Brooks, C.

Fang, F.

Jordan, J.M.

Kraus, V.B.

Renner, J.B.

Stabler, T.

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Poster Presentations – Biomarkers S59
formation), TRACP (osteoclast number), and GAG release (cartilage
turnover). Overall cell viability was monitored using the dye Alamar Blue.
Passive release from metabolically inactive femur heads was measured
as background.
Results: Stimulation of the femur heads with RANKL, PTH, Il-1a and
OSM + TNF-a led to an increase in CTX-I release. Adding GM6001 to
OSM + TNF-a abrogated the release of CTX-I. Femur heads stimulated
with Il-1a, OSM + TNF-a and OSM + TNF-a + GM6001 induced an
increase in sGAG release. CTX-II release was increased by RANKL, PTH,
Il-1a, OSM + TNF-a and IGF-I. CTX-II release from the OSM + TNF-
a condition was abrogated when treated with GM6001. The osteoclast
marker TRACP increased when stimulated by RANKL, PTH, Il-1a, OSM +
TNF-a, OSM + TNF-a + GM6001. PIINP release was reduced when
stimulating with Il-1a, OSM + TNF-a, OSM + TNF-a + GM6001, whereas
PTH and IGF-I increased PIINP release.
Conclusions: We have established a whole tissue model for osteoarthri-
tis consisting of both cartilage and bone, and which is highly responsive
to both catabolic and anabolic stimulation. This is useful for testing
potential treatments for OA interfering with more than one aspect of
the pathological situation. Further it allows for investigating interactions
between cartilage and bone cell types.
Hopefully, future treatments for OA may be better identiﬁed after the
establishment of such a system for drug screening.
106 LOCALIZATION OF MMP- AND AGGRECANASE-GENERATED
NEO-EPITOPES IN OA ARTICULAR CARTILAGE
A-C. Bay-Jensen1, B. Wang2, C. Christiansen1, J. Wang2, Q. Zheng2,
M.A. Karsdal1, P. Qvist1. 1Nordic Bioscience A/S, Herlev, DENMARK,
2Nordic Bioscience A/S, Beijing, CHINA
Purpose: MMP- and aggrecanase-mediated degradation of the cartilage
matrix, and aggrecan in particular, have been extensively studied in
cartilage explants as these two families of proteases are the most impor-
tant proteolytic enzymes involved in cartilage turnover. Catabolic stimu-
lated bovine explants cultures have been reported to release aggrecan-
fragments carrying aggrecanase-mediated neo-epitopes (e.g. the amino
acid sequence 374ARGSV. . . ) in the early phases, while the release
into the supernatant of fragments expressing the MMP-mediated neo-
epitopes (such as 342FFGVG. . . ) is delayed until the later stages. We
therefore wanted to investigate if this separation in time was reﬂected
in the differential localization of the aggrecan fragments in the cartilage
tissue. The overall aim of the study was to determine the localization of
aggrecan-related neo-epitopes in human sections of articular cartilage.
Methods: Human OA articular cartilage was obtained from knee replace-
ment surgery. Full depth biopsies were isolated from areas proximal
to lesions, followed by ﬁxing in paraformaldehyde, decalciﬁcation and
parafﬁn embedding. Sequential cartilage section were immunohistochem-
ically stained for presence of MMP and aggrecanase-mediated neo-
epitopes using monoclonal antibody AF28, BC-3 and 1H11 recognizing
the 342FFGVG. . . , the 374ARGSV. . . and the NITEGE373, respectively.
Results: IHC staining of the cartilage sections aggrecanase-mediated
aggrecan fragments in the proximity of the chondrocytes in the upper
zones. However, aggrecan fragments generated by aggrecanases – BC-3
and 1H11 – were found at the interface between the upper zone and the
superﬁcial layer. Since the superﬁcial layer is still present at this part
of the biopsies (low degree of erosion) it could depict the presence
of aggrecanases-mediated fragments at earlier stages of disease. In
contrast, MMP-mediated fragments were mainly observed in the upper-
mid zone where the superﬁcial layer was lost (high degree of erosion),
speciﬁcally in the surrounding of clusters.
Conclusions: Our immunehistochemistry results support earlier reports,
that the release of aggrecan fragments into the supernatant of the
catabolic stimulated explants cultures showed a bi-phasic pattern with
aggrecanase-mediated release at early time points and MMP-mediated
release of aggrecan fragments at the later stages. We speculate that
MMP and aggrecanases activity is related to disease states more than to
speciﬁc sites. Furthermore that MMP-mediated degradation is related to
areas with high cellular activity (e.g. clusters). The molecular mechanism
and sequence of events is still unclear, but current study gives some
direction to which path to follow.
107 WHOLE BLOOD LEAD (Pb) LEVELS AND PUTATIVE
OSTEOARTHRITIS BIOMARKERS IN AFRICAN AMERICAN
AND CAUCASIAN MEN: THE JOHNSTON COUNTY
OSTEOARTHRITIS PROJECT
J.M. Jordan1, F. Fang2, C. Brooks3, J.B. Renner1, T. Stabler4,
V.B. Kraus4. 1University of North Carolina School of Medicine, Chapel
Hill, NC, USA, 2StatWorks, Inc., RTP, NC, USA, 3Brevard College,
Brevard, NC, USA, 4Duke University Medical Center, Durham, NC, USA
Purpose: Lead (Pb) is a heavy metal that affects many aspects of
bone including osteoclasts, osteoblasts, and calcium and vitamin D
metabolism, and may have effects on cartilage as well. We have reported
associations between whole blood Pb levels and radiographic OA severity
in men and women, and with selected OA biomarkers in women. The
purpose of this analysis was to examine associations between putative
OA biomarkers and blood Pb levels in African American and white men.
Methods: A total of 333 men in the Johnston County OA Project Met-
als Exposure Sub-study (mean age 64.8 (10.7) years, 38.4% African
American) had available demographic and clinical data and whole blood,
serum, or urine samples for whole blood Pb and biomarkers assessments.
Whole blood Pb was measured by inductively coupled plasma mass
spectrometry at the Inorganic Toxicology laboratory, Division of Labora-
tory Sciences, National Center for Environmental Health, CDC, Atlanta,
Georgia. Urine C-telopeptide fragments of type II collagen (CTX-II), cross
linked N telopeptide of type I collagen (NTX-I), serum hyaluronic acid
(HA), cartilage oligometric matrix protein (COMP), and type II procollagen
(CPII) were measured by commercially available kits. Natural logarithm
(ln) transformation was used to produce distributions close to normality
for blood Pb and biomarkers. Spearman correlation coefﬁcients were
calculated between ln Pb and ln of each biomarker. Analysis of covariance
models were used to examine associations between blood Pb levels and
the 5 chosen biomarkers with ln transformed biomarkers as outcomes,
adjusting for age, race, and BMI. Effect modiﬁcation between ln Pb
and race were examined, with signiﬁcance deﬁned by p-values <0.1 for
interaction terms.
Results: Median Pb levels were 2.2 ug/dL (0.5−25.1) and were higher in
African American men than white men (p< 0.0001). In bivariate associa-
tions, ln Pb was correlated with ln CTX-II (r = 0.12, p = 0.035) and ln COMP
(r = 0.20, p< 0.0002), but not with ln NTX-I, ln HA, or ln CPII (r = 0.02,
0.0008, and 0.13, respectively, p> 0.13). In adjusted models, ln Pb was
associated with mean ln CTX-II (p = 0.024) and COMP (p=0.0001). There
were no notable race and ln Pb interactions.
Conclusions: Mean blood Pb levels were associated with urine CTX-II
and serum COMP, but not serum HA or CPII in both African American
and white men. These data suggest that Pb may have an effect on type II
collagen and non-collagenous matrix proteins. Potential effects of Pb in
the pathogenesis of OA, then, are likely to be related to alterations in
these factors, but less likely to effects on synovial inﬂammation.
108 AUROTHIOMALATE INHIBITS COX-2 EXPRESSION AND
PGE2 PRODUCTION IN CHONDROCYTES BY INCREASING
MKP-1 EXPRESSION AND DECREASING p38 AND JNK
PHOSPHORYLATION
R. Nieminen, R. Korhonen, E. Moilanen. Medical School, University of
Tampere, FINLAND
Purpose: Disease-modifying anti-rheumatic drugs (DMARDs) suppress
inﬂammation, and retard cartilage degradation and bone erosion in arthri-
tis. The molecular mechanisms of action of many traditional DMARDs are
not known in detail. Inducible prostaglandin synthase (cyclooxygenase-2,
COX-2) is highly expressed in OA and RA cartilage and it produces
high amounts of proinﬂammatory prostanoids in the joint. The signaling
mechanisms involved in the up-regulation of COX-2 in chondrocytes are
not known in detail. In the present study we investigated the effects
of DMARDs on mitogen-activated protein kinase (MAPK) pathways and
MAP kinase phosphatase-1 (MKP-1) in immortalized H4 chondrocytes*.
Methods: PGE2 production was measured by RIA, protein expression
was measured by Western blot and mRNA expression was measured by
quantitative PCR.
Results:We investigated the effects of traditional DMARDs on MKP-1 ex-
pression in chondrocytes. Unlike the other tested compounds (cyclosporin
A, hydroxychloroquine, leﬂunomide, its active metabolite A771726,
methotrexate and sulfasalazine), aurothiomalate was found to enhance
MKP-1 expression. Aurothiomalate inhibited Il-1b-induced COX-2 expres-
sion and prostaglandin E2 (PGE2) production by destabilizing COX-2
mRNA as did p38 MAPK inhibitor SB203580. Interestingly, aurothiomalate


107 WHOLE BLOOD LEAD (Pb) LEVELS AND PUTATIVE OSTEOARTHRITIS BIOMARKERS IN AFRICAN AMERICAN AND CAUCASIAN MEN: THE JOHNSTON COUNTY OSTEOARTHRITIS PROJECT 

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107 WHOLE BLOOD LEAD (Pb) LEVELS AND PUTATIVE OSTEOARTHRITIS BIOMARKERS IN AFRICAN AMERICAN AND CAUCASIAN MEN: THE JOHNSTON COUNTY OSTEOARTHRITIS PROJECT

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