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(Received for publication, August 29, 1995; and in revised form, October 2,
1995) From the
The cause and consequence of altered proteoglycans in
atherosclerosis are poorly understood. To determine whether
proteoglycans affect monocyte binding, we studied the effects of
heparin and proteoglycan degrading enzymes on THP-1 monocyte adhesion
to subendothelial matrix (SEM). Monocyte binding increased about 2-fold
after SEM was treated with heparinase. In addition, heparin decreased
monocyte binding to fibronectin, a known SEM protein, by 60%. These
data suggest that SEM heparan sulfate inhibits monocyte binding to SEM
proteins. We next examined whether lysolecithin, a constituent of
modified lipoproteins, affects endothelial heparan sulfate proteoglycan
(HSPG) production and monocyte binding. Lysolecithin (10-200
µM) decreased total
One characteristic of the atherosclerotic lesion is the presence
of lipid-rich macrophages termed foam cells. Many of these cells are
descendants of blood monocytes that have accumulated within the
subendothelial space and, in this location, have ingested lipoprotein
lipid(1) . In animal models, elevated amounts of circulating
lipoproteins can lead to migration of monocytes into the vessel wall (2, 3) . There have been a number of important
advances in our understanding of monocyte binding to endothelial cells
(EC) ( Within the vessel wall, monocytes may
adhere to SEM adhesion proteins including collagen, fibronectin (FN),
laminin, and vitronectin(8) . These proteins contain domains
(such as Arg-Gly-Asp) that can interact with monocyte cell surface
integrins(9) . Among these protein-protein interactions, the
ones between monocyte VLA-4 and FN and between monocyte MAC-1 and
fibrinogen have been postulated to be of particular
importance(10) . Apart from the adhesion proteins, a
significant proportion of the SEM is composed of proteoglycans (PG),
negatively charged polysaccharides that play an important role in
several cellular processes(11, 12, 13) . Each
proteoglycan molecule contains glycosaminoglycan (GAG) carbohydrate
chains and a core protein. Although SEM PG contain three classes of GAG
(heparan sulfate, chondroitin sulfate, and dermatan sulfate), heparan
sulfate is by far the most abundant, and HSPG accounts for about 80% of
SEM PG produced by cultured EC(14) . Three species of HSPG were
identified in cultured bovine aortic EC, one of which, perlecan, has a
core protein size of 400 kDa and is associated with basement
membrane(15) . The composition and content of vessel wall PG
change during development of the atherosclerotic
lesion(16, 17, 18, 19, 20) .
Arterial wall dermatan sulfate and chondroitin sulfate content increase
during atherogenesis; dermatan sulfate and chondroitin sulfate have
been postulated to retain LDL within the vessel wall(21) . In
contrast, there is a decrease in HSPG in atherosclerotic
vessels(19, 20) . The significance of this decrease in
HSPG has not been addressed. The present investigation was
undertaken to understand the significance and the biochemical
mechanisms of these alterations in SEM PG. The specific questions we
asked were whether a decrease in HSPG facilitates monocyte binding to
SEM and whether the HSPG levels are altered when EC are exposed to
lipoproteins and atherogenic lipids such as lysolecithin. Lysolecithin
is a component of atherogenic lipoproteins such as oxidized LDL and
For size fractionation experiments
Figure 1:
Effect of heparinase and
chondroitinase on monocyte binding to aortic EC and SEM. THP-1
monocytes were labeled as described under ``Experimental
Procedures.'' Confluent monolayers of endothelial cell or SEM in
48-well culture dishes were incubated with 1 unit/ml of heparitinase or
chondroitinase (Chond'ase) in 0.5 ml of DMEM-BSA for 1 h
at 37 °C. Control cells were incubated in DMEM-BSA. Cells and SEM
were washed three times with DMEM-BSA. Labeled monocytes were added in
DMEM-BSA, and binding was carried out for 1 h under cell culture
conditions. Unbound monocytes were removed by washing three times with
DMEM-BSA, and bound radioactivity was assessed by treatment with 0.1 N NaOH/0.1% SDS. Values represent average of two triplicate
experiments ± S.D.
The effect of similar treatments on monocyte binding to
SEM was then assessed. Treatment of SEM with 1 unit of heparinase
increased the number of monocytes bound to SEM to 170% of control (Fig. 1). Chondroitinase treatment of SEM had no effect.
Different concentrations of heparinase were then used to treat EC and
SEM (Fig. 2). At 3 units/ml concentration, monocyte binding to
SEM was increased by 2.2-fold, whereas monocyte binding to EC was not
altered. These data suggest that HSPG in SEM inhibit monocyte binding
to SEM and that their removal by heparinase increases monocyte binding.
In separate experiments treatment of
[
Figure 2:
Effect of different concentrations of
heparinase on monocyte binding to EC and SEM. EC and SEM were incubated
with various concentrations of heparinase for 1 h at 37 °C as
described under Fig. 1. Labeled monocytes were added and binding
was assessed. The values represent the averages of triplicate
experiments ± S.D.
Figure 3:
Monocyte binding to control and protein
coated plates: effect of heparin. 24-well plates were coated either
with 1 µg/ml fibronectin or 5 µg/ml albumin in borate buffer,
pH 10, for 4 h at room temperature. Plates were washed five times with
phosphate-buffered saline and incubated with labeled monocytes in the
presence and absence of 100 units/ml heparin for 1 h at 37 °C.
Unbound monocytes were removed by washing, and bound monocytes were
released by NaOH/SDS and counted. Values represent average of two
triplicate experiments.
Figure 4:
Effect of lipoproteins and lysolecithin on
endothelial proteoglycan metabolism. EC were labeled with
Figure 5:
Effect of LDL and lysolecithin on monocyte
adhesion to SEM. EC were incubated with 500 µg/ml LDL in DMEM
containing 10% lipoprotein-deficient serum or 50 µM lysolecithin (Lyso) in DMEM containing 10% fetal bovine
serum for 16 h. Medium was removed, and cells were washed four times
with DMEM-BSA. SEM was prepared from control and experimental cells as
described. Labeled monocytes were added, and binding was assessed.
Heparinase treatment of SEM was carried out as described in the legend
to Fig. 1.
To
examine whether the increased monocyte binding to SEM in
lysolecithin-treated cells was due to a decrease in the SEM HSPG, we
tested whether heparinase would increase monocyte adhesion to SEM from
lysolecithin-treated cells. Heparinase (1 unit/ml) as expected
increased monocyte adhesion to control SEM by 65%. In
lysolecithin-treated cells, however, no further increase was observed
with heparinase treatment. These data suggest that the
lysolecithin-induced decrease in HSPG resulted in increased monocyte
binding, and, for this reason, the increase was not enhanced by
heparinase treatment.
Figure 6:
ECCM from lysolecithin-stimulated cells
contains HSPG degrading activity. Confluent monolayers of EC in T-75
culture flasks were incubated for 16 h in DMEM with 10% FBS with or
without 50 µM lysolecithin, and ECCM from untreated and
lysolecithin-treated cells (lysoECCM) was collected and
filtered. EC in 48-well culture plates were labeled either with
Further experiments were
done to confirm the presence of heparanase activity in lyso-ECCM.
Several investigators studied the effects of proteases on the large
HSPG and showed that the heparan sulfate chains are asymmetrically
attached to core protein and treatment with different proteases leads
to association of [
Figure 7:
Lyso-ECCM contain a heparanase-like
activity.
The present studies were performed to understand the
contribution of SEM HSPG to monocyte adhesion. Our data suggest that
SEM HSPG function as a negative regulator of monocyte adhesion to SEM.
Removal of SEM HSPG increased monocyte binding by approximately 2-fold.
Several matrix proteins have been shown to bind to GAG. This led to the
suggestion that cell surface PG play a role in cell-substrate binding (31, 32, 33, 34) . Sanders and
Bernfield (33) showed that mammary epithelial cells have at
least two distinct cell surface receptors for FN. These cells have a
trypsin-resistant molecule that binds to Arg-Gly-Asp sequences and a
trypsin labile HSPG that binds to the carboxyl-terminal heparin-binding
domain of FN. Other studies have suggested that cell adhesion through
PG may be an auxiliary mechanism that complements a more specific
integrin-mediated adhesion(35, 36) . Several studies
have also shown that GAG inhibit integrin-mediated cell binding to FN (35) or to perlecan core protein itself(37) . Vessel
wall contains several basement membrane adhesion proteins including
collagen, FN, and laminin. Studies have shown that FN is made by aortic
cells and rapidly incorporated into extracellular matrix (38) .
Immunohistochemical studies have also shown that FN is present
throughout the vessel wall of normal and atherosclerotic vessels and
have suggested that FN may promote monocyte chemotaxis and play a role
in the pathogenesis of
atherosclerosis(39, 40, 41) . Hence in our
studies we employed FN as a representative of SEM adhesion proteins and
as a candidate for monocyte binding. Our results show that GAG inhibit
monocyte binding to FN. This inhibition could be due to effects on
monocyte integrin-mediated binding or monocyte surface HSPG-mediated
binding, both of which are known to be inhibited by GAG. This may also
be true for other adhesion proteins present in SEM, such as collagen
and laminin, which also contain both GAG and integrin-binding domains.
Thus, in the present studies SEM HSPG inhibition of monocyte binding
may, in part, be due to HSPG GAG binding to some of these proteins
thereby affecting monocyte binding. Lysolecithin has been implicated
in several of the atherogenic effects of modified lipoproteins
including induction of chemotaxis and expression of EC adhesion
proteins(22, 28) . Our studies demonstrate another
potentially atherogenic action of lysolecithin, increasing monocyte
adhesion to SEM. The extent of the observed increase in monocytes
adhering to SEM can be explained by the loss of HSPG alone, i.e. the increase in monocyte binding due to lysolecithin treatment was
similar to that found after heparinase treatment of SEM from control
cells, a 2-fold increase. Moreover, because heparinase treatment of SEM
from lysolecithin-treated cells did not lead to a further increase in
monocyte adherence, it is likely that the lysolecithin effect is
entirely due to the reduction of SEM HSPG. Heparanase is produced by
several mammalian cells including neutrophils, platelets, and tumor
cells(42, 43, 44) . Tumor cell heparanase has
been implicated in tumor metastasis(45) . Although EC have been
postulated to produce heparanase like enzymes in situations such as
wound healing and angiogenesis(46) , such an activity has not
been demonstrated. Matrix-degrading metalloproteases are found in
atherosclerotic vessels and are synthesized by lesion
macrophages(47, 48) , and these enzymes are implicated
in extracellular matrix remodeling during atherogenesis. Although such
proteases could also alter proteoglycans(49) , our data are
most consistent with the production of a heparanase-like activity by
lysolecithin-stimulated cells. The amount of sulfate-labeled proteins,
predominantly PG, were decreased in the SEM but not in cells after
lysolecithin treatment. This was associated with the presence in
lyso-ECCM of an activity that released sulfate but not labeled amino
acids from the SEM. Furthermore, this activity was inhibited by
heparin, a known heparanase inhibitor, and the The identity of the endothelial heparanase is not clear.
Hoogewerf et al.(25) recently made a very interesting
observation that platelet heparanases belong to the CXC
chemokine family of peptides that include connective tissue-activating
peptide-III and neutrophil-activating peptide-2. These peptides have
molecular masses of 8-10 kDa and are different from the platelet
heparanase previously characterized(43) . These chemokine
family of heparanases are also different from those produced by tumor
cells(50) . Although endothelial cells are not known to produce
connective tissue-activating peptide-III and neutrophil activating
peptide-2(51) , it is possible that under specific stimulus (e.g. exposure to lysolecithin) endothelial cells may produce
these chemokines. In our preliminary experiments using size
fractionation of ECCM we were not able to detect the activity in the
fraction with a molecular weight of <30,000 (not shown). Experiments
to identify whether endothelial heparanase belong to the chemokine
family are currently underway. Exposure of EC to LDL (500 µg/ml)
for 24 h neither increased monocyte adhesion to SEM nor changed
proteoglycan metabolism. In one previous study, incubation of EC with
high concentrations of LDL for 48 h caused a 50% decrease in basement
membrane HSPG(52) . It is, however, conceivable that the LDL
underwent oxidation during the long course of incubation leading to the
generation of products such as lysolecithin. How lysolecithin perturbs
EC is unclear. It should be noted that the effects of lysolecithin in
the present studies were observed in presence of serum containing
media, suggesting that such effects are possible in vivo in
the environment of the subendothelial intima. Recent studies have shown
that alterations in endothelial function by lysolecithin are mediated
by activation of protein kinase C (53, 54) . In
addition, lysolecithin has been shown to inhibit calcium influx in
aortic EC(55) . We are currently investigating whether similar
mechanisms operate with respect to its actions on EC HSPG metabolism. In summary our studies provide evidence for a protective role for
vessel wall HSPG, i.e. preventing monocyte retention in the
intima. Based on our data we hypothesize that in normal intima the
basement membrane proteins such as FN, laminin, and collagen are masked
by HSPG present in SEM. This masking prevents the binding and retention
of monocytes within the intima. Our data also suggest an additional
atherogenic role for lysolecithin, i.e. to modulate
subendothelial HSPG. Upon exposure to lysolecithin EC are
``activated,'' resulting in the secretion of SEM HSPG
degrading (heparinase-like) activity. This, we postulate, decreases SEM
HSPG and increases monocyte-SEM interaction. In vivo such an
effect could increase retention of monocytes within the arterial wall,
allowing them to convert into macrophage-rich foam cells. Removal of
SEM HSPG, apart from facilitating monocyte retention, may have other
atherogenic consequences. It may increase arterial permeability leading
to further increase in lipoprotein movement and retention. SEM contains
HSPG bound growth factors(56, 57) . When released,
these factors stimulate smooth muscle cells to migrate and proliferate
in the intima. Thus, our results for the first time demonstrate that
endothelial cells produce a heparanase activity under specific
stimulus, and we postulate that dysregulation of the endothelial
heparanase could play an important role in the pathophysiology of
atherosclerosis.
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 29760-29765
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
SO
in SEM
(20-75%). 2-fold more monocytes bound to SEM from lysolecithin
treated cells than to control SEM. Heparinase treatment did not further
increase monocyte binding to lysolecithin-treated SEM. HSPG degrading
activity was found in medium from lysolecithin-treated but not control
cells. SO
-labeled products obtained from
labeled matrix treated with lysolecithin-conditioned medium were
similar in size to those generated by heparinase. These data suggest
that lysolecithin-treated endothelial cells secrete a heparanase-like
activity. We hypothesize that decreased vessel wall HSPG, as occurs in
atherogenic conditions, allows increased monocyte retention within the
vessel and is due to the actions of an endothelial heparanase.
)and monocyte movement across the endothelial barrier.
Monocytes are attracted by chemotactic signals released during the
initial stages of atherogenesis(4) , which then attach to the
vascular endothelium through specific adhesion molecules. In vitro studies have identified three molecules, intracellular adhesion
molecule 1 (CD 54), E-selectin (endothelial-leukocyte adhesion
molecule-1), and vascular cell adhesion molecule, that are inducible on
the endothelial surface and mediate monocyte
binding(5, 6, 7, 8, 9) .
After monocytes bind to endothelium, they migrate through intercellular
junctions and come in contact with SEM. They are then retained within
the vessel wall and, in this location, are thought to acquire large
amounts of lipid via their uptake of oxidatively modified
lipoproteins(1) .
migrating very low density lipoproteins and has been postulated
to be an important causal agent of atherosclerosis(22) . Our
results provide initial evidence that SEM HSPG play an important role
in preventing monocyte binding to SEM and that their levels are altered
in response to specific stimuli. Furthermore, we show that decreases in
SEM HSPG with lysolecithin are associated with the production of a
heparanase-like activity by EC.
Materials
L-
-Lysophosphatidylcholine (lysolecithin, cat.
no. L 1381) from bovine brain was purchased from Sigma. L-[4,5-
H]Leucine (147 Ci/mmol) and
[S]sulfate aqueous solutions were obtained from
Amersham Corp. Heparinase and heparitinase were purchased from either
Sigma or Sekagaku America Inc. (Bethesda, MD). Chondroitin ABC lyase
was from Sekagaku America Inc. 1 unit will form 0.1 µmol of
unsaturated uronic acid/h.
Cells
Monocytes
THP-1 cells were purchased
from the American Type Culture Collection (Rockville, MD) and grown in
RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal bovine
serum (Gemini Bioproducts Inc., Calabasas, CA).Endothelial Cells
Bovine aortic EC were
isolated and cultured as described(23) . The cells (10-15
passages) were grown in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies,
Inc.).Subendothelial Matrix
Confluent EC
monolayers were grown in 24- or 48-well culture dishes (Falcon, Becton
Dickinson, Lincoln Park, NJ). SEM was prepared as described
previously(24) . EC monolayers were washed three times with
phosphate-buffered saline and incubated for 5 min in a solution
containing 20 mM NHOH and 0.1% Triton X-100 at
room temperature. Detached cells were removed by washing three times
with phosphate-buffered saline followed by three times with DMEM
containing 3% bovine serum albumin (DMEM-BSA).Monocyte Binding
Monocytes were incubated with DMEM-BSA lacking leucine for 30
min prior to labeling. Approximately 100 µCi of
[H]leucine was added to 1 
10
cells and incubated for another 2 h under cell culture
conditions. Labeled cells were centrifuged at 800 rpm for 5 min to
remove the label. The cells were then washed four times with DMEM-BSA
and suspended in DMEM-BSA. Suspended cells were then added to either
monolayers of EC or SEM in 48-well plates (2-4 10
cells/well). Binding was continued for 1 h at 37 °C. The
spontaneous release of radioactivity under these conditions was about
5%. Unbound monocytes were removed by washing four times with DMEM-BSA,
and bound radioactivity was extracted by incubating in 0.1 N NaOH containing 0.1% SDS for 30 min at 37 °C and counted. The
number of monocytes bound to endothelial monolayer (approximately 30%
of cells added) was about 2-3-fold more than that bound to SEM.
In some experiments monocyte binding was carried out in DMEM containing
10% calf serum. For enzyme treatments, EC monolayers or SEM were
incubated for 1 h at 37 °C with different concentrations of
heparinase/heparitinase or chondroitinase and washed, and then
monocytes were added. To test the effect of lysolecithin, EC were
incubated with 50 µM lysolecithin (from a 10 mM stock solution in 100% ethanol) for 16 h under cell culture
conditions. Control cells received an equal amount of ethanol. SEM was
prepared from control and lysolecithin-treated cells as described
above.Metabolic Labeling
EC were labeled with [S]sulfate for
24-48 h to label cellular PG. Cell-associated PG were assessed by
removing cells with NH
OH/Triton X-100 as described above.
SEM PG were extracted either by incubation with 0.1 N NaOH/SDS
or with 6 M guanidine hydrochloride for 4 h. Alternatively,
SEM was incubated with heparinase/heparitinase, and released
radioactivity was measured to assess heparan sulfate. Total EC proteins
were labeled by incubating cells with [H]leucine
for 2 h at 37 °C. Unincorporated amino acid was removed, and total
protein synthesis was assessed either by extraction of cells with
NaOH/SDS or by precipitation with 10% trichloroacetic acid.Measurement of HSPG Degrading Activity of EC
Conditioned Medium
Confluent monolayers of EC in T-175 culture flasks were
incubated for 16 h in DMEM with 10% FBS with or without 50 µM lysolecithin. Subsequently, ECCM was collected and filtered. EC
monolayers and SEM in 48-well culture plates were labeled with
[H]leucine or SO
.
Labeled cells and SEM were incubated with ECCM prepared from control
and lysolecithin-treated cells for 4 h at 37 °C, and released label
was counted.SO
labeled matrix in 6-well plates were
incubated with guanidine hydrochloride, heparinase/heparitinase,
trypsin (0.25 mg/ml), proteinase K (2 mg/ml), or lyso-ECCM for 4 h at
37 °C. Released SO
products were size
fractionated either by centrifuging through a Centricon-100 (molecular
weight cut-off, 100,000) or Centricon-30 (molecular weight cut-off,
30,000) (Amicon) according to Hoogewerf et al.(25) .
Labeled material in the filtrate (bottom) and retentate (top) was
measured by scintillation counting.
Effect of Heparinase and Chondroitinase Treatment
of EC and SEM on Monocyte Binding
To examine whether
removal of HSPG from EC or from SEM affects monocyte binding, the
number of monocytes binding to control and heparinase-treated EC and
SEM was studied. Treatment of EC with 1 unit of heparinase did not
significantly change the amount of THP-1 monocytes bound to EC (Fig. 1). In control experiments, heparinase at this
concentration decreased I-lipase binding to EC by
>60%(23) . In some experiments, a small, <20%, decrease
in the number of monocytes adherent to EC was found with chondroitinase
treatment.
H]leucine-labeled SEM with heparinase did not
lead to a release of radioactivity, suggesting the absence of proteases
in the heparinase preparation (data not shown).
Effects of Heparin on Monocyte Binding to
FN
Endothelial cells produce several matrix adhesion
proteins that can potentially play a role in monocyte
binding(8) . These include collagen type IV, FN, laminin, and
vitronectin, all of which contain domains that can bind to GAG. In the
following experiment we used FN as a model SEM protein and examined
whether the addition of a GAG such as heparin would affect monocyte
adherence to FN. Plates were coated with FN (5 µg/ml), and monocyte
binding to control plates and BSA- or FN-coated plates was assessed in
the presence and the absence of 10 units/ml heparin. FN-coated plates
bound 10-fold more monocytes than control or BSA-coated plates (Fig. 3). The number of monocytes bound to the FN-treated plates
was decreased by >60% in presence of heparin. Binding to control and
BSA-coated plates was not affected by heparin treatment. Furthermore
heparin also inhibited monocyte binding to heparinase-treated SEM by
approximately 50% (data not shown). These results suggest that one
potential mechanism by which GAG can inhibit monocyte binding to matrix
is by interacting with SEM adhesion proteins, e.g. as shown
here fibronectin, and interfering with monocyte binding.
Effect of LDL and Lysolecithin on Endothelial HSPG
Metabolism
Atherosclerotic vessels have altered
proteoglycan levels (16, 17, 18, 19) and some in
vitro studies have shown that LDL affect cellular PG
metabolism(26, 27) . We therefore tested whether LDL
or lysolecithin, a component of modified lipoproteins, alters EC PG
metabolism. PG production was assessed by growing cells in media
containing [S]sulfate. Incubation of EC with LDL
(500 µg/ml) or lysolecithin (50 µM) had no significant
effect on the amount of cell-associated PG (Fig. 4A).
Similarly, LDL had no significant effect on the amount of
SO
incorporated into SEM PG. In contrast,
lysolecithin decreased SEM PG by 55% (Fig. 4B);
heparinase-releasable PG (HSPG) decreased by 60% (Fig. 4B, inset). These data demonstrate that
lysolecithin primarily decreased the amount of HSPG. At 25-200
µM concentration, lysolecithin decreased SEM PG by
25-70% (Fig. 4C). Direct analysis by
SDS-polyacrylamide gel electrophoresis and autoradiography of SO
-labeled SEM proteins revealed decreased SO
incorporation into a large (>500 kDa)
proteoglycan in lysolecithin-treated cells (Fig. 4D).
To determine whether the lysolecithin altered cellular metabolism, e.g. by causing cell toxicity, total protein synthesis was
assessed. [H]Leucine incorporation into cellular
proteins was not affected by lysolecithin treatment.
SO
for 24-48 h as described under
``Experimental Procedures.'' Labeled EC were incubated with
500 µg/ml LDL in DMEM containing 10% lipoprotein deficient serum or
50 µM lysolecithin (Lyso) in DMEM containing 10%
fetal bovine serum for 16 h. Media were removed, and cells were washed
five times with DMEM-BSA. Cell associated and SEM PG were assessed as
described under ``Experimental Procedures.'' A, cell
associated. B, SEM. Inset, lysolecithin treatment
decreases heparinase-releasable SEM PG. SEM from
[S]sulfate-labeled cells was incubated with 1
unit/ml heparinase for 1 h at 37 °C. The released radioactivity
from control and lysolecithin treated cells was counted. Values
represent average of two triplicate experiments. C, effect of
different concentrations of lysolecithin on SEM PG.
SO
-labeled endothelial cells were incubated
with 10-200 µM lysolecithin in DMEM with 10% FBS for
16 h, and SEM was prepared. Total PG were isolated by 6 M guanidine hydrochloride and counted. The values represent the
averages of triplicate experiments. D, SDS-PAGE and
autoradiography of SO
-labeled SEM PG. Labeled
SEM PG from control and lysolecithin-treated EC were extracted with 6 M guanidine HCl and analyzed on 3-12% gels. The amount
of radioactivity in a large MW HSPG (molecular mass, >500 kDa) is
decreased in lysolecithin-treated cells.
Lysolecithin Increases Monocyte Binding to
SEM
Because lysolecithin decreased the amount of SO
incorporated into SEM PG, we next tested
whether lysolecithin treatment altered monocyte binding to SEM.
Exposure of EC to lysolecithin (50 µM) increased monocyte
adhesion to EC by approximately 2-fold, in agreement with published
studies(28) . In addition, lysolecithin treatment increased the
number of monocytes adhering to SEM by 1.8-fold (Fig. 5).
Treatment of EC with LDL had no significant effect on the binding of
monocytes either to EC (not shown) or SEM (Fig. 5).
EC Secrete a HSPG Degrading Activity in Response to
Lysolecithin Treatment
Decreased SEM HSPG in
lysolecithin-treated cells could result from either 1) degradation of
HSPG by the secretion of a protease or a heparanase or 2) decreased
synthesis of SEM HSPG. To determine if ECCM from lysolecithin-treated
cells contain HSPG degrading activity, ECCM was prepared from control
and lysolecithin-treated cells as described under ``Experimental
Procedures'' and tested for its ability to release radioactivity
from SO
- (labeled PG) or
[H]leucine-labeled (total proteins) SEM (Fig. 6). Compared with control ECCM, lyso-ECCM released
>2-fold more S radioactivity from SEM. The addition of
lysolecithin to control ECCM did not affect the
SO
release. The amount of S radioactivity released by
lyso-ECCM in different experiments varied from 40-60% of the
counts released by heparinase treatment (not shown). Lyso-ECCM did not
increase the amount of [
H]leucine released. The
addition of heparin (100 units/ml) to lyso-ECCM abolished the releasing
activity. Similarly, addition of suramin, another heparanase inhibitor,
also inhibited the lyso-ECCM activity (not shown). This suggested that
the S release was due to heparanase-like activity and not
due to the actions of a general protease.
SO
or with [H]leucine,
and labeled SEM was prepared as described under ``Experimental
Procedures.'' Labeled SEM was incubated with medium alone, ECCM
alone, ECCM containing 50 µM lysolecithin, Lyso-ECCM, or
Lyso-ECCM containing 100 units/ml heparin for 2 h at 37 °C, and
released label was counted. The values represent the averages of
triplicate experiments. The amount of radioactivity released by medium
or ECCM was <10% of the total radioactivity present in
SEM.
S]GAG in fragments with
molecular masses of 130-200 kDa(29, 30) . To
determine if lyso-ECCM contained a protease- or heparanase-like
activity, SEM was incubated with lyso-ECCM, heparinase (2 unit/ml),
trypsin (0.25 mg/ml), or proteinase K (0.5 mg/ml) for 4 h at 37 °C.
The released
S radioactivity (degradation products) was
analyzed by size fractionation (25) as described under
``Experimental Procedures'' (Fig. 7). About
90-95% of the total extractable PG were found to have molecular
masses of >100 kDa and did not go through Centricon-100 (Fig. 7, GnHCl bar). Approximately 80-90% of the
radioactivity released by either heparinase or lyso-ECCM passed through
a Centricon-100 (molecular weight, <100,000). In contrast only
20-25% of trypsin and proteinase K released radioactivity was
filtered. In addition, 45-50% of the heparinase or lyso-ECCM
released radioactivity passed through a Centricon-30 membrane,
indicating the presence of products with a molecular mass of <30
kDa. Thus, our data suggest that lysolecithin treatment of EC
stimulates the secretion of a heparanase-like activity that in turn
decreases SEM HSPG.
S-labeled SEM was prepared as described under
``Experimental Procedures.'' Labeled SEM was incubated for
2-4 h at 37 °C with 6 M guanidine hydrochloride (GnHCl), 0.25 mg/ml trypsin, 0.5 mg/ml proteinase K (Prot.K), 2 units/ml heparinase (Hep'ase), or 1
ml of lyso-ECCM (LysoECCM). Released radioactivity was
collected and counted. Trypsin and proteinase K released approximately
80% of the total
S (guanidine HCl-releasable) in the
matrix. Approximately 20-30,000 cpm of the released radioactivity
from different treatments was filtered through a Centricon-100
(molecular weight cut-off (MW), 100,000) or a Centricon-30
(molecular weight cut-off, 30,000), and the amount of radioactivity
that passed through (molecular weight less than 100,000 and 30,000,
respectively) was assessed. The values represent the averages of two
different experiments that were within 10% variation. Approximately 50%
of the total radioactivity released by Lyso-ECCM and heparinase was in
products of molecular weight <30,000.
SO
degradation products were similar to those released by
heparinase. It should be noted that obtaining heparanase in active form
in ECCM was not always possible. It is not clear whether the enzyme is
rapidly inactivated in the medium or bound to PG and not easily
released.
)
We thank Dr. Lata Paka for assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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