© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
The Heparin-Binding Proteins Apolipoprotein E and Lipoprotein Lipase Enhance Cellular Proteoglycan Production
From the Departments of Medicine (J.C.O., S.P., L.P., Y.K., M.J.B., I.J.G.) and Pediatrics (Y.-Y.H., R.J.D.) and the Institute of Human Nutrition, Columbia University College of Physicians & Surgeons, New York, NY; the Department of Pathology (W.D.W.), Wake Forest University School of Medicine, Winston-Salem, NC; the University of Tokyo School of Medicine (N.Y.), Tokyo, Japan; and the Departments of Medicine and Biochemistry (T.M.), Rush Medical College, Chicago, Ill.
Correspondence to Dr Joseph C. Obunike, Division of Preventive Medicine, BB 906, Department of Medicine, Columbia University, 630 W 168th St, New York, NY 10032.
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Abstract |
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Abstract—Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with 35[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [3H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non–apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.
Key Words: macrophages • atherosclerosis • glycosaminoglycans • chondroitin • heparan
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Introduction |
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ApoE and lipoprotein lipase (LPL) are key proteins in the regulation of lipoprotein metabolism.1 2 3 4 Their actions are interrelated: VLDL and chylomicron removal from the bloodstream requires both of these proteins. LPL hydrolyzes triglyceride in the lipoprotein core and creates a remnant lipoprotein. ApoE mediates clearance of the remnant particles by associating with the LDL receptor and the LDL receptor–related protein on hepatocytes. LPL can also function as an LDL receptor–related protein ligand.5 6 7 Animals that have a gene deletion for apoE have high levels of cholesterol-rich remnants in their bloodstream and extensive lipid-loaded atherosclerotic lesions.8 9 LPL deficiency results in severe hypertriglyceridemia and neonatal demise in mice.10 11
LPL is synthesized primarily in muscle and adipose tissue, and apoE is made in the liver. Both of these proteins are also expressed in several other cell types, including macrophages.12 13 14 Macrophage LPL provides fatty acids for energy.15 Macrophage apoE may facilitate the unloading of cholesterol from cells, thereby promoting the antiatherogenic process of reverse cholesterol transport.16 17 18 19
ApoE and LPL bind to heparin and proteoglycans (PGs) with high affinity. PGs are important constituents of cell membranes and the extracellular matrix20 21 and consist of a core protein to which linear, pendant chains of negatively charged polysaccharides, termed glycosaminoglycans (GAGs), are attached. The 3 major GAG classes in the vessel wall are heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS). PG synthesis requires addition of long GAG chains followed by enzymatic sulfation of these GAGs at different N and O groups. A number of enzymes involved in this process have been cloned.22 23 24 25 26 27 Like PG synthesis, PG catabolism appears to require the participation of several enzymes, including proteases, endoglycosidases, exoglycosidases, and sulfatases.28 29 30 31 32 33 There appears to be an initial, slow internalization of cell-surface PGs followed by a 2-step degradation31 32 33 ; only the second of the 2 steps is inhibited by lysosomal inhibitors. It is unclear whether this process is regulated by ligands that associate with PGs on the cell surface or within the cells.
In previous studies from our laboratory,34 35 we showed that the lipoproteins and LPL that were associated with cell-surface PGs were degraded much more slowly than through the usual receptor-mediated processes. By assessing the rate of turnover of pericellular PGs, we concluded that at least a portion of the LPL and lipoprotein degradation appeared to occur via the uptake and remodeling of cell-surface PGs. Because LPL interaction with PGs is facilitated by low pH,36 we postulated that complexes of PGs and their associated proteins might be more resistant to lysosomal, degradative enzymes. To test this hypothesis, in the present report we studied whether LPL and apoE affect the production of PGs by cells. Because lipoprotein retention within arteries and the actions of growth factors that affect smooth muscle cell proliferation involve vessel wall PGs, we postulated that alterations in the cellular production of PGs by apoE and LPL would modulate atherogenesis.
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Methods |
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Cells
J774 Macrophages
J774 cells were stably transfected to express a wild-type, human apoE cDNA as described previously in detail.18 37 The cells were prepared by cotransfection of the apoE cDNA expression vector containing the human metallothionine IIA promoter with pSV2-neo by calcium phosphate coprecipitation in the presence of 10 mmol/L NH4Cl. This cell line constitutively expresses a human apoE cDNA (E3 isoform). Cells were maintained in 400 µg/mL G418 (Geneticin, Life Technologies) and grown in 10% fetal bovine serum (Gemini Bioproducts Inc) in Dulbecco’s modified Eagle’s medium (DMEM) until the start of the described experimental incubations.
Fibroblasts
ApoE-expressing fibroblasts were derived from F111 rat embryonal
fibroblasts.38 Cells were cotransfected with a human apoE
cDNA expression vector18 and a pß-hygro vector
conferring hygromycin resistance by calcium phosphate–mediated DNA
precipitation.39 Cells were selected, maintained, and
grown as mentioned above.
CHO Cells
Chinese hamster ovary (CHO)-K cells were maintained in DMEM/F-12
supplemented with 10% fetal bovine serum. Plasmid pCMV-LPL, in which
LPL cDNA was fused to the Rc/CMV vector, was used to transfect CHO
cells by using the DNA–calcium phosphate coprecipitation method as
previously described.40 LPL-transfected cells were
maintained in the same medium with addition of G418. To confirm that
the cells were producing LPL, LPL activity was measured before each
experiment by using a glycerol triolein emulsion41 as
previously described.35
Transfection of the amino-terminal 17% of apoB (apoB17) was performed by using a calcium phosphate–DNA coprecipitate containing 20 g of pCMV5-apoB17-DAF plasmid DNA, 1 g of SV2-neo selection maker, and 20 mol/L chloroquine.42 Cells were selected, maintained, and grown as mentioned above. CHO cells producing apoA1 were generously provided by Dr L. LeCureux (The Upjohn Company, Kalamazoo, Mich).
PG Production
PGs in nontransfected and transfected cells were labeled by
incubating them for 20 hours in DMEM containing 10% fetal bovine serum
and 50 µCi/mL [35S]sulfate. Cultured medium,
pericellular (trypsin-releasable), and intracellular PGs were
collected, extensively dialyzed, and assessed in triplicate cultures as
previously described.35 Total PG radioactivity was
measured after precipitation with 3 volumes of absolute ethanol
containing sodium acetate (0.8 g/L) for 18 hours at -20°C. Samples
were spun at 2000 rpm for 1 hour, the supernatant was removed, and the
pellet was solubilized in 0.5N NaOH. The radioactivity in 1 mL of the
aqueous phase was determined by using 3.5 mL of scintillation fluid
(Hydrofluor, National Diagnostic) in a model 1800
liquid scintillation counter (Beckman Instruments).
PG Synthesis
Macrophages were labeled with 50 µCi/mL
[35S]sulfate for 30 minutes to 4 hours. At the
end of each time period, pericellular, intracellular, and secreted PGs
were collected and dialyzed extensively. Total PG radioactivity was
measured as described above.
Assessment of HSPG and DS/CSPG
Aliquots of dialyzed PGs from the 3 pools were treated with 0.05
U of chondroitinase ABC in enzyme buffer containing 0.01 mol/L
N-ethylmalemide, 0.07 mmol/L pepstatin, 0.001 mol/L
PMSF (protease inhibitors), 1 mg/mL BSA, and CS (50 µg/mL
as a carrier) and then incubated for 20 hours at 37°C. HSPG
radioactivity was measured after ethanol precipitation as described
above. DS/CSPG was determined by subtracting HSPG from the total
PG.
GAG Identification and Size Estimation
For GAG radiolabeling, cells were incubated with medium
containing 30 µCi [35S]sulfate and 30 µCi
[3H]glucosamine per milliliter at 37°C for 20
hours. PGs from different pools were collected and dialyzed as
described above. Protein-free GAG chains were isolated as previously
described.43 44 Aliquots (2 mL) of dialyzed PGs were
treated with 200 µL of 10N NaOH for 18 hours at 26°C with constant
shaking and then neutralized with 10N HCl. To remove core proteins, the
samples were adjusted to 7 mol/L urea and loaded onto a column
containing 1 mL DEAE cellulose that was washed with 3 bed volumes of 7
mol/L urea, 0.1% Triton X-100, and 0.2 mol/L NaCl in 0.05 mol/L Tris,
pH 7.2. 3H-labeled GAGs were eluted with a
solution of 7 mol/L urea, 0.1% Triton X-100, and 1 mol/L NaCl in 0.05
mol/L Tris, pH 7.2. Urea was removed by dialysis against 10 mol/L Tris
and 0.1% Triton X-100, pH 7.0.
For size analysis, 3H-labeled GAGs from the pericellular pool were chromatographed on a 1.5x90-cm column of Sepharose 6B (Pharmacia) and eluted with 0.2 mol/L NaCl at a flow rate of 11.5 mL/h. Fractions of 0.4 mL were collected and analyzed for radioactivity. Average peak elution position (Kav) of the sample GAG (Ve) was calculated from the equation Kav=(Ve-Vo)/(Vt-Vo), where Vo equals the column void determined by the elution position of LDL, and Vt equals the column total bed volume determined by the elution position of bromophenol blue.45
GAGs of known molecular weights were used to generate a standard curve for molecular weight, and Kav45 molecular weights of the sample GAGs were estimated on the basis of Kav values relative to standard GAGs.
Human Recombinant ApoE
Bacterial recombinant human apoE (apoE3/E3 isoform) was provided
by Biotechnology General (Rehovot, Israel) and isolated as previously
described.46 This apoE has similar physical and biological
properties to native human plasma apoE3.46
Antibodies
Anti-human monoclonal apoE antibody, which interacts with the
LDL receptor–binding site of apoE, was kindly provided by Dr Linda
Curtiss (The Scripps Research Institute, La Jolla, Calif).
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Results |
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Production of PGs by Transfected Cells
We first examined whether apoE expression by J774 macrophages altered PG production. After incubating the cells for 20 hours in [35S]sulfate-containing medium, the amounts of radiolabel in the medium, pericellular PGs, and intracellular PGs were assessed. As shown in Figure 1A
, apoE-expressing cells had 57% more
label secreted into the medium pool, 65% more label in the
pericellular pool, and 155% more label in the intracellular pool of
PGs.
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To determine whether apoE expression would also enhance PG
production by fibroblasts, cells were labeled as mentioned
earlier. As shown in Figure 1B
, apoE expression by fibroblasts
led to an even greater increase in radiolabeled PGs. The
apoE-expressing fibroblasts contained 
174% more PGs in the medium
pool, 151% more PGs in the pericellular pool, and 185% more
35S labeled PGs in the intracellular pool.
Therefore, apoE expression was associated with a greater amount of
sulfate-labeled PGs in both kinds of cells.
Because cells produce several different classes of PGs, PG-degrading
enzymes were used to assess the relative increase in HSPG and DS/CSPG.
As shown in Figure 2, after 20 hours of
labeling, there was a 71% increase in medium HSPG. Similarly, the
amount of sulfate label in DS/CSPG was 185% greater in the
apoE-producing cells. Therefore, both HSPG and DS/CSPG were secreted in
greater amounts from apoE-synthesizing cells.
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PG Synthesis in ApoE-Transfected Cells
The increase in cellular and pericellular PGs and the increase in
secretion of PGs into the medium could have resulted from an increase
in synthesis rate or a decrease in PG degradation. To test whether
expression of apoE increased PG synthesis, control and apoE-expressing
macrophages were incubated with
[35S]sulfate for up to 4 hours to assess newly
synthesized PGs in different pools. No difference in label
incorporation into PGs was noted in the 3 pools during the first hour
of incubation (Figure 3). However, later
in the incubation, apoE-expressing cells contained more radiolabeled
PGs. Similarly, differences in label in the secreted, pericellular, and
intracellular PGs were noted beginning at 2 hours, so that at 4 hours
the amount of label found in the apoE-expressing cells was 3-fold more
in the medium (Figure 3A), 1.6-fold more in the pericellular PGs
(Figure 3B), and 2-fold more in the intracellular PGs (Figure 3C). These data suggest that apoE increased either the sulfation
or the total amount of GAGs. It should be noted that the labeling
procedure of sulfation of GAG chains occurs in the Golgi. This process
has different kinetics than that of protein synthesis; this could
explain the time required to observe a difference in radiosulfate
incorporation.
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Comparison of ApoE Effects on GAG Production
The above data show that apoE increased the amount of sulfate
label in cellular, pericellular, and medium PGs. To determine whether
this was due to sulfation or an increase in the amount or length of the
GAG chains, we assessed whether the ratio of glucosamine to sulfate
label was similar in control and apoE-expressing cells. For this
experiment, apoE-expressing macrophages were doubly labeled
with [35S]sulfate and
[3H]glucosamine overnight, and the 3 pools of
PGs were isolated. The ratio of the 2 labels was the same in the
apoE-expressing and control cells (data not shown). Therefore, there
was an increase in total GAGs and not a selective increase in
sulfation.
To assess whether the increase in GAGs was due to more or longer
chains, after the PG core proteins were eliminated, GAG length was
measured by elution position on a Sepharose 6B gel filtration
column and compared with published calibration
curves.45 The gel filtration profile of the pericellular
[3H]GAG is shown in Figure 4. Longer GAG chains were found in the
pericellular pool from the apoE-expressing macrophages than
from non–apoE-expressing cells; the average chain length increased
from 7 to 28 kDa. In the first part of the Results, we indicated that
the medium PG increase was 174% (Figure 1), so the increase in
GAG size can account for probably all of the increase observed in GAG
production. Thus, apoE either increased the chain length or
protected the GAGs from degradative processes.
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Effects of ApoE Expression on PG Degradation
One possibility is that the association of apoE protects GAGs from
degradation. To study degradation, apoE- and non–apoE-expressing J774
macrophages were labeled for 20 hours, the label was removed,
and sulfate-labeled PGs in the 3 pools were assessed during a chase
period. Pericellular PG loss was, if anything, more rapid in the
apoE-producing cells. As shown in Figure 5, at 30 minutes, 49% of the
pericellular sulfate label was lost from the control cells, but 56% of
the label was lost from the apoE-expressing cells; at 4 hours, 26% of
the original label remained in the pericellular pool of controls and
19% in the pericellular pool of the apoE-expressing cells (Figure 5B). Moreover, as shown previously, there was a marked increase
in secretion of PG into the medium. Therefore, it appeared that in the
apoE-expressing cells, the larger intracellular and pericellular PG
pools led to more PG secretion into the medium (10 890 950 versus
212 502 680 counts per minute per 3x106 cells;
Figure 5A). This suggests that apoE increased the amount of GAGs
either because longer chains were synthesized or because fewer newly
synthesized GAG chains were partially degraded.
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Effects of LPL Expression on PG Production
Because more PG was produced by apoE-expressing cells, we next
tested whether another heparin-binding protein, LPL, would increase
cellular PG production. PG production was assessed in
control and LPL-overexpressing CHO cells (Figure 6). LPL-transfected CHO cells had 3-fold
more sulfate-labeled PGs in cells and in the pericellular pool after a
20-hour incubation. As noted for the apoE-expressing cells, secretion
of labeled PGs was much greater in the LPL-expressing cells. PG
secretion into the medium at the end of a 20-hour incubation was 185%
more in the LPL-expressing cells.
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To determine whether the increase in PG production observed in
LPL-transfected cells was due to transfection, we assessed PG
production in CHO cells transfected with apoA1 and apoB17
(Figure 7). PGs secreted into the medium
(Figure 7A), in the pericellular pool (Figure 7B), and in
the intracellular pool (Figure 7C) by apoA1- and
apoB17-transfected cells were similar to control cells and >1.8- to
2-fold less than LPL-expressing cells. Because an increase in PG
production was observed only in LPL-expressing cells but not in
apoA1- or apoB17-expressing cells, these data suggest that transfection
does not cause increased PG synthesis in cells.
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Effects of Exogenous ApoE, ApoE Antibody, and LPL on PG
Catabolism
We next determined whether the increased GAG secretion into the
medium was due to association of the heparin-binding proteins with
cell-surface PGs. ApoE-transfected and nontransfected
macrophages were incubated with either apoE or LPL, and the
turnover of pericellular PGs and accumulation of medium PGs were
assessed. The hypothesis was that the binding of these proteins to the
cell-surface PGs would protect them from intracellular degradation, so
that more of them would be secreted into the medium. Human recombinant
apoE (5 µg/mL) with or without 10 µg/mL anti-human apoE
antibody was added to control and apoE-expressing macrophages,
and PG production after 4 hours was assessed. As expected,
apoE-expressing cells produced more PGs (>3-fold) than did
non–apoE-expressing cells (Figure 8). However, the addition of
neither apoE nor anti-human apoE antibody altered PG production
in the medium, pericellular pool, and intracellular pool. Moreover,
when apoE-containing lipid emulsion particles were incubated with the
cells, PG production was not altered (data not shown).
Therefore, it appears that only intracellular production of
apoE affected PG production.
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It had been hypothesized that association of proteins with cell-surface
PGs would alter PG metabolism.47 Our data
concerning the addition of apoE to cells do not appear to support this
conclusion. To further test this, LPL, a stronger heparin-binding
protein, was added to macrophages. This addition led to no
change in the rate of turnover of pericellular, sulfate-labeled PGs
Figure 9. Therefore, when LPL was bound
to cell-surface PGs, it did not alter their degradation.
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Discussion |
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The present data show that PG-binding proteins modulate PG structure. ApoE expression increased the amount of GAGs associated with PGs in macrophages and fibroblasts; LPL-expressing CHO cells produced more radiolabeled PGs than did control cells. In both cell types, PG levels increased in the cellular, pericellular, and medium pools after the cells were labeled with [35S]sulfate. Similar increases were found when the cells were labeled with [3H]glucosamine. Thus, the amount of carbohydrate in GAGs was increased. Analysis of GAG chain length showed that they were longer in apoE-synthesizing cells.
Why should these PG-binding proteins increase GAG chain length? Either they increased GAG synthesis or decreased its degradation. Data confirming 1 of these processes were not obtained, in part because the complexity of PG assembly complicates analysis of radioisotope labeling studies. Increased synthesis by apoE-producing cells was noted after only 1 hour of labeling. This could have occurred if newly synthesized GAGs were protected from intracellular degradation. Alternatively, the apoE might have bound to nascent GAGs and thereby increased their length.
The enzymes involved in the intracellular PG degradation pathways and their locations are well defined. Studies from other cell types suggest that the initial cleavage of HS GAG is carried out by endoglycosidases that primarily exist in nonlysosomal (chloroquine-insensitive) compartments.31 32 48 The HS intermediates generated by these endoglycosidases are further cleaved, by a chloroquine-sensitive endoglycosidase activity, to smaller oligosaccharides that are rapidly degraded in lysosomes by endoglycosidases. ApoE and LPL might protect GAGs in a chloroquine-sensitive/lysosomal or a low-pH/endosomal compartment, because protein association with GAGs can be greater in more acidic pH. Earlier studies showed that this was true for the LPL association with endothelial cell-surface PG,36 and it was postulated that this was the reason that endothelial cells do not degrade LPL. An alternative to this degradation hypothesis is that apoE and LPL increased the chain length or protected GAGs from the intracellular degradation that occurs during intracellular transport of newly synthesized PGs.
In vitro studies from this and other laboratories have suggested that
binding of LPL and basic fibroblast growth factor (bFGF) by HS can
protect it from digestion by purified heparanases.49 50
When LPL was preincubated with endothelial HSPG and
then subjected to heparanase digestion, LPL protected the fragments of
HS from heparanase digestion.49 Other studies showed that
the conversion of 85-kDa HS chains to short 6-kDa HS by CHO cell
heparanase was inhibited by bFGF.50 A 30- to 60-molar
excess of bFGF completely blocked heparanase actions. Our gel
filtration data on GAG chain length are consistent with the
hypothesis that apoE and LPL blocked GAG degradation. The GAGs in the
pericellular pool of apoE-expressing cells were 7 to 28 kDa longer
than the GAGs from control macrophages. It is conceivable that
apoE binding protects GAGs from endogenous GAG-degrading
activities.
Perhaps the most important aspect of these studies is the observation that apoE and LPL increased the amount of PG in the medium. There are several pathways responsible for PG delivery to the medium. A pool of newly synthesized PGs is directly secreted from cells. Second, some internalized pericellular PG is delivered into the medium. A third pathway is via release of cell-surface PGs through the actions of extracellular enzymes, a process referred to as "shedding."32 51 This process may involve degradation of core proteins by proteases or, in the case of glycosylphosphatidylinositol-anchored PGs, by cell-surface, phosphatidylinositol-specific phospholipase C. The protease cleaves the transmembrane core proteins, thus releasing the ectodomains with attached GAGs. Our data are more consistent with apoE’s affecting the first pathway of PG secretion. This is based on the observation that exogenous apoE or LPL, which can associate with pericellular PGs, failed to increase PG levels in the medium. A portion of the newly synthesized PGs in late Golgi vesicles may fuse with endosomes and lysosomes and be degraded, instead of going through the secretory pathway. However, in the presence of bound apoE or LPL, this degradation after fusion may be prevented.
How could apoE protect GAGs in the lysosomal compartment if apoE itself can be degraded by lysosomal proteases? Previous studies have shown that apoE is relatively resistant to intracellular degradation after endocytosis.52 By gel filtration and ultracentrifugation analyses, Chen et al53 recently showed that apoE aggregates at low pH and that this phenomenon may in part be the reason for the insensitivity of apoE to protease degradation. This aggregation can be overcome by increasing the pH.
A final observation from our studies relates to the metabolism of cell-surface PGs to which ligands have been attached. We had previously shown34 that when LPL became associated with the cell surface, some of the uptake and degradation of LPL and its associated lipoproteins appeared to occur exclusive of lipoprotein receptors. Much of this high-capacity but kinetically slower pathway could be accounted for by the turnover of cell-surface PGs; the LPL and lipoproteins were merely bystanders in this process. Because both transmembrane (ie, syndecans) and glycosyl-phosphatidylinositol-anchored PGs are internalized and lead to LPL degradation,35 54 55 there did not appear to be any special role for the transmembrane region of syndecan in this process. Syndecan clusters and is phosphorylated, but the importance of this molecule in ligand and PG metabolism is unknown.51 It has been hypothesized that ligand association with syndecans will cause clustering and thereby alter syndecan metabolism.47 Our studies comparing the turnover of pericellular PGs in the presence and absence of exogenously added LPL do not support this view; LPL association with PGs did not alter PG turnover rates.
A number of PG-mediated processes are thought to be central to the development of the atherosclerotic plaque and may be involved in lipid accumulation, cell proliferation, matrix remodeling, and thrombosis. Both proatherosclerotic and antiatherosclerotic actions of PGs have been described. After crossing the endothelial barrier, apoB-containing lipoproteins may be retained within the arterial wall by their interaction with PGs,56 and smooth muscle cell growth is correlated with the amount of cell-surface PGs.57 Moreover, by sequestering growth factors such as bFGF, matrix PGs prevent growth factor stimulation of cells.58 Matrix metalloproteinases are activated by a PG-bound factor,59 and PG activation of antithrombin and heparin cofactor II60 61 is thought to prevent thrombosis on and within the artery. Finally, PGs in the subendothelial matrix can prevent the association of monocytes62 and Lp(a)63 with matrix adhesive proteins. Thus, depending on the type of PG and the cell of origin, increases in PGs induced by apoE or LPL could modulate pathological responses within the vessel.
In summary, we provide data that 1 process that regulates GAG production by cells is the coproduction of PG-binding proteins. These proteins lead to longer GAG chains. Increases in GAG production and secretion may alter a number of in vivo processes. Because growth factors and cytokines bind to PGs, actions of the former might be modified by altered matrix GAGs. ApoE production by macrophages appears to reduce atherosclerosis in atherosclerosis-prone mice.19 64 65 Perhaps this occurs in part by modulation of the amount or type of GAG that is present within the artery.
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Acknowledgments |
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This work was funded by grants HL-03323 (to J.C.O.), HL-45095 (to I.J.G.), HL-40404 (to R.J.D.), and HL-39653 (to T.M.) from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md. S.P. is an investigator of the American Heart Association, New York City Affiliate. We are indebted to Dr Lyold LeCureux from Upjohn Company (Kalamazoo, Mich) who provided us with apoA1 CHO cells. We wish to thank Tommy Katopodis for maintenance of cell cultures.
Received September 3, 1998; accepted June 17, 1999.
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