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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Clinical Biochemistry, Surgery,
and Medicine and the Center for Research, Prevention and Treatment of
Atherosclerosis, Hebrew University-Hadassah Medical Centers,
Jerusalem, Israel; Department of Pathology and Laboratory Medicine and
the Department of Medicine, University of Pennsylvania, Philadelphia;
Division of Endocrinology, Diabetes and Metabolic Diseases, Department
of Medicine, Thomas Jefferson University, Philadelphia, PA; and
the Division of Pulmonary and Critical Care, Department of Medicine,
University California at Los Angeles.
Inflammation may contribute to the pathogenesis of atherosclerosis.
On the basis of previous reports that human atherosclerotic lesions
contain Atherosclerosis is a multifactorial disorder
that develops over decades under the influence of both genetic and
environmental factors. Retention of low-density lipoproteins (LDL)
within the walls of blood vessels is a pathognomonic feature and
perhaps the central cause of the disease.1 In accordance
with this, elevated plasma levels of LDL resulting from a deficiency in
the expression of LDL-receptor (LDL-R),2 mutations in LDL
that impair its ability to bind to this receptor,3,4 or
mutations in the receptor itself5 are associated
with an increased risk of atherosclerotic vascular disease. Failure to
endocytose and degrade LDL via the LDL-Rs increases not only its plasma
concentration but also its residence time in the vasculature, thereby
permitting it to undergo oxidative and nonoxidative (reviewed in
Williams and Tabas6) modifications. Modified LDL is taken
up by a number of "scavenger" receptors expressed by macrophages
and other arterial cell types contributing to the formation of foam
cells.3,7 Cytokines and proteolytic enzymes released from
foam cells may contribute to smooth muscle cell proliferation, intimal
thickening, aneurysm formation, and plaque
instability.8
Inflammation within the arterial wall (see Ross8 for
review) also contributes to the initiation and propagation of
atherosclerotic vascular disease. For example, some atherosclerotic
lesions and inflammatory lesions share a number of histologic
features.8,9 Persistent vascular infection with certain
DNA viruses,10,11 bacteria,12 and
chlamydia13; allograft rejection14; and biologically active molecules released from modified LDL6,8 may stimulate localized inflammation of the arterial wall, predisposing a person to accelerated lipoprotein retention and atherosclerotic vascular disease. Induction of adhesion receptors for leukocytes on
endothelial cells has been implicated in the early stages of the
disease.15,16 In addition, elevated levels of acute phase reactants, such as C-reactive protein, fibrinogen, and
interleukin-6 correlate with the risk of clinical events and with the
response to therapies such as aspirin.17 Furthermore, a
correlation between the number of circulating granulocytes and
prevalence of atherosclerosis has also been identified in many
studies.18-20
Neutrophil-derived ( Importantly, Materials
Binding of LDL to cells
Experiments were performed to determine whether LDL underwent oxidation in the presence of defensin under the conditions used to measure cell binding and catabolism. To do so, fibroblasts were incubated with 50 nmol/L unlabeled LDL in PBS containing 0.25% BSA in the presence or absence of 10 µmol/L defensin for 4 hours at 4°C, after which the amount of oxidized LDL in the media was measured.40 This experiment was performed twice, each time in triplicate (n = 6). Binding of defensin to cells and to LDL Defensin was iodinated and the specific binding to cells was measured as described previously.30,33 In some experiments, binding was measured in the presence of anti-LDL-R or control immunoglobulin. LDL or BSA (10 nm each) was incubated with 125I-defensin (0 to 5 µmol/L) for 1 hour at 37°C. The mixture was then applied to a Bio-Gel P-30 polyacrylamide gel (Bio-Rad, Hercules, CA), and the radioactivity in the excluded volume (ie, MW exceeding approximately 40 kd) was measured. Specific binding of defensin to LDL was defined as the difference between the BSA-associated and LDL-associated radioactivities.Internalization and degradation of LDL LDL-R-deficient (FH) fibroblasts were incubated with 100 nmol/L 125I-LDL in serum-free medium in the presence or absence of 10 µmol/L defensin for 2 hours at 4°C to permit surface binding without catabolism. The cells were washed 4 times with PBS-BSA; fresh medium without ligands was then added; and the incubations were continued at 37°C for the indicated times followed by chilling to 4°C. Glycine buffer, pH 3.0, at 4°C was added, and the acid-elutable radioactivity was measured as a marker of cell-surface-associated ligand. The difference between the acid eluted and the residual acid-resistant radioactivity was taken as an estimate of internalized ligand that had not been degraded.50 The cell supernatant was removed, and the trichloroacetic acid (TCA) (10% final concentration) soluble radioactivity was measured. Cell-specific degradation was calculated from the difference between cell-associated degradation and spontaneous degradation of ligand in cell-free wells measured in parallel. TCA-precipitable radioactivity in the media was quantified as an indication of retroendocytosis or desorption from the cell surface. To examine the temperature dependence of binding and degradation, HUVECs were incubated with 125I-LDL (100 nmol/L) and defensin (10 µmol/L) for 3 hours at 4°C. The cells were washed 4 times with PBS-BSA and either kept at 4°C or warmed to 37°C for an additional 4 hours. Glycine, pH 3.0, was added, and the residual cell-associated radioactivity was measured. In other experiments, XT or
wild-type CHO cells were incubated for 18 hours at 37°C with 10 nmol/L 125I-LDL in the presence or absence of 10 µmol/L
defensin and the presence or absence of 100-fold molar excess unlabeled
LDL, and the specific degradation of labeled ligand was measured as
described above.
Defensins stimulate the binding of 125I-LDL to HUVECs We examined the effect of -defensin on the interaction of
125I-LDL with vascular cells. Defensins stimulate the
binding of 125I-LDL to HUVECs (Figure
1A). Maximal binding of
125I-LDL was increased approximately 5-fold in the presence
of 10 µmol/L defensin. with little change in the concentration of
125I-LDL required for half maximal binding (22 nmol/L vs 40 nmol/L). Defensin stimulated the binding of 125I-LDL to
human SMCs and fibroblasts to a similar extent (not shown). Stimulation
of LDL binding by defensin increased approximately 10-fold at 37°C
compared with 4°C (not shown) and depended on the concentration of
defensin present (Figure 1B). Stimulation was apparent at a defensin
concentration of 2.5 µmol/L and reached its half-maximal effect at
4.3 µmol/L, which is within the plasma concentration
attained in vivo during severe inflammation.25,26 More
than 85% of the binding of 125I-LDL in the presence of
defensin was inhibited by excess LDL (Figure
2) or by preincubation of
125I-LDL/defensin with a monoclonal antidefensin antibody
(Figure 2). Incubation of 125I-LDL with defensin under
these conditions did not increase lipoprotein oxidation when
measured directly.
On the basis of these results, we investigated the hypothesis that defensin and LDL form complexes that bind to cells as a unit. To address this possibility, 125I-defensin in varying concentrations was incubated with 10 nmol/L LDL, and the radiolabeled protein that bound to LDL was isolated by size-exclusion chromatography. Defensin bound to LDL in a dose-dependent and saturable manner, first apparent at a defensin concentration of 10 nmol/L (ie, 1:1 molar ratio with LDL) and reaching half-maximal binding at a defensin concentration of approximately 1.2 ± 0.04 µmol/L (mean ± SD; n = 3). Preincubation of HUVECs with saturating concentrations of defensin33 stimulated binding of 125I-LDL to the same extent as when both ligands were present simultaneously. This outcome suggests that LDL can bind to cells either as a soluble preformed complex with defensin or directly to cell-associated defensin. The LDL-R does not mediate defensin-stimulated LDL binding We next examined the cellular binding sites for defensin and for defensin/LDL complexes. We began by determining if defensin binds to the LDL-R, thereby enhancing the cell-surface binding of LDL through the formation of a bridge between the lipoprotein and the receptor. Consistent with this hypothesis, an anti-LDL-R antibody that blocks LDL binding was able to inhibit the binding of 125I-defensin to HUVECs by 43% ± 7% (mean ± SD, n = 3), whereas nonimmune rabbit immunoglobulin (Ig)G did not affect defensin binding. Similarly, the binding of 125I-defensin to FH fibroblasts lacking LDL-R was 39% ± 6% (mean ± SD, n = 3) of binding to wild-type cells. Binding of 125I-defensin to FH cells was not inhibited by anti-LDL-R antibody. These data indicate that a substantial fraction of cell-surface binding of defensin depends on LDL-R.We then directly examined whether defensin enhances the binding of LDL
to LDL-R. Several lines of evidence indicated that this was not the
case. First, in the presence of defensin, 125I-LDL binds to
FH and wild-type fibroblasts to the same extent (Figure
3). Second, r-RAP, which inhibits ligand
binding of LDL to several LDL-R family members,44
inhibited the binding of 125I-LDL to HUVECs by 60 ± 15%
(mean ± SD, n = 3) in the absence of defensin, but had no
effect on the augmented binding of LDL in the presence of defensin.
Third, binding of 125I-LDL to wild-type fibroblasts in the
presence of defensin was not inhibited by anti-LDL-R blocking
antibody, whereas binding of LDL was inhibited 80% ± 11% (mean ± SD, n = 3); nonimmune rabbit IgG did not affect LDL binding in the
presence or absence of defensin. Taken together, these results indicate
that neither LDL-R nor LDL-R family members mediate the increased
surface binding of LDL in the presence of defensin.
Defensin increases the binding and degradation of LDL We then examined the effect of defensin on the binding and degradation of 125I-LDL. To do so, we first examined FH cells in view of the finding that defensins stimulate the binding of 125I-LDL in an LDL-R-independent manner. Consistent with this finding, defensin augmented the degradation of 125I-LDL by FH fibroblasts (Figure 4). The t1/2 for disappearance of LDL from the surface in the presence of defensin was approximately 2 to 2.5 hours. For comparison, we examined the internalization of 125I-LDL by wild-type fibroblasts in the presence or absence of defensin. In the presence of defensin, the t1/2 of 125I-LDL internalization was the same as that obtained with FH cells, whereas in the absence of defensin the t1/2 was 9 to 13 minutes (not shown). Thus, the time course of LDL internalization by wild-type and FH fibroblasts was the same in the presence of defensin and was too slow for internalization by LDL-R, which, like other coated-pit receptors, mediates internalization with a t1/2 of 5 to 15 minutes. Rather, the internalization of LDL in the presence of defensin is more consistent with the behavior of ligands bound to syndecan proteoglycans.48 Furthermore, cells grown in the presence or absence of LDL to modulate their expression of LDL-R bound and degraded similar amounts of defensin/LDL (not shown).
Heparan sulfate-containing proteoglycans mediate defensin-stimulated LDL degradation Heparan sulfate-containing proteoglycans (HSPGs) have recently been implicated in the binding and degradation of LDL.6,48 Therefore, we investigated the possibility that defensin and/or defensin/LDL complexes bind to and are internalized through HSPGs. Binding of defensin/125I-LDL complexes to HUVECs was inhibited approximately 75% by 0.2 U/mL heparin (data not shown), a concentration well below that required to inhibit LDL binding to LDL-R and that had no effect on LDL binding in absence of defensin. Binding of 125I-defensin to XT
CHO cells, which lack heparan- and chondroitin-sulfate-containing proteoglycans, was reduced 33 ± 11% (mean ± SD; n = 3)
relative to wild-type cells. Furthermore, degradation of
125I-LDL in the presence of defensin (10 µmol/L) by
XT cells was reduced more than 80% at 4 hours compared
with wild-type cells (Figure 5). In
addition, degradation of 125I-LDL in the absence of
defensin was reduced more than 4-fold in the WT cells, whereas
degradation by XT cells was essentially the same in the
presence and absence of defensin (not shown).
XT
This study demonstrates that Several lines of evidence indicate that the binding of defensin/LDL to the vascular cells is not mediated via the LDL-R or LDL-R family members.2 First, binding of defensin/LDL to fibroblasts was not inhibited by anti-LDL-R antibodies or by r-RAP.44 Second, defensins stimulate the endocytosis and degradation of 125I-LDL by LDL-R-deficient FH fibroblasts and wild-type cells to the same extent. Rather, our data indicate that defensin forms stable complexes with LDL in solution and on cell surfaces. The resultant defensin/LDL complexes bind to heparan sulfate-containing proteoglycans including syndecan-1.6,48,51,52 The time course of internalization of defensin/LDL, which is measured in hours, is also inconsistent with the kinetics of LDL-R-mediated internalization, but is consistent with the rate of internalization and degradation of LDL by syndecan HSPGs.48 Binding of defensin/LDL to cells is also inhibited by low concentrations (0.2 U/mL) of heparin and is markedly reduced on cells lacking HSPGs. Taken together, these data indicate that defensin promotes surface binding, endocytosis, and degradation of LDL via HSPGs. Our data indicate that defensin, which contains 4 arginine residues on its molecular surface, binds to LDL-R and therefore would be predicted to competitively inhibit the binding of LDL.23 Binding of defensin to fibroblasts is itself partially inhibited by anti-LDL-R antibody, and binding of defensin to LDL-R-deficient fibroblasts is reduced compared with wild-type cells. This interpretation is consistent with the finding that contact between cationic residues in apoB of LDL is required for optimal binding to LDL-R.38,53 Defensin bound to LDL-R appears unavailable to bind LDL, in contrast to defensin bound to HSPGs, which binds LDL quite well. Thus, in the presence of defensin, binding of LDL to the LDL-R is likely to be competitively inhibited, whereas binding to HSPGs is promoted. Diversion of LDL from the LDL-R to HSPGs by defensin may slow the degradation of the lipoprotein31 and subject it to oxidation and other modifications. Thus, the studies reported here provide insight into one mechanism by which leukocyte defensins may modulate LDL metabolism and thereby the development of atherosclerosis. We have previously observed that defensin is readily detected in
atherosclerotic human coronary and cerebral arteries.30,34 During systemic infection,
Submitted December 28, 1999; accepted March 27, 2000.
Supported in part by grants HL58107 and HL60169 from the National Institutes of Health, grant 960105000 from the American Heart Association, and a grant from the Joint Research Fund of the Hebrew University and Hadassah University Hospital. During part of this work, K.J.W. was an Established Investigator of the American Heart Association and Genentech.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Abd Al-Roof Higazi, Department of Pathology and Laboratory Medicine, 513A Stellar-Chance, 422 Curie Blvd, Philadelphia, PA 19104; e-mail: higazi{at}mail.med.upenn.edu.
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© 2000 by The American Society of Hematology.
|
-defensins stimulate proteoglycan-dependent
catabolism of low-density lipoprotein by vascular cells: a new class of
inflammatory apolipoprotein and a possible contributor to
atherogenesis
Top
Abstract
Introduction
Stop
(single-letter amino acid code)
mutation.
) of 10 µmol/L defensin, and the cell-associated
radioactivity was measured. Specific binding was defined as the
difference between cell-associated radioactivity in the presence and
the absence of 100-fold molar excess unlabeled LDL. (B) HUVECs were
incubated with 125I-LDL (50 nmol/L) in the presence of the
indicated concentrations of defensin under the experimental conditions
described in panel A, and the specific binding was determined. The
mean ± SEM of 3 experiments is shown.
) or
absence (
) of defensin was measured. The cell supernatant was
removed and the TCA-soluble radioactivity in the presence (
) or
absence (×) of defensin was measured. The experiment shown is
representative of 3 experiments so performed.
