Báo cáo khoa học: Sulfated polysaccharides inhibit the catabolism and loss of both large and small proteoglycans in explant cultures of tendon doc

Sulfated polysaccharides inhibit the catabolism and loss
of both large and small proteoglycans in explant cultures
of tendon
Tom Samiric, Mirna Z. Ilic and Christopher J. Handley
School of Human Biosciences, La Trobe University, Melbourne, Australia
The role of tendons is to absorb and transmit force
generated by muscles to bones, the basis of joint move-
ment. Tendons are fibrous connective tissues that are
sparsely populated with cells that are responsible for
the synthesis and maintenance of the extracellular mat-
rix. Type I collagen is the major macromolecule pre-
sent in the extracellular matrix of tendon, comprising
over 50% of the dry weight and providing the tissue
with its tensile strength. The remainder of the extracel-
lular matrix is made up of proteoglycans and noncolla-
genous proteins [1,2]. We and others have shown that
the small proteoglycans, predominantly decorin, con-
stitute 80% of the total proteoglycan content in the
proximal ⁄ tensional regions of tendon [3–5]. The core
proteins of small proteoglycans are characterized by
leucine-rich repeat structures that have been implicated
in the binding of these proteoglycans to collagen [6].
The large proteoglycans aggrecan and versican are
found in similar proportions in tendon, and together
make up almost 20% of the total proteoglycan content
of the tissue [5]. These proteoglycans can bind to hya-
luronan and form hydrated aggregates within the
extracellular matrix or interact with other matrix mac-
romolecules and contribute to the organization of the
extracellular matrix [7].
We have previously shown in both cartilage and

fibrous connective tissues such as tendon and ligament
that the loss of newly synthesized large proteoglycans
from the matrix occurs at a faster rate than that of
small proteoglycans [8–10]. In arthritic diseases, the
large proteoglycans are one of the first matrix compo-
nents to be degraded and lost from the articular carti-
lage and this is associated with impairment of the
biomechanical function of the tissue [11]. An increased
rate of aggrecan degradation can be attributed to pro-
teolytic cleavage at specific sites along the core protein.
Keywords
aggrecan; calcium pentosan polysulfate;
decorin; heparin; proteoglycan; tendon
Correspondence
T. Samiric, School of Human Biosciences,
La Trobe University, 3086, Victoria, Australia
Fax: +61 394795784
Tel: +61 394793417
E-mail:
(Received 10 May 2006, accepted 2 June
2006)
doi:10.1111/j.1742-4658.2006.05348.x
This study investigated the effects of two highly sulfated polysaccharides,
calcium pentosan polysulfate and heparin, on the loss of newly synthesized
proteoglycans from the matrix of explant cultures of bovine tendon. The
tensional region of deep flexor tendon was incubated with [
35
S]sulfate for
6 h and then placed in culture for up to 15 days. The amount of radiolabel
associated with proteoglycans lost to the medium and retained in the mat-

rix was determined for each day in culture. It was shown that both sulfated
polysaccharides at concentrations of 1000 lgÆmL
)1
inhibited the loss of
35
S-labeled large and small proteoglycans from the matrix and concomitant
with this was a retention of chemical levels of proteoglycans in the explant
cultures. In other explant cultures that were maintained in culture in the
presence of both agents for more than 5 days after incubation with [
35
S]sul-
fate, inhibition of the intracellular catabolic pathway was evident, indica-
ting that these highly sulfated polysaccharides also interfered with the
intracellular uptake of small proteoglycans by tendon cells.
Abbreviations
ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; CaPPS, calcium pentosan polysulfate; DMEM, Dulbecco’s
modified Eagle’s medium; GnHCl, guanidinium chloride.
FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS 3479
We and others have shown that the predominant activ-
ity responsible for the degradation of aggrecan in con-
nective tissues in vitro is aggrecanase, which has been
shown to be attributable to a number of proteinases
of the ADAM-TS family of proteinases namely
ADAMTS-1, -4 and -5 [12–17]. In contrast, the degra-
dation of small proteoglycans in cartilage, ligament and
tendon appears to occur at a slower rate, even in the
presence of catabolic agents [8,10,18]. This may suggest
that the interaction of the small proteoglycans with the
surface of the collagen fibrils may protect these pro-
teoglycans from proteolysis during the early phases of

connective tissue degradation [18]. We have shown that
in ligament and tendon, the loss of small proteoglycans
from the tissue involves either the loss of intact or par-
tially degraded core proteins, or internalization and
subsequent degradation by the cells [10,19].
A number of highly sulfated polysaccharides have
been shown to slow the degradation of articular carti-
lage in animal models of osteoarthritis [20]. One such
polymer is pentosan polysulfate, a sulfated polyxylan
that has been shown to modify both the symptoms
and the progression of osteoarthritis in both humans
and animals [21]. We and others have shown that the
calcium salt of pentosan polysulfate (CaPPS) will
directly inhibit aggrecanase activity by interacting with
aggrecanase proteinases and will suppress aggrecanase-
mediated degradation of newly synthesized aggrecan
by cartilage explant cultures [22,23]. Heparin, which is
recognized for its anticoagulant properties, is another
highly sulfated polysaccharide that has been shown to
have inhibitory effects on aggrecanase activity [22–24].
Although these highly sulfated polysaccharides have
been shown to suppress the catabolism of proteogly-
cans by cartilage [25], the effects of these agents on
joint fibrous connective tissues such as tendon where
the small proteoglycans are predominant are lacking.
In order to elucidate further the mechanisms regulating
proteoglycan loss from the tissue, we have investigated
the effects of two highly sulfated polysaccharides,
CaPPS and heparin, on the degradation of newly syn-
thesized proteoglycans from the extracellular matrix of

bovine tendon explant cultures.
Results
Measurement of loss of
35
S-labeled
proteoglycans from bovine tendon explant
cultures with varying concentrations
of CaPPS or heparin
The proximal region of deep flexor tendon was incuba-
ted with 200 lCiÆmL
)1
[
35
S]sulfate at 37 °C for 6 h as
described in the Experimental procedures. The tissue
was then maintained in Dulbecco’s modified Eagle’s
medium (DMEM) in the presence of 0, 100 and
1000 lgÆmL
)1
CaPPS or heparin for 10 days. After
predetermined times in culture, the tissue was extracted
with 4 m GnHCl for 72 h at 4 °C followed by 0.5 m
NaOH as described in the Experimental procedures.
The percentage of
35
S-labeled proteoglycans remaining
in the matrix was determined and plotted as a function
of time in culture. Figure 1A shows that CaPPS inhib-
ited the catabolism of
35

S-labeled proteoglycans in
these cultures in a dose-dependent manner. On day 10
of the culture period, 60% of
35
S-labeled proteogly-
cans remained in the matrix of cultures that had been
%
5
3
-
alS
e
p
lbd
g
t
or
eo
y
le
s
a
c
r
n
i
n
in
i
am

e

t
ng
me
h
xi
r
t
a
0
10
20
30
40
50
60
70
80
90
100
Days in Culture
012345678910
0
10
20
30
40
50
60

70
80
90
100
CaPPS
Heparin
A
B
Fig. 1. Kinetics of loss of
35
S-labeled proteoglycans by bovine ten-
don explant cultures maintained in DMEM in the presence of vary-
ing concentrations of CaPPS or heparin. The proximal region of
bovine deep flexor tendon was incubated with [
35
S]sulfate for 6 h
and maintained in DMEM in the presence of 0 (d), 100 (.), or
1000 (s) lgÆ mL
)1
CaPPS (A) or in the presence of 0 (d), 100 (.),
or 1000 (s) lgÆmL
)1
heparin (B) for 10 days. The percentage of
35
S-labeled proteoglycans remaining in the matrix of tendon cul-
tures at each day after incubation with [
35
S]sulfate was determined
as described in the Experimental procedures. The positive error
bars represent the range of duplicate samples over the 10 days.

Inhibition of proteoglycan catabolism in tendon T. Samiric et al.
3480 FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS
maintained in DMEM alone. However, in cultures
maintained in DMEM containing 100 and
1000 lgÆmL
)1
CaPPS, the percentage of
35
S-labeled
proteoglycans remaining in the matrix by day 10 of
culture was 65 and 72%, respectively. This was also
observed in repeated experiments using tissue from dif-
ferent animals. Figure 1B shows that in the presence
of heparin, the catabolism of
35
S-labeled proteoglycans
in these cultures was also inhibited. By day 10 of the
culture period, 62% of
35
S-labeled proteoglycans
remained in the matrix of cultures that had been main-
tained in DMEM alone. However, in cultures main-
tained in DMEM containing 100 and 1000 lgÆmL
)1
heparin, the percentage of
35
S-labeled proteoglycans
remaining in the matrix after 10 days in culture was 77
and 79%, respectively. This was also observed in other
cultures. In addition, these data indicate that heparin

was more potent than CaPPS in inhibiting the loss of
35
S-labeled proteoglycans from the matrix of tendon
explants.
The rate of incorporation of [
3
H]serine into proteins
of tendon explant cultures was measured on days 5
and 10 of the culture period in the conditions above
(Table 1). The addition of CaPPS or heparin, at con-
centrations of up to 1000 lgÆmL
)1
, to the culture
medium did not affect the rate of incorporation of
[
3
H]serine into protein on days 5 and 10, indicating
that neither sulfated polysaccharide had an adverse
affect on cellular metabolism. This lack of effect on
the cellular metabolism has also been observed in
explant cultures of articular cartilage maintained in both
agents at concentrations of up to 1000 lgÆmL
)1
[25].
The endogenous glycosaminoglycan levels present in
the matrix of tendon cultures maintained in the
absence or presence of CaPPS or heparin on days 5
and 10, as determined by hexuronate, are shown in
Table 2. The levels of endogenous glycosaminoglycans
in the matrix on day 10, represented by the chemical

pools of both large and small proteoglycans, were
greater in cultures maintained in DMEM containing
1000 lgÆmL
)1
CaPPS or heparin. Heparin appeared to
inhibit the loss of tissue glycosaminoglycans to a
greater extent than CaPPS in these cultures.
Effects of polysulfated polysaccharides on the
loss of
35
S-labeled large and small proteoglycans
from bovine tendon explants
In order to determine whether highly sulfated polysac-
charides inhibited the loss of either
35
S-labeled large or
small proteoglycans from bovine tendon matrix, the
hydrodynamic size of
35
S-labeled proteoglycans present
in tendon matrix at various times during culture
maintained in DMEM alone or DMEM containing
1000 lgÆmL
)1
CaPPS or heparin was determined. Tis-
sue from the experiment described in Fig. 1 was
extracted on days 0, 2, 4, 6, 8, and 10 with 4 m GnHCl
for 72 h at 4 °C as described in the Experimental pro-
cedures. These samples were subjected to size-exclusion
chromatography on a column of Sepharose CL-4B

eluted under dissociative conditions as described in the
Experimental procedures (data not shown). From this,
the percentage of
35
S-labeled large and small proteo-
glycans remaining in the matrix at various times after
incubation of tendon with [
35
S]sulfate was determined
as previously described [10].
Figure 2 shows that CaPPS (Fig. 2A) and heparin
(Fig. 2B) inhibited the loss of both the
35
S-labeled
large and small proteoglycans from the extracellular
matrix. On day 10 of the culture period, 10% of
Table 1. Incorporation of [
3
H]serine into macromolecules of bovine
tendon explants maintained in DMEM containing varying concentra-
tions of CaPPS or heparin. The proximal region of bovine deep
flexor tendon was maintained in DMEM alone or DMEM containing
100 or 1000 lgÆmL
)1
CaPPS or heparin. Tissue cultures were
incubated with 30 lCiÆmL
)1
[
3
H]serine for 2 h on days 5 and 10, as

described in Experimental procedures. The values represent the
median ± range of duplicate samples.
Treatment
[
3
H]Serine incorporation
(dpm ⁄ mg wet weight of
tissue per 2 h)
Day 5 Day 10
DMEM alone 1593 ± 748 1356 ± 57
DMEM +100 lgÆmL
)1
CaPPS 1654 ± 499 1064 ± 168
DMEM +1000 lgÆmL
)1
CaPPS 1822 ± 219 1224 ± 129
DMEM +100 lgÆmL
)1
heparin 1708 ± 371 1452 ± 522
DMEM +1000 lgÆmL
)1
heparin 1498 ± 456 1390 ± 284
Table 2. Hexuronate concentration of bovine tendon explants main-
tained in DMEM alone or DMEM containing 1000 lgÆmL
)1
CaPPS
or heparin. The proximal region of bovine deep flexor tendon was
maintained in DMEM alone, or DMEM containing 1000 lgÆmL
)1
CaPPS or heparin for up to 10 days. Tissue was extracted on day 5

and 10 of the culture period with 0.5
M NaOH as described in
Experimental procedures. The values represent the median ± range
of duplicate samples.
Treatment
Hexuronate concentration
(lg ⁄ 100 mg wet weight of
tissue)
Day 0 Day 5 Day 10
DMEM alone 114 ± 8 60.4 ± 18 62.4 ± 2
DMEM +1000 lgÆmL
)1
CaPPS – 75.7 ± 5 76.2 ± 2
DMEM +1000 lgÆmL
)1
heparin – 86.5 ± 8 102.9 ± 22
T. Samiric et al. Inhibition of proteoglycan catabolism in tendon
FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS 3481
35
S-labeled large proteoglycans remained in the matrix
of cultures that had been maintained in DMEM alone
but, in cultures maintained in DMEM containing
1000 lgÆmL
)1
of either CaPPS (Fig. 2A) or heparin
(Fig. 2B), the percentage of
35
S-labeled large proteo-
glycans remaining in the matrix by day 10 of culture
was 45%. This indicates that with the inclusion of

these sulfated agents there was significant inhibition
(40%) of rate of loss of
35
S-labeled large proteogly-
cans from the matrix by the end of the culture period.
Similarly, by day 10 of culture, 70% of
35
S-labeled
small proteoglycans remained in the matrix that had
been maintained in DMEM alone, but in cultures main-
tained in DMEM containing 1000 lgÆmL
)1
of either
CaPPS (Fig. 2A) or heparin (Fig. 2B), the percentage
of
35
S-labeled small proteoglycans remaining in the
matrix by day 10 of culture was 80%. This indicates
that these sulfated polysaccharides inhibited the loss of
the radiolabeled small proteoglycans by 33%.
Analysis of radiolabeled
35
S-proteoglycan core
proteins remaining in the matrix
To determine the effects of CaPPS and heparin, at con-
centrations of 1000 lgÆmL
)1
, on the proteolytic pro-
cessing of
35

S-labeled proteoglycans remaining in the
tendon matrix after 9 days in culture, the
35
S-labeled
proteoglycans core protein fragments were isolated on
anion-exchange chromatography prior to being ana-
lyzed on SDS ⁄ PAGE followed by fluorography.
Figure 3 shows that a high molecular weight band
above 200 kDa, representing the large proteoglycans, is
retained in the matrix after 9 days in culture in the
presence of CaPPS (Fig. 3A) or heparin (Fig. 3B). This
further indicates that these highly sulfated agents inhi-
bit the loss of
35
S-labeled large proteoglycans.
Effects of polysulfated polysaccharides on the
intracellular catabolism of
35
S-labeled small
proteoglycans of tendon explant cultures
Previous work has shown that approximately one-half
of the
35
S-labeled small proteoglycans that are lost from
the tendon matrix are taken up by the cells and degra-
ded [10]; the following experiment was undertaken to
determine the effects of CaPPS and heparin, at concen-
trations of 1000 lgÆmL
)1
, on the intracellular catabol-

ism of
35
S-labeled small proteoglycans by bovine tendon
explant cultures. After incubation with [
35
S]sulfate as
described in the Experimental procedures, tissue was
maintained in culture in DMEM alone for 5 days to
allow time for loss from the cultures of the large
35
S-labeled proteoglycans and any unincorporated
[
35
S]sulfate from the tissue [10]. Cultures were then
maintained for a further 10 days in either DMEM alone
or DMEM containing 1000 lgÆmL
)1
CaPPS or heparin.
Culture medium was collected daily and the tissue
extracted at the end of the culture period with 4 m
GnHCl followed by 0.5 m NaOH as described in the
Experimental procedures. Both medium and tissue
extract samples were analyzed for the presence of
35
S-labeled macromolecules and free [
35
S]sulfate by
size exclusion chromatography using Sephadex G-25
columns, as we have previously shown that the
Fig. 2. The effects of CaPPS and heparin on the percentage of

each
35
S-labeled proteoglycan species remaining in the matrix of
bovine tendon cultures. For the experiment outlined in the legend
of Fig. 1, the percentage of
35
S-labeled large and small proteogly-
cans remaining in the tissue at each time after incubation of bovine
tendon with [
35
S]sulfate was determined in (A) DMEM alone [large
(s) and small (,) proteoglycans] or DMEM containing
1000 lgÆmL
)1
CaPPS [large (d) and small (.) proteoglycans], or (B)
DMEM alone [large (s) and small (,) proteoglycans] or DMEM
containing 1000 lgÆmL
)1
heparin [large (d) and small (.) proteogly-
cans]. The percentage of
35
S-labeled proteoglycans remaining in
the matrix of tendon cultures at each day after incubation with
[
35
S]sulfate was determined as described in Experimental proce-
dures. The positive error bars represent the mean range of dupli-
cate samples over the 10 days.
Inhibition of proteoglycan catabolism in tendon T. Samiric et al.
3482 FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS

continued presence of free [
35
S]sulfate in the medium
throughout the culture period results from intracellular
degradation of small proteoglycans [10].
Figure 4A shows that the rate of [
35
S]sulfate appear-
ing in the medium was suppressed by the inclusion of
the sulfated polysaccharides. At the end of the culture
period, both CaPPS and heparin reduced the appear-
ance of [
35
S]sulfate in the culture medium by 5 and
11%, respectively. It was also shown that the rate of
release of
35
S-labeled small proteoglycans into the cul-
ture medium was increased by 5 and 10% in the
presence of either CaPPS or heparin, respectively
(Fig. 4B). These results indicate that these agents sup-
press intracellular degradation of
35
S-labeled small
proteoglycans, whilst simultaneously increasing the loss
of
35
S-labeled small proteoglycans into the culture
medium. These highly sulfated polysaccharides had lit-
tle effect during this time period on the total levels of

35
S-labeled small proteoglycans that remained in the
extracellular matrix of the tendon cultures (Fig. 4C).
200
97
68
43
Stds (kDa)
ABC
Fig. 3. Analysis of
35
S-labeled proteoglycan core proteins present in
the matrix or medium of tendon explant cultures in the presence or
absence of 1000 lgÆmL
)1
CaPPS and heparin. Newly synthesized
35
S-labeled proteoglycans remaining in the matrix of tendon explant
cultures after 9 days in culture were isolated as described in the
Experimental procedures and digested with chondroitinase ABC
and keratanase, prior to electrophoresis on a 4–15% polyacryla-
mide ⁄ SDS gel. The gel was subjected to fluorography as described
in the Experimental procedures. Lanes show peptides present in
tissue matrix after 9 days of culture in (A) medium alone, (B) med-
ium containing 1000 lgÆmL
)1
CaPPS, and (C) medium containing
1000 lgÆmL
)1
heparin. The amount of sample applied to each lane

corresponds to 5000 dpm
35
S-radioactivity.
5 6 7 8 9 10 11 12 13 14 15
Accumula itve crep atne fo eg
rf[ ee
53
S]sulf pa etairaepgn
in c eht ult rum eidemu
0
10
20
30
40
50
60
70
80
90
100
5 6 7 8 9 101112131415
Ac ucmulaitve rep ce egatn fo
53
S-l lebaedrp
ot lgoeycansr eleas de
into t huc elturm e edi mu
0
10
20
30

40
50
60
70
80
90
100
Days in Culture
56789101112131415
%
53
S-lebale rp dot lgoeycasn
rema ni
n
igni t he m atr xi
0
10
60
70
80
90
100
B
C
A
Fig. 4. Effects of CaPPS and heparin on the rate of formation of
[
35
S]sulfate and release of
35

S-labeled proteoglycans from bovine
tendon explant cultures. The proximal region of the bovine deep
flexor tendon was incubated with [
35
S]sulfate as described in the
Experimental procedures and then maintained in DMEM for
5 days prior to analysis. Tissue was subsequently cultured in
DMEM alone (d), DMEM containing 1000 lgÆmL
)1
CaPPS (s),
or DMEM containing 1000 lgÆmL
)1
heparin (.). The culture med-
ium was collected daily and analyzed for the presence of
[
35
S]sulfate and
35
S-labeled proteoglycans. From this data, (A) the
rate of release of [
35
S]sulfate into the medium, (B) the rate of
release of
35
S-labeled small proteoglycans into the medium, and
(C) the percentage of
35
S-labeled small proteoglycans remaining
in the matrix were determined as described in the Experimental
procedures.

T. Samiric et al. Inhibition of proteoglycan catabolism in tendon
FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS 3483
Discussion
This study investigated the effects of two highly sulfat-
ed polysaccharides, CaPPS and heparin, on the cata-
bolism of both newly synthesized large and small
proteoglycans in tendon explant cultures. Previous
studies have showed that both agents inhibit aggrecan
loss from explant cultures of articular cartilage [23].
However, this is the first study to demonstrate that
highly sulfated polysaccharides are also able to inhibit
proteoglycan catabolism in a connective tissue other
than articular cartilage.
This study showed that both CaPPS and heparin
inhibited the loss of
35
S-labeled large proteoglycans in
tendon explants by 40%. Furthermore, the chemical
pool of large proteoglycans was also shown to be
inhibited as indicated by hexuronate levels as shown in
Table 2. The predominant, large proteoglycans present
in the extracellular matrix of tendon have been shown
to be aggrecan and versican [3–5]. We have previously
analyzed the catabolic products of the large proteogly-
cans present in the extracellular matrix of fresh and
cultured tendon, as well as medium originating from
tendon cultures [10]. It was evident that the catabolism
of aggrecan by tendon can be solely attributed to the
action of aggrecanase, as all the catabolic products of
this proteoglycan that were isolated were derived from

the cleavage of the core protein at aggrecanase clea-
vage sites [10]. In the case of versican, it appears that
the catabolism may involve the action of other matrix
proteinases as well as that of aggrecanase [10]. It has
been demonstrated that CaPPS and heparin inhibit sol-
uble aggrecanase activity as well as recombinant aggre-
canase proteinases, and it is therefore likely that the
outcome of the inhibition of aggrecanase activity in
tendon is the retention of large proteoglycans within
the extracellular matrix of the tissue [22–27].
The mechanism by which highly sulfated polysaccha-
rides inhibit aggrecanase proteinases remains unclear.
The ADAM-TS family of proteinases contain one or
more thrombospondin type-1 motifs that are able to
bind to sulfated glycosaminoglycans. It has been dem-
onstrated in ADAM-TS4 that the interaction between
the thrombospondin type-1 motifs and glycosamino-
glycans of aggrecan core protein play a critical role in
the recognition of specific cleavage sites within the core
protein [28,29]. It is possible that when highly sulfated
polysaccharides bind to these thrombospondin type-1
motifs this results in the inhibition of proteolytic activ-
ity. It should be pointed out that we have demonstra-
ted that the inhibition of aggrecanase is dependent on
the degree of sulfation of polysaccharides, as chondro-
itin or keratan sulfate glycosaminoglycans had no
effect on soluble aggrecanase activity whereas heparin
and heparin sulfate are inhibitory [25]. This relation-
ship between the degree of sulfation of polysaccharides
and inhibitory activity towards aggrecanase suggests

that an ionic interaction may be responsible for the
inhibition of aggrecanase by polyanionic molecules
[23]. Apart from direct inhibition of aggrecanases,
CaPPS or heparin may also be inhibiting aggrecanase
activity by altering the nuclear events within tendon
fibroblasts that result in decreased expression of aggre-
canase activity. Studies have shown that sodium pento-
san polysulfate and heparin are internalized by cells
and are able to modulate gene expression of matrix
proteinases at the transcriptional level [30–32]. It has
also been suggested that highly sulfated polysaccha-
rides may regulate aggrecanase activity by suppressing
the activation of aggrecanase in a similar manner to
that suggested for glucosamine [33].
In the case of versican, it is likely that CaPPS and
heparin inhibit the catabolism of this proteoglycan by
suppressing aggrecanase activity as described above
and by inhibiting the activity of other matrix protein-
ases since it has been shown that pentosan polysulfate
is an inhibitor of serine proteinases as well as a num-
ber of metalloproteinases that include MMP-1 (inter-
stitial collagenase) and MMP-3 (stromelysin-1) [21].
This study showed that both CaPPS and heparin
inhibited the loss of
35
S-labeled small proteoglycans in
tendon explants by 33%. Based on chemical analysis,
we have previously shown that 85% of the small
proteoglycans present in tendon is made up of decorin,
and the remainder consists of mainly biglycan [5]. This

work also showed that biglyan is lost from the tendon
matrix intact, whereas decorin is lost from the tissue
either intact or as the result of partial proteolytic clea-
vage [5]. Furthermore, using radiolabeled techniques,
we have shown that a pool of small proteoglycans are
taken up by tendon cells and degraded intracellularly
[10]. In the present study, we showed that when tendon
explant cultures were incubated with [
35
S]sulfate for
6 h then maintained in culture for 5 days to allow for
the radiolabeled large proteoglycans to be lost, expo-
sure of the cultures to either CaPPS or heparin resul-
ted in a reduced rate of cellular uptake and subsequent
degradation of
35
S-labeled small proteoglycans as
measured by the appearance of free [
35
S]sulfate in the
medium (Fig. 4A). Inhibition of endocytosis of small
proteoglycans by heparin has been reported by previ-
ous investigators in cultured skin fibroblasts, however,
intracellular degradation of these proteoglycans was
not affected by heparin [34–36]. In these cultures, the
exposure of tendon to CaPPS or heparin did not affect
the levels of radiolabeled small proteoglycans retained
Inhibition of proteoglycan catabolism in tendon T. Samiric et al.
3484 FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS
in the matrix of the tissue but resulted in more radio-

labeled small proteoglycans being lost from the tissue
presumably from the pool of small proteoglycans that
was destined to be internalized by tendon cells
(Fig. 4B,C). This is consistent with our previous work
using tendon and ligament indicating that if the pool
of radiolabeled small proteoglycans that was destined
for intracellular degradation is inhibited, then this pool
of small proteoglycans is lost from the tissue [10,19].
The work presented in this paper supports previous
observations that show that highly sulfated polysaccha-
rides are able to inhibit the loss of proteoglycans from
articular cartilage [25]. However, it is evident in tendon
that higher concentrations of these agents are required
to observe effects compared with cartilage [25]. The pre-
sent study found that both CaPPS and heparin, at a
concentration of up to 1000 lg ÆmL
)1
, did not have an
adverse impact on cellular metabolism as assessed by
the incorporation of [
3
H]serine into matrix macromole-
cules of tendon explant cultures. Although highly sulfat-
ed polysaccharides have the potential to cause adverse
effects that are related to their anticoagulant and fibrin-
olytic activities [37,38], complications involving con-
nective tissues following administration of these
compounds have not been reported [21]. However, it is
possible that the inhibition of the catabolism of prote-
oglycans in tendon and other fibrous connective tissues

such as ligament may result in an increase in the matrix
levels of these proteoglycans that would contribute to
the increased stiffness of the tissue. Nevertheless, the
work described in this paper indicates that highly sulfat-
ed polysaccharides have the potential to modulate levels
of proteoglycans in joint connective tissues in disease.
Experimental procedures
Materials
DMEM, Eagle’s nonessential amino acids, penicillin and
streptomycin were from CSL (Melbourne, Victoria, Austra-
lia). Sephadex G-25 (as prepacked PD-10 columns) was
from Pharmacia (Uppsala, Sweden) and 0.20 lm Minisart
Ò
microporous membrane filters were from Sartorius
(Goettingen, Germany). Amicon
Ò
Ultra-4 centrifugal filter
devices (molecular weight cut-off¼10 000) were purchased
from Millipore Corporation, Bedford, MA, USA, and
Amplify was from Amersham Pharmacia (Amersham,
Buckinghamshire, UK). Heparin (from porcine intestinal
mucosa) was purchased from Celsus Laboratories (Cincin-
nati, OH, USA). Calcium pentosan polysulfate was a gift
from Arthropharm Pty Ltd (Sydney, Australia). Aqueous
solutions of [
35
S]sulfate (carrier-free) and [
3
H]serine
(27.00 CiÆmmol

)1
) were from DuPont New England Nuc-
lear (Boston, MA, USA). Chondroitinase ABC (protease-
free from Proteus vulgaris; EC 4.2.2.4) was from ICN
Biochemicals (Costa Mesa, CA, USA). Keratanase (from
Pseudomonas sp.; EC 3.2.1.103) was obtained from Sigma
Chemical Co. (St Louis, MO, USA). Adult bovine meta-
carpophalangeal joints were obtained from a local abattoir.
Preparation of solutions containing sulfated
polysaccharides
The sulfated polysaccharides used for these experiments
were dissolved in DMEM at concentrations up to
20 mgÆmL
)1
. Each solution was then filtered through a ster-
ile 0.20 lm Minisart
Ò
microporous membrane filter before
being added to the culture medium to achieve the appropri-
ate final concentration.
Maintenance of tendon in explant culture
The proximal region of deep flexor tendon was dissected
from a single metacarpophalangeal joint obtained from an
adult steer. The dissected tendon was chopped into small
pieces and incubated in sulfate-free medium (1 g of tissue
per 10 mL medium) containing 200 lCiÆmL
)1
[
35
S]sulfate at

37 °C for 6 h, as previously described [10]. After washing
the tissue exhaustively in culture medium to remove most
of the unincorporated radioisotope, duplicate samples con-
taining 100 ± 20 mg of tissue were distributed into individ-
ual sterile plastic vials containing 4 mL of DMEM with
varying concentrations of CaPPS or heparin (0, 100 or
1000 lgÆmL
)1
) for 10 days. The culture medium used for
these experiments was DMEM containing 1 mg glu-
coseÆmL
)1
and supplemented with organic buffers and
amino acids [39]. The medium was collected and replaced
daily with fresh media. The collected medium was stored at
)20 °C in the presence of proteinase inhibitors [40]. After
predetermined times in culture, the tissue was rinsed several
times with NaCl ⁄ P
i
to remove residual exogenous sulfated
glycosaminoglycans that had been present in the culture
media, before being extracted with 4 m GnHCl at 4 °C for
72 h, followed by 0.5 m NaOH at 21 °C for 24 h. Individ-
ual experiments were repeated at least three times using tis-
sue from different animals. In some experiments tissue was
extracted with 0.5 m NaOH to determine the amounts of
glycosaminoglycans remaining in the matrix.
Measurement of turnover of
35
S-labeled

proteoglycans in tendon maintained in culture in
medium containing varying concentrations of
polysulfated polysaccharides
To determine the proportion of
35
S-labeled proteoglycans
remaining in the matrix of tendon cultures on each day
after incubation with [
35
S]sulfate, 0.5 mL aliquots of the
T. Samiric et al. Inhibition of proteoglycan catabolism in tendon
FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS 3485
medium fractions, GnHCl and NaOH extracts were applied
to columns of Sephadex G-25 (PD-10 columns) equilibrated
and eluted with 4 m GnHCl, 0.1 m Na
2
SO
4
,50mm sodium
acetate, 0.1% (v ⁄ v) Triton X-100, pH 6.1. The rate of loss
of
35
S-labeled proteoglycans from the matrix of explant cul-
tures (which eluted in the excluded volume fractions on
Sephadex G-25 columns) was calculated from the amount
of
35
S-labeled macromolecules that appeared in the medium
on each day of culture and that remaining in the matrix at
the end of the culture period [41]. From these data, the per-

centage of
35
S-labeled proteoglycans remaining in the mat-
rix on each day was determined and plotted as a function
of time in culture [10].
Separation of
35
S-labeled proteoglycans
remaining in the matrix of tendon explant
cultures by size exclusion chromatography
Aliquots (1 mL) of the GnHCl extracts obtained from tis-
sue after predetermined times in culture were applied to a
column of Sepharose CL-4B (1.3 · 87.0 cm) equilibrated
and eluted with 4 m GnHCl, 50 mm sodium acetate buffer,
0.1% Triton X-100, pH 5.8. Fractions of 1 mL were collec-
ted at a flow rate of 6 mL Æh
)1
and assayed for
35
S-radioac-
tivity. From these data, the percentage of
35
S-labeled large
and small proteoglycan species remaining in the matrix at
different times in culture was determined [9].
Measurement of incorporation of [
3
H]serine into
macromolecules by tendon maintained in culture
in medium containing varying concentrations of

sulfated polysaccharides
Bovine tendon was distributed into individual vials
(100 mg tissue per vial) containing 4 mL of DMEM and
varying concentrations (0, 100 and 1000 lgÆmL
)1
) of heparin
or CaPPS. The tissue was maintained in culture for 10 days
and the medium from each vial was replaced daily. The treat-
ment groups were maintained as duplicates and explants were
incubated with [
3
H]-serine on days 5 and 10 of the culture
period. The tissue was preincubated for 1 h in 2 mL of fresh
DMEM, followed by 2 h incubation in DMEM containing
[
3
H]-serine (30 lCiÆmL
)1
). The rate of incorporation of
[
3
H]serine into macromolecules by the explant cultures was
determined for each experimental group as previously des-
cribed and expressed as disintegrations per minute (dpm) ⁄ mg
wet weight per 2 h [9].
Measurement of hexuronate concentration of
tendon explants in the presence or absence of
1000 lgÆmL
)1
CaPPS or heparin

The glycosaminoglycan concentration of the NaOH
extracts after predetermined times as described above was
determined by assaying the extracts for hexuronate as
described by Bitter and Muir in 1962 using glucuronic
acid as standard [42]. Interference with the hexuronate
assay by the polysulfated polysaccharides that were added
to the culture media was minimized by thoroughly rinsing
the tissue with NaCl ⁄ P
i
prior to extraction with NaOH
[25].
Analysis of
35
S-labeled proteoglycan core
proteins remaining in the matrix of tendon
explant cultures
Tissue was dissected from a single metacarpophalangeal
joint and incubated with [
35
S]sulfate for 6 h as described
above. The tissue was maintained in DMEM alone or
DMEM containing 1000 lgÆmL
)1
CaPPS or heparin for
9 days. The culture medium was replaced daily. At the end
of the culture period, the tissue was extracted with 4 m
GnHCl as described above. Proteoglycans were isolated
from tissue extracts by ion-exchange chromatography on
Q-Sepharose as described previously [5].
Purified samples were concentrated in H

2
O using
Amicon
Ò
Ultra-4 centrifugal filter devices as described by
the manufacturer. Concentrated samples were resuspended
and re-concentrated in 0.1 m Tris ⁄ 0.1 m sodium acetate,
pH 7.0, and digested with chondroitinase ABC (0.0375 U)
and keratanase (0.075 U) at 37 °C for 24 h in the pres-
ence of proteinase inhibitors [43]. Digested samples were
then concentrated, and finally resuspended and then
exchanged in H
2
O using the filter devices as described
above. Samples were then lyophilized and subjected to
electrophoresis on 4–15% gradient polyacrylamide ⁄ SDS
slab gels. The gels were fixed in a solution of 30% (v ⁄ v)
methanol and 10% (v ⁄ v) acetic acid for 30 min, soaked
in Amplify for a further 30 min, dried and exposed to
X-ray film at )20 °C for 4 weeks.
Measurement of intracellular degradation of
35
S-labeled small proteoglycans in the presence
or absence of CaPPS or heparin
In order to determine the rate of intracellular degradation
of
35
S-labeled small proteoglycans, bovine tendon from a
single metacarpophalangeal joint was incubated with
[

35
S]sulfate for 6 h as described above and then maintained
in DMEM alone for 5 days to allow for the loss of the
35
S-
labeled large proteoglycans from the tissue cultures. After
5 days, the tissue was maintained in culture in the presence
or absence of 1000 lgÆmL
)1
CaPPS or heparin for a further
10 days. The rate of intracellular proteoglycan catabolism
by tendon explants was determined from the amount of ra-
diolabeled sulfate appearing in the medium on each day as
described previously [10].
Inhibition of proteoglycan catabolism in tendon T. Samiric et al.
3486 FEBS Journal 273 (2006) 3479–3488 ª 2006 The Authors Journal compilation ª 2006 FEBS
Acknowledgements
This work was supported by a grant from Faculty of
Health Sciences, La Trobe University and the Arthritis
Foundation of Australia.
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