for aggrecanolysis in these
bovine cartilage injury models. This appears to contrast with the finding
that ADAMTS-4 protein and MT4MMP are increased in abundance and
appear to be responsible for aggrecanolysis in bovine cartilage explants
treated with interleukin-1 [10]. Ongoing analyses of both tissue and
medium compartments with multiple quantitative assays (ELISA),
determination of the activation status of the proteinases present, and
siRNA strategies are focused on identifying definitively the relative
importance of the different aggrecanases (ADAMTS-1,4,5,8,9,15) in
cartilage degradation in different species and different biological
environments.
day 4
day 16
Control
Injury
Co-culture
Injury +
Co-culture
Figure 1: Cartilage injury and co-culture with joint capsule result in an
increase in ADAMTS-5 in the tissue.
Control
Injury
Co-culture
Inj+Co-cult
80 kD
60 kD
Figure 2: Cartilage injury and co-culture with joint capsule cause
accumulation of aggrecanase generated aggrecan fragments in the tissue
stained with an antibody to the G1 domain of aggrecan.
REFERENCES: [1] Gelber+, Ann Intern Med 133:3211-8, 2000. [2]
Chen+, JOR 17:870-9. 1999. [3] Jubb+, Arth and Rheum 23:545-55,
1980. [4] Lee+, Arth and Rheum 52:2386-95, 2005. [5] Lee+, Trans
ORS 51st #1032, 2005. [6] Yutani+, J Bone Miner Metab 17:7-10, 1999.
[7] Vankemmelbeke+, Eur J Biochem 268:1259-69, 2001. [8] Glasson+,
Nature 434:644-648, 2005. [9] Stanton+, Nature 434:648-652, 2005.
[10] Patwari+, Osteoarth and Cart 13:269-77, 2005.
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 0214