Akagi, R.

Akatsu, Y.

Endo, J.

Fukawa, T.

Hoshi, H.

Katsuragi, J.

Mukoyama, S.

Muramatsu, Y.

Takahashi, K.

Takahisa, S.

Watanabe, A.

Yamaguchi, S.

Yamamoto, Y.

Elsevier - Publisher Connector 

Abstracts / Osteoarthritis and Cartilage 22 (2014) S57–S489S144We have taken advantage of ENCODE consortium which provides
information about tissue-speciﬁc histone modiﬁcations, particularly
enrichment of mono-methylation of histone H3 lysine 4 (H3K4me1)
and acetylation of histone H3 lysine 27 (H3K27Ac) associated with
active enhancer regions to explore the presence of novel enhancers in
the Acan gene. We selected 8 cis acting sequences including the pre-
viously described enhancer at -11kb (Han and Lefebvre, 2008), based on
evolutionarily conserved sequences and chromatin modiﬁcation.
Results: We cloned all eight isolated regions upstream of the silent
hsp68 minimal promoter and the lacZ reporter gene, We show that four
of these sequences express in cartilage in transgenic mice at E15.5.
Three of these are new enhancers that are comparable in their temporal
and spatial expression to that already described enhancer at -11kb.
Moreover, we show that these sequences can differentiate between
chondrocytes and other aggrecan expressing cells.
Some of these enhancers contain potential binding sites for Sox9,
consistent with its described role as a regulator of Acan expression.
Conclusions: We have made several lines of several nocel enhancers in
the Acan gene and we are investigating their role in maintenance of
adult cartilage. The requirement for multiple enhancers are discussed
and potential use of these sequences to target speciﬁc tissues is being
investigated.
234
COMPARATIVE ANALYSIS OF GENE EXPRESSION BETWEEN
CARTILAGE AND MENISCI IN EARLY-PHASE EXPERIMENTAL
OSTEOARTHRITIS
J. Endo, Sr., S. Takahisa, A. Watanabe, S. Yamaguchi, R. Akagi,
Y. Muramatsu, S. Mukoyama, Y. Akatsu, J. Katsuragi, T. Fukawa,
Y. Yamamoto, H. Hoshi, K. Takahashi. Chiba Univ., Chiba, Japan
Purpose: Pathogenesis of knee osteoarthritis (OA) is still largely
unknown but secondary OA gaining much interest in terms of patho-
genesis and intervention. Primal initial trauma such as ligament tear,
resulting in joint instability, unnatural articulation, causes eventual
development of histopathologic abnormality as secondary OA. Some
researchers stressed the importance of meniscal degeneration in OA
development. Whether the association between meniscal and cartilage
degeneration in OA is purely due to the mechanical property of the
meniscus, or whether meniscal cells secrete enzymes and inﬂammatory
mediators that contribute to degeneration of other joint tissues, are
unclear. It would not be too much to say the integrity of the meniscus is
vital in maintaining healthy articular cartilage and prevention of
degenerative changes in the knee. Despite the clear link between
meniscal deterioration and the development of OA, there have been
relatively few studies investigating the molecular mechanisms of
meniscal degeneration compared with cartilage. In this study, we
analyzed the gene expression using microarray technique for the pur-
pose of researching OA pathogenesis. We focus on evaluating molecular
changes in very early phase of OA in ACLT in rats in order to further our
understanding of the relationship between cartilage and meniscal
degradation.
Methods: OA was surgically induced in 10 week old male Wister rats
by ACLT surgery. Articular cartilage from the femoral condyles and
bilateral menisci were separately dissected of 6 ACLT and 6 sham
operated rats. Each specimen composed of 3 knees was pooled for
averaging and yielding enough RNA for subsequent analyses. Con-
sequently, total of 8 RNA samples (2 ACLT and 2 sham femoral carti-
lage, 2 ACLT and 2 sham medial menisci) were extracted. Affymetrix
3’GeneChips arrays were used to assess genome-wide changes in gene
expression. Probe sets were ﬁltered using the criterion of a minimum
1.5-fold change in differential gene expression between ACLT operated
and sham operated in both cartilage and meniscus, respectively.
Subsequently, the genes over 1.5-fond change were analyzed using the
Database for Annotation, Visualization and Integrated Discovery
(DAVID) for cluster analysis (http://david.abcc.ncifcrf.gov/). Five rats in
each group were underwent the same surgical protocol and sac-
rigﬁced 3 weeks after surgery for histological and immunohis-
tochemical analysis.
Results: Histologically, 3 weeks after ACLT operated rats showed
slight ﬁbrillation in bilateral surface of cartilage and menisci. The
proteoglycan content appeared almost normal in ACLT operated rats
since no loss of Safranin O staining was observed comparing to shamoperated rats in both of cartilage and menisci. Of the 36,685 tran-
scripts detectable by microarray, 641 (1.74%) and 651 (1.77%) tran-
scripts in ACLT cartilage, 1439 (3.92%) and 1773 (4.83%) transcripts in
ACLT menisci were up-regulated by more than 1.5 fold change
comparing to sham operated, respectively. Cluster analysis using
DAVID showed genes related to OA were found in ACLT menisci such
as response to stimulus, angiogenesis, and apoptosis. Representative
proteases including Adamts1,2,3,4, Mmp1,2,12,13,14,19, and extra
cellular matrix genes including Versican(Vcan), lumican (Lum), syn-
decan 1 (Sdc1), and prostaglandin endoperoxide synthase2 (Ptgs2)
were up-regulated in menisci but were not up-regulated in cartilage.
Expression of type II collagen (Col2a1) was not affected in both car-
tilage and menisci in ACLT operated rats as well as sham operated
rats. Immunohistochemical analysis revealed that MMP-13, Adamts-4
and type II collagen-related neoepitope (C2C) were present in bilat-
eral menisci of ACLT operated rats, however no staining was found in
cartilage.
Conclusions: We evaluated molecular changes in very early phase
of OA by microarray analysis in terms of the relationship between
cartilage and meniscal degradation in surgically-induced OA models
in rats. It has been unclear in the previous studies that meniscal
and cartilage degeneration in OA is purely due to the mechanical
property of the menisci, or cellular reaction of meniscal cells where
secretion of enzymes or inﬂammatory mediators that contributes to
degeneration of other joint tissues. Our results indicated that
molecular changes occurred in menisci preceded those occurred in
cartilage in very early phase of surgically-induced OA models. And
this suggested the possibility of interventions targeting menisci in
early phase of OA might be a new strategy to prevent OA
development.
d Cartilage 22 (2014) S57–S489 S145Abstracts / Osteoarthritis anFig. 1. Ă235
STRAIN DIVERSITY OF ARTICULAR CARTILAGE DEGENERATION
INDUCED BY OVARIECTOMY AND FORCED RUNNING IN MICE
J. Yamada y, K. Tsuji z, K. Miyatake y, Y. Matsukura y, A. Kahaer y,
I. Sekiya x, T. Muneta y. yDept. of joint surgery and sports medicine Tokyo
Med.&dental Univ., Tokyo, Japan; zDept. of Cartilage Regeneration Tokyo
Med.&dental Univ., Tokyo, Japan; xCtr. for stem Cell and Regenerative
Med. Tokyo Med.&dental Univ., Tokyo, Japan
Purpose: Generally, osteoarthritis (OA) is a multifactorial disease with
factors such as humoral factors, mechanical stress and racial disparities. In
mice, it is a possibility that the different susceptibility to osteoarthritis due
to differences in strains. We previously reported a non-invasive exper-
imentalOAmodel, inwhichwe showed that synergistic effects of extensive
treadmill exercise and ovariectomy (OVX) on articular cartilage degener-
ation in Balb/c mice(refer Miyatake OARSI2012). Histological and immu-
nohistochemical analysis indicated that exercise and OVX synergistically
induced synovitis. In this study, to examine the effect of racial and ethnic
disparities on articular cartilage homeostasis in mice, we employed the
sameexperimentalprotocol andexaminedarticular cartilagedegeneration
in C57BL/6Jmice.We investigated the differences between the two groups
Methods: We employed the same protocol with previously reported.
Brieﬂy, 28 female C57BL/6J mice (8 weeks) were randomly divided into
OVXor SHAMgroups.Moreoverwedivided them into Cage orRungroups.
Run groups were subjected to treadmill exercise for 6 weeks for :60km.
While Cagegroupswere left in cage ad libitum.After 6weeks,both left and
right knee joints were harvested. Left knee joints were sliced into 5mm-
serial sagittal sections for histology and immunohistochemistry. To eval-
uate the loss of proteoglycan from articular cartilage in safranin-O stain,
we developed modiﬁed Mankin scoring system. To further compare the
severity of articular cartilage damage between the four groups, we ana-
lyzed type II collagen expression in articular cartilage. H&E slides were
used to evaluate synovial activation by scoring thickening of the synovial
lining and cellular inﬂux into joint cavity and synovium. Further we
evaluated macrophage inﬁltration in F4/80 stain. Right knee joints were
subjected for mCTanalyses. Bothmetaphysial and epiphyseal bone volumeper tissue volume (BV/TV) were calculated using Tri/3D-BONE software.
We compared the results of Balb/c and C57BL/6J mice.
Results: Safranin-O dyeability of articular surface was quite similar
between the 4 groups (Fig.1a). Modiﬁed Mankin score indicated no sig-
niﬁcant difference between the 4 groups (Fig.1b). To further compare the
severity of articular cartilage damage, we analyzed type II collagen
expression in articular cartilage (Fig.1c). As shown in the ﬁgure, type II
collagen expression in both femoral and tibial articular cartilage was
comparable between the 4 groups. Fig.2a shows H/E staining of sagittal
sectionsof theknee joint. The synoviumbecame thickenedandadensityof
cells was high in SHAMþRun, OVXþCage and OVXþRun groups. The
synovitis score of SHAMþRun, OVXþCage and OVXþRun groups were
signiﬁcantly higher than that of SHAMþCage group in Fig.2b. As shown in
Fig.2a, F4/80 immunostaining of the joint tissue revealed that synovial
hyperplasia and macrophage inﬁltration into synovial membrane were
signiﬁcantly enhanced in the SHAMþRun and OVXþRun groups(black
arrows). Micro CT pictures showed no apparent alteration in the bone
shape and osteophyte formation between the four experimental groups
(Fig.3a). In parallel with previous reports, we observed signiﬁcant reduc-
tion in BV/TV in OVXþCage group in epiphysis (Fig.3b). Forced running for
6weeks partially reversed BV/TV in the OVXmice (Fig.3b,3c).In this study,
the effects of exercise andOVXonbonehomeostasiswere on the same line
between the two mice lines.But we showed no synergistic effects on
extensive treadmill exercise andOVXonarticular cartilage degeneration in
C57BL/6J mice. We investigated the differences between the Balb and C57
mice. Firstly, we focused on the difference of bone parameters. The BV/TV
of Balb mice was higher 1.27-fold as compared to that of C57 mice in
metaphysis aswithprevious reports.Moreover the subchondralboneplate
of Balb mice was signiﬁcantly higher 1.04-fold as compared to that of C57
mice in only OVXþRun group. The difference in bone parameters of both
mice may affect some on the phenotype of articular cartilage. Secondary,
we noted the inﬂuence of the difference of inﬂammatory response. The
synovitis score of each groupwashigher inBalbmice about 1.07w1.90 fold
as compared to thatof C57mice. Further the F4/80 score ofOVX-Rungroup
washigher inBalbmice2.14-fold ascomparedwithC57mice. Thedifferent
sensitivity in inﬂammatory response in synovial tissues may have some
impacts on articular cartilage.
Conclusions: Our experimental model suggests that cartilage metab-
olism is partially dependent on the strain differences.


Comparative analysis of gene expression between cartilage and menisci in early-phase experimental osteoarthritis 

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Comparative analysis of gene expression between cartilage and menisci in early-phase experimental osteoarthritis

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