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J Biol Chem, Vol. 274, Issue 36, 25913-25920, September 3, 1999
From the The presence of a lipoprotein profile
with abundance of small, dense low density lipoproteins (LDL), low
levels of high density lipoproteins (HDL), and elevated levels of
triglyceride-rich very low density lipoproteins is associated with an
increased risk for coronary heart disease. The atherogenicity of small,
dense LDL is believed to be one of the main reasons for this
association. This particle contains less phospholipids (PL) and
unesterified cholesterol than large LDL, and the apoB-100 appears to
occupy a more extensive area at its surface. Although there are
experiments that suggest a metabolic pathway leading to the
overproduction of small, dense LDL, no clear molecular model exists to
explain its association with atherogenesis. A current hypothesis is
that small, dense LDL, because of its higher affinity for
proteoglycans, is entrapped in the intima extracellular matrix and is
more susceptible to oxidative modifications than large LDL. Here we
describe how a specific reduction of approximately 50% of the PL of a
normal buoyant LDL by immobilized phospholipase A2
(PLA2) (EC 3.1.1.4) produces smaller and denser particles
without inducing significant lipoprotein aggregation (<5%). These
smaller LDL particles display a higher tendency to form nonsoluble
complexes with proteoglycans and glycosaminoglycans than the parent
LDL. Binding parameters of LDL and glycosaminoglycans and proteoglycans
produced by human arterial smooth muscle cells were measured at near to
physiological conditions. The PLA2-modified LDL has about 2 times higher affinity for the sulfated polysaccharides than control
LDL. In addition, incubation of human plasma in the presence of
PLA2 generated smaller LDL and HDL particles compared with
the control plasma incubated without PLA2. These in
vitro results indicate that the reduction of surface PL
characteristic of small, dense LDL subfractions, besides contributing
to its small size and density, may enhance its tendency to be retained
by proteoglycans.
The low density lipoproteins
(LDL)1 with density 1.019 to
1.063 g/ml in humans are characterized by the presence of a major apolipoprotein, the apoB-100, and a variable lipid complement. This
operationally defined density range can contain subclasses of particles
of different size and density caused by dissimilar contents of core and
surface lipids (1, 2). Much interest is focused on the metabolic and
genetic factors leading to the presence of LDL profiles in which the
majority of particles centers around small, dense particles (LDL-III)
with diameters of 25.5 to 24.2 nm and densities between 1.040 and 1.060 g/ml, the phenotype B. Subjects with such phenotype have significantly
higher susceptibility to CHD, when compared with subjects that have
most of their LDL as large particles (LDL-I and LDL-II) with diameters
between 25.5 and 27.5 nm and densities between 1.025 and 1.040 g/ml,
the phenotype A (3-5). Excess LDL-III is a marker of insulin
resistance and type II diabetes, and it occurs almost always in
combination with moderate hypertriglyceridemia, above 2 mM
or 180 mg/dl, and low levels of HDL (6, 7). Because its association
with CHD, this lipoprotein pattern has been also termed the atherogenic lipoprotein phenotype (ALP) (8). The susceptibility to develop the ALP
appears associated with genes in chromosomes 19, 11, and 16, and
heredity controls about 50% of its expression. The rest of the
expression is dependent on hormonal and life style factors (3).
The metabolic reasons for the appearance of an excess of LDL-III are
under intense exploration, and several steps in the lipolytic conversion of VLDL and intermediate density lipoproteins have been
postulated to be responsible for its production in conjugality with the
action of cholesterol ester transport protein. One hypothesis, supported on metabolic studies, proposes that overproduction of large
TG-rich VLDL causes an increase in the exchange of TG for cholesterol
esters (CE) between this particle and LDL leading to a momentary
increase of TG in LDL. The resulting TG-rich LDL become a good
substrate for the action of hepatic lipoprotein lipase, which also has
a phospholipase activity, and possibly for the endothelial lipoprotein
lipase. This produces a small dense particle with a reduced content of
core CE and of the surface components PL and UC (9). However, when
subfractions of LDL with increasing density are isolated from normal
subjects a similar trend in decreased content of surface UC and PL and
core CE per apoB-100 particle is observed (2, 9). Therefore the
phenotype B can be considered the result of exaggerated accumulation in plasma of small, dense particles that may exist at lower concentration in normal men and women with the A phenotype. Two, nonexclusive, hypotheses have been considered to be responsible for the increased atherogenicity of small, dense LDL and its association with
cardiovascular disease. One is its preferential entry and retention in
the arterial wall, especially at sites of lesion development. This is
supported by in vitro experiments (10, 11). The other, also
based on in vitro results, suggests that small, dense LDL is
more susceptible to oxidative and hydrolytic modification than buoyant,
large LDL once entrapped in the arterial intima at sites of lesion
progression (12, 13).
The reasons why small, dense LDL are better retained in lesions than
buoyant LDL are not clear. However, our laboratory showed ex
vivo that smaller subfractions of human LDL bind with higher affinity to human arterial chondroitin sulfate proteoglycans (CSPG). The smallest LDL showed a significantly lower content in surface PL and
UC than large LDL. Owing to the lower content in the surface lipids,
the apoB-100 was estimated to have up to 45% larger area to cover at
the particle surface compared with the largest LDL subfraction. We
suggested that this could expose more of the PG-binding segments of the
apoB-100 (14). Additionally, human macrophages internalized and
degraded significantly more of the small, dense LDL with high affinity
for CSPG than the larger low affinity subfractions (14). Recently,
Anber et al. (15, 16) found that subjects with the ALP and
with high levels of LDL-III also show a higher affinity for human
arterial CSPG. These results suggest that the entrapment of LDL-III by
CSPG in the intima may contribute to its atherogenicity. As mentioned,
the smaller size of centrifugally separated small, dense LDL or that of
the small, dense LDL separated by its affinity for CSPG results mainly
in a lower content of PL and UC at the surface monolayer (2, 9, 14).
However, the apparent causality of a reduction in surface PL or UC on
the increased affinity for PG and glycosaminoglycans (GAG) remains to
be established. To explore these potential relationships, we studied
the effect on size, density, and affinity for GAG and PG of an in
vitro reduction of surface PL of LDL by immobilized PLA2.
Materials--
PLA2-agarose, decorin, biglycan,
deuterium oxide, HEPES, free fatty acid-free BSA, cetyl pyridinium
chloride, Oil Red O, Fat Red 7B, and toluidine blue O were purchased
from Sigma. C6S and dermatan sulfate was bought from Seikagaku Co.
(Tokyo, Japan). NEFA-C kit was from Wako Chemicals GmbH (Neuss,
Germany). NuSieve agarose 3:1 and GelBond films were from FMC
Bioproducts (Rockland, ME). Salts and other buffer substances or
detergents were of analytical grade and purchased from Merck
(Darmstadt, Germany). All the water used was filtered through a
MilliPore® Milli-Q system and was of high purity (resistivity >18
megohm cm PLA2 Modification of LDL--
LDL was isolated from
healthy male donors by sequential centrifugation as described (17). LDL
containing 10 µM butylated hydroxytoluene (BHT) was
equilibrated in 10 mM HEPES buffer, pH 7.4, containing 140 mM NaCl, 4 mM CaCl2, 2 mM MgCl2, and 50 mg/ml nonesterified fatty acid
(NEFA)-free BSA (750 µM) was added to sequester the NEFA
liberated by PLA2. Reaction mixtures (1 ml) containing 1 mg
of LDL (apoB-100 protein) (2 µM) and 0.1 unit of
PLA2-agarose were incubated at 37 °C for the indicated
time points. The reaction was stopped by removing the
PLA2-agarose by a brief centrifugation at 10,000 × g, and the supernatant was collected. The activity of
PLA2 was monitored by measuring the release of NEFA using a
commercial kit (NEFA-C, Wako Chemicals).
Characterization of PLA2 LDL--
Charge alterations
of LDL were evaluated by agarose gel electrophoresis. A 0.7% agarose
gel was prepared with running buffer (0.05 M barbital
buffer, pH 8.5). Ten µl of the PLA2-LDL (1 mg/ml) and
control LDL (incubated without PLA2) was loaded on the gel, and electrophoresis was run for 3 h at 60 V with buffer
recirculation and cooling. The gel was fixed in 60% ethanol for 2 h, and after air-drying the lipids were stained with 0.015% Oil Red O
and 0.06% Fat Red 7B in the fix solution.
The effects of PLA2 on LDL density were analyzed by density
gradient centrifugation. Continuous density gradients (1.006-1.116 g/ml) were prepared using D2O as described (18). The
lipoproteins were placed at the bottom of each gradient. After
centrifugation at 20 °C for 46 h at 35,000 rpm (SW 40 rotor,
XL-90 Ultracentrifuge, Beckman), 0.5-ml fractions were collected by
bottom displacement with the use of a fraction recovery system
(270-331580, Beckman). The cholesterol content in the collected
fractions was monitored by the CHOD-PAP method (19) with reagents
bought from Roche Molecular Biochemicals (absorbance determined at 500 nm).
The LDL particle size of the peak fractions after D2O
gradient centrifugation was measured by nondenaturing polyacrylamide gel electrophoresis on 2-16% gradient gels as described (20).
Cholesterol and phospholipid distribution profiles were measured with a
size exclusion high performance liquid chromatography system, SMART,
with a Superose 6 PC 3.2/30 column (Amersham Pharmacia Biotech). The
chromatographic system was linked to an air-segmented continuous flow
system for on-line post-derivatization analysis of total cholesterol
and phospholipids using enzymatic colorimetric reagents. The SMART
system was connected to a sample injector (Gina 50, Gynkotek HPLC,
Germering, GmbH). Elution buffer consisted of 0.01 M Tris,
0.03 M NaCl, pH 7.40, and the flow rate was 35 µl/min.
The on-line flow system was equipped with a peristaltic pump, flow rate
0.7 ml/min, and a coil for 8 min of incubation at 37 °C. The
absorbance was measured at 500 nm with a UV-visible detector (Jasco
UV-970, Jasco International Co, LTD, Japan). Data were integrated with
a Chromeleon chromatography data system (Gynkotek HPLC, Germering,
GmbH). The distribution of lipoproteins was continuously measured as
cholesterol or phospholipids by using the enzymatic reagents
reconstituted in buffer at double the amount suggested by the
manufacturer. Cholesterol was measured with a kit from Roche Molecular
Biochemicals (cholesterol MPRI 1442341) and phospholipids with a kit
from Wako Chemicals (phospholipids B 990-54009 E). Each run lasted 60 min, and the sample size used was 10 µl. The integrated area is
expressed in molar concentrations.
LDL were re-isolated, after incubation with or without
PLA2, by D2O ultracentrifugation as described
below. All lipid classes in LDL were analyzed with a modification of
the high performance liquid chromatography developed by Homan and
Anderson (21). The column was an Alltech, Allsphere Si, 5 µm,
150 × 4.6 mm. In the first solvent n-heptane was used
instead of tetrahydrofuran. The detector was an evaporative light
scattering 500 from Alltech (Deerfield, IL). The external standards
used were prepared by Larodan Fine Chemicals (Malmö, Sweden). The
high performance liquid chromatography system was from Gynkotek GmbH,
equipped with a Chromaleon software (Munich, Germany).
LDL Precipitation Assay--
Analysis of interactions between
PLA2-LDL and GAG at low ionic strength was done essentially
as described (16, 22). Briefly, LDL (1 mg/ml) modified by
PLA2 for the indicated time points was diluted to 0.1 mg of
apoB/ml with buffer 10 mM HEPES, 2 mM
CaCl2, 4 mM MgCl2, pH 7.2. This
gives a final NaCl concentration of 14 mM. Four µg of GAG
was added to 100 µl of LDL, and the samples were incubated for 30 min
at room temperature. The precipitate formed was collected by
centrifugation at 10,000 rpm for 30 min at 4 °C. The supernatants
were removed and the pellets dissolved in 10 mM HEPES
buffer, pH 7.4, containing 1 M NaCl. The cholesterol in the
pellets was measured according to the CHOD-PAP method with reagents
bought from Roche Molecular Biochemicals. In experiments with
proteoglycans (decorin and biglycan), the LDL was diluted with buffer
10 mM HEPES, 4 mM CaCl2, 2 mM MgCl2, 20 mM NaCl, pH 7.4. This
gives a higher final NaCl concentration (32 mM); otherwise the same protocol was used as for the GAG.
Gel Mobility Shift Assay--
Analysis of interactions between
PLA2-LDL and C6S at physiological salt concentrations was
done with the following modification of a described procedure (23, 24).
First, the PLA2-LDL and native LDL were reisolated by
ultracentrifugation using a 10 mM HEPES buffer, pH 7.4, prepared with D2O with a final density of 1.082 g/ml. After
centrifugation at 20 °C for 3 h at 260,000 × g
(rotor TLA 120.2, Optima TLX Ultracentrifuge, Beckman), the top
fraction containing LDL was collected by aspiration. A constant amount
of C6S (0.5 µg) was incubated for 1 h at room temperature with
increasing concentrations of LDL in a final volume of 20 µl of 10 mM HEPES buffer, pH 7.4, containing 140 mM
NaCl, 4 mM CaCl2, 2 mM
MgCl2. Two µl of glycerol was added, and the samples were
loaded on a 0.7% agarose gel (NuSieve 3:1) prepared with running
buffer containing 10 mM HEPES, 2 mM
CaCl2, and 4 mM MgCl2, pH 7.2. Electrophoresis was run for 1 h at 60 V with buffer recirculation and cooling. The gel was fixed in 0.1% cetyl pyridinium chloride in
70% ethanol for 2 h. After air drying, the gel was stained with
0.1% toluidine blue O in HAc:ethanol:H2O (0.1:5:5) for 30 min and destained in the same solution without toluidine blue O for 30 min. The gel was scanned using a DuoScan (Agfa, Mortsel, Belgium) and
the intensity of the bands representing the free C6S was quantified
using a KS 400 version 2.0 image analyzing system (Carl Zeiss,
Oberkochen, Germany). The intensity was linearly proportional to the
amount of GAG in the range 0-0.5 µg of C6S (r2 = 0.99).
Binding parameters were also measured with the gel mobility shift assay
using radiolabeled proteoglycans synthesized by human arterial smooth
muscle cells. 35S- and 3H-labeled proteoglycans
were isolated by ion exchange chromatography from conditioned medium of
human arterial smooth muscle cell as described (25). This preparation
of total proteoglycans contains mainly chondroitin sulfate and dermatan
sulfate. Each reaction contained a constant amount of labeled
proteoglycans (measured as counts/min) and increasing concentrations of
LDL in the same way as described above. However, the final NaCl
concentration in each reaction was slightly lower (105 mM
NaCl) than stated above for C6S. The quantification of the dried gels
was done by digital autoradiography using a DAR signal analyzing system
(Berthold Laboratories, Wilband, Germany).
Incubation of Human Plasma with PLA2--
Plasma was
collected from 9 healthy volunteers (5 female and 4 male) after
overnight fasting. The plasma was diluted 1:4 with phosphate-buffered
saline, and BHT was added to a final concentration of 10 µM. For each donor 1 ml of diluted plasma was incubated with or without (control) 0.5 units of PLA2-agarose at
37 °C for 20 h. The reaction was stopped by removing the
PLA2-agarose by a brief centrifugation at 10,000 × g, and the supernatant was collected. The generation of NEFA
was measuring using a commercial kit (NEFA-C, Wako Chemicals). Aliquots
(0.7 ml) from the control plasmas and the PLA2-modified
plasmas were taken, and the following parameters were determined by
proton NMR spectroscopy (LipoMed Inc. Raleigh, NC) as described (26,
27): lipoprotein subclass distribution, average lipoprotein sizes,
total TG, total cholesterol, VLDL TG, LDL cholesterol, LDL particle
concentration, and HDL cholesterol.
Statistical Evaluation and Nonlinear Regression
Analysis--
Mean and standard deviation of measurements, nonlinear
regression of binding parameters, and Student's t tests for
determinations of statistical significance were evaluated with the
computer software GraphPad PrismTM version 2.0 (GraphPad
Software Inc. San Diego, CA).
Incubation of LDL with PLA2 at physiological
conditions results in hydrolysis of LDL phospholipids. The
PLA2-derived NEFA were quantified as a function of the
incubation time, and the results are shown in Fig.
1. Incubation of LDL without
PLA2-agarose did not generate any detectable amounts of
NEFA under the conditions used in this study. The electrophoretic
mobility of the PLA2-LDL was compared with LDL incubated in
parallel without PLA2. As shown in Fig.
2 the mobility of the
PLA2-LDL was slightly modified and the lipoprotein migrated
as a more diffuse band. Although the amount of BSA used should
sequester most of the NEFA produced, we attributed this mobility change
to some residual NEFA that remained LDL-bound. No difference in
mobility was detected between native LDL (kept at 4 °C) and LDL
incubated without PLA2. Isoelectric focusing did not show
any significant change in the isoelectric point comparing native LDL
and LDL incubated with PLA2 for 3 and 14 h (data not
shown). To rule out the possibility of oxidation of the lipoproteins,
the content of conjugated dienes was analyzed at 234 nm in the
PLA2-LDL and compared with native LDL. No increase in the
content of conjugated dienes was found in the PLA2-LDL (data not shown). This is, however, expected since the LDL solutions always contained 10 µM BHT.
To evaluate the effect of PLA2 hydrolysis of LDL-PL on its
density, the lipoproteins were fractionated using density gradient centrifugation in D2O. The cholesterol content in the
collected fractions was determined, and representative gradient
profiles are shown in Fig. 3. In the
experiment presented in Fig. 3 the peak density of LDL increased from
1.036 to 1.041 g/ml after 3 h PLA2 modification and to
1.046 g/ml after 14 h. There was no indication of aggregation
based on turbidity measurements at 430 nm in any of the collected
fractions (data not shown). The denser, PLA2-modified LDL
were also of smaller size than native LDL. In the nondenaturing
polyacrylamide gel electrophoresis in Fig.
4, the size of the lipoprotein particles
decreased from 27.0 to 26.6 nm and to 26.5 nm after incubation with
PLA2 for 3 and 14 h, respectively. No aggregated
lipoproteins were observed in the gel. The 17 nm band in each lane
represents thyroglobulin that was used as internal standard and added
together with the sample buffer prior to loading the gel.
The degree of possible LDL aggregation due to PLA2
lipolysis was further analyzed using size exclusion chromatography.
Control LDL and LDL incubated with PLA2 for 14 h were
applied to a Superose 6 column, and the elution profiles were measured
as cholesterol, and phospholipid content was continuously recorded.
Representative elution profiles are shown in Fig.
5. To indicate the resolution of the
SMART system, human plasma profiles are also included in the figures.
In the PLA2-LDL there was only a slight tendency of LDL
aggregation, and after repeated injections using different PLA2-LDL preparations it could be concluded that the degree
of aggregation was consistently low (<5%). The phospholipid profiles (Fig. 5B) show a reduction of about 50% in the phospholipid
content in the LDL incubated with PLA2 for 14 h
compared with the control. On the other hand, there is an increase in
the content of choline in the albumin fraction supporting a role of
albumin as a lysophosphatidylcholine transporter.
Phospholipase A2 Modification of Low Density
Lipoproteins Forms Small High Density Particles with Increased Affinity
for Proteoglycans and Glycosaminoglycans*
§,¶,
Wallenberg Laboratory for
Cardiovascular Research,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hydrolysis of LDL by PLA2.
LDL containing 10 µM BHT was equilibrated in 10 mM HEPES buffer, pH 7.4, containing 140 mM
NaCl, 4 mM CaCl2, 2 mM
MgCl2, and 50 mg/ml NEFA-free BSA. One-ml reaction mixtures
containing 1 mg of LDL and 0.1 units of PLA2-agarose was
incubated at 37 °C for the indicated time points. The reaction was
stopped by removing the PLA2-agarose by a brief
centrifugation at 10,000 × g, and the supernatant was
collected. The activity of PLA2 was monitored by measuring
the release of NEFA using a commercial kit (NEFA-C, Wako Chemicals). No
detectable amounts of NEFA were generated in the controls incubated
without PLA2. The data points represents average ± S.E. of 3-5 independent experiments using three different LDL
preparations.
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[in a new window]
Fig. 2.
Agarose gel electrophoresis of
PLA2-LDL. Ten µl of PLA2-LDL (1 mg/ml)
and control LDL (incubated without PLA2) was loaded on a
0.7% agarose gel prepared with running buffer (0.05 M
barbital buffer, pH 8.5). After electrophoresis, the gel was fixed and
stained as described under "Experimental Procedure." The incubation
time with (+) or without ( 
) PLA2 is indicated in the
figure. Native LDL (kept at +4 °C) is abbreviated Nat.
The LDL migrated from the cathode (bottom) to the
top (anode), and the positions of the slots are indicated by + and .
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Fig. 3.
Deuterium oxide gradient fractionation of
PLA2-LDL. Density gradients (1.006-1.116 g/ml) were
prepared using D2O. Lipoproteins were placed at the
bottom of each gradient. After centrifugation at 20 °C
for 46 h at 35,000 rpm, 0.5-ml fractions were collected by bottom
displacement. The cholesterol content in the collected fractions was
monitored by the CHOD-PAP method with reagents bought from Roche
Molecular Biochemicals (absorbance determined at 500 nm). Native LDL,
open circles; PLA2-LDL 3 h, filled
circles; PLA2-LDL 14 h, open squares;
and the density gradient, dashed line.
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Fig. 4.
Determination of LDL particle size. The
LDL article size of the peak fractions after D2O gradient
centrifugation was measured by nondenaturing polyacrylamide gel
electrophoresis on 2-16% gradient gels. The gel was stained with
Coomassie Brilliant Blue, and the particle size was determined using
molecular weight standards (Std). Native LDL, lane
1; PLA2-LDL 3 h, lane 2; and
PLA2-LDL 14 h, lane 3.
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Fig. 5.
Lipoprotein profiles determined by size
exclusion chromatography. LDL (1 mg/ml) was incubated for 14 h at 37 °C in the presence (PLA2-LDL) or
absence (Ctrl-LDL) of PLA2-agarose. Cholesterol
(A) and phospholipid (B) distribution profiles
were then measured with a SMART chromatography system described under
"Experimental Procedures." Included in each figure is also the
lipoprotein profile of human plasma, and the various peaks in the
profiles are designated VLDL, LDL, and HDL for simplicity, even though
it is clear that the separation is determined primarily by the size of
the lipoproteins.
A lipid analysis of LDL before and after incubation with or without
PLA2 for 14 h showed that the action of
PLA2 is highly specific and almost only degrades the PC
component present in the LDL (Fig. 6).
The lipoproteins used for the analysis were reisolated from the
reaction buffer by D2O ultracentrifugation. After
incubation of LDL for 14 h with PLA2 about 50% of the
PC was degraded. The lipid content in native LDL and LDL incubated for
14 h at 37 °C without PLA2 (Fig. 6,
Ctrl-LDL) was similar.
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The hydrolysis of LDL-PL by PLA2 caused an increase in the
affinity of the lipoprotein for GAG and PG when measured by LDL precipitation at low ionic strength or by gel mobility shift assay at
near to physiological ionic conditions. Fig.
7 shows the increase of complex formation
of the PLA2-LDL with C6S in correlation to the degree of PL
hydrolysis. Incubation of native LDL with C6S under these conditions
led only to formation of small amounts of nonsoluble complexes.
Experiments using dermatan sulfate gave similar results (data not
shown). PLA2-modified LDL incubated with the proteoglycans
decorin and biglycan also formed more nonsoluble complexes than native
LDL (Fig. 8). More nonsoluble complexes were formed with biglycan than with decorin probably due to the higher
content of GAG in biglycan compared with decorin (two and one CS/DS
chain, respectively). Gel mobility shift assay was used to study the
interactions of PLA2-LDL and C6S at conditions in which
reversible complexes are formed. PLA2-modified lipoproteins were incubated with C6S, and the complexes formed were separated from
the free GAG by agarose gel electrophoresis. Similar experiments were
carried out with metabolically labeled extracellular matrix PG secreted
by human arterial smooth muscle cells. Table
I summarize the Kd
values for the interactions of PLA2-LDL and C6S and smooth
muscle cell-derived PG. It can be observed that the affinity for C6S
increased about 2 times after 14 h of PLA2
modification, and similar results were obtained with smooth muscle cell
PG.
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To investigate the effects of PLA2 activity on lipoproteins under more physiological conditions, we incubated diluted human plasma with immobilized PLA2. The PLA2 activity was measured as the release of NEFA, and the results are shown in Table II. There was a significant increase in the NEFA concentration after incubation with PLA2 compared with the control. In addition, the parameters analyzed by proton NMR spectroscopy were average lipoprotein sizes, total TG, total cholesterol, VLDL TG, LDL particle concentration, and LDL and HDL cholesterol. Table II summarizes the results from the NMR analysis. The average LDL and HDL sizes decreased after incubation of the plasmas with PLA2, whereas the average VLDL size did not change significantly. There were no differences in total TG, VLDL TG, LDL cholesterol, and LDL particle concentration between plasma incubated with or without PLA2. However, there was a statistically significant decrease in the total cholesterol, and it appeared to be due to a decrease in HDL cholesterol specifically. By using the proton NMR technology, it is possible to divide the total VLDL fraction in 6 discrete subclasses, with the largest being VLDL6 and then in descending order to VLDL1. In the same way LDL and HDL is divided into 3 and 5 subclasses, respectively. Fig. 9 shows the relative distribution of the subfractions after incubation of plasma with PLA2. The data are presented as percentage of each subclass in relation to the total lipoprotein class. It can be observed that there is a shift in the LDL fractions toward smaller particles after incubation with PLA2 compared with the control, especially the level of LDL3 decreases and LDL1 increases. Similar results were found in the HDL fractions where the significant changes were found in subclass HDL4 and HDL2, respectively. No clear differences were observed in the VLDL fractions.
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The molecular or metabolic reasons for the association between increased risk of CHD and the presence of high concentrations of small, dense LDL remain to be established. It is not obvious why a reduced number of PL and UC molecules at the surface monolayer and an altered exposure of the apoB-100 could be more atherogenic than the situation with a larger LDL. One possibility is the dissimilar affinity of different LDL subfractions for extracellular and pericellular proteoglycans in arterial cells. Olsson et al. (28) carried out competition experiments in which the LDL binding to fibroblasts was competed with synthetic peptides with specific sequences of apoB-100 that are responsible for PG binding (29). They showed that small, dense LDL requires higher concentrations of the competing peptides than more buoyant LDL for its detachment from the fibroblast surface (28). Galeano et al. (30) recently found that small, dense LDL has a reduced affinity for the LDL receptor and an increased affinity for nonreceptor associated binding sites than large LDL. These nonreceptor associated binding sites are made mostly of heparan sulfate PG at the cell surface. These researches concluded that the atherogenicity of small, dense LDL could be related to a decreased hepatic clearance and to a higher anchoring to proteoglycans of the extracellular matrix in arteries. Results with rabbit aortic segments also indicate that small, dense LDL is better retained in the extracellular intima than more buoyant LDL (11). Increased residence time or retention of LDL is an early marker of atherosclerotic lesion progress (31-33).
Our laboratory found that human arterial PG can discriminate LDL subfractions, binding more efficiently those small, dense LDL that were poor in PL and UC at its surface monolayer. These subfractions have therefore more surface area for the apoB-100 to cover (14). The results presented here indicate that the reduction of PC in the surface monolayer of LDL by PLA2 in vitro reduces the size and increases the density of the particle to values close to those observed in LDL-III. By using the data from the lipid analysis of PLA2-LDL presented in Fig. 6 and knowing that each particle contains at most one apoB-100 molecule, it is possible to estimate the surface area covered by the apoB-100. The calculations and assumptions needed for this estimation have been outlined in detail previously (14). The data presented in this study show that a reduction of about 50% of the PC content after PLA2 modification of LDL would, according to these calculations, result in a 25% decreased surface area. Such new free area should be covered by a more extended apoB-100 in the PLA2-LDL assuming also that there is no reorganization of the polar phase/nonpolar phase lipid class distribution (2, 14).
The changes in surface area covered by PL are associated with an increase in the affinity of the PLA2-LDL for PG and GAG. This interaction depends strongly on ionic interactions between arginine- and lysine-rich segments of the apoB-100 with the sulfated groups of the GAG (29). When LDL is incubated with PLA2 in the presence of excess albumin, there is little change on its surface-charge balance (34). Recently, Öörni et al. (35) showed, using NMR techniques, that a rapid hydrolysis of LDL-PL by PLA2 caused a substantial increase in the exposure of normal lysine residues but not of active lysines per apoB-100. Under their experimental conditions the authors showed that PLA2 modification induced aggregation of LDL particles. As a consequence the number of active lysines per LDL aggregate increased and caused a stronger binding to PG columns. In our experiments we could only detect a very low degree of lipoprotein aggregation (<5%). However, the enzyme treatment caused an increase in the affinity for PG and GAG, evaluated by two methods (Figs. 7 and 8 and Table I). Our results are in line with a recent report demonstrating that LDL as well as lipoprotein(a) show an increased binding to subendothelial matrix after PLA2 modification without lipoprotein aggregation (36).
LDL subfractions that are poor in PL and UC exist in plasma, and hypothetically the described increased affinity for intima PG may cause its retention in this compartment. Once there, the increased residence time may give the opportunity for oxidative and hydrolytic modifications of LDL that may condition its further processing by cells (37, 38). This possibility is supported by studies describing how PLA2-modified LDL is more susceptible to free radical-mediated oxidation (39, 40). Additionally, secretory nonpancreatic PLA2, active toward LDL, is present in the extracellular matrix of human lesions (41-44). This enzyme may further reduce the surface components of LDL in the intima, causing increased affinity for the matrix PG and leading to aggregation (35, 38). This hypothesis is supported by reports showing that apoB-100 containing particles isolated from normal human arterial intima have a reduced PC content (45). Also, the particles and aggregates from human lesions show a reduced content of PC and appear enriched in sphingomyelin (46, 47). A similar result was obtained during the analysis of apoB-containing particles from lesions of Watanabe rabbits (48).
The action of phospholipases on the surface monolayer of LDL depends on the surface concentration of the substrates and on the surface pressure of the monolayer (49, 50). The surface monolayer of LDL appears to have a high lateral pressure that does not allow the association of other apolipoproteins but apoB-100 (2, 51). It is possible that a reduction of PC by PLA2 or hepatic lipase in plasma or by PLA2 in the intima may potentiate the action of other enzymes like sphingomyelinase because of the increase in the surface concentration of sphingomyelin and the reduction in surface pressure. Recent results about the increased hydrolysis of the sphingomyelin of LDL retained in the intima of rabbit aorta after treatment with human nonpancreatic secretory PLA2 support these ideas (52). These results indicate that a further reduction in the intima of the LDL surface-polar components by lipases could make its entrapment irreversible by the aggregation and increased affinity for extracellular PG (35).
Biglycan and decorin are proteoglycans present in the arterial wall (53). Recently, biglycan was reported to be found in lipid-enriched areas of human coronary atherosclerotic lesions co-localized with apoB-100 (54). Additionally, in vitro experiments have shown that biglycan and decorin are able to interact with apoB-100-containing lipoproteins (54, 55). In the present study we demonstrate that hydrolysis of PL in LDL by PLA2 increased significantly the capacity of LDL to form nonsoluble complexes with biglycan and decorin.
The potential role of secretory PLA2 in atherosclerosis has recently been studied in transgenic mice expressing human secretory group IIa PLA2 (56, 57). It was demonstrated that these animals exhibited significant atherosclerotic lesions both when maintained on a high and low fat diet and that human secretory group IIa PLA2 was present in the lesions. In our experiments when incubating human plasma with PLA2 we found, besides formation of smaller LDL and HDL particles, that there was a significant decrease in HDL cholesterol. This observation is in line with the data from the studies on the transgenic mouse model. The authors speculated that this finding could be due to an altered effect of lecithin:cholesterol acyltransferase in the plasma (56). However, further studies are necessary to elucidate the molecular mechanisms behind this apparent relationship.
The results presented here show that a reduction of the PL moiety of
LDL contributes to make the lipoprotein particle smaller and denser
without inducing aggregation. These alterations may contribute to the
atherogenicity of the LDL subclass by increasing its retention by
arterial intima PG and its further hydrolytic and oxidative
modifications. The products of these modifications may contribute to
the extracellular accretion of LDL remnants and its uptake by
macrophages (58, 59). Taken together, these findings are consistent
with the hypothesis that PLA2 modification of
apoB-containing lipoproteins may contribute to the pathogenesis of
atherosclerosis by enhancing their trapping in the artery wall.
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ACKNOWLEDGEMENTS |
|---|
We thank Dr. Johannes Hulthe and Aira Lidell for their help with LDL particle size determinations.
| |
FOOTNOTES |
|---|
* This work was supported by the Swedish Society for Medical Research Project 970133, the Swedish Heart and Lung Foundation Projects 61538 and 63503, the Swedish Medical Research Council Project 4531, King Gustaf Vs 80 years foundation, and AstraZeneca, Mölndal, Sweden.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden. Tel.: 46 31 3422933; Fax: 46 31 823762; E-mail: Peter.Sartipy@wlab.wall.gu.se.
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ABBREVIATIONS |
|---|
The abbreviations used are: LDL, low density lipoproteins; HDL, high density lipoproteins; TG, triglyceride; VLDL, very low density lipoproteins; CHD, coronary heart disease; PL, phospholipids; UC, unesterified cholesterol; PLA2, phospholipase A2; GAG, glycosaminoglycans; PG, proteoglycans; ALP, atherogenic lipoprotein profile; CE, cholesterol esters; C6S, chondroitin 6-sulfate; NEFA, nonesterified fatty acids, BHT, butylated hydroxytoluene; BSA, bovine serum albumin; CSPG, chondroitin sulfate proteoglycans.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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S. D. Proctor and J. C.L. Mamo Intimal Retention of Cholesterol Derived From Apolipoprotein B100- and Apolipoprotein B48-Containing Lipoproteins in Carotid Arteries of Watanabe Heritable Hyperlipidemic Rabbits Arterioscler. Thromb. Vasc. Biol., September 1, 2003; 23(9): 1595 - 1600. [Abstract] [Full Text] [PDF]
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K. Hanasaki, K. Yamada, S. Yamamoto, Y. Ishimoto, A. Saiga, T. Ono, M. Ikeda, M. Notoya, S. Kamitani, and H. Arita Potent Modification of Low Density Lipoprotein by Group X Secretory Phospholipase A2 Is Linked to Macrophage Foam Cell Formation J. Biol. Chem., August 2, 2002; 277(32): 29116 - 29124. [Abstract] [Full Text] [PDF]
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V. Llorente-Cortes, M. Otero-Vinas, E. Hurt-Camejo, J. Martinez-Gonzalez, and L. Badimon Human Coronary Smooth Muscle Cells Internalize Versican-Modified LDL Through LDL Receptor-Related Protein and LDL Receptors Arterioscler. Thromb. Vasc. Biol., March 1, 2002; 22(3): 387 - 393. [Abstract] [Full Text] [PDF]
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 E. Hurt-Camejo, G. Camejo, H. Peilot, K. Oorni, and P. Kovanen Phospholipase A2 in Vascular Disease Circ. Res., August 17, 2001; 89(4): 298 - 304. [Abstract] [Full Text] [PDF]
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J. K. Hakala, K. Oorni, M. O. Pentikainen, E. Hurt-Camejo, and P. T. Kovanen Lipolysis of LDL by Human Secretory Phospholipase A2 Induces Particle Fusion and Enhances the Retention of LDL to Human Aortic Proteoglycans Arterioscler. Thromb. Vasc. Biol., June 1, 2001; 21(6): 1053 - 1058. [Abstract] [Full Text] [PDF]
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 N. D. Oakes, P. G. Thalén, S. M. Jacinto, and B. Ljung Thiazolidinediones Increase Plasma-Adipose Tissue FFA Exchange Capacity and Enhance Insulin-Mediated Control of Systemic FFA Availability Diabetes, May 1, 2001; 50(5): 1158 - 1165. [Abstract] [Full Text]
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 G. Embery, R. Hall, R. Waddington, D. Septier, and M. Goldberg Proteoglycans in Dentinogenesis Critical Reviews in Oral Biology & Medicine, January 1, 2001; 12(4): 331 - 349. [Abstract] [Full Text] [PDF]
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K. Öörni, M. O. Pentikäinen, M. Ala-Korpela, and P. T. Kovanen Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions J. Lipid Res., November 1, 2000; 41(11): 1703 - 1714. [Abstract] [Full Text]
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 C. J. Packard, D. S.J. O'Reilly, M. J. Caslake, A. D. McMahon, I. Ford, J. Cooney, C. H. Macphee, K. E. Suckling, M. Krishna, F. E. Wilkinson, et al. Lipoprotein-Associated Phospholipase A2 as an Independent Predictor of Coronary Heart Disease N. Engl. J. Med., October 19, 2000; 343(16): 1148 - 1155. [Abstract] [Full Text] [PDF]
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P. T. Kovanen and M. O. Pentikainen Secretory Group II Phospholipase A2 : A Newly Recognized Acute-Phase Reactant With a Role in Atherogenesis Circ. Res., March 31, 2000; 86(6): 610 - 612. [Full Text] [PDF]
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H. Peilot, B. Rosengren, G. Bondjers, and E. Hurt-Camejo Interferon-gamma Induces Secretory Group IIA Phospholipase A2 in Human Arterial Smooth Muscle Cells. INVOLVEMENT OF CELL DIFFERENTIATION, STAT-3 ACTIVATION, AND MODULATION BY OTHER CYTOKINES J. Biol. Chem., July 21, 2000; 275(30): 22895 - 22904. [Abstract] [Full Text] [PDF]
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