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INTRODUCTION |
Proteoglycans (PG),1
important constituents of vascular cell membranes and extracellular
matrix (1, 2), consist of a core protein to which long chains of
negatively charged polysaccharides termed glycosaminoglycans (GAG) are
attached. The three major PG classes in the vessel wall are heparan
sulfate (HS), chondroitin sulfate, and dermatan sulfate. HSPG play an
important role in the regulation of various vascular functions. They
bind and promote lipoprotein lipase activity, the key enzyme in the
hydrolysis of triglyceride-rich lipoproteins (3). Basic fibroblast
growth factor, a potent mitogen and angiogenic factor, requires the
presence of cell surface HSPG or exogenous heparin to bind to its high affinity cell signaling receptor (4). In addition, HSPG potentiate the
thrombin-inhibiting actions of antithrombin (5).
A reduction in arterial HS and heparin has been observed under
conditions of inflammation and atherosclerosis as well as with increased age (6-14). The age-dependent decrease in HS is
more pronounced in atherosclerotic tissues than in normal tissues (10, 11). An inverse correlation between the amount of cholesterol in the
lesion and the concentration of HS was observed in human aortas. More
importantly, this negative correlation was observed in both normal and
atherosclerotic vessels. 4-5-fold more cholesterol was found in
vessels that have 50% less HS.
The negative relationship between HS and atherosclerosis is not
surprising because arterial HS is known to inhibit smooth muscle cell
(SMC) proliferation and promote antithrombin activity (5, 15-17). In
addition, our recent studies show that HS masks subendothelial proteins
such as fibronectin and prevents lipoproteins such as lipoprotein(a)
and monocytes from associating with the matrix (18, 19). Thus, an
increase in vascular HSPG could be athero-protective. Although the
decrease of HSPG in atherosclerosis has been known for several years,
it is not clear how this occurs. In vitro, a reduction in
subendothelial HSPG was observed when endothelial cells were exposed to
moderately oxidized LDL or lysolecithin, a product of lipoprotein
oxidation. This reduction in subendothelial HSPG was found to be caused
by secretion, by endothelial cells, of a HSPG-degrading heparanase
activity (18, 19). During these studies an initial observation was made
that HDL blocked the oxidized LDL- and lysolecithin-mediated decreases
in HSPG (19). We now show that HDL, more specifically the apoE
component of HDL, stimulates endothelial HS synthesis.
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MATERIALS AND METHODS |
[35S]Sulfate aqueous solutions were from Amersham
Pharmacia Biotech. Heparinase I and heparitinase (heparinase III) and
chondroitin ABC lyase were purchased from Seikagaku America Inc.
(Bethesda, MD). Receptor-associated protein (RAP) was kindly provided
by Dr. D. K. Strickland (Bethesda, MD). For experiments with apoE either a recombinant apoE3 (kindly provided by Dr. M. Al-Haideri of
Columbia University (20)) or a commercially obtained apoE3 (Calbiochem)
was used.
Lipoproteins--
VLDL (d < 1.006), LDL
(d < 1.063), and HDL (d = 1.0-1.21)
were isolated from fresh human plasma in the presence of EDTA by sequential ultracentrifugation and dialyzed against phosphate-buffered saline containing 0.5 mM EDTA (20). For mouse HDL, blood
was drawn by retroorbital bleeding from wild type (C57BL/6J), apoE-null (genetic background C57BL/6J), and apoA-I-null mice; HDL was isolated as above.
Cells--
Bovine aortic endothelial cells were isolated and
cultured as described (21). The cells (5-15 passages) were grown in
minimal essential medium (MEM) containing 10% fetal bovine serum (Life Technologies). The subendothelial matrix was prepared as described previously (18) by incubation for 5 min in a solution containing 20 mM NH4OH and 0.1% Triton X-100 at room temperature.
Metabolic Labeling--
Endothelial PG were radiolabeled with
either [35S]sulfate or [3H]glucosamine for
the indicated time periods. Cell-associated PG were assessed by
removing the cells with NH4OH/Triton X-100 as described
above. Subendothelial matrix PG were extracted by incubation with 6 M guanidine hydrochloride for 4 h. Alternatively, the
subendothelial matrix was incubated with either heparinase/heparitinase or chondroitinase, and released radioactivity was measured to assess HS
and chondroitin sulfate/dermatan sulfate PG. Total endothelial cell
proteins were labeled by incubating the cells with
[3H]leucine for 16 h at 37 °C. Protein synthesis
was assessed either by extraction of cells with 0.1 N NaOH
and 0.1% SDS for 1 h or by precipitation with 10%
trichloroacetic acid.
To study the effects of HDL and other lipoproteins, endothelial cells
were incubated in culture medium containing
35SO4 and the indicated concentrations of the
lipoprotein or apoE (free or in dimyristoylphosphatidylcholine (DMPC)
vesicles; see Ref. 56) for 16 h. Cell and matrix PG were assessed
as above.
DEAE-Cellulose Chromatography of Proteoglycans--
To determine
changes in PG, DEAE-cellulose chromatography was performed as described
previously (21, 22). Endothelial cells were labeled with
[3H]leucine, and labeled proteins were extracted using 10 mM Tris-HCl buffer, pH 7.4, containing 1% octyl glucoside,
1% CHAPS, 0.1 mM each EDTA and phenylmethylsulfonyl
fluoride, and 1 µg/ml leupeptin. Equal amounts of labeled proteins
from control and HDL-treated cells were loaded on a DEAE-cellulose
column previously equilibrated with HEPES buffer containing 0.15 M NaCl and 0.1% Tween 80. Stepwise elution was done with
HEPES buffer containing 0.25 M and 0.5 M NaCl.
GAG Analysis--
The relative incorporation of
[3H]glucosamine and 35SO4 was
determined in isolated GAG. To prepare GAG chains, aliquots of purified PG were incubated with 0.10 volume of 10 N NaOH for 18 h at 26 °C with constant shaking and then neutralized with 10 N HCl. Samples were dialyzed, and protein-free GAG were
purified by DEAE-cellulose chromatography. Chondroitin sulfate/dermatan
sulfate-GAG were degraded by treatment with chondroitin ABC lyase (0.1 unit) treatment as described previously (22). To determine the size of
the GAG, gel filtration on Sepharose 6B was performed using 0.2 M NaCl as eluant as described previously (22).
To determine heparin-like sequences in the subendothelial matrix,
35SO4-labeled matrix prepared from control and
HDL-treated cells was incubated with 1 unit/ml of heparitinase for
2 h at 37 °C. Released material (containing degraded and
undegraded HS) was then passed through a PD-10 gel filtration column.
Radioactivity eluting in the void volume was determined as undigested
heparin-like HS. The undegraded material was further subjected to low
pH nitrous acid treatment as described previously (22). Briefly, 1 ml
of PD-10 void volume material was incubated with an equal volume of a
mixture containing 20% butyl nitrite in 1 M HCl. The
mixture was incubated for 2 h, and the digested material was gel
filtered on a PD-10 column.
Labeling of Monocytes and Heparin-binding Proteins and Binding to
the Subendothelial Matrix--
THP-1 monocytes were labeled with
[3H]leucine (100 µCi/1 × 107 cells)
for 2 h at 37 °C. The label was removed, and cells were washed
three times with MEM-BSA and suspended in MEM-BSA. Suspended cells were
added to the subendothelial matrix prepared from control or HDL-treated
endothelial cells in 24-well plates (2-4 × 105
cells/well) and incubated 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 MEM-BSA, and bound
radioactivity was extracted by incubation in 0.1 N NaOH and
0.1% SDS for 1 h.
Antithrombin and lipoprotein lipase were iodinated using the enzymes
lactoperoxidase and glucose oxidase as described previously (23).
Iodinated proteins were purified by heparin-agarose chromatography, and
proteins were eluted with 1.5 M NaCl. 5 µg of iodinated
protein was incubated with subendothelial matrix in MEM-BSA for 2 h at 37 °C. Unbound protein was removed, and bound radioactivity was determined as above.
Smooth Muscle Cell Proliferation--
Rat aortic SMC were kindly
provided by Dr. L. Rabbani (Department of Medicine, Columbia
University). SMC were grown in basal medium supplemented with growth
factors (Clonetics). To determine the effects of HS on SMC
proliferation, cells were plated at low density (8 × 104/well) and cultured for 3 days in the presence or
absence of matrix HS. Matrix HS was isolated from
[3H]glucosamine-labeled control and HDL-treated
endothelial cells. Equal amounts of glucosamine radioactivity from
control and HDL-treated cells were used. On the 4th day, the cell
number was counted with a hemacytometer. Net growth was determined by
subtracting the final cell number from the initial cell number. The
percentage of SMC growth inhibition was calculated by using the average
of each triplicate with the formula
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(Eq. 1)
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In another experiment, SMC were cultured in the above conditions
for 3 days, and the cells were then labeled with
[3H]thymidine for 6 h; radioactivity incorporated
into the DNA was determined. For this experiment HS isolated from
nonlabeled cells was used.
Determination of 35SO4 Incorporation in
Mouse Tissues--
Control and apoE-null mice (4 weeks old, three
each) were injected intraperitoneally with 100 µCi of
35SO4 in 100 µl of saline. Mice were
sacrificed after 4 h, tissues were perfused (through the left
ventricle) with PBS, and liver and heart (together with the proximal
aorta) were removed. Tissues were homogenized (Polytron) for 30 s
in ice-cold HEPES pH 7.0 buffer containing 4 M urea, 0.5%
CHAPS, 0.05 M NaCl, 1 mM each phenylmethylsulfonyl fluoride and benzamidine, and 5 µg/ml leupeptin. Homogenates were centrifuged (14,000 rpm, 20 min), and the supernatants were dialyzed extensively to remove free sulfate. Aliquots of dialyzed
supernatants were counted, and radioactivity was expressed per mg of
tissue protein.
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RESULTS |
HDL Stimulates Endothelial PG Production--
To determine the
effects of HDL on PG, confluent monolayers of endothelial cells were
incubated with HDL for 16 h. A dose curve of HDL versus
35SO4 incorporation in cells, matrix (Fig.
1A), and medium (Fig. 1B) is shown. 35SO4 incorporation in
all three pools, secreted, cellular, and matrix, increased by
50-100%. An increase of 43% with 250 µg of HDL protein and an
increase of 83% with 500 µg of HDL protein was observed in matrix.
HDL also increased [3H]leucine incorporation into
proteins by 15-20% (not shown). However, the increase in PG sulfate
shown in Fig. 1 was found after normalizing for protein. These data
suggest that HDL stimulated sulfate incorporation into PG. It should be
noted that these changes were found using levels of HDL which are
within the normal plasma concentrations of HDL protein (150-180 mg/dl
fasting plasma (24)).

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Fig. 1.
HDL stimulates endothelial PG. Confluent
monolayers of endothelial cells in 24-well plates were incubated with
the indicated concentration of HDL in growth medium (MEM and 10% fetal
bovine serum) containing [35S]sulfate (50 µCi/well) for
16 h under culture conditions. Sulfate-labeled PG in the cells and
subendothelial matrix (panel A) and in the medium
(panel B) were determined. Values represent the mean ± S.D.
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HDL Increases Endothelial Cell PG Synthesis--
To determine
whether the HDL-mediated increase in sulfate incorporation was caused
by increased synthesis, cells were labeled with
35SO4 for different time periods in the
presence of HDL (1,000 µg/ml). HDL increased sulfate incorporation by
25% in 2 h and by 37% in 6 h in cells (Fig.
2A) and matrix (Fig.
2B). When endothelial PG were first labeled with
35SO4 and chased in cold medium for different
time periods in the presence or absence of HDL, HDL did not alter the
turnover rates of PG (not shown). These data suggest that HDL-mediated
increases in 35SO4 incorporation were primarily
the result of increased synthesis.

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Fig. 2.
HDL increases PG synthesis. Confluent
monolayers of endothelial cells in 24-well plates were incubated with
the indicated concentration of HDL in growth medium containing
[35S]sulfate (50 µCi/well) at 37 °C for different
time periods. Sulfate-labeled PG in the cell layer and subendothelial
matrix compartments were determined. Values represent the mean ± S.D.
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HDL Did Not Alter PG Core Proteins but Increased Sulfation of
GAG--
To determine if increased 35SO4
incorporation was caused by increased PG core proteins or increased GAG
synthesis, cells were labeled either with [3H]leucine (to
label proteins) or with [3H]glucosamine (to label GAG).
To differentiate [3H]leucine incorporation into PG core
proteins versus other cellular proteins, DEAE-cellulose
chromatography was performed. In different experiments we showed that
under the conditions ~90% of bound 35SO4
radioactivity was eluted at salt concentrations > 0.25 M NaCl. When equal amounts of cellular proteins were loaded
on DEAE, the amount of radiolabel eluted at 0.5 M NaCl was
not different (Fig. 3, 2,992 cpm in
control cells versus 3,079 cpm in HDL-treated cells).
Although this suggests that total core proteins were not altered,
whether HDL altered core proteins of specific PG (such as perlecan and
syndecans, which may not reflect in total core proteins) remains to be
determined.

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Fig. 3.
HDL effects on PG core proteins.
Confluent monolayers of endothelial cells were incubated with growth
medium containing [3H]leucine (100 µCi/well) and with
(1,000 µg/ml) or without HDL for 16 h under culture conditions.
3H-Labeled proteins in the cell layer were extracted and
subjected to DEAE-cellulose chromatography and eluted with buffers
containing 0.25 M NaCl and 0.5 M NaCl. The
majority of labeled proteins eluted at 0.25 M NaCl. Total
radioactivities eluting at 0.5 M NaCl (containing mostly
PG) were similar in control and HDL-treated endothelial cells.
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HDL increased endothelial GAG ([3H]glucosamine
incorporation into PG, Fig.
4A), by 34% in cells and by
41% in matrix. Thus, the increase in 35SO4
incorporation into PG was, in part, the result of increased GAG
synthesis. Endothelial cells primarily synthesize HS; consistent with
this >75% of total GAG was resistant to chondroitin ABC lyase (which
removes chondroitin and dermatan sulfates) treatment (not shown).
Enrichment in GAG could be caused by either an increased number of GAG
or an increased chain length. Sizing analysis of isolated GAG by
Sepharose 6B gel filtration chromatography did not show changes in the
chain length of GAG isolated from control and HDL-treated cells (not
shown). We then compared the increases in sulfation and GAG (expressed
as sulfate cpm/glucosamine cpm) (Fig. 4B). Although HDL
increased glucosamine (Fig. 4A), the GAG were relatively
more sulfated in HDL-treated cells as indicated by an increase in the
sulfate:glucosamine ratio. A 2.5-fold increase in the sulfation of
cellular GAG and an increase of 1.5-fold in the sulfation of matrix GAG
was observed.

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Fig. 4.
Panel A, HDL effects on GAG production.
Confluent monolayers of endothelial cells were incubated with growth
medium containing [3H]glucosamine (25 µCi/well) and
with (1,000 µg/ml) or without HDL for 16 h under culture
conditions. 3H-Labeled PG in cell layer and subendothelial
matrix compartments were determined. Values represent the mean ± S.D. Panel B, HDL increases sulfation of GAG.Endothelial
proteoglycans were labeled with [3H]glucosamine or
[35S]sulfate in the presence or absence of the indicated
concentrations of HDL. Labeled PG in the cell layer and subendothelial
matrix compartments were determined, and the ratio of 35S
to 3H was determined. HDL increases the sulfate:glucosamine
ratio, suggesting increased sulfation of GAG.
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Anti-atherogenic and Anti-thrombogenic Properties of HS from
HDL-treated Endothelial Cells--
Several anti-atherogenic actions of
HS require specific sulfate groups. Studies from this laboratory showed
that subendothelial HS inhibition of monocyte binding require sulfation
(18). Acceleration of antithrombin-induced proteinase inhibition by HS
requires a pentasaccharide with 3-O sulfate on the internal
glucosamine (2). The anti-proliferative activity of arterial HS appears
to require sequences rich in 2-O sulfated uronic acid (25).
Similarly, HS that contains 2-O iduronic acid has a high
affinity for lipoprotein lipase (26). These sequences are generally
localized to heparin-like domains in HS (resistance to heparitinase).
In our experiments we found a 1.7-fold increase in these
heparitinase-resistant but nitrous acid-sensitive heparin-like
sequences in matrix (cpm/well in a six-well plate, 10,532 ± 765 in control versus 17,986 ± 1,289 in HDL-treated cells).
To determine the consequences of increased matrix HS, matrix was
prepared from control and HDL-treated endothelial cells and incubated
either with [3H]leucine-labeled monocytes or
125I-labeled antithrombin III. The number of monocytes
binding to the HDL matrix was decreased by approximately 49%,
suggesting that increased HSPG inhibited monocyte interactions with
subendothelial matrix (Fig. 5). In
contrast, antithrombin III binding to matrix was increased by 43% in
HDL-treated cells compared with control matrix. These data suggest an
increase in specific HS (containing 3-O sulfates).

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Fig. 5.
Effect of increased matrix HSPG on monocyte
and antithrombin binding. THP-1 monocytes were labeled with
[3H]leucine (100 µCi/1 × 107 cells)
for 2 h at 37 °C. Label was removed, and the cells were washed
three times with MEM-BSA and suspended in MEM-BSA. Suspended cells were
added to matrix prepared from control or HDL-treated endothelial cells
in 24-well plates (2-4 × 105 cells/well) and
incubated for 1 h at 37 °C. Unbound monocytes were removed by
washing four times with MEM-BSA, and bound radioactivity was extracted
by incubation in 0.1 N NaOH and 0.1% SDS for 1 h.
Antithrombin was iodinated using the lactoperoxidase/glucose oxidase
method and purified by heparin-agarose chromatography. 5 µg of
iodinated protein was incubated with matrix prepared from control and
HDL-treated cells for 2 h at 37 °C. Unbound protein was
removed, and bound radioactivity was determined.
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We next tested the ability of the matrix HS to inhibit SMC
proliferation, a key event in the development of atherosclerosis. SMC
were cultured in the presence or absence of matrix HS isolated from
control and HDL-treated endothelial cells. Cell proliferation was
assessed either by counting the number of cells (Fig.
6) or by assessing
[3H]thymidine incorporation. Endothelial HS was
normalized for glucosamine concentration before adding to SMC.
Incubation of SMC with HS isolated from chlorate (an inhibitor of
sulfation)-treated endothelial cells did not significantly inhibit SMC
growth, suggesting the requirement for sulfated HS. Heparin, a known
inhibitor of SMC proliferation, at 50 units/ml inhibited SMC
proliferation by 42 ± 6%. HS prepared from HDL-treated
endothelial cells showed most inhibition (82 ± 5%), better than
heparin and control HS (56 ± 6%). These data suggest that HDL
treatment increased antiproliferative HS in endothelial cells.

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Fig. 6.
HS isolated from HDL-treated endothelial
cells is a potent inhibitor of SMC proliferation. SMC were plated
at low density (8 × 104/well) and cultured for 3 days
in medium alone (None) or media containing 50 units/ml
heparin, matrix HS isolated from 25 µM chlorate-treated,
control, and HDL-treated endothelial cells. On the 4th day, the cell
number was determined. The percentage of SMC growth inhibition was
calculated as described under "Materials and Methods."
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HDL Effects Require ApoE--
We next tested whether HDL effects
on PG sulfation require apoA-I and/or apoE. HDL was isolated from wild
type (WT), apoA-I-null, and apoE-null mice and tested for its ability
to stimulate sulfation. Equal amounts of HDL-protein (100 µg/ml) were
used. HDL isolated from control and apoA-I-null mice increased
35SO4 incorporation into cellular PG. HDL from
apoE-null mice, in contrast, failed to stimulate endothelial PG
sulfation (Fig. 7A). Similar
results were obtained when apoE HDL was removed from human HDL by
heparin-Sepharose chromatography; the E-deficient HDL did not increase
PG sulfation (data not shown). To determine further if this lack of
stimulation was caused by the absence of apoE, apoE was added to
apoE-null HDL (Fig. 7A). The addition of 5 µg of
apoE restored the ability of E-null HDL to stimulate PG sulfation to
mouse WT HDL levels. At 10 µg of apoE, E-null HDL increased 35SO4 incorporation by 2-fold. These data show
that apoE is required to stimulate PG sulfation.

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Fig. 7.
Panel A, apoE is required for HDL
stimulation of endothelial HS. HDL was isolated from wild type
(C57BL/6J, denoted WT), apoE-null (E(0)), and
apoA-I-null (AI(0)) mice. Confluent monolayers of
endothelial cells in 24-well plates were incubated in growth medium
containing [35S]sulfate (50 µCi/well) and different
HDLs (100 µg/ml) for 16 h under culture conditions.
Sulfate-labeled PG in the cell layer was determined. For add-back
experiments, E(0) HDL was preincubated with purified apoE (5 and 10 µg) for 30 min before adding to endothelial cells. Sulfate-labeled PG
in the cell layer was determined. Values represent the mean ± S.D. Panel B, effects of apoE and apoE-containing
particles on 35SO4
incorporation. Confluent monolayers of endothelial cells in 24-well
plates were incubated in 35SO4-containing
growth medium for 37 °C for 16 h. The medium contained one of
the following: purified apoE (5 µg/ml) or DMPC vesicles or apoE in
DMPC vesicles (E-DMPC) or VLDL (200 µg/ml) or HDL (500 µg/ml). Sulfate-labeled PG in cell layer were determined.
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We further examined the effects of apoE in emulsions and in lipid free
form. ApoE was able to stimulate endothelial HS production in both free
form and DMPC emulsions (Fig. 7B). These data suggest that
apoE actions do not require other HDL components. Surprisingly, however, VLDL failed to stimulate HS production despite containing similar or greater amounts of apoE (determined by SDS-polyacrylamide gel electrophoretic analysis; not shown).
We next tested whether apoE would stimulate
35SO4 incorporation in other cells. Unlike in
endothelial cells, incubation of J774 macrophages (which do not
synthesize apoE) or human skin fibroblasts with apoE (10 µg/ml) did
not alter PG production (Table I).
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Table I
Effect of apoE on 35SO4 incorporation in different cell
types
Aortic endothelial cells or J774 macrophages or fibroblasts were grown
to confluence and incubated in growth medium containing 25 µCi/ml
35SO4 with (10 µg/ml) or without apoE for 16 h
under culture conditions. Cellular proteoglycans were determined as
described under "Materials and Methods."
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Endothelial Cell Surface Molecules Involved in ApoE
Actions--
We next determined the role of lipoprotein receptors in
mediating apoE actions. Competition with lipoproteins and receptor antagonists were used (Fig. 8). LDL or
VLDL neither stimulated HS production (closed bars) nor
inhibited HDL-mediated stimulation (open bars). Similar
results were obtained with LDL receptor antibody. The 39-kDa RAP at
concentrations that others have used to inhibit LDL receptor-related
protein (5-10 µg) did not inhibit an HDL-mediated increase in
35SO4 incorporation. However, at a higher dose
(20 µg/ml) RAP inhibited apoE-mediated HS production by >60%. The
requirement for higher doses of RAP could be caused by the longer
incubation times (16 h) required for HS stimulation in the current
experiments. Nevertheless, these data suggest that RAP-sensitive
pathways are involved in apoE function. In different experiments
heparin increased 35SO4 incorporation by
25-30%. HDL effects, however, were less pronounced in the presence of
heparin. These data indicate that cell surface HSPG may partly
contribute to apoE actions.

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Fig. 8.
Effects of competitors and receptor
antagonists on the apoE-mediated increase in
35SO4 incorporation. HDL effects on
35SO4 incorporation in endothelial cells were
assessed as described in Fig. 7B in the presence of the
following competitors: LDL receptor antibody (IgG, 1 µg/ml), LDL (500 µg/ml), VLDL (200 µg/ml), RAP (10 and 20 µg/ml), and heparin (50 units/ml). Sulfate label in the cell layer was determined. Values
represent the mean ± S.D.
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PG in ApoE-null Mice--
To test whether apoE would affect PG
sulfation in vivo, 35SO4
incorporation was determined in WT and apoE-null mice. 4-week-old mice
were injected with 35SO4, and incorporation
into PG was determined after 4 h as described under "Materials
and Methods." 35SO4 incorporation was not
different in livers of WT and apoE-null mice (Fig.
9). However, hearts (containing part of
the aorta) from apoE-null mice had ~40% reduction in
35SO4 incorporation compared with hearts from
WT mice. These data suggest that apoE also stimulates sulfation of PG
in vivo.

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Fig. 9.
35SO4 incorporation
in tissues of apoE-null mice. Control and apoE-null mice (4 weeks
old, three each) were injected intraperitoneally with 100 µCi of
35SO4 in 100 µl of saline. Mice were
sacrificed after 4 h, tissues were perfused with
phosphate-buffered salineS, and liver and heart (together with proximal
aorta) were removed. Tissues were homogenized (Polytron) for 30 s
in ice-cold HEPES (pH 7.0) buffer containing 4 M urea,
0.5% CHAPS, 0.05 M NaCl, 1 mM each
phenylmethylsulfonyl fluoride and benzamidine, and 5 µg/ml leupeptin.
Homogenates were centrifuged (14,000 rpm, 20 min), and the supernatants
were dialyzed extensively to remove free sulfate. Aliquots of dialyzed
supernatants were counted, and radioactivity was expressed per mg of
tissue protein.
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DISCUSSION |
Loss of endothelial HS has been postulated to lead to several
pathological events, in particular to events related to atherosclerosis (15-19, 27-31). These include 1) altered endothelial permeability, 2)
increased cell migration through blood vessel walls, 3) thrombin generation, 4) increased monocyte binding to the subendothelial matrix,
5) increased lipoprotein retention, 6) increased SMC proliferation, and
7) increased susceptibility to bacterial infection. HS has also been
shown to inhibit matrix metalloproteinase activity (32), involved in
plaque rupture (33), and to regulate the bioavailability of basic
fibroblast growth factor activity (34). Agents that decrease
endothelial HSPG include lipopolysaccharide and tumor necrosis
factor-
(35), homocysteine (36), lysolecithin, and oxidized LDL (18,
19). Thus, a decrease in HS may be a general inflammatory reaction.
The present studies show that HDL increases endothelial HS and thus
could decrease the occurrence of the above mentioned events. HDL has
several anti-atherogenic effects, and epidemiological studies inversely
correlated HDL with atherosclerosis (37). HDL facilitates reverse
cholesterol transport from peripheral tissues to liver (38). In
addition, platelet-activating factor acetyl hydrolase and paraoxonase
enzymes associated with HDL can metabolize and reduce the content of
biologically active oxidized lipids in oxidized LDL (39, 40). Our
current results show yet another mechanism by which HDL can be
anti-atherogenic, i.e. by increasing sulfation of
endothelial HSPG.
HDL increased both glucosamine and sulfate incorporation into PG
without significantly affecting the total core proteins. Although total
core proteins have not changed based on the elution of
[3H]leucine-labeled proteins from DEAE-cellulose, it is
conceivable that HDL increased specific PG core proteins which remains
to be determined. The ratio of sulfate to glucosamine was increased by
HDL, suggesting that the GAG were more sulfated in HDL-treated endothelial cells. The increase in sulfation does not appear to be due
to apoE binding to HS and preventing its degradation because incubation
of 35SO4-labeled endothelial cells with HDL did
not prevent degradation. Thus, HDL treatment may have increased
sulfation by affecting sulfotransferases. Several enzymes involved in
the sulfation of HSPG have been characterized (41-45). These include
glucosamine N-, 3-O, and 6-O
sulfotransferases and uronic acid 2-O sulfotransferase. It
is conceivable that HDL increased the activities of one or more of
these enzymes. Regulation of sulfation and sulfotransferases in
endothelial cells is a poorly studied area. This is surprising considering the several known anti-atherogenic effects of endothelial heparin and HS. Evidence has recently been put forward that a deficiency of endogenous heparin or heparin-like substances predisposes to atherosclerosis (46, 47), and heparin administration has been shown
to increase sulfation of endothelial HS (48).
Subendothelial HS contains substantial amounts of highly sulfated
blocks (referred to as heparin-like) (49, 50). These are resistant to
heparitinase (heparinase III) digestion. Data in Figs. 4 and 5 support
the conclusion that HDL increased sulfated HS. Antithrombin binding is
largely restricted to heparin chains containing glucosamine
N and 3-O sulfates (51). In our experiments antithrombin binding was increased to matrix prepared from HDL-treated cells, suggesting an increase in
GlcNSO3(3-OSO3)-heparins. Second, HS prepared
from HDL-treated matrix inhibited SMC proliferation, suggesting an
increase in HS sequences containing 2-O uronic acids (25).
An increase in 2-O iduronic acid-containing HS was also confirmed by increased binding of lipoprotein lipase (1.6-fold, not shown).
Experiments with HDL prepared from apoE and apoA-I knockout mice
suggest that apoE is required for stimulation of sulfation. This was
confirmed further by apoE add-back experiments and apoE emulsion
experiments. Lipid-free apoE also stimulated endothelial HS production.
It is, however, conceivable that apoE acquired cellular lipid during
the 16-h incubation. Nevertheless, these data clearly show that apoE is
required for HS stimulation. Although containing apoE, VLDL failed to
stimulate HS. Whether this is because of conformational changes in VLDL
apoE remains to be determined.
Although apoE is known to play a key role in lipoprotein clearance,
recent studies from several groups indicate that its anti-atherogenic effects go beyond its role in remnant clearance. Shimano et
al. (52) and Bellosta et al. (53) expressed apoE in the
vessel and decreased atherosclerosis in apoE-null mice without
significant changes in plasma lipoproteins. Recently, Fazio et
al. (54), by transplanting apoE-null macrophages into normal
C57BL6 mice, increased atherosclerosis. How apoE protects the vessel
wall from accumulating lipoproteins is not clear. This may in part be
the result of its ability to facilitate removal of cholesterol from the
cells of arterial walls (37).
Other studies have also suggested possible anti-atherogenic roles for
apoE-HDL, including inhibition of lipase-mediated LDL retention (55)
and inhibition of platelet aggregation through the nitric oxide pathway
(56). Studies from humans as well as mice suggest that low apoE as well
as apoE-HDL are important risk factors for vascular disease (57-60).
Our observation that apoE-HDL increases sulfation of HS offers an
alternative explanation for the anti-atherogenic effects of
vascular/macrophage apoE. Atherosclerotic vessels have decreased HS,
and we showed that removal of subendothelial HSPG resulted in a
2-10-fold increase in the binding of atherogenic lipoproteins such as
lipoprotein(a) and monocyte-macrophages (18, 19). Consistent with this,
our present data show that increasing HSPG by HDL treatment decreased
the number of monocytes binding to the subendothelial matrix. In
addition, an apoE-HDL-mediated increase in vascular HSPG can prevent
SMC proliferation in the subendothelial space.
The mechanism of apoE-HDL-mediated stimulation of sulfation is not
clear. ApoE is known to affect cell signaling by increasing cAMP and
cGMP levels (56). Preliminary experiments showed that 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor (1-5 mM), did not inhibit apoE actions (not shown). It appears
that the apoE-mediated effects are cell-specific as no increase in 35SO4 incorporation was observed in either
macrophages or fibroblasts. Data from WT and apoE-null mice further
support this. Hearts (with proximal aorta) but not livers showed a
significant decrease in 35SO4 incorporation in
apoE-null mice. This may be caused by differences in the tissue
metabolism of lipoproteins or endothelial cell heterogeneity (61).
However, further experiments are needed to prove that the HS decrease
is a direct effect of apoE deficiency. It is conceivable that vascular
cell-specific surface molecules mediate apoE actions. ApoE has several
known receptor molecules, including LDL receptor, LDL-receptor related
protein, VLDL receptor, and proteoglycans. Endothelial cells are not
known to express LDL receptor-related protein (62). Our data showed
that LDL receptor antibody, excess LDL or VLDL did not inhibit
apoE-mediated HS stimulation. RAP, a LDL receptor family antagonist,
inhibited apoE actions at high doses. VLDL receptor, which is abundant
on endothelial cells, appears to be one potential candidate for
mediating apoE actions (62). It is also conceivable that RAP, by direct
interaction with cell surface HSPG, inhibited apoE binding (63).
However, RAP binding to HSPG is controversial (64). Heparin also
inhibited apoE actions, suggesting that HSPG, either directly or
indirectly facilitate binding to RAP-sensitive receptors and are
involved in the mediation of apoE actions. Experiments with apoE-null
mice also showed decreased 35SO4 incorporation
only in heart but not liver. Although this is surprising considering
the fact that liver is an endothelial cell-rich organ, one possible
reason for this is the lack of VLDL receptors or other potential
RAP-sensitive receptors.
In summary, our data show that apoE HDL enhances sulfation of
endothelial PG and suggest a novel mechanism by which apoE can be
anti-atherogenic. These effects were seen within the physiological concentrations of HDL and within the levels of apoE found in HDL (24).
Our data, in addition, offer an alternative explanation for the
anti-atherogenic effects of macrophage apoE. After synthesis and
secretion from macrophages, apoE or apoE particles can act locally and
stimulate endothelial HSPG. Increased HSPG can inhibit subsequent
accumulation of lipoproteins and monocytes and inhibit subendothelial
SMC proliferation. Thus, identification of specific apoE peptides that
can stimulate endothelial cell heparin production may have important
therapeutic application.