(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 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.
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) ()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) .
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
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.
For size fractionation experiments 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.
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
[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).
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 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.
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 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.
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 [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.
Figure 7:
Lyso-ECCM contain a heparanase-like
activity. 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.
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 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.
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.