Barbe, U.

Bausero, P.

Berenbaum, F.

Blaise, R.

Brou, C.

Corvol, M.

Mahjoub, M.

Rannou, F.

Salvat, C.

Savouret, J-F.

Sellami, S.

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233 NOTCH PATHWAY: ITS IMPLICATION IN ARTICULAR CHONDROCYTE DEDIFFERENTIATION COULD BE MEDIATED BY MMP13 

S108 Osteoarthritis and Cartilage Vol. 16 Supplement 4
In contrast, cytomegalovirus promoter-driven Sp1 overexpression further
enhanced Il-1-induced ADAMTS-4 expression. Expression of constitutive
ADAMTS-5 was not affected by any of the agents.
Conclusions: These results provide pharmacological and genetic evi-
dence for the importance of Sp1 in ADAMTS-4 gene regulation by Il-1.
232 CHONDROITIN SULFATE MODULATES THE
MITOCHONDRIAL ACTIVITY OF HUMAN ARTICULAR
CHONDROCYTES
F.J. Blanco1, M.J. Lo´pez-Armada1, B. Cillero-Pastor1, B. Carame´s1,
E. Montell2, J. Verge´s2, F. Galdo1. 1Osteoarticular and Aging Research
Laboratory. Biomedical Research Center. INIBIC-Hospital Universitario
Juan Canalejo, Corun˜a, SPAIN, 2Laboratorios Bioiberica Farma,
Barcelona, SPAIN
Purpose: Human articular chondrocytes originating from osteoarthritic
cartilage present an altered mitochondrial activity due to an alteration in
the mitochondrial membrane potential. This mitochondrial alteration can
cause an increment of free radicals which may lead to a rise in the number
of dead chondrocytes in the cartilage and a decrease in protoglycans in
the extracellular matrix.
Objectives: To analyse the effect of Chondroitin Sulfate (CS) on the
mitochondrial activity of osteoarthritic chondrocytes.
Methods: Human articular chondrocytes were isolated from osteoarthritic
cartilage obtained by prosthetic surgery. Chondrocytes were cultivated
and stimulated with 10 ng/ml TNFa, during different time intervals (6,
12, 24 and 48 hours), which caused depolarisation of the mitochondrial
membrane. This depolarization was then measured using ﬂow cytometry.
Subsequently chondrocytes were stimulated with different concentrations
of CS (10, 50, 100, 150, 200 and 500mg/ml) at different time intervals
(6, 12, 24 and 48 hours), which enables us to analyze the capacity that
the drug has in slowing down mitochondrial depolarization induced by
TNF. Effects of CS on mitochondrial function were also validated via ATP
synthesis.
Results: The concentrations of CS that were used did not alter the
membrane potential at 6, 12 and 24 hours. However CS concentrations
greater then 50 mg/ml at 48 hours showed a signiﬁcant increase in the
red/green ratio suggesting that CS induces a state of mitochondrial hyper-
polarisation in osteoarthritic chondrocytes (50mg/ml = 135%; 100mg/ml =
148%; 150mg/ml = 156%; 200mg/ml = 164% and 500mg/ml = 171% vs
control 100%). TNFa induced a signiﬁcant reduction in the red/green ratio
at 12 hours = 41%; 24 hours = 41% and 48 hours = 39%. At 6 and
12 hours CS did not decrease the effect of TNFa on the mitochondrial
membrane potential. However CS reduced the effect of TNFa on the
mitochondrial depolarization at 24 and 48 hours. At 24 hours the effective
concentrations used were: CS 150 mg/ml = 58%; 200 mg/ml = 64% and
500mg/ml = 64% vs. TNFa 41%. At 48 hours the effective concentrations
used were: CS 10 mg/ml = 58%; 50 mg/ml = 60%; 100 mg/ml = 60%;
150mg/ml = 65%; 200 mg/ml = 66% and 500 mg/ml = 73% vs. control
39%). CS at a 200mg/ml concentration increased the basal synthesis of
ATP produced by articular chondrocytes in cultures (Control = 0.55 vs.
200ml = 0.65).
Conclusions: CS concentrations greater than 50mg/ml and at 48 hours
increases the red/green ratio, reducing the effect of TNFa on the mi-
tochondrial depolarisation and increases the synthesis of ATP. All this
data could have a relation to the anti apoptotic effects produced in
chondrocytes described by CS.
233 NOTCH PATHWAY: ITS IMPLICATION IN ARTICULAR
CHONDROCYTE DEDIFFERENTIATION COULD BE
MEDIATED BY MMP13
M. Mahjoub1, R. Blaise2, C. Salvat2, U. Barbe2, C. Brou3, M. Corvol4,
J-F. Savouret4, F. Rannou4, S. Sellami1, P. Bausero2, F. Berenbaum2.
1La Rabta Hospital, Tunis, TUNISIA, 2CNRS UMR 7079, Universite´
Pierre et Marie Curie, Paris, FRANCE, 3CNRS URA 2582, Institut
Pasteur, Paris, FRANCE, 4INSERM UMR-747, Universite´ Paris
Descartes, Paris, FRANCE
Purpose: Members of Notch family receptors act as membrane-tethered
transcription factors that are tightly associated with cell fate decisions
while controling differentiation, proliferation and apoptosis.
Upon ligand binding, Notch molecules undergo proteolytic cleavages by
the g-secretase complex that lead to the activation of the transcription
of Notch target genes. Although, the molecular mechanism of Notch
activation is well characterized, further analyses in an appropriate cel-
lular context will provide new insight into several diseases such as
osteoarthritis (O.A). We therefore examined correlations between Notch
pathway activation and regulation of metalloproteases (MMPs) as well as
chondrocyte markers expressions, during phenotypic modulation process
of chondrocytes, as it occurs in O.A.
Methods:We used serial monolayer primary cultures of immature murine
articular chondrocytes (MACs) as an in-vitro model of the events which
occur during phenotypic modulation. MACs were cultured with or without
a Notch inhibitor, and transfected with different notch expressing vectors.
Cells were used for quantitative RT-PCR and immunoblotting analyses to
characterize Notch molecules and to determine their impact on chondro-
cytes markers and MMPs expressions.
Results: Differentiation chondrocyte markers (Type II collagen, Aggre-
can) are down regulated during passages. Inversely, the dedifferentiation
marker, type I collagen is up regulated during MACs culture.
Concomitantly, Notch ligands and target genes are up regulated. In the
presence of a g-secretase inhibitor, DAPT, we observed a diminution
of type II collagen degradation, but without variation of Col2a1 gene
expression. In order to elucidate type II collagen protection mechanisms,
we studied the expression of MMPs with or without Notch inhibitor.
We demonstrated that expression of MMP13 is regulated in a Notch-
dependant manner while MMP2 expression is not modiﬁed. Using con-
stitutively active forms of Notch1 receptor or a dominant negative form
of the Notch1 transcriptional co-activator (CSL), we demonstrate a direct
effect of Notch in MMP13-dependent Type II collagen degradation.
Conclusions: These data suggest that Notch signalling could be critical
for the MMP13-dependent degradation of type II collagen during pheno-
typic modulation of chondrocytes occurring during O.A.
234 CARTILAGE CALCIFICATION AND BMP SIGNALING IN
EARLY HAND OSTEOARTHRITIS
M.H. Stradner1, M. Asslaber2, H. Angerer1, D. Setznagl1, F.C. Fu¨rst1,
H. Denk2, W.B. Graninger1. 1Department of Rheumatology, Medical
University of Graz, Graz, AUSTRIA, 2Institute of Pathology, Medical
University of Graz, Graz, AUSTRIA
Purpose: The aim of this project was to reveal basic morphological and
biochemical changes in early hand osteoarthritis of the proximal inter-
phalangeal (PIP) joint.
Methods:We explanted affected and normal PIP joints of human dissect-
ing room cadavers with Bouchard’s nodes according to the rules of our
local ethic committee. The joint specimens were ﬁxed in formalin, decal-
ciﬁed in EDTA and embedded in parafﬁn. Sections were stained Safranin
O and H&E. Furthermore immunohistochemistry for bone morphogenetic
protein (BMP)-2, -4, and -6 was performed.
Results: Histological examination of macroscopically normal PIP joints
from patients with manifest Bouchard’s nodes of other PIP joints revealed
tidemark duplication proteoglycan loss and cartilage calciﬁcation. This
ﬁnding was especially prominent at the dorso-lateral edges of both the
caput of the proximal phalanx and the basis of the phalanx media. At
these sites the remaining cartilage lining in some sections was reduced
to less than 80mm. Using immunohistochemistry we found marked BMP-6
expression in both articular cartilage and adjacent ligament tissue. BMP-2
was expressed to a lesser content in cartilage, whereas BMP-4 was not
detected at all.
Conclusions: We suggest that cartilage calciﬁcation of the dorso-lateral
edges of the PIP joints is the ﬁrst morphological step in the pathogenesis
of Bouchard’s nodes. As BMP signalling is associated with cartilage os-
siﬁcation, the ﬁnding of BMP expression in the vicinity of these sites may
imply a role of BMP signalling in the development of hand osteoarthritis.
235 PROLONGED EXPOSURE TO LEPTIN INDUCES A
CATABOLIC EFFECT IN NORMAL AND OSTEOARTHRITIC
CARTILAGE
R. Dryer-Minnerly, V. Stojanovic-Susulic. Centocor, Radnor, PA, USA
Purpose: Obesity is a major risk factor in development and progression
of Osteoarthritis (OA). Although there is an established link between OA
and the biomechanical factors associated with obesity, there undoubtedly
exists a metabolic component that inﬂuences the disease in the non-
weight bearing joints, such as hands. The correlation of leptin levels with
body mass index (BMI), coupled with ﬁndings of increased leptin in syn-
ovial ﬂuid of OA patients, suggests a role of this cytokine-like, pleiotropic
hormone in the progression of OA. In this study, we investigated the


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233 NOTCH PATHWAY: ITS IMPLICATION IN ARTICULAR CHONDROCYTE DEDIFFERENTIATION COULD BE MEDIATED BY MMP13

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