Human Coronary Smooth Muscle Cells Internalize Versican-Modified LDL Through LDL Receptor–Related Protein and LDL Receptors
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. Anti–LDL 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 receptor–related 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 receptor–related protein (fused LDL).
- vascular smooth muscle cells
- LDL receptor–related protein
- cholesteryl ester accumulation
- antisense oligodeoxynucleotides
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.1–3⇓⇓ Interestingly, it has been demonstrated that LDL binds and is retained by versican-like PGs secreted by human vascular smooth muscle cells (VSMCs)3–5⇓⇓ and macrophages.6,7⇓ The binding between LDL and versican or biglycan seems to be enhanced in the presence of lipoprotein lipase8 or phospholipase A2.9,10⇓ The relevance of the interaction between LDL and PG in vivo has been demonstrated (1) by the isolation of complexes apoB-100–chondroitin sulfate (CS), which is rich in negatively charged GAGs from human arteries1; (2) by the colocalization of apoB in the intima with CS-rich regions11,12⇓; 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 particles5 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 receptor–related 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-triglyceride–rich 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.
For an expanded Methods section, please refer to the online supplement (which can be accessed at http://atvb.ahajournals.org).
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⇓
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.27–29⇓⇓ 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 acid–reactive 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 ODN–treated 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⇓
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 000g, 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).
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 anti–LDL 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 anti–LDL receptor antibody. However, the anti–LDL 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 ODN–treated 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 ODN–treated VSMCs but not in sense ODN–treated 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).
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 ODN–treated VSMCs did not (Figure 3D, b). Sense ODN–treated 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.
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 versican–to–protein 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 vortexing18 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 heterogeneity3,33⇓ 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 ODN–treated 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.
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 d’Hebró 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; revision accepted November 6, 2001.
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