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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2007-2019
By
From the Departments of Pathology and Laboratory Medicine, Medicine,
Cell Biology, and Pediatrics, University of Pennsylvania, Philadelphia,
PA; the School of Life and Health Sciences, University of Delaware,
Newark, DE; the Department of Biochemistry, Queen's University,
Kingston, Ontario, Canada; the Division of Cardiovascular Medicine,
Stanford University, Stanford, CA; the Division of Pulmonary and
Critical Care, Department of Medicine, University of California at Los
Angeles, Los Angeles, CA; and the Department of Clinical Biochemistry,
Hebrew University-Hadassah Medical Centers, Jerusalem, Israel.
Retention of lipoproteins within the vasculature is a central event
in the pathogenesis of atherosclerosis. However, the signals that
mediate this process are only partially understood. Prompted by
putative links between inflammation and atherosclerosis, we previously
reported that
THE DEVELOPMENT OF atherosclerosis
results from an interplay among diverse factors, including endothelial
cell denudation and activation, adherence and activation of platelets,
hyperlipidemia, oxidation of lipoproteins, infiltration of the vessel
wall by macrophages and their conversion to foam cells, and smooth
muscle cell proliferation and migration, among others.1
Although many prospective and cross-sectional studies demonstrate that
elevated plasma levels of low-density lipoprotein (LDL) and lipoprotein (a) [Lp(a)]1 are risk factors for atherosclerosis (eg,
see Bostom et al2 and Assmann et al3), the
overall complexity of the process may help to explain why no single
factor correlates with disease incidence or severity in all
individuals.4 Indeed, dyslipoproteinemia and other known
risk factors have been estimated to account for only approximately half
of the interindividual variability in the risk of developing
atherosclerosis.5,6
Plasma levels of Lp(a) and other lipoproteins may not correlate with
pathogenic events more closely, because additional factors regulate
their interaction with the vasculature. The rate of lipoprotein transport into vessel walls exceeds the rate of egress or
catabolism.7,8 This suggests that factors contributing to
the retention of lipoproteins in vessel walls may contribute to
atherogenesis.5,9,10 Vascular retention may be a
prerequisite for subsequent injurious events such as monocyte
migration,11 lipoprotein oxidation, uptake by scavenger
receptors, generation of foam cells, and the induction of smooth muscle
cell proliferation. In accord with this concept, reducing vascular
retention time of lipoproteins may help to limit oxidant-induced
vascular injury.12 Yet, considerable gaps remain in our
understanding of how lipoproteins such as Lp(a) accumulate in
atherosclerotic vessels13.
Lp(a) closely resembles LDL in its content of cholesterol,
phospholipid, and apolipoprotein B-100, but differs by the presence of
an attached glycoprotein, known as apoprotein (a)
[apo(a)], which invests the molecule with additional
properties. Apo(a) is a modular protein composed of multiple tandem
repeats of a sequence that closely resembles plasminogen kringle IV at
its amino terminus followed by sequences that closely resemble kringle V and the protease domain of plasminogen.14 All apo(a)
isoforms contain 10 distinct classes of kringle IV repeats. The
variable number (<10 to >50 copies) of type IV-2 repeats is
responsible for the size heterogeneity among Lp(a)
isoforms.15 The sequence of kringle IV type 10 (alternately
designated KIV-37) resembles that of plasminogen kringle IV most
closely, especially with respect to the composition of its
lysine-binding site (LBS; see below). Apo(a) binds to apoB initially
through noncovalent associations involving kringle IV types
6-816 and subsequently by covalent binding through
Cys4057 in kringle IV-9.17
Lp(a) binds to fibrin, fibronectin, proteoglycans, and cell
surfaces18-21 through kringle (LBS)-dependent and, perhaps,
kringle-independent processes.22 Residues in kringle IV-37
that contribute to binding have been identified through the study of
primate homologues,23 recombinant kringles,24
natural polymorphisms in the human protein,23,25 and, more
recently, the study of transgenic mice expressing apo(a) variants.26,27 Apo(a) contains binding sites for fibrin
that are masked in the Lp(a) particle but are exposed by proteolytic digestion.23 Lp(a) competes with plasminogen for binding to fibrin and to cells18,19,28 and inhibits plasmin-dependent fibrinolysis directly.29,30 Inhibition of plasmin formation may contribute to the terminal thrombotic complications associated with
plaque rupture and impede the activation of latent transforming growth
factor Notwithstanding this knowledge, the mechanism by which the retention of
Lp(a) in the vasculature is regulated is only partially understood. One
insight in this process comes from the emerging appreciation of the
relationship between inflammation and atherosclerosis (see
Ridker33 and Tracy et al34 for review). For
example, macrophages play a central role in the development of
atherosclerosis.1,35 In addition, plasma concentrations of
ferritin, interleukin-6, fibrinogen, and C-reactive protein have been
identified as independent risk factors in the development of ischemic
events and the probability of response to certain forms of
therapy.36 A correlation between neutrophil numbers and
atherosclerosis has also been observed in numerous studies (eg,
Weijemberg et al37 and Zalokar et al38). Neutrophil activation leads to diverse biochemical changes, including the generation of hydrogen peroxide and oxygen radicals and the release
of lysosomal enzymes that may alter vascular function.
Activated neutrophils also release We have previously reported that Materials
Defensin.
Defensins (human neutrophil peptides 1 and 2) were prepared from human
plasma and radiolabeled as described previously40,50; each
protein migrated as a single band on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rabbit
polyclonal and mouse monoclonal anti-HNP-1, which recognize HNP-2 and
HNP-3, but not HNP-4 or HD-5, were prepared as described.53
Lipoproteins.
Lipoproteins were isolated from human plasma by ultracentrifugation at
1.21 g/mL. Lp(a) was isolated using lysine-sepharose chromatography.
The fraction that binds to lysine-sepharose was brought to 7.5% CsCl
(wt/wt) and centrifuged at 50,000 rpm for 27 hours to separate Lp(a)
from triglyceride-rich material. LDL was purified from the fraction
that did not bind to the lysine sepharose, as described.54
All lipoprotein preparations were filtered under sterile conditions,
aliquoted into plastic tubes, and stored under N2 at
4°C without exposure to light in 0.01% Na2EDTA, 0.1%
NaN3, and 1 mmol/L benzamidine until use. The purity of
Lp(a) and the apparent molecular weight (Mr) of the apo(a) was determined by SDS-PAGE (3% to 9% acrylamide gradient gel) stained
with Coomassie blue under reduced and nonreduced
conditions.54 Most studies were performed using Lp(a)
containing apo(a) (Mr ~500 kD) on SDS-PAGE. Native apo(a) was
isolated from Lp(a) as described.55 Lp(a) and apo(a) were
radiolabeled with 125I using a modification of the iodine
monochloride method54,56 to a specific activity of 3.1 × 105 cpm/pmol. In some experiments, Lp(a) was
oxidized as described.57 The oxidation state of the Lp(a)
was monitored by measuring thiobarbituric acid reactive
substances.58 The migration of oxidized and nonoxidized 125I-Lp(a) was analyzed on 0.5% native agarose gels
(Paragon Lipoprotein "Lipo" electrophoresis kit; Beckman
instruments, Inc, Fullerton, CA) by autoradiography. All preparations
studied were free of oxidized Lp(a) (see Fig 4). To measure the effect
of defensin on the oxidation state of Lp(a),
125I-oxidized or nonoxidized Lp(a) (20 nmol/L) was
incubated in the presence or absence of defensin (10 µmol/L) in
Tris-buffered saline (TBS)/4 mmol/L calcium, and 0.01%
Tween 20 (TBS/Ca/T) buffer containing 0.1% bovine serum albumin (BSA)
for 1 hour at 37°C. Migration of the labeled proteins under native
conditions on 0.5% agarose gel was analyzed using autoradiography.
Recombinant apo(a) [r-apo(a)], 17K (kringles; Mr ~525 kD), was
prepared as described.59
Antibodies.
Rabbit polyclonal antisera against human TSP-1 was the kind gift of Dr
Jack Lawler (Brigham and Womens Hospital, Boston, MA). The IgG
fractions of this antisera and normal rabbit sera were isolated using
protein-G agarose (Life Technologies, Gaithersburg, MD; catalogue no.
15920-010). Human plasma fibronectin was purchased from Life
Technologies (catalogue no. 33016-023); affinity-purified polyclonal
sheep IgG antihuman vitronectin was from Enzyme Research Laboratories,
Inc (Indianaoplis, IN; catalogue no. SAVN-AP); affinity-purified rabbit
IgG antihuman fibronectin was from Sigma Chemical Co (St Louis, MO;
catalogue no. F-3648); and bovine lung heparin was from Sigma
(catalogue no. H-9133).
Cell Culture and Isolation of Extracellular Matrix (ECM)
Binding of Lp(a) to ECM or Coated Fibronectin
Binding of Defensin to Lp(a) Binding of defensin to Lp(a) and its components was measured using gel filtration, surface plasmon resonance, and immunoelectron microscopy.Gel filtration. Gel filtration experiments were performed under conditions similar to those used to measure binding of Lp(a)/defensin to cell matrices. Specifically, 125I-HNP-1 (0.019 to 10 µmol/L) was incubated alone or in the presence of 10 nmol/L Lp(a) or BSA in TBS/Ca/T (final volume, 60 µL) for 1 hour at 37°C. The proteins were passed over a Bio-spin 30 column (Bio-Gel P-30 polyacrylamide gel, catalogue no. 732-6006; Bio-Rad, Hercules, CA) and the radioactivity in the excluded volume was measured; small proteins like defensin (3.5 kD) are expected to be retained in the these columns (exclusion limit, ~40 kD). Binding of 125I-HNP-1 to BSA measured in parallel was minimal and was subtracted from each data point as a control for nonspecific interactions. Surface plasmon resonance. Binding kinetics of defensin to Lp(a) and to its components was measured using a B2000 optical biosensor (Biacore AB, Uppsala, Sweden).63 This method detects binding interactions in real time and enables association and dissociation rate constants to be estimated. For these studies, Lp(a), native apo(a), LDL, and recombinant apo(a) were coupled to CM5-research grade sensor chip flow cells (Biacore AB) using standard procedures.64 The sensor surface was activated for 7 minutes with an equimolar mixture of 0.1 mol/L N-hydroxysuccinimide/N-ethyl-N'-[3-(dimethylamino) propyl] carbodiimide hydrochloride (Pierce, Rockford, IL) 1:1 (vol/vol) at a flow rate of 5 µL/min. Protein was then immobilized to the sensor surface at a flow rate of at least 10 µL/min, and the unreacted amine groups were blocked with 1 mol/L ethanolamine, pH 8.5. Sensor surfaces were coated with ligands (15 µg/mL) in 10 mmol/L NaAc buffer, pH 4.0. Newly immobilized ligand surfaces were equilibrated with PBS, pH 7.4, 0.005% Tween-20 (binding and running buffer). Surfaces were regenerated after each binding interaction by short pulses (20 to 30 seconds) of 10 mmol/L NaAc buffer, pH 2.5 to 3.0, or 5 mmol/L HCl, pH 2.5. Binding of 0.019 to 10 µmol/L defensin (HNP-1 or HNP-2) was measured at 25°C at a flow rate of 30 µL/min. The bulk shift due to interaction of defensin with similarly activated/inactivated uncoated chips was subtracted from the binding signal at each concentration to correct for nonspecific interactions. Immobilized BSA was used as an additional control for protein binding. Electron microscopy. Rotary-shadowed samples were prepared by spraying a dilute solution of protein (final concentration, ~40 µg/mL) in a volatile buffer (0.05 mol/L ammonium formate at pH 7.4) and 70% glycerol onto freshly cleaved mica and shadowing with tungsten followed by deposition of a carbon film in a vacuum evaporator (Denton Vacuum Co, Cherry Hill, NJ).67,68 Ratios of Lp(a) to defensin were generally 1:10 or 1:22, and samples were incubated for 30 minutes at 22°C. A 10× excess of rabbit anti-HNP-1 sera was incubated for an additional 30 minutes at 22°C. All experiments were repeated several times and many micrographs were taken of randomly selected areas to ensure that the results were reproducible and representative. All of the specimens were examined in a Philips 400 electron microscope (Philips Electronic Instruments Co, Mahwah, NJ), usually operating at 80 kV. Confocal laser scanning microscopy.
Confocal laser scanning microscopy was used to examine the effect of
defensin on the endocytosis of Lp(a). Subconfluent monolayers of HUVEC
were grown on fibronectin-coated glass coverslips overnight. The cells
were washed 3 times with Dulbecco's modified Eagle's medium (DMEM;
Life Technologies) supplemented with 1% BSA. Defensin (10 µmol/L),
Lp(a) (40 nmol/L), or defensin plus Lp(a) was added in the same media
for 1 hour either at 4°C or at 37°C for 30 minutes. Cells
treated with defensin or defensin/Lp(a) in this manner were greater
than 95% viable as judged by exclusion of trypan blue. The cells were
rinsed in PBS to remove unbound ligands and fixed in 2%
paraformaldehyde in PBS for 10 minutes, followed by cold methanol
( Formation of Foam Cells J774 murine macrophages were cultured in DMEM supplemented with 2 mmol/L glutamine and 10% fetal calf serum (FCS) in 24 Multiwell dishes (Falcon, Plymouth, UK). The cells were washed free of FCS and the medium was replaced with DMEM containing 200 nmol/L native or oxidized Lp(a) in the presence or absence of 10 µmol/L defensin (HNP-2). 3H-oleic acid (OA; New England Nuclear, Boston, MA) was added (100 µmol/L) as an oleate:albumin complex (3:1 molar ratio) for 24 hours at 37°C and the incorporation of 3H-OA was measured. To do so, the medium was collected, the cells were washed 4 times with PBS, and 1 mL absolute ethanol was added for 2 hours. The ethanol extract was separated by thin-layer chromatography on silica gels (Analtech, Inc, Newark, DE). Bands were identified with iodine and isolated, and the radioactivity in the cholesterol ester fraction was counted.
We previously reported that leukocyte defensins promote the binding of Lp(a) to cultured human endothelial cells and smooth muscle cells without a concomitant increase in degradation. We investigated 2 possible mechanisms to explain these findings: ie, that defensin promotes the binding and retention of Lp(a) on vascular matrices and/or that defensin promotes the binding of Lp(a) to cellular receptors but protects it from degradation. Defensin Promotes Binding of Lp(a) to Endothelial Cell Matrix We asked first whether defensin promotes the binding and retention of Lp(a) by vascular matrices. Binding of Lp(a) (20 nmol/L) to HUVEC matrix was stimulated approximately 40-fold by 10 µmol/L defensin (Fig 1A). Binding of 125I-Lp(a) in the presence of defensin [defensin/Lp(a)] was inhibited greater than 80% by 20-fold molar excess unlabeled Lp(a) (Fig 1A). Defensin did not stimulate the binding of 125I-Lp(a) in the absence of matrix (not shown). Defensin promoted 125I-Lp(a) binding to HUVEC matrix in a dose-dependent and saturable manner (Fig 1B). Binding of Lp(a) approached saturation at a defensin concentration of 10 µmol/L, consistent with previous results using intact cells.48,50 Identical results were obtained using oxidized 125I-Lp(a) or Lp(a) containing the apo(a) isoform (Mr ~350 kD) and when matrices derived from HVSMC were studied (not shown). The binding of defensin/125I-Lp(a) to matrix was essentially irreversible. Whereas approximately 30% of the bound ligand dissociated from the matrix over the initial 3 hours, there was no further loss over the succeeding 72 hours (Fig 2). Thus, defensin caused a sustained net 40-fold increase in matrix-bound Lp(a).
Formation of Stable Complexes Between Defensin and Lp(a)
Characterization of Defensin/Lp(a) Binding to Matrix
Effect of Defensin on Subcellular Distribution of Lp(a) and Foam
Cell Formation
Inflammatory changes within the vessel wall may promote the retention
of Lp(a) and the development of atherosclerosis. In accord with this
possibility, we have found that Submitted December 8, 1998; accepted May 4, 1999.
Supported in part by National Institutes of Health Grant No. HL5810.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Douglas B. Cines, MD, Department of
Pathology and Laboratory Medicine, 513 A Stellar-Chance, 422 Curie
Blvd, Philadelphia, PA 19104; e-mail: dcines{at}mail.med.upenn.edu.
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TOP
ABSTRACT
INTRODUCTION
-defensins released by neutrophils are present in
human atherosclerotic lesions and promote the binding of lipoprotein(a)
[Lp(a)] to vascular cells without a concomitant increase in
degradation. We have now tested the hypothesis that this accumulation
results from the propensity of defensin to form stable complexes with
Lp(a) that divert the lipoprotein from its normal cellular degradative
pathways to the extracellular matrix (ECM). In accord with this
hypothesis, defensin stimulated the binding of Lp(a) to vascular
matrices approximately 40-fold and binding of the reactants to the
matrix was essentially irreversible. Defensin formed stable,
multivalent complexes with Lp(a) and with its components, apoprotein
(a) and low-density lipoprotein (LDL), as assessed by
optical biosensor analysis, gel filtration, and immunoelectron
microscopy. Binding of defensin/Lp(a) complexes to matrix was inhibited
(>90%) by heparin and by antibodies to fibronectin (>70%), but
not by antibodies to vitronectin or thrombospondin. Defensin increased
the binding of Lp(a) (10 nmol/L) to purified fibronectin more than
30-fold. Whereas defensin and Lp(a) readily traversed the endothelial
cell membranes individually, defensin/Lp(a) complexes lodged on the
cell surface. These studies demonstrate that 
(TGF
-defensins, a family of closely
related peptides that comprise approximately 5% of their total protein
content.
/
80°C until use. ECM-coated plates were used within 1 week of
preparation, with no change in activity. In other experiments, 96-well
plates (Immulon 2; Dynatech Labs Inc, Chantilly, VA) were incubated
with purified fibronectin (5 µg/mL in TBS/Ca2+) for 12 hours at 4°C and washed as described above before use.
-aminocaproic acid [
, 
) or 20-fold
molar excess cold Lp(a) (
) for 1 hour at 37°C, and
the bound radioactivity was measured. The mean ± SD of 3 experiments
is shown. This corresponds to an increase in bound Lp(a) from a mean of
2.9 fmol in the absence of defensin to 96 fmol in the presence of
defensin. The mean ± SD of 3 experiments is shown. (B)
125I-Lp(a) (10 nmol/L) was incubated with HUVEC matrix in
the presence of the indicated concentrations of defensin for 1 hour at
37°C, and the bound radioactivity was measured. The data are
expressed relative to the binding of 125I-Lp(a) alone. The
mean ± SD of 3 experiments is shown.
) were incubated
with HUVEC matrix as described in the legend to Fig 
