© 1999 American Heart Association, Inc.
Vascular Biology |
Proteoglycans Contribution to Association of Lp(a) and LDL With Smooth Muscle Cell Extracellular Matrix
From the Wallenberg Laboratory, Sahlgren's University Hospital, Göteborg, S-41345, Sweden (U.L., E.H.-C., G.O., P.S., G.C., O.W.); and the Astra Hässle AB, Preclinical Research Laboratories, Mölndal, S-431 83, Sweden (G.C.).
Correspondence to Germán Camejo, Astra Hässle AB, Preclinical Research Laboratories, Mölndal, S-431 83, Sweden. E-mail german.camejo{at}hassle.se.astra.com
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Abstract—Lp(a) interference with fibrinolysis could contribute to atherothrombosis. Additionally, accumulation of Lp(a) and LDLs, could lead to cholesterol deposition and foam cell formation in atherogenesis. The interactions between Lp(a) and LDL could cause their entrapment in the extracellular matrix of lesions. We found that association of Lp(a) with matrix secreted by cultured human arterial smooth muscle cells increased 2 to 3 times the subsequent specific binding of radioactive LDL. Chondroitin sulfate proteoglycans seem responsible for formation of the specific matrix-Lp(a) and matrix-LDL aggregates. The proteoglycans appeared also to participate in a cooperative increase of radioactive LDL binding to matrix pretreated with Lp(a). In the matrix preincubated with LDL,
50% of the additional lipoprotein was bound by ionic interactions.
In the matrix preincubated with Lp(a), 20% of the additional LDL was
held by ionic bonds, and the rest was held by strong nonionic
associations. Binding analysis in
physiological solutions confirmed that
chondroitin sulfate-rich proteoglycans from the smooth muscle
cell matrix have a high affinity for Lp(a) and LDL. The results provide
an explanation to the observed localization of Lp(a) and LDL in the
extracellular matrix of arterial lesions and suggest a
mechanism for their cooperative accumulation there.
Key Words: lipoprotein(a) • LDL • smooth muscle cells • extracellular matrix
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Lp(a) is an LDL-like particle distinguished by an apo(a) attached to apoB100 by a disulfide bridge and by noncovalent associations. Circulating levels of Lp(a) are an independent risk factor for atherosclerosis-related cardiovascular disease.1 2 Lp(a) contribution to atherothrombosis may be caused by its competition with plasminogen for binding at sites of fibrin deposition, thus inhibiting plasmin formation and clot lysis.3 4 A second hypothesis for the atherogenicity of Lp(a) suggests that it has special affinity for components of the extracellular intima. Therefore, together with other apoB100 containing lipoproteins, Lp(a) could be deposited at sites of lesion development.5 6 7 Lp(a) and LDL accumulate in the extracellular matrix of human lesions at regions rich in proteoglycans from where they can be extracted and measured.5 Furthermore, there is preferential retention of Lp(a) in the intima of atherosclerotic lesions when compared with normal regions.8 Lp(a) may interact with one or several of the intima extracellular matrix proteins and proteoglycans (PGs) by its apoB100 and apo(a) apolipoproteins.9 Recently, Hughes et al10 and Boonmark et al11 reported on the important role that lysine-binding sites in apo(a) kringles 4–10 may have for binding to the arterial wall in human-apoB transgenic mice. The apoB100 in Lp(a) could be another ligand for interacting with components of intima extracellular matrix. It contains glycosaminoglycan-binding, positively charged sequences, in its surface that may associate with intima PGs.7 Twenty years ago Dahlen et al12 suggested that glycosaminoglycans (GAGs) of the intima may be involved in the specific retention of Lp(a). More recently, Bihari-Varga et al13 concluded that Lp(a) may complex with arterial GAGs by the interaction of its apoB100 moiety and not by means of its apo(a). This conclusion is supported by the fact that the apo(a) does not have GAG-binding consensus sequences, such as those present in apoB100 or apolipoprotein E.14 15 Another possibility for Lp(a) association with the extracellular matrix is suggested by results in which the extracellular matrix was treated with exogenous lipoprotein lipase (LPL) and sphingomyelinase (SML). This was found to increases the binding of LDL and Lp(a), probably by formation of ternary complexes Lp(a)-LPL-GAG or Lp(a)-SML-GAG and the equivalents with LDL.16 17 Extracellular matrix fibronectin was also proposed as an Lp(a)-binding agent in arteries if unmasked of heparan PGs.18 An additional possibility is that, after specific noncovalent association of Lp(a) with LDL, this complex may associate with intima components. This idea is supported by findings from Trieu et al19 20 showing that Lp(a) and LDL can form noncovalent complexes and that a specific segment in apoB100, 3304-3317, is responsible for the association with kringle 36. However, to our knowledge no data about a cooperative effect between Lp(a) and LDL on their binding to the extracellular matrix of arterial cells is available. Smooth muscle cells are probably the main contributors of macromolecular matrix components of the arterial intima and are prominently involved in atherogenesis.9 21 We have explored the interactions of LDL and Lp(a) in a model of extracellular matrix secreted by human arterial smooth muscle cells (HASMCs).22 The results indicate that there is a cooperative binding of LDL and Lp(a) to the matrix and that the GAGs of the PGs present mediate most of this effect.
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Chemicals
Guanidine-HCl, Triton X-100, N-ethylmaleimide, epsilon aminocaproic-acid, benzaminidine-HCl, phenylmethylsulfonylfluoride, cetylpyridinium bromide, ethylaminihexanoic acid, cell culture tested BSA, HEPES buffer, and trypsin-EDTA were purchased from Sigma. Chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), and heparan sulfate were purchased from Seikagaku Co. Cyanogen activated sepharose, PD-10 columns, Lysine sepharose 4B columns, and Hi-Trap Q columns were from Pharmacia Fine Chemicals. The immunoreaction kit for Lp(a) was from Boehringer Mannheim. Collagen I was purchased from Collaborative Biomedical Products. Cell culture media, antibiotics, FCS, and culture vessels were from Flow Laboratories. Na2[35S]SO4,(25 to 40 Ci/mg); L-4,5-[3H]leucine,(120 to 190 mCi/mmol), 125I-iodine monochloride, and hyperfilms for autoradiography were from Amersham International. Other salts and solvents were of analytical grade and bought from Merck. NuSieve agarose was from FMC BioProducts.
Plasma Lipoproteins
LDL (d=1.019 to 1.063 g/mL) and HDL
(d=1.063 to 1.21 g/mL) were isolated by differential
ultracentrifugation from fresh plasma containing
Na2EDTA and with adjustment of densities with
KBr.23 Lipoprotein preparations were stored at 2°C
to 4°C in KBr and 1 mg/mL EDTA until used. Lipoproteins were used
within a week after preparation. Immediately before use, the
lipoproteins were equilibrated in the desired buffer solution using
PD-10 gel filtration columns. The number of LDL particles was
calculated with an electroimmunoassay for apoB100.24
Lipoprotein(a) was obtained from plasma pools from 3 to 4 healthy
donors containing a minimum of 210 mg/L apo(a). The plasma lipoprotein
fraction with d=1.063 to 1.21 g/mL was used as the Lp(a)
source. Lp(a) was purified by affinity chromatography
on lysine-sepharose 4B with the use of 
-amino caproic acid to elute
the bound lipoprotein.25 The identity and purity of
each preparation of LDL and Lp(a) were verified by agarose gel
electrophoresis and by immunodiffusion against polyclonal antibodies
against human apoB100, Lp(a), apoA1, apoE, and serum albumin.
The binding experiments required appreciable amounts of Lp(a);
therefore, we used pooled preparations from 3 to 4 individuals that
contained different isoforms. The amount of Lp(a) in the binding
experiments was estimated from the apoB100 content measured by
electroimmunoassay.26 LDL was labeled with
125I-iodide monochloride to a specific activity
of 50 cpm/ng of protein.27
Arterial Smooth Muscle Cells Extracellular Matrix
and Proteoglycans
Extracellular matrix (ECM) produced by confluent cultured HASMCs
was prepared as described.16 22 Briefly, confluent HASMC
cultures in collagen-pretreated 48-well plates were used. The
subcellular layer of ECM was exposed by dissolving the cell layer by a
sequential treatment with 0.5% triton X-100 in PBS followed by 25
mmol/L NH4OH in PBS. The remaining ECM layer
remained firmly attached, covered the bottom of the wells, and under
phase microscopy was free of nuclear and other cellular debris.
Extracellular matrix proteoglycan, rich in chondroitin sulfate and made
mostly of biglycan and labeled with
35SO4, was prepared and
characterized from batches of HASMC cultured to confluence in
75-cm2 bottles as described.28
Solid Phase Binding of LDL and Lp(a) to ECM
Three types of competition experiments were carried out to
evaluate the association of Lp(a) and LDL to the ECM. In all cases the
ECM-covered wells were first treated for 1 hour at 37°C with a
blocking buffer to diminish nonspecific binding. The blocking buffer
contained 10 mmol/L HEPES, 20 mmol/L NaCl, 5 mmol/L
CaCl2, 2 mmol/L MgCl2,
0.2% BSA, pH 7.4. In preliminary experiments we found that
radioiodination of Lp(a) caused a rapid time-dependent decrease in its
affinity for ECM. Also, incorporation of nonpolar fluorescent
labels into Lp(a), as the octadecyl indocarbocyanines, was found to
modify the binding to the extracellular matrix, and the probe rapidly
exchanged with the LDL in competition experiments. The use of
covalent–bound fluorescent probes was not possible because
changes in charged amino acid side chains of the apolipoproteins also
modify its interaction with GAGs.7 Therefore, in all 3
types of experiments to be described, LDL was used as the radioactive
probe to avoid labeling the Lp(a). The association of LDL to the ECM
after radiolabeling ECM was more stable than that of Lp(a). In the
first competition experiment a fixed amount of
125I-LDL (150 000 cpm, 3 µg apoB
protein, or 6 pmol) was added to the ECM-containing wells together with
increasing amounts of unlabeled LDL and Lp(a) in binding buffer that
was similar to the blocking buffer but contained no albumin.
After incubation for 2 hours at 37°C and removal of unbound ligands,
the wells were rapidly washed 3 times with cold binding buffer and the
bound 125I-LDL dissolved in 0.2 M NaOH and
counted. This was the only type of experiment in which unlabeled Lp(a)
was used as a competitor because of the large amounts required to run a
complete displacement curve (2 to 4 mg). In the other 2 types of
experiments to be described unlabeled LDL was used as the displacing
molecule. To evaluate if associations between LDL or Lp(a) with the ECM
could affect further binding of LDL a second type of competition
experiment was performed. The blocked ECM wells were pretreated with
0.2 nmol/well of either unlabeled Lp(a) or LDL, measured as apoB
protein. After 2 hours preincubation at 37°C the unbound LDL or Lp(a)
were removed and the wells were rapidly washed once with cold binding
buffer. After the preincubation, a constant amount of
125I-LDL (150 000 cpm, 6 pmol apoB/well) was
incubated with increasing amounts of unlabeled LDL in binding buffer
for 2 hours at 37°C. After removal of unbound ligands, the wells were
washed 3 times with cold binding buffer. The third type of competition
experiment was similar to the previous one but included a pretreatment
of the ECM with chondroitinase ABC. Solutions of 0.16 U/mL of the
enzyme were applied to digest the chondroitin and dermatan sulfate GAGs
of the ECM overnight at 37°C.22 The wells were washed 3
times with cold binding buffer, treated with blocking buffer, and
preincubated with unlabeled Lp(a) or LDL (0.2 nmol apoB/well). The
wells were then washed and used for competition experiments with a
constant amount of 125I-LDL and increasing
concentrations of unlabeled LDL or Lp(a). Radioactivity in the samples
was followed with a gamma counter (Ria-Gamma, Wallac).
To study the nature of the association of LDL with ECM pretreated with Lp(a) or LDL a binding and extraction experiment according to Moscatelli29 was performed. In this experiment the ECM-containing wells were preincubated with unlabeled Lp(a) or LDL and increasing amounts of labeled LDL were added. After incubation for 2 hours at 37°C the wells were rapidly washed 3 times with cold binding buffer. The amount of 125I-LDL attached to the matrix was discriminated into a loosely bound and a tightly bound fraction. The first was detached by adding a hypertonic buffer made of 10 mmol/L HEPES, 2 mmol/L Na2-EDTA, 500 mmol/L NaCl, pH 7.4 to the wells for 30 minutes at 37°C . This buffer should dissociate mostly ionic complexes promoted by divalent cations. The fraction of labeled LDL that remained bound by other forces (tightly bound) was collected by dissolving the ECM in 0.2 mol/L NaOH.29 30
Fluid Phase Binding of Lp(a) to ECM Proteoglycans
Direct binding of Lp(a) and LDL with the versican-like PGs
secreted by the HASMC was evaluated using an agarose gel mobility shift
assay. This assay was developed for evaluation of reversible binding of
lipoproteins to PGs at physiological ionic
strength.31 In brief, a constant amount of
35S-labeled chondroitin sulfate-rich PGs,
containing 0.4 to 0.5 µg/mL of GAGs and 8000 to 10 000 cpm was mixed
with increasing concentrations of Lp(a) or LDL. The lipoproteins, GAGs,
and PGs were dissolved in "fluid phase binding buffer" that was
made of 10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L
CaCl2, 2 mmol/L MgCl2,
pH 7.4. After incubation for 2 hours at room temperature, the samples
were made 10% vol/vol in glycerol to increase the density and 10
µL was applied to 0.7% agarose gels. The agarose was prepared in
buffer made of 10 mmol/L HEPES, 2 mmol/L
CaCl2, and MgCl2, pH
7.2.
After the electrophoretic step, the gels were fixed in 0.1% cetylpyridinium bromide for 16 hours, dried, and the radioactive PGs bands were evaluated in a digital autoradiograph (Berthold Laboratories, Wilband, Germany) and by autoradiography.
Data Analysis
All experiments were run in triplicate and repeated at least
once with LDL from 2 different individuals, 2 different pools of Lp(a),
and 2 different cell preparations. The binding experiments for each
preparation were run in triplicate cell culture wells and in duplicate
electrophoresis gels. Statistical analysis, mean±SD and
nonlinear curve fitting was done with the PRISM program of GraphPad
Software Inc.32
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Solid Phase Competition Experiments
The ECM in each well retained 0.08 pmol of the 6 pmol added 125I-LDL when no unlabeled ligands were present (Figure 1
). In the
experiments, designed as simple competition of radioactive LDL with
unlabeled Lp(a) or LDL, the Lp(a) always showed a very unusual
displacement curve. At the lowest concentration of Lp(a) used, there
was a net increase in the binding of 125I-LDL,
and even at the highest concentration there was little displacement of
radioactive LDL (Figure 1). The displacement curve with
unlabeled LDL was more predictable with a 65% displacement obtained at
the highest concentration of LDL. The data suggested that that Lp(a)
increased the binding of radioactive LDL to the matrix. To explore this
possibility further, a second type of competition experiment was
carried out. In these experiments the ECM-containing wells were
preincubated with unlabeled Lp(a) or LDL before adding the
125I-LDL probe followed by increasing amounts of
unlabelled LDL as a competitor. With no competitor, 2 to 3 times more
125I-LDL was bound to the Lp(a)-treated ECM than
to the LDL-treated one. Such increased binding of radioactive LDL by
pretreatment with Lp(a) could be competed by increasing amounts of
unlabeled LDL (Figure 2). However, 2 to 3
times more unlabeled LDL was required to compete 50%
(IC50) off the displaceable labeled LDL in the
Lp(a)-treated ECM than in the LDL-treated ECM.
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) or
Lp(a) (
), measured as apoB protein. After washing, a constant amount
of 125I-LDL was added to each well (6 pmol/well,
The possible contribution of the extracellular matrix GAGs on the
association of the lipoproteins was explored in ECM that was treated
with chondroitinase ABC before preincubation with Lp(a) or LDL. This
caused 50% to 60% reduction of the increased
125I-LDL binding related to the preformed
lipoproteins–ECM complexes, indicating that chondroitin sulfate GAGs
are responsible for the majority, but not all, of the effect of
pretreatment of ECM with Lp(a) (Figure 3A) or LDL (Figure 3B). The
binding of radioactive LDL, before and after chondroitinase ABC
treatment, was also competed by unlabeled LDL. This suggests that the
residual excess binding was also specific for LDL. The
IC50 for the competition curves were not
different. As indicated by the size of the standard deviations in the
figures, for each batch of lipoprotein and ECM preparation, the data
points have acceptable dispersions. However, the net amount of LDL
bound to the matrix varied with the batch of ECM and lipoprotein
preparation. To allow for an easier comparison of the effects of
chondroitinase treatment on the ECM preincubated with Lp(a) and LDL,
the results in Figure 3 are expressed as a percentage of the
initial radioactive LDL bound.
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The nature of the LDL interaction with the ECM preincubated with Lp(a)
or LDL was investigated by separation of the LDL retained into a
loosely (Figure 4A) and a tightly bound
fraction (Figure 4B). The results in Figure 4A and 4B
show again that more radioactive LDL was immobilized to the
Lp(a)-pretreated ECM than to the LDL-pretreated ECM. Additionally, the
data indicate that, at near saturation, >80% of the radioactive LDL
bound to the Lp(a)-preincubated ECM was in a tightly bound form (Figure 4B). Whereas on the LDL-preincubated ECM, only 40% of the total
bound LDL (tightly plus loosely bound, empty circles in Figure 4A and 4B) LDL was in the tightly bound form (Figure 4B).
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Lp(a) Interactions With ECM Proteoglycans in Fluid Phase
The described experiments in Figure 3
indicated that
chondroitin sulfate and dermatan sulfate GAGs were responsible for
>1/2 the increased LDL binding associated with pretreatment of
ECM with Lp(a) or LDL. To test if, at physiological
ionic conditions, Lp(a) or LDL could interact directly with the
chondroitin sulfate-rich PGs isolated from the ECM, we used
electrophoretic mobility assays. The fraction isolated from smooth
muscle cell ECM and used here is made mostly of biglycan. In the
conditions selected the interaction took place in the fluid phase. The
electrophoretic step was designed to measure complex formation by the
change in electrophoretic mobility of the PGs induced by association
with the Lp(a) or LDL (Figure 5). The
patterns obtained indicated that, at low concentrations of LDL and
Lp(a), associations were formed that depleted the radioactivity of the
band corresponding to the free versican-like PG (most anodic band).
Increasing the lipoprotein concentration led to changes in the
electrophoretic behavior of the PGs. This suggested that associations
were formed that retarded the mobility of subclasses of PGs until part
of them was not capable of entering the gel at high LDL and Lp(a)
concentrations (Figure 5). The experiments allowed us to measure
an apparent Kd of the lipoproteins for the
PG. The affinity of LDL for the versican was higher than that of Lp(a),
Kd=27 nmol/L and 180 nmol/L, respectively.
The amount of PG used in the incubation corresponded to 50 mol/L,
assuming a molecular weight of 200 000kDa for biglycan. Therefore, it
can be roughly estimated that 1 to 2 molecules of LDL were sufficient
to bind the PGs whereas 4 to 6 times more Lp(a) molecules were
required.
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Part of the atherogenicity of Lp(a) may be caused by its accumulation at sites of lesion development because of its special affinity for components of the intima extracellular matrix.10 12 13 Although Lp(a) particles are generally much less abundant in plasma than those containing only apoB100, their relative content in human lesions is not very different. This suggests that there should be an efficient trapping mechanism in the arterial intima for Lp(a).5 13 17 33 Most of the Lp(a) and apoB particles in lesions are detected in the extracellular intima and can be extracted from it as partially modified structures.5 33 34 In vivo studies confirm that, in the intima of lesions, there is increased retention of Lp(a) particles compared with normal regions.8 Recently, extensive Lp(a) deposits in lesions of transgenic mice expressing the human lipoprotein were described.11 Our experiments explored additional mechanisms that could contribute to the high affinity of Lp(a) for the extracellular intima.
Progressing lesions have an intima rich in chondroitin sulfate proteoglycans of the versican type with a special affinity for apoB100-containing particles. This may be related to secretion of PGs with larger chondroitin sulfate chains after smooth muscle cell migration from the media and proliferation at the intima.31 35 Another source of proteoglycans, in this case heparan PGs, with special affinity for apoB lipoproteins, could be macrophages accumulating in growing lesions.36 Fibronectin is another ECM macromolecule prominent in the intima of lesions that is product of migrating and proliferating smooth muscle cells.9 Fibronectin is another ECM protein that binds Lp(a). This protein may bind Lp(a) by means of the apoB100 and through specific sequences in the apo(a).37 Also laminin and fibrinogen bind recombinant apo(a).10 18 38 Lp(a) and LDL can also associate with macromolecules residing temporally in the extracellular and pericellular matrix, such as lipoprotein lipase or sphingomyelinase.16 17 Additionally, apo(a) has specific sequences in its kringle 4 that recognize the apoB sequence 3304–3317 by ionic interactions of high affinity (10-8 M).20 This suggests the possibility that, in the intima fluid phase, associations of Lp(a) and LDL can be formed in different states of aggregation. These hypothetical complexes may have restricted capacity to return to the circulation across the endothelial barrier. This may lead to its focal accumulation and eventual associations with the arterial intima.19 20 39 An alternate, but not exclusive, possibility may be that cooperative associations are formed at the surface of extracellular matrix fibril structures.
Our results, obtained with ECM secreted by HASMCs, suggest that Lp(a),
after interacting with chondroitin sulfate proteoglycans, increased the
further binding of apoB-containing lipoproteins to the matrix. Once
bound to the matrix, Lp(a), and to a lesser extent, LDL, created new
binding sites, probably by self-aggregation. The competition curves
(Figure 2) suggest that the affinity of LDL for the ECM-Lp(a)
complex is 2 to 3 times greater than for the ECM-LDL complex
(IC50=6.1x10-8 and
2.8x10-8 mol/L, respectively). The maximal
binding of LDL to the ECM-Lp(a) was also 2 to 3 times greater than for
ECM-LDL. Dissimilar types of noncovalent bonds may be responsible for
these differences in affinity and capacity. This idea is supported by
the experiments showing that 80% of the LDL bound by ECM presaturated
with Lp(a) was tightly bound, probably by hydrophobic associations
(Figure 4). The apoB100 peptide 3284-3317, identified by Trieu
et al20 as the main segment responsible for the
hydrophobic association between Lp(a) and LDL, was not an effective
competitor in our model of ECM-Lp(a)-LDL aggregates (data not shown).
It is then possible that the preliminary binding of Lp(a) to the ECM
leads to complex formation with additional components of the
apolipoproteins and to fusion of the particles that the peptide cannot
compete. Insoluble aggregates of Lp(a) with LDL in calcium-containing
buffers have been described.39 Also, the association with
arterial PGs induces large aggregates of LDL in
calcium-containing buffers.7 40
The interaction of Lp(a) with GAGs of the proteoglycans appears
responsible for about half of the ECM-Lp(a)-LDL or ECM-LDL-LDL
aggregation phenomenon observed. The remaining effect may be
contributed by other components of the ECM or GAGs resistant to
the chondroitinase ABC treatment (Figure 3). The fact that, in
the competition experiments, no total displacement was achieved with
Lp(a) (Figure 1) or LDL (Figures 2 and 3)
indicates that the association with the ECM is not a fully reversible
process. Furthermore, it indicates that other nonspecific associations
are present. This complicates the quantitative characterization of
the solid phase system in terms of association constants, and therefore
we only have used the concept of IC50 to compare
the curves. The fluid phase binding experiments using electrophoretic
band shift showed complex formation of Lp(a) with the PGs secreted by
smooth muscle cells at physiological ionic
conditions (Figure 5). These results support the conclusion that
chondroitin sulfate PGs in the ECM secreted by the arterial
smooth muscle could be important contributors to the binding of Lp(a)
and LDL, and to the possible cooperativity of this association. The
apparent affinity of the PG-LDL associations was 6 to 7 times higher
than that of PG-Lp(a) association, but both were in the nanomolar
range. Therefore, in the experiments with pretreatment of ECM, Lp(a)
and LDL may have easily saturated the initial ECM binding sites. The
subsequent enhancement of LDL binding could be then mediated by
associations with the preformed complexes of ECM-proteoglycans and the
lipoproteins. Bihari-Varga et al measured the binding capacity of
arterial PGs and GAGs for LDL and Lp(a) by titration with
increasing concentrations of the purified polysaccharides at
low ionic strength and measuring turbidity. These researchers concluded
that the binding capacity at saturation of Lp(a) for
arterial PGs and GAGs was 4 times higher than that of
LDL.12 However they did not report the values for the
Kd. On the other hand, our band shift
procedure expresses the affinity in terms of an apparent
Kd and measures the interaction at
physiological ionic strength. This discrepancy
should be evaluated with a procedure that allows evaluation of
Kd and maximal binding at
physiological salt concentrations using similar PGs
and GAGs. One aspect not explored in the present experiments is the
possibility that Lp(a) isoforms with different numbers of kringles may
have dissimilar affinity for PGs. We used pools of plasma to prepare
Lp(a) and therefore our preparations contained different isoforms. In
conclusion, our results and those from the cited literature suggest
that several mechanisms are likely involved in the entrapping of Lp(a)
and LDL in the matrix of the intima. We speculate that Lp(a) and LDL
may contribute to their focal accumulation at sites of progressing
lesions by its interaction with chondroitin sulfate proteoglycans
secreted by smooth muscle cells. Once formed, the PG-Lp(a) or PG-LDL
complexes may serve as additional anchoring sites for other apoB
lipoproteins. These hypothetical cooperative effects of Lp(a) and LDL
in lesions could be related to the observed additive effect of these 2
apoB lipoproteins as risk factors for atherosclerotic
disease.41
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Acknowledgments |
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This work was supported by grants from the Swedish Heart and Lung Foundation (projects No. 61026 and 61358) and from Astra Hässle AB.
Received May 27, 1998; accepted September 2, 1998.
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