Differential Role of Heparan Sulfate Proteoglycans on Aggregated LDL Uptake in Human Vascular Smooth Muscle Cells and Mouse Embryonic Fibroblasts
Objective— Low density lipoprotein (LDL) receptor–related protein (LRP) binds and internalizes aggregated LDL (agLDL) in human vascular smooth muscle cells (VSMCs). To analyze the contribution of proteoglycans (PGs) to agLDL uptake in human VSMCs, in wild-type mouse embryonic fibroblasts (MEF line), and in LRP-deficient mouse embryonic fibroblasts (PEA13 line).
Methods and Results— PGs in the medium and cellular and extracellular matrix have been isolated by metabolic radiolabeling with [35S]Na2SO4 and characterized by selective digestion with heparinase I and III (4 U/mL each) and chondroitinase ABC (2 U/mL). To examine the contribution of PGs and LRPs to agLDL internalization, nonexpressing and LRP-expressing cells, treated or not with polysaccharidase, were incubated with agLDL (25, 50, and 100 μg/mL) for 18 hours. In human VSMCs, agLDL was unable to induce cholesteryl ester (CE) accumulation in antisense LRP-oligodeoxynucleotide–treated cells, and heparan sulfate (HS)-PG depletion leads to a reduction of the CE accumulation. In mouse fibroblasts, PEA13 compared with MEF showed lower, but still considerable, CE accumulation, and HS-PG depletion almost completely inhibited CE accumulation.
Conclusions— In MEF, HS-PGs can function alone as receptors that bind and internalize agLDL in the absence of LRP, but in human VSMCs, although HS-PGs facilitate agLDL binding to the cells, LRP is essential for agLDL internalization.
- heparan sulfate proteoglycans
- human vascular smooth muscle cells
- mouse embryonic fibroblasts
- aggregated LDL
- LDL-related protein
In the intima, proteoglycans (PGs) are in the pericellular space of the endothelial and smooth muscle cells and are also the major constituents of the extracellular matrix (ECM).1 Vascular smooth muscle cells (VSMCs) secrete most chondroitin sulfate (CS)-PGs associated with hyaluronan, a glycosaminoglycan (GAG). Indeed, versican (a CS-like PG) is the main PG structuring the ECM.2–4⇓⇓ Several studies demonstrate that CS-PGs act as sites for apoB-100 lipoprotein retention by the interaction between positively charged heparin-binding domains on apoB-100 or apoE and negatively charged GAG chains of the PGs.2,4⇓ The GAGs of versican induce alterations in the LDL particle that lead to the formation of fused and aggregated LDL (agLDL).5,6⇓ VSMCs also synthesize heparan sulfate (HS)-PGs, which can be secreted (perlecan) or shed from the cell surface (perlecan, syndecan, or glypican).7,8⇓ In contrast to CS-PGs, which play a major role in LDL retention and modification in arterial intima, HS-PGs may act as potential receptors for atherogenic lipoproteins9–11⇓⇓ or facilitate the uptake of ligands by a process called ligand transfer to lipoprotein receptors, such as the LDL receptor–related protein (LRP).12–16⇓⇓⇓⇓ We previously demonstrated that LRP binds and internalizes agLDL in human VSMCs.6,17⇓ The aim of the present study was to analyze the contribution of PGs and LRPs on agLDL internalization by human VSMCs and fibroblasts. HS-PGs and CS-PGs from the medium and from cellular and ECM fractions have been characterized by metabolic radiolabeling and selective digestion of PGs with heparinase I and III (HSI&III)18,19⇓ and chondroitinase ABC (ChABC),20 respectively. To examine the contribution of PGs and LRPs on agLDL internalization, human VSMCs, which do not express LRP (antisense LRP-oligodeoxynucleotide [ODN]–treated VSMCs),6,17⇓ and LRP-deficient mouse embryo fibroblasts (PEA13 line)21 in parallel with LRP-expressing cells (either treated or not with polysaccharidase) were incubated with increasing concentrations of agLDL (25, 50, and 100 μg/mL). We have found that in VSMCs, CS-PGs are the major component of cellular matrix and ECM. In contrast, HS-PGs are more abundant than CS-PGs in cellular and ECM fractions of wild-type mouse embryonic fibroblasts (MEF line). Although HSI&III and ChABC treatment completely degrades HS-PGs and CS-PGs, respectively, only HS-PG cleavage has consequences for agLDL internalization in both cell types. However, there are marked differences in the role of HS-PGs between human VSMCs and fibroblasts. In fibroblasts, HS-PGs alone can function as receptors that bind and internalize agLDL in the absence of LRP. In contrast, in human VSMCs, although HS-PGs facilitate the agLDL binding to the cells, LRP is essential for agLDL internalization.
Cell culture medium and reagents were from GIBCO Laboratories. MEF (CRL-2214) and PEA13 (CRL-2216) fibroblasts were from American Type Culture Collection. Benzamidine-HCl, Triton X-100, ε-amino caproic acid, guanidinium-HCl, BSA, cetylpyridinium chloride, heparinase I (heparin lyase I, EC 18.104.22.168), heparinase III (heparin lyase III, heparitinase I; EC 22.214.171.124), and ChABC (ChABC lyase, EC 126.96.36.199) were from Sigma Chemical Co. HiTrap Q ion exchange columns and [35S]Na2SO4 (100 mCi/mmol) were from Amersham Pharmacia Biotech. Bicinchoninic acid protein assays were from Pierce. Four percent to 12% Tris-glycine gels and Sypro Ruby protein gel staining were from Bio-Rad, and EN3HANCE (NEF981G) was from NEN Life Sciences.
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 that we described previously.6,17⇓ Explants were incubated at 37°C in a humidified atmosphere of 5% CO2. Outgrown cells were suspended in a solution of trypsin/EDTA and subcultured. They grew in monolayers in DMEM supplemented with 20% FCS, 2% human serum, 2 mmol/L l-glutamine, 100 U/mL penicillin G, and 100 μg/mL streptomycin. VSMCs were used between passages 2 and 6. Nonexpressing LRP human VSMCs were obtained by treatment of the cells with antisense LRP-ODNs as previously described.6,17⇓
MEF and PEA13 were grown in DMEM supplemented with 10% FCS, 2 mmol/L l-glutamine, 100 U/mL penicillin G, and 100 μg/mL streptomycin, as previously described.21
Cells grown on coverslips were treated or not with heparinase I (4 U/mL) or ChABC (2 U/mL) at 37°C for 2 hours. They were then fixed with glutaraldehyde (1.6%), washed, cryoprotected in 10% methanol, and cryofixed by projection against a copper block cooled by liquid nitrogen (−196°C) with the use of Cryovacublock de Reichert-Jung (Leica). The frozen samples were stored at −196°C in liquid nitrogen until subsequent use. Samples were freeze-dried and coated with platinum and carbon by using a freeze-etching unit (model BAF 060, BAL-TEC). A rotatory shadowing of the exposed surface was made by evaporating 10 nm of carbon evaporated at a 75° angle. The replica was separated from the coverslip by immersion in 38% hydrofluoric acid, washed twice in distilled water, and digested with 5% sodium hypochlorite for 5 to 10 minutes. Finally, the replicas were washed several times in distilled water, broken into small pieces, and picked up on copper grids coated with plastic for electron microscopy. All electron micrographs were obtained by using an electron microscope (Hitachi HU-600), operating at 75 kV.
Radiolabeling and Digestion of PGs
Cells were synchronized in medium containing 0.2% FCS for 2 days. Then the medium was removed, and fresh DMEM (10% FCS) containing 20 μCi/mL [35S]Na2SO4 was added and maintained for 3 days to biosynthetically label PGs as previously described.22 CS-PGs and HS-PGs were digested by adding a mixture of HSI&III (4 U/mL each) or ChABC (2 U/mL), respectively, to the incubation media for 2 or 18 hours. Control cells without PG enzymatic digestion were processed in parallel.
Isolation of PGs
After the 3 days of labeling, the culture medium from cells treated or not with enzymes for 2 or 18 hours in the absence or presence of agLDL (100 μg/mL) were transferred to tubes. Protease inhibitors were added to a final concentration of 10 mmol/L EDTA, 10 mmol/L ε-amino caproic acid, and 1 mg/mL benzamidine-HCl, and the medium was stored at −20°C until use.
Cells were washed with PBS containing 50 μg/mL heparin for 30 minutes at room temperature. The heparin-containing buffer was removed, and the cells were washed 3 times with PBS without heparin and dissolved by 2 extractions (5 mL each) of buffer containing 1% Triton X-100, 0.l5 mol/L NaCl, 10 mmol/L Tris, 5 mmol/L MgCl2, 2 mmol/L EDTA, 0.255 mmol/L dithiothreitol, and 1 μmol/L AEBSF, pH 7.2. After incubation for 30 minutes under gentle shaking, the cellular extract was removed and stored at −20°C until use. The remaining matrix was washed with PBS and solubilized by 2 extractions (5 mL each) of 8 mol/L urea, 2 mmol/L EDTA, 0.5% Triton X-100, and 20 mmol/L Tris-HCl (pH 7.5) containing protease inhibitors (1 mg/mL benzamidine-HCl and 10 mmol/L ε-amino-n-caproic acid). The wells were left overnight at 4°C before the ECM extract was collected with a cell scraper.23
The culture media and cellular and ECM extracts were dialyzed against binding buffer (8 mol/L urea, 2 mmol/L EDTA, 0.5% Triton X-100, and 20 mmol/L Tris-HCl, pH 7.5) for 48 hours at 4°C and were then chromatographed on a HiTrap Q (5-mL) column equilibrated with binding buffer at a flow rate of 5 mL/min. The 35S-labeled PG-containing fractions were collected after elution with a linear NaCl gradient (0.25 to 3 mol/L NaCl) and dialyzed at 4°C against water.
PG Characterization by SDS-PAGE
Equal amounts of protein from VSMCs and fibroblasts were loaded on a precasted 4% to 12% Tris-glycine gels, and SDS-PAGE was run for 2 hours at 60 V. The proteins were then fixed with methanol/acetic acid, stained with Sypro Ruby protein gel stain, and observed under UV light to control the equal protein loading.
The gels were impregnated with EN3HANCE before drying. The dried gels were placed for autoradiography at −80°C for 14 days before they were developed.
LDL Preparation and Modification
Human LDLs (density 1.019 to 1.063 g/mL) were obtained from pooled sera of normocholesterolemic volunteers, isolated by sequential ultracentrifugation, and dialyzed. The model system of agLDL was generated by vortexing as previously described.6,19,24⇓⇓
Determination of Intracellular Cholesterol Content
Arrested VSMCs or fibroblasts were untreated or treated with HSI&III (4 U/mL each) or ChABC (2 U/mL) for 2 hours before the addition of increasing concentrations of agLDL (25, 50, 100 μg/mL) to the incubation medium containing the enzymes. After 18 hours, cells were exhaustively washed and harvested into 1 mL of 0.10 mol/L NaOH. Lipid extraction and thin-layer chromatography (TLC) were performed as previously described.6,17,24⇓⇓ The spots corresponding to free cholesterol and cholesteryl esters (CEs) were quantified by densitometry against the standard curve of cholesterol and cholesterol palmitate, respectively, by using a computing densitometer (Molecular Dynamics).
Characterization of PGs Synthesized by Human VSMCs and MEF
Human VSMCs and MEF were metabolically labeled with [35S]Na2SO4 for 3 days, and PGs from the medium and cellular and ECM fractions were purified from untreated or enzyme-treated cells. Enzymatic treatment was performed by incubating the cells with HSI&III (4 U/mL each) or ChABC (2 U/mL) for 2 hours. To test the maintenance of the enzymatic activity during the agLDL incubation period, we also tested the pattern of PGs after 18 hours in the absence or presence of agLDL. Online Figure I (available at http://www.ahajournals.org) shows the elution patterns of PGs synthesized by untreated cells and cells treated enzymatically for 2 hours. In human VSMCs and MEF, PGs eluted as a single peak between 1 and 1.5 mol/L NaCl. The majority of synthesized [35S]GAGs were cell-associated (58% in human VSMCs and 56% in MEF). Approximately 36% and 40% of the newly synthesized [35S]GAGs were secreted in the cellular medium in human VSMCs and MEF, respectively, and the remaining newly synthesized GAGs were in the ECM (7% in human VSMCs and 4% in MEF). As shown in the Table, HS-PGs seem to be absent from the cell medium of both cell types. The percentage of HS-PGs in the cell and ECM fractions is higher in MEF compared with human VSMCs (32.5±1.5% versus 7.5±2.5%, respectively, in the cell fraction; 59±2% versus 24±3.5%, respectively, in the ECM). On the contrary, the percentage of CS-PGs is higher in all the fractions from human VSMCs compared with those obtained from MEF (57±3% versus 24.5±11.5%, respectively, in medium; 76±3% versus 34.5±8.5%, respectively, in the cell fraction; and 63±7% versus 52.5±4.5%, respectively, in the ECM). These results indicate significant differences in the GAG composition of PGs synthesized by human VSMCs and MEF. No statistically significant differences in GAG chromatographic patterns were observed between 2 and 18 hours of enzymatic treatment (in the absence or presence of agLDL) in any cell type.
The autoradiographic analysis of the eluted PGs (Figure 1) revealed differences in the pattern of bands susceptible to being degraded by HSI&III and ChABC between human VSMCs and MEF. In human VSMCs, there is a defined band at the beginning of the polyacrylamide gel in the cell and ECM fractions that was degraded by HSI&III treatment and that, according to its size, could be perlecan.25,26⇓ In MEF, the main bands degraded by HSI&III seem to be mostly syndecans.27 Additionally, there are high amounts of bands that are susceptible to being degraded by ChABC; these bands were especially abundant in the cell fraction and were different in size for human VSMCs and MEF. Protein loading was determined to be equal for enzymatically treated and untreated cells.
As shown in Figure 2, the pericellular matrix was observed as a tangled network of thin filaments in both VSMCs (Figure 2A) and MEF (Figure 2B). Heparinase I treatment disrupted the pericellular matrix in human VSMCs (Figure 2C) and MEF (Figure 2D). Similar photographs were obtained by ChABC treatment of the cells.
Role of PGs and LRPs on agLDL Internalization in Human VSMCs and MEF
To reveal the role of HS-PGs and CS-PGs on agLDL internalization, HS-PGs and CS-PGs were selectively degraded in human VSMCs and MEF. It has been previously demonstrated that heparinase treatment did not influence LRP-binding capacity in fibroblasts.28 agLDL internalization experiments were performed by incubating LRP-expressing VSMCs and MEF and non–LRP-expressing cells (antisense LRP-ODN–treated VSMCs and PEA13, respectively), either enzymatically treated or not, with increasing concentrations of agLDL for 18 hours.
We have previously demonstrated that the increase in CE content observed in VSMCs reflects the cholesterol that enters the cell as LDL.24 An initial period of prolonged cell surface contact, facilitated by cell PGs, in which CE hydrolysis exceeds protein degradation (selective uptake), has previously been described in macrophages.29 A similar process cannot be excluded in CE accumulation induced by agLDL in human VSMCs and fibroblasts. As shown in Figure 3A, although agLDL induced a high intracellular cholesterol accumulation in a dose-dependent manner in human VSMCs (from 44.87±1.77 μg CE per milligram protein at 25 μg/mL to 81.81±1.6 μg CE/mg protein at 100 μg/mL), agLDL was unable to induce CE accumulation in antisense LRP-ODN–treated VSMCs, in agreement with previous results.6,17⇓ HS-PG depletion leads to a reduction in the CE accumulation derived from agLDL at each analyzed concentration (31.68±2 versus 44.87±1.77 μg CE/mg protein at 25 μg/mL, 45.9±3 versus 64.72±2.82 μg CE/mg protein at 50 μg/mL, and 51.1±3 versus 81.81±1.6 μg CE/mg protein at 100 μg/mL). Taken together, these results indicate that LRP is essential for agLDL internalization in VSMCs and that HS-PGs facilitate the process. In mouse fibroblasts, the mechanism seems to be different, inasmuch as PEA13 showed significantly lower, but still considerable, CE accumulation compared with MEF (32±6 versus 44.58±5 μg CE/mg protein, respectively, at 25 μg/mL; 47±8 versus 77.5±3.06 μg CE/mg protein, respectively, at 50 μg/mL; and 46±9 versus 95.51±4 μg CE/mg protein, respectively, at 100 μg/mL; Figure 3B). In addition, HS-PG depletion almost completely inhibited the CE accumulation induced by agLDL in MEF and PEA13. CS-PG depletion, in contrast to HS-PG depletion, did not show any significant effect on CE accumulation derived from agLDL in any cell type.
As previously shown, agLDL (100 μg/mL) induced a CE accumulation of 81.81±1.6 μg CE/mg protein in human VSMCs and 95.51±4 μg CE/mg protein in MEF. Considering the contribution of pathways independent of LRPs and HS-PGs in human VSMCs (4±0.75 μg CE/mg protein) and MEF (7±1.5 μg CE/mg protein), LRP alone is responsible for intracellular CE accumulation in HSI&III-treated VSMCs (51.1±2.46 μg CE/mg protein) and in HSI&III-treated MEF (18±7 μg CE/mg protein), ≈58% and 12% of CE accumulation in human VSMCs and MEF, respectively. In the same way, considering the role of HS-PGs alone as responsible for intracellular CE accumulation in antisense LRP-ODN–treated VSMCs (4.59±1.0 μg CE/mg protein) and PEA13 (46±9 μg CE/mg protein), HS-PGs would account for ≈1% and 41% of the CE accumulation in human VSMCs and MEF, respectively.
By subtracting LRP-mediated CE accumulation and HS-PG–mediated CE accumulation from the total CE accumulation, a percentage of 26% in human VSMCs and 25% in MEF can be ascribed to an accumulation that is accomplished by a cooperative mechanism (both pathways).
Online Figure II (available at http://www.ahajournals.org) shows photomicrographs of representative untreated and HSI&III-treated human VSMCs and MEF incubated with agLDL. Pictures were taken after the first wash with PBS to eliminate agLDL that was not bound. As shown, untreated human VSMCs (online Figure IIA) or untreated MEF (Figure IIB) had many aggregates of LDL bound (arrows) on the cell surface. In contrast, HSI&III-treated VSMCs (Figure IIC) had less aggregate bound, and HSI&III-treated MEF did not show any aggregate bound (Figure IID). As observed in the photographs, HSI&III treatment did not induce changes in the morphology of any cell type.
We recently demonstrated that LRP is responsible for agLDL uptake in human VSMCs. These cells have very high levels of LRP expression and are unable to accumulate cholesterol from agLDL in the absence of LRP.6,17⇓ Because it has been proposed that PGs may play a role in the internalization of certain LRP ligands in hepatic and arterial cells,8–16⇓⇓⇓⇓⇓⇓⇓⇓ we explored the role of HS-PGs and CS-PGs on agLDL internalization in human VSMCs and MEF by degrading PGs with specific polysaccharidase. Chromatographic and autoradiographic results indicate some differences in HS-PG and CS-PG composition between VSMCs and MEF, especially in the cellular and ECM fractions. The amount and molecular weight of CS-PGs were higher in VSMCs than in MEF. HS-PGs were more abundant in MEF compared with human VSMCs. Additionally, although perlecan was the main HS-PG associated with VSMCs, syndecans were most abundant in MEF. Our results are in agreement with the pattern of PGs previously described in fibroblasts20 and VSMCs.30,31⇓ For further analysis, we have assumed that synthesized PGs by the parental MEF line and the selected PEA13 line are identical. HSI&III and ChABC treatment completely degraded HS-PGs and CS-PGs, respectively, and also disrupted the pericellular matrix in both cell types, but only HS-PGs cleavage has consequences on agLDL internalization by the cells. These results support a specific role of HS-PGs on cell uptake mechanisms, in agreement with previous studies describing the role of HS-PGs in the arterial wall.8 However, there are differences in the role of HS-PGs on agLDL internalization in VSMCs and fibroblasts. According to our results, HS-PGs can bind and internalize agLDL in the absence of LRPs, as demonstrated in PEA13 fibroblasts. HS-PGs seem to be essential for CE accumulation in fibroblasts, although LRPs can facilitate agLDL internalization. Our results are in agreement with the presence of an LRP-independent pathway involving HS-PGs as receptors in fibroblasts10,12⇓ and in macrophages.9,10,14⇓⇓ However, in human VSMCs, HS-PGs do not play a role as receptors for agLDL because they do not internalize agLDL in the absence of LRPs. LRPs alone internalize most of the agLDLs, although as in fibroblasts, there is a certain synergism between LRPs and HS-PGs. The HS-PG and LRP cooperation that we observed for agLDLs in human VSMCs and fibroblasts has been previously described for a wide variety of LRP ligands.12–16⇓⇓⇓⇓ The differential role of HS-PGs on agLDL internalization by fibroblasts and human VSMCs could be partially related to the higher amount and species of HS-PGs associated with fibroblasts compared with human VSMCs. In fibroblasts, the main HS-PGs associated with the cell fraction are syndecans, which are very efficient in internalization processes, whereas the main HS-PG associated with VSMCs is perlecan, which has been described to be fairly inefficient at internalizing bound material, but it cooperates with LDL receptor family members.25 Additionally, in the fibroblast membrane, with a sparser LRP distribution, HS-PGs seem to be indispensable for the binding of agLDL, a multimeric ligand that likely requires extensive binding to many cell surface molecules at once. In contrast, the sheer quantity of LRPs on the VSMC cell membrane17 may be sufficient for agLDL binding. Smaller ligands, such as tissue factor pathway inhibitor, can be internalized through the LRP independently of HS-PGs in fibroblasts, 32 suggesting that the nature of the ligand might also be important in determining the relative role of LRPs and HS-PGs in ligand internalization.
Therefore, an important role is played by HS-PGs in intracellular cholesterol accumulation in human VSMCs and MEF. However, the differences in HS-PG contribution to agLDL internalization make extrapolation from one cell type to another not suitable for target-specific cell internalization mechanisms. The main mechanism for agLDL internalization in human VSMCs is mediated by LRPs.
This work has been partially funded by MSO, Spain 99/0907, FISS 01/0354, Fundación Investigación Cardiovascular Catalana-Occidente, and Fundación Mapfre Medicina. Dr Otero-Viñas is a fellow of Fundación Investigación Cardiovascular. The authors thank the Heart Transplant Team of the Division of Cardiology and Cardiac Surgery of Hospital Santa Creu i Sant Pau and Blood Bank of Hospital Vall d’Hebron, Barcelona, for their collaboration. Electron microscopy experiments were performed in Serveis Cientifico-Tecnics of the University of Barcelona. The authors also thank Rosa Mendoza by her technical help.
↵*These authors contributed equally to the present study.
Received June 18, 2002; revision accepted August 2, 2002.
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