Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:387-393
doi: 10.1161/hq0302.105367
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:387.)
© 2002 American Heart Association, Inc.
Human Coronary Smooth Muscle Cells Internalize Versican-Modified LDL Through LDL ReceptorRelated Protein and LDL Receptors
Vicenta Llorente-Cortés;
Marta Otero-Viñas;
Eva Hurt-Camejo;
José Martínez-González;
Lina Badimon
From the Cardiovascular Research Center (V.L.-C., M.O.-V., J.M.-G., L.B.), IIBB-CSIC, Institut de Recerca del Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, and Wallenberg Laboratory for Cardiovascular Research (E.H.-C.), Göteborg University, Göteborg, Sweden.
Correspondence to Prof Lina Badimon, IIBB-CSIC, C/Jordi Girona 18-26, 08034 Barcelona, Spain. E-mail lbmucv{at}cid.csic.es
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Abstract
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Versican-like proteoglycans are the main component of the intimal
extracellular matrix interacting with low density lipoprotein
(LDL). The aim of this study has been to investigate the receptors
involved in versican-modified LDL uptake by human vascular smooth
muscle cells (VSMCs). We have found that versican-LDL interaction
leads to the following: (1) monomeric LDL particles that are
similar in size and electrophoretic mobility to native LDL but
that have a higher capacity to induce intracellular cholesteryl
ester (CE) accumulation and (2) fused LDL particles similar
in size to those obtained by vortexing. The precipitable fraction
of versican-LDL, composed of 50% monomeric and 50% fused LDL
particles, induced a dose-response increase in the CE content
of VSMCs. AntiLDL receptor antibody decreased the CE
accumulation derived from monomeric LDL particles by 88±3%
and that derived from the total precipitable fraction by 45±3%.
Inhibition of LDL receptorrelated protein expression
by antisense oligodeoxynucleotides reduced the CE accumulation
derived from the precipitable fraction by 65±2.8%, whereas
it did not produce any effect on the CE accumulation derived
from monomeric LDL. These results suggest that versican-LDL
induces CE accumulation in human VSMCs by the LDL receptor (monomeric
particles) and LDL receptorrelated protein (fused LDL).
Key Words: vascular smooth muscle cells LDL receptorrelated protein versican cholesteryl ester accumulation antisense oligodeoxynucleotides
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Introduction
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Vascular proteoglycans (PGs) have a common structure of a core
protein to which glycosaminoglycan (GAG) chains are covalently
attached. The content of versican-like PG, the main PG structuring
the extracellular matrix, is high in regions prone to lesion
development and increases with lesion progression.
13 Interestingly, it has been demonstrated that LDL binds and is
retained by versican-like PGs secreted by human vascular smooth
muscle cells (VSMCs)
35 and macrophages.
6,7 The binding
between LDL and versican or biglycan seems to be enhanced in
the presence of lipoprotein lipase
8 or phospholipase A
2.
9,10 The relevance of the interaction between LDL and PG in vivo
has been demonstrated (1) by the isolation of complexes apoB-100chondroitin
sulfate (CS), which is rich in negatively charged GAGs from
human arteries
1; (2) by the colocalization of apoB in the intima
with CS-rich regions
11,12; and (3) by the formation of complexes
ex vivo between LDL and CS-PGs extracted from arterial wall.
13,14 Versican is one of the PGs with the highest binding affinity
for plasma LDL. It has been described that GAGs accelerate proteolytic
and oxidative modification of the particles
5 and can also induce
LDL aggregation and fusion of the LDL particle under certain
incubation conditions.
15 Fused LDLs, remarkably similar to those
found in the arterial wall,
16,17 can be obtained by vortexing.
18 We have recently demonstrated that modified LDL particles generated
by vortexing are taken up through the LDL receptorrelated
protein (LRP) in human VSMCs.
19 LRP is also the receptor that
mediates the binding and internalization of other modified lipoproteins,
such as apoE-enriched VLDL,
20 lipoprotein lipase-triglyceriderich
lipoprotein complexes,
21,22 Lp(a),
23 and chylomicron remnants.
24 Although LRP is expressed in normal vessels and atherosclerotic
lesions, LRP expression increases in rabbit atherosclerotic
lesions; furthermore, LRP seems to play a role in the development
of atherosclerotic lesions.
25,26 In the present study, we report
on the characteristics of changes induced in the LDL particle
by the interaction with versican, the effect of versican-LDL
on the cholesteryl ester (CE) content of VSMCs, and the involvement
of LRP in the uptake of versican-LDLs in human VSMCs. Our results
indicate that versican interaction with LDL leads to the following:
(1) monomeric LDL particles (similar to native LDL [nLDL] in
electrophoretic mobility and electron microscopy size) that
enter the cells through the LDL receptor but are able to induce
CE accumulation and (2) fused LDL particles (similar in size
to those particles obtained by vortexing) that are internalized
through the LRP. These results indicate that versican increases
LDL atherogenicity.
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Methods
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Materials
For an expanded Methods section, please refer to the online
supplement (which can be accessed at http://atvb.ahajournals.org).
VSMC Culture
Primary cultures of human VSMCs were obtained from human coronary arteries of explanted hearts at transplant operations performed at the Hospital de la Santa Creu i Sant Pau. VSMCs were obtained by a modification of the explant technique, as we have described previously.27,28
LDL Preparation
Human LDLs (density 1.019 to 1.063 g/mL) were obtained from pooled sera of normocholesterolemic volunteers, isolated by sequential ultracentrifugation, and dialyzed as previously described.27 Vortexed LDL was prepared by vortexing LDL in PBS at room temperature.2729 The aggregates formed after vortexing (LDLs in fused form, precipitable fraction) can be separated from the nonaggregated LDLs (nonprecipitable fraction) by centrifugation at 10 000g for 10 minutes16,29
Preparation of Versican-LDL
Versican was isolated from pig aortas according to procedures previously described.10,30,31 The GAG composition of the aortic PGs varied between 50% and 65% for chondroitin-6-sulfate, between 10% and 25% for chondroitin-4-sulfate, and between 10% and 20% for dermatan sulfate. This variation was more related to the original composition of GAGs in the aorta than to the isolation procedure. The GAG-to-protein ratio varied between 7:3 and 6:4. The molecular size of the CS PG preparation by high-performance liquid chromatography and the GAG composition indicated that this preparation of CS PGs consisted mainly of the versican type of PG. The interaction between versican and LDL was carried out after the equilibration of LDL and versican in a solution containing 5 mmol/L HEPES, 20 mmol/L NaCl, 4 mmol/L CaCl2, and 2 mmol/L MgCl2, pH 7.2. LDL and versican in the protein (proportion 100:1) were incubated for 2 hours at 37°C. Versican-LDL, like vortexed LDL, did not show any change in thiobarbituric acidreactive substance content from nLDL (data not shown). The precipitable fraction was separated from the nonprecipitable fraction by centrifugation at 10 000g for 10 minutes16,29 and resuspended in a solution containing 5 mmol/L HEPES, 150 mmol/L NaCl, 4 mmol/L CaCl2, and 2 mmol/L MgCl2, pH 7.2.
Characterization of Vortexed LDL and Versican-LDL
The precipitable and nonprecipitable fractions of versican-LDL, compared with nLDL and the precipitable and nonprecipitable fractions of vortexed LDL, were analyzed by agarose gel electrophoresis and electron microscopy.
For transmission electron microscopy, the different LDLs were negatively stained with 2% uranyl acetate for 1 minute and were observed in a Hitachi 600 AB transmission electron microscope. Images were digitalized with a Bioscan Gatan camera. The estimation of the particle diameter was performed by using a software program (IMAT) designed by the central Services of the University of Barcelona.
To eliminate large LDL aggregates, the precipitable fraction of versican-LDL was filtered through a 0.22-µm filter, and the particles in the filtrate were characterized by agarose gel electrophoresis and nondenaturing acrylamide gradient gel electrophoresis (GGE). GGE was performed according to Nichols et al,32 with small modifications. Two solutions at 2% and 16% were prepared by using a stock solution of acrylamide and bis-acrylamide (30% total, 5% cross-linker) and mixed by using 2 P-1 peristaltic pumps (Pharmacia). The different fractions from versican-LDL (5 µL at 0.5 to 1 mg/mL) were preincubated for 15 minutes with 10 µL Sudan black (0.1% [wt/vol]) in ethylene glycol and 5 µL saccharose (50% [wt/vol]). Ten microliters of this mixture was electrophoresed at 4°C for 30 minutes at 20 V, 30 minutes at 70 V, and 16 hours at 100 V. Bands were scanned by densitometry at 595 nm, and LDL size was determined by using a plasma pool containing LDL particles of known size (22.9±0.5, 24.5±0.2, 26.2±0.2, and 28.4±0.4 nm) as a standard. The diameter of standard LDL particles was assessed by electron microscopy.
Determination of Free and Esterified Cholesterol Content
This procedure is discussed online (http://atvb.ahajournals.org).
RT-PCR and Western Blot
This procedure is discussed online (http://atvb.ahajournals.org).
LRP ODN Treatment
VSMCs were treated with antisense or sense LRP oligodeoxynucleotides (ODNs, 10 µmol/L), as previously described.19 Then, nLDL (precipitable and nonprecipitable fractions) from vortexed LDL and versican-LDL (40 µg/mL) were added to nontreated and to antisense ODN and sense ODNtreated VSMCs 12 hours before ending the second 24 hours of the arresting period. Then, the cells were exhaustively washed and harvested into 1 mL of 0.10 mol/L NaOH. The determination of free cholesterol (FC) and CE content was performed as previously described.27,28
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Results
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Characterization of Versican-LDL
After the incubation of LDLs with versican, the turbidimetry
of the LDL preparation (680 nm) increased from undetectable
levels to 0.20 relative units, indicating the presence of LDL
aggregates. Versican-LDLs, vortexed LDLs, and nLDLs were centrifuged
(10 000
g, 10 minutes) in parallel, and the precipitable and
nonprecipitable fractions were analyzed by agarose gel electrophoresis
(
Figure 1) and electron microscopy (
Figure 2). Particles of
the nonprecipitable fraction of vortexed LDL or versican-LDL
had an electrophoretic mobility similar to that of nLDL (
Figure 1A).
The precipitable fraction of versican-LDL was composed
of LDL particles with an electrophoretic mobility similar to
that of nLDL and of particles that did not enter into the agarose
gel (remaining at the gel origin), whereas the precipitable
fraction of vortexed LDL was composed only of particles that
did not enter into the agarose gel. To further characterize
the monomeric particles of the precipitable fraction of versican-LDL,
we filtered this fraction through a 0.22-µm filter and,
thus, obtained a new fraction (filtrate). Agarose gel electrophoresis
showed that the filtrate was composed of monomeric particles
with an electrophoretic mobility that was the same as that for
nLDLs (
Figure 1A). GGE assays (
Figure 1B) showed that the filtrate
was composed of monomeric particles with a diameter of

26.2
nm. The relative proportion of LDL in the filtrate was estimated
as 50±2% of the precipitable LDL (either in protein or
cholesterol). Electron microscopic analysis (
Figure 2) revealed
nLDL as monomeric particles (particle diameters ranged between
17 and 30 nm,
Figure 2E) and the precipitable fraction of vortexed
LDL as fused LDL (particle diameters ranged between 77 and 160
nm;
Figure 2A and 2F). In contrast, the precipitable fraction
of versican-LDL contained a mixture of fused LDL (particle diameters
ranged between 92 and 166 nm;
Figure 2C and 2G) and monomeric
particles that were similar to those in the nonprecipitable
fraction of versican-LDL and vortexed LDL (
Figure 2D and
Figure 2B,
respectively) and to nLDL (
Figure 2E).

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Figure 1. A, Agarose electrophoresis of nLDL, vortexed LDL, and versican-LDL. Vortexed LDL and versican-LDL were generated as described in Methods. After centrifugation at 10 000g, 1 aliquot from the precipitable fraction and 1 aliquot from the nonprecipitable fraction were applied to the electrophoresis gel. Arrow indicates electrophoretic origin. B, Nondenaturing acrylamide GGE of nLDL and versican-LDL. The precipitable fraction was filtered through a 0.22-µm filter, and the filtrate was applied beside the total precipitable and the nonprecipitable fraction into the GGE. The arrows indicate the position of bands corresponding to standard LDL of known diameter size.
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Figure 2. Electron microscopy of nLDL and nonprecipitable and precipitable fractions of vortexed LDL and versican-LDL. Samples were negatively stained and observed by electron microscopy as described in Methods. A, Precipitable fraction from vortexed LDL. B, Nonprecipitable fraction from vortexed LDL. C, Precipitable fraction from versican-LDL. D, Nonprecipitable fraction from versican-LDL. E, nLDL. F, Doubly magnified section from panel A. G, Doubly magnified section from panel C. m indicates monomeric; f, fused; and a, aggregated.
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Effect of Versican-LDL on VSMC Cholesterol Content
VSMCs were incubated in parallel with increasing concentrations of nLDL and the precipitable and nonprecipitable fractions of vortexed LDL and versican-LDL (20, 40, and 60 µg/mL). CE content increased in VSMCs incubated with the different types of LDL, whereas FC content of the VSMCs remained unaltered (see Figure I, which can be accessed online at http://atvb.ahajournals.org). nLDL induced a slight increase in CE content. However, monomeric particles that compose the nonprecipitable fraction of versican-LDL and vortexed LDL, although similar in size to nLDL, were able to induce a CE accumulation that was higher than that of nLDL. The precipitable fraction of versican-LDL induced a significant dose-dependent CE accumulation that was close to that induced by vortexed LDL (see online Figure I).
Cell Surface Binding of Versican-LDL
VSMCs were incubated with nLDL, with the precipitable and nonprecipitable fractions of vortexed LDL and versican-LDL (40 µg/mL), and simultaneously with different ligands, such as lactoferrin (ligand for the LDL receptor and the LRP), polyinosinic acid (ligand for the scavenger receptor), and galactose and fetuin (ligands for the asialoglycoprotein receptor. As shown in the online Table (which can be accessed at http://atvb.ahajournals.org), polyinosinic acid, galactose, and fetuin did not change CE accumulation levels. In contrast, lactoferrin produced a strong inhibition of the CE accumulation derived from all the lipoproteins tested. These results indicate that scavenger receptors or asialoglycoprotein receptors were not involved in versican-LDL uptake by VSMCs. To investigate whether the LDL receptor was involved in the uptake of the different fractions of versican-LDL, VSMCs were incubated with these fractions and increasing concentrations of antiLDL receptor antibody. As shown in Figure II (which can be accessed online at http://atvb.ahajournals.org), CE accumulations induced by nLDL (27.21±4.5 µg CE per milligram protein), by the filtrate from the precipitable fraction of versican-LDL (47.36±5.2 µg CE per milligram protein), or by the nonprecipitable fraction of versican-LDL (44.52±2.2 µg CE per milligram protein) were almost abrogated by antiLDL receptor antibody. However, the antiLDL receptor antibody only partially inhibited (45±3% inhibition at 25 µg/mL) the CE accumulation induced by the precipitable fraction of versican-LDL (80.54±2 µg CE per milligram protein).
Effect of Antisense LRP ODNs on CE Accumulation From Versican-LDL
To determine the role of LRP on versican-LDL uptake, we tested the effect of versican-LDL on LRP ODNtreated VSMCs. The commercial anti-LRP antibody used to detect LRP in Western blot analysis was not able to inhibit LRP function (data not shown). Because other types of antibodies were not available, we used a molecular approach to test LRP function. LRP mRNA transcription was blocked by an antisense ODN previously designed by us.19 LRP mRNA expression (see online Figure IIIA, which can be accessed at http://atvb.ahajournals.org) and LRP protein expression (see online Figure IIIB) decreased by 83±4.6% and 70±8.58%, respectively, in antisense ODNtreated VSMCs but not in sense ODNtreated VSMCs. LDL receptor mRNA expression (62.6±0.7 arbitrary units) was not altered by either antisense or sense LRP ODN treatment. As shown in Figure 3A and 3B, antisense LRP treatment reduced the CE accumulation derived from the precipitable fraction of vortexed LDL and versican-LDL by 70.8±1.4% and 65.3±3.5%, respectively, but it did not show any effect on CE accumulation derived from the nonprecipitable fraction of versican-LDL or vortexed LDL (Figure 3C).

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Figure 3. Effect of LRP ODN treatment on the CE accumulation derived from vortexed LDL and versican-LDL in VSMCs. VSMCs were treated with ODNs as explained in Methods. Then, VSMCs were exhaustively washed and harvested for measurement of FC and CE. A, Thin-layer chromatography showing the FC and CE bands corresponding to VSMCs treated with antisense ODN (AS) or sense ODN (S) against nontreated VSMCs (control) and incubated with the precipitable fraction. B, Bar graphs showing quantification of the CE bands in untreated VSMCs (open bars) and in antisense (solid bars) and sense (hatched bars) LRP ODNtreated VSMCs incubated with the precipitable fraction. C, Bar graphs showing the quantification of the CE bands in VSMCs incubated with the nonprecipitable fraction. Results are expressed as micrograms cholesterol per milligram protein and are shown as mean±SEM of 3 independent experiments. D, Effect of ODN anti-LRP antisense on versican-fused LDL binding by VSMCs. VSMCs were treated with LRP ODN, as explained in Methods (a, untreated VSMCs with versican-fused LDL bound [arrows] to the cell surface; b, antisense LRP ODNtreated VSMCs with no versican-fused LDL; and c, sense ODNtreated VSMCs with versican-fused LDL bound [arrows] to the cell surface). During the final 12 hours of the arresting period, versican-LDL (40 µg/mL) was added to the incubation medium. Then, VSMCs were washed with PBS and photographed (original magnification x100). Arrows indicate versican-fused particles bound to the cell surface.
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Figure 3D shows microphotographs of representative cells after incubation of control and ODN-treated VSMCs with versican-LDL. Pictures were taken after the first PBS wash to eliminate free versican-LDL (not bound). As shown, untreated VSMCs (Figure 3D, a) had versican-fused LDL bound (arrows) on the cell surface, whereas antisense LRP ODNtreated VSMCs did not (Figure 3D, b). Sense ODNtreated VSMCs (Figure 3D, c) also had versican-fused LDL bound to the cell surface. Treatment of VSMCs with ODNs did not induce morphological changes in VSMCs.
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Discussion
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Secreted versican molecules occupy the tridimensional network
of the intimal extracellular space and are highly expressed
in human arteries with high susceptibility to atherosclerosis.
2 Our results demonstrate that LDL-versican interaction produces
structural changes in the LDL particle. Versican was able to
induce LDL fusion in a very short incubation time (2 hours),
with few molecules of versican (protein versicantoprotein
LDL ratio 1:100) and under physiological conditions as reported
by Camejo and colleagues,
30,31 who described structural alterations
in the apoB-100 surface structure by interaction with CS-PGs.
The obtained fused particles are similar in size to those obtained
by vortexing
18 and those described in atherosclerotic lesions.
16,17 The high capacity of versican to induce LDL fusion seems to
be due to the high affinity of VSMC-secreted versican for LDL.
3 Fused LDLs generated by incubation with versican, contrary to
those generated by vortexing, were not completely separated
from monomeric LDLs by low-speed centrifugation. In fact, LDL
vortexing induced such strong surface changes on LDLs that most
LDL particles underwent fusion,
18 allowing a clear separation
of monomeric and fused LDL particles. In contrast, the LDL modification
induced by versican depends on the strength of versican-LDL
interaction, which seems to be driven by the structural characteristics
of the LDL.
1 In our experimental conditions, the formation of
versican-LDL complexes was facilitated by using a low ionic
strength buffer. Afterward, the precipitable fraction was resuspended
in a physiological buffer with increased ionic strength, favoring
separation of versican from LDL. The reversibility of the aggregation
process, facilitated by the incubation conditions, and LDL heterogeneity
3,33 might explain the presence of monomeric and a few small LDL
aggregates besides fused LDL particles in the precipitable fraction
of versican-LDL.
The monomeric particles that precipitate are similar to those that remain in the nonprecipitable fraction, according to their electrophoretic mobility, size, and capacity to induce CE accumulation. Although monomeric particles were able to induce higher CE accumulation than were nLDLs, they were similar in size and electrophoretic mobility to nLDLs. In fact, the uptake of monomeric LDL particles was a saturable process that could be completely inhibited by LDL receptor antibodies, indicating that these particles enter the cell through LDL receptors. Our results are in agreement with those obtained by Hurt-Camejo et al34 and Hurt et al35 and differ from those of Vijayagopal and colleagues,36,37 who proposed that the LDL receptor is not involved in the uptake of PG-LDL complexes. The difference could be due to the nature of the complexes formed in the different studies; Vijayagopal et al, different from Hurt-Camejo et al, Hurt et al, or the present study, used a high ratio of PG to LDL and a buffer that stabilizes the complexes LDL-PG in the incubation media. The increase of CE accumulation induced by monomeric versican-LDL over nLDL could be explained by the selectivity of versican for small dense LDL particles.38
In contrast to the uptake of the nonprecipitable fraction, the uptake of the precipitable fraction, composed of monomeric and fused particles, involved a nonsaturable process. Because monomeric particles represent 50% of the LDL in the precipitable fraction, they are responsible for
40% of the CE accumulation induced by the precipitable fraction. Furthermore, the percentage of CE inhibition by LDL receptor antibodies fully corresponds to that induced by monomeric LDLs. According to our results, scavenger and asialoglycoprotein receptors are not involved in the CE accumulation derived from versican-LDL uptake. In contrast, we have demonstrated the involvement of LRP on versican-LDL internalization by the marked decrease of the LDL binding and CE accumulation derived from versican-LDL in antisense LRP ODNtreated VSMCs. Because this treatment has no effect on the CE accumulation derived from monomeric LDL, the observed 65% reduction would correspond to the CE accumulation induced by fused LDL. These results are in agreement with the high capacity of LRP to bind and internalize fused LDLs generated by vortexing,19 which have a size similar to that of versican-fused LDLs.
In summary, we demonstrate that versican PGs have a very high capacity to induce fusion of LDL particles that are internalized through LRP in VSMCs. The LRP involvement on the internalization of versican-fused LDLs further enhances the importance of LRP as a lipoprotein receptor involved in VSMC foam cell formation. Although monomeric LDLs are also able to induce CE accumulation after interaction with versican with use of the LDL receptor, this receptor is only moderately expressed in the vascular wall cells, whereas LRP is highly expressed in normal and atherosclerotic lesions.25,26 Because versican is one of the main PGs interacting with LDL, and LRP is one of the main lipoprotein receptors in the arterial wall, the uptake of versican-fused LDL through LRP is likely one of the main mechanisms contributing to VSMC LDL internalization.
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Acknowledgments
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This study was partially funded by FIS99/0907, FIC-Catalana
Occidente, and BIOMED BMH4-CT96-0134. We thank the Heart Transplant
Team of the Division of Cardiology and Cardiac Surgery, Blood
Bank of the Vall dHebró Hospital. We thank Dr
Jordi Ordoñez Llanos from the Biochemistry Department
of the Hospital Santa Creu i Sant Pau for his help with LDL
characterization. The authors also thank Olga Bell for technical
assistance. Electron microscopy studies were performed at the
Central Services of the University of Barcelona.
Received November 6, 2001;
accepted November 6, 2001.
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