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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:55.)
© 2002 American Heart Association, Inc.

Vascular Biology

Proteoglycans Synthesized by Arterial Smooth Muscle Cells in the Presence of Transforming Growth Factor-ß1 Exhibit Increased Binding to LDLs

Peter J. Little; Lisa Tannock; Katherine L. Olin; Alan Chait; Thomas N. Wight

From the Cell Biology of Diabetes Laboratory (P.J.L.), Baker Medical Research Institute, Melbourne, Victoria, Australia; the Division of Metabolism, Endocrinology and Nutrition (L.T., K.L.O., A.C.), Department of Medicine, and the Department of Pathology (T.N.W.), University of Washington, Seattle; and The Hope Heart Institute (T.N.W.), Seattle, Wash.

Correspondence to Thomas N. Wight, PhD, The Hope Heart Institute, 1124 Columbia Ave, Suite 783, Seattle, WA 98104. E-mail twight{at}hopeheart.org

	Abstract

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The "response-to-retention" hypothesis of atherogenesis states that atherogenic lipoproteins, such as low density lipoprotein (LDL), are retained in vessels by proteoglycans and undergo proatherosclerotic modifications. Transforming growth factor (TGF)-ß1 has been identified in atherosclerotic vessels and has been shown to stimulate the synthesis of chondroitin sulfate– and dermatan sulfate–containing proteoglycans by arterial smooth muscle cells (ASMCs), but whether it promotes lipid retention has not been addressed. We investigated whether TGF-ß1 modulates the biosynthesis of proteoglycans by ASMCs in a manner that promotes binding to LDL. Proteoglycans isolated from TGF-ß1–treated ASMCs exhibited enhanced binding to native LDL compared with the binding of proteoglycans isolated from control cultures (K_d 18 µg/mL LDL versus 81 µg/mL LDL, respectively). The increase in proteoglycan-LDL binding caused by TGF-ß1 could be attributed primarily to the glycosaminoglycan portion of the proteoglycans, since the glycosaminoglycan chains liberated from the core proteins of these proteoglycans synthesized in the presence of TGF-ß1 exhibited increased LDL binding as well. Furthermore, glycosaminoglycan chains initiated on xyloside (an initiator of glycosaminoglycan synthesis) in the presence of TGF-ß1 were longer and displayed enhanced binding to LDL compared with the LDL binding of xyloside-initiated glycosaminoglycan chains from control cultures. These results indicate that TGF-ß1 promotes LDL-proteoglycan interaction primarily by its effects on the glycosaminoglycan synthetic machinery of the ASMCs. Therefore, this study supports a proatherogenic role for TGF-ß1.

Key Words: proteoglycans • glycosaminoglycans • smooth muscle cells • transforming growth factor-ß1 • lipoproteins

	Introduction

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Cardiovascular disease resulting from coronary artery atherosclerosis is the major cause of morbidity and mortality in industrialized countries.^1–3 The biochemical and metabolic mechanisms responsible for the initiation and progression of atherosclerosis are not fully understood. Retention of lipoproteins by extracellular matrix molecules in the vascular wall is believed to play an important role in atherogenesis.^4–7 The "response-to-retention" hypothesis states that apoB- and apoE-containing lipoproteins bind to and are retained by vascular matrix molecules, particularly proteoglycans.⁵ The interaction of proteoglycans and lipoproteins predominantly occurs as an ionic interaction between negatively charged residues on the proteoglycans and positively charged residues on the apoproteins or via bridging molecules such as apoE and lipoprotein lipase. The retention of lipoproteins is increased by measures that increase the number of negatively charged residues on the glycosaminoglycan chain, either by an increase in chain length or increases in the degree of sulfation (see reviews^4–8).

Transforming growth factor (TGF)-ß1 is a cytokine that is increased in atherogenesis and has been shown to promote atherosclerotic lesion formation.^9–14 TGF-ß1 influences many of the events that contribute to lesion development. For example, TGF-ß1 increases the proliferation of arterial smooth muscle cells (ASMCs) under certain conditions¹¹ and can affect cell differentiation and migration.¹⁵

In addition, TGF-ß1 affects the production of extracellular matrix components, such as proteoglycans, that contribute to atherosclerotic lesion progression.^16–18 For example, TGF-ß1 increases the synthesis by ASMCs of two proteoglycans, versican and biglycan,^17,18 that accumulate in lesions of atherosclerosis.^19–23 In addition, these two proteoglycans have been localized to lipid-rich regions of atherosclerotic plaques along with TGF-ß1,¹³ suggesting a role for TGF-ß1 in lipid retention.

To determine whether TGF-ß1 affects proteoglycan-lipoprotein interactions and to identify the mechanism(s) responsible, we have assessed the capacity of proteoglycans produced by ASMCs in the presence or absence of TGF-ß1 to bind lipoproteins. We demonstrate that compared with proteoglycans isolated from ASMCs that were not treated with TGF-ß1, proteoglycans produced by TGF-ß1–treated ASMCs exhibit enhanced binding to native human LDL. Furthermore, we present data to indicate that the effect of TGF-ß1 on proteoglycan-lipoprotein interactions is mainly through its effect of modulating the glycosaminoglycan synthetic machinery of the ASMC. The present study supports a proatherogenic role for TGF-ß1 through its ability to promote proteoglycan binding to LDL.

	Methods

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Human recombinant TGF-ß1 and methyl ß-D-xylopyranoside (xyloside) were obtained from Sigma Chemical Co. [³⁵S]Sulfate was from ICN Biomedicals. Cell culture materials were from GIBCO-BRL. All other chemicals were obtained from Sigma, unless otherwise specified.

Cell Culture
ASMCs isolated from monkey (Macaca nemestrina) thoracic aorta were kindly provided by the laboratory of Elaine Raines, University of Washington, Seattle, and cultured as previously described.²⁴ Cells were grown in DMEM with 5.6 mmol/L glucose supplemented with pyruvate, nonessential amino acids, 4 mmol/L glutamine, antibiotics, and 5% calf serum. Cells were used between passages 6 and 18.

Determination of Proteoglycan and Xyloside-Initiated Glycosaminoglycan Synthesis
Cells were seeded at 60 000 cells per well into 12-well cell culture dishes in DMEM (5.6 mmol/L glucose) containing 10% serum. The medium was changed on the following day, and cells were confluent by day 3 or 4. Cells were made quiescent by changing to DMEM with 0.1% serum and incubating for 48 hours. Cells were radiolabeled with 50 µCi/mL [³⁵S]sulfate (to label glycosaminoglycan chains) or 60 µCi/mL [³⁵S]methionine (to label core proteins) in the presence or absence of TGF-ß1 (2 ng/mL) for 24 hours. Earlier studies showed that this concentration provided maximal stimulation of proteoglycan synthesis by monkey ASMCs.^17,18 To determine whether TGF-ß1 influenced glycosaminoglycan synthesis independent of core protein synthesis, TGF-ß1–treated cultures were supplemented with 0.5 mmol/L xyloside during the 24-hour labeling period.²⁵ Xyloside acts as an artificial acceptor for galactosyltransferase in the initiation of glycosaminoglycan synthesis and results in increased synthesis and secretion of free glycosaminoglycan chains and decreased synthesis of intact proteoglycans.²⁵ At the conclusion of the incubation period, culture medium (50 µL) from each well was spotted in duplicate onto paper (Whatman 3M, Whatman Int), which was then washed (5 times) in cetylpyridinium chloride (CPC) solution (1% in 0.05 mol/L NaCl). The paper was air-dried, cut into segments, and counted in a scintillation vial with Instagel (Packard Corp) for determination of CPC-precipitable counts as a measure of glycosaminoglycan synthesis.²⁶

Proteoglycan Isolation and Characterization
Proteoglycans from triplicate wells of 12-well plates were passed over DEAE-Sephacel (0.25 mL) and washed with "low salt buffer" (0.25 mol/L NaCl, 8 mol/L urea, 2 mmol/L Na₂EDTA, and 0.5% Triton X-100, pH 7.5). Proteoglycans were eluted with "high salt buffer" (3 mol/L NaCl, 8 mol/L urea, 2 mmol/L Na₂EDTA, and 0.5% Triton X-100, pH 7.5) 5 times at 0.3 mL. Aliquots (10 µL) were counted to locate the fractions containing proteoglycans, which were then combined. Aliquots of the pools were precipitated with ethanol by using chondroitin sulfate as a carrier and finally dissolved for SDS-PAGE in 20 µL of buffer (8 mol/L urea and 2 mmol/L Na₂EDTA, pH 7.0).²⁷

Pooled proteoglycans eluted from DEAE-Sephacel minicolumns were treated with 1 mol/L sodium borohydride in 50 mmol/L NaOH for 4 hours at 45°C to release glycosaminoglycan chains from core proteins by reductive ß elimination.^17,18 The reaction was terminated by neutralization with glacial acetic acid. The glycosaminoglycans obtained from untreated and TGF-ß1–treated cells were prepared for SDS-PAGE, hydrodynamic sizing, and gel mobility shift assays. Proteoglycans and glycosaminoglycans were subjected to electrophoresis (SDS-PAGE) by using a 4% to 12% gradient acrylamide gel for proteoglycans and a 4% to 16% gradient acrylamide gel for glycosaminoglycans with a 3.5% stacking gel according to the procedure of Laemmli.²⁸ Apparent molecular weights were estimated by comparisons to ¹⁴C protein standards (GIBCO-BRL Life Technologies Inc).

Hydrodynamic sizes of the DEAE-purified proteoglycans and glycosaminoglycans were determined by molecular sieve chromatography on analytical Sepharose CL-2B or CL-6B columns (0.7x110 cm), respectively, equilibrated in 8 mol/L urea buffer with 0.5% Triton X-100.^17,18,27 Eluted fractions (1 mL) were collected, and radioactivity was determined by liquid scintillation counting. The elution position of the free radioisotope was used as a marker for the total volume, and the void volume was determined by the elution position of [³H]DNA. Assessment of glycosaminoglycan molecular weight was made by chromatography on Sepharose CL-6B columns by using the standard curve of Wasteson.²⁹

Proteoglycan-Lipoprotein Binding Assay
LDLs (density 1.019 to 1.063 g/mL) were separated from plasma from normal human volunteers by preparative ultracentrifugation in a Beckman VTi 50 vertical rotor (Beckman Instruments) and purified by sequential density gradient ultracentrifugation.³⁰ LDL was stored in the dark at 4°C in 1 mol/L EDTA until it was used in the gel mobility shift assay.

Radiolabeled proteoglycans were isolated as described above and concentrated by using Centricon concentrators (Millipore Corp). Cellulose filtration membranes with cutoffs of 50 and 10 kDa were used for proteoglycans and free glycosaminoglycans, respectively. The sample was diluted with HEPES buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L CaCl₂, and 2 mmol/L MgCl₂, pH 7.4) and recentrifuged. The concentration and dilution process was repeated 4 times.

The interaction of proteoglycans with lipoproteins was assessed by using a gel mobility shift assay.³¹ Briefly, fixed amounts of radiolabeled proteoglycans, glycosaminoglycans, or core proteins were incubated with increasing concentrations of LDL for 1 hour at 37°C in the HEPES buffer. The reaction mixture was subjected to electrophoresis at 60 V for 3 hours at 4°C in 0.7% NuSieve Agarose Gel (FMC Bioproducts) in circulating running buffer (10 mmol/L HEPES, 2 mmol/L CaCl₂, and 4 mmol/L MgCl₂, pH 7.2). The gels were fixed with CPC (0.1% [wt/vol] in 70% ethanol) for 1 hour and air-dried. Radioactivity was assessed by using PhosphorImager (Molecular Dynamics) screens. The images were analyzed by using Optiquant proprietary software. The amount of complexed versus unbound proteoglycans or glycosaminoglycans in each lane was assessed, and the percent bound was calculated as the proportion of the radioactivity retained at the origin of the gel relative to the total radioactivity in each lane. Values for K_d and B_max from the electrophoretic data were determined with the SAAM II modeling program (SAAM Institute) by using the Michaelis-Menten equation.

To determine whether core proteins of proteoglycans had bound to LDL, [³⁵S]methionine-labeled proteoglycans were isolated as described above. Samples were dialyzed into 0.1 mol/L Tris HCl, 10 mmol/L calcium acetate, and 18 mmol/L sodium acetate, pH 7.0. Glycosaminoglycan chains were digested from aliquots of control and TGF-ßl–stimulated proteoglycans by digestion with 0.7 U/mL chondroitin ABC lyase (ICN), 20 U/mL heparinase I, and 20 U/mL heparinase II in the presence of protease inhibitors for 6 hours at 37°C. Additional aliquots were treated in parallel in the absence of enzymes. The digestion was dialyzed into gel mobility shift buffer by using MOPS (20 mmol/L MOPS, 140 mmol/L NaCl, 5 mmol/L CaCl₂, and 2 mmol/L MgCl₂, pH 7.4), and binding to lipoproteins was determined by a gel mobility shift assay as described above. SDS-PAGE confirmed that the digestion of glycosaminoglycan chains was complete (data not shown).

	Results

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Previous studies with ASMCs cultured from monkey (Macaca nemestrina) aortas have shown that TGF-ß1 increases mRNA transcripts and core protein for versican and biglycan.^17,18 Additionally, TGF-ß1 causes elongation of the chondroitin sulfate and dermatan sulfate chains attached to versican and biglycan core proteins. To determine whether proteoglycans produced by TGF-ß1–stimulated cells have altered the binding affinity for LDL, the total pools of media proteoglycans from untreated and TGF-ß1–treated ASMCs were purified and concentrated by using a combination of DEAE ion-exchange and molecular-sieve chromatography. Subsequently, the radiolabeled proteoglycans were mixed with increasing concentrations of LDL, and complex formation was evaluated by an electrophoretic gel mobility shift assay (Figure 1A and 1B).

View larger version (62K): [in this window] [in a new window]   Figure 1. TGF-ß1 treatment of vascular smooth muscle cells increases the binding of proteoglycans to LDLs. Quiescent ASMCs were untreated (A) or treated with 2 ng/mL TGF-ß1 (B) for 24 hours in the presence of [35S]SO4. Proteoglycans in the media were purified and concentrated on DEAE-Sephacel ion-exchange columns and exchanged into HEPES buffer. Fixed amounts of proteoglycans were incubated with increasing concentrations of native human LDL, and the binding was analyzed by a gel mobility shift assay as described in Methods. The percent bound was calculated as a ratio of amount of radioactivity retained at the origin to the total radioactivity per lane. Panel C shows that proteoglycans (PGs) synthesized in the presence of TGF-ß1 (solid circles, solid line) demonstrate higher binding affinity than do control proteoglycans (open circles, dashed line; P<0.002). LDL conc. indicates LDL concentration. The figure shown is representative of 3 independent experiments that gave similar results.

Analysis of the gels demonstrates that proteoglycan-lipoprotein complexes form at lower concentrations of LDL with proteoglycans from the TGF-ß1–treated ASMCs compared with proteoglycans from control ASMCs (Figure 1A and 1B). Michaelis-Menten analysis of the binding curves fitted the data with correlation coefficiets >0.95. The total binding was nearly identical in control and TGF-ß1–treated preparations and approximated 100% (Figure 1C). The K_d value for binding of the control proteoglycan pool was 81 µg/mL of LDL (K_d 1.47x10^-7 mol/L apoB), assuming a molecular mass of 550 000 Da for apoB, and the K_d for binding of proteoglycans from the TGF-ß1–treated cells was 18 µg/mL LDL (3.27x10^-8 mol/L apoB, P<0.002). The lower K_d value indicates higher affinity binding of the proteoglycans synthesized by ASMCs treated with TGF-ß1.

To determine whether the glycosaminoglycan chains were responsible for binding to LDL, the glycosaminoglycan chains were chemically released from the core proteins of the proteoglycans, and their binding to LDL was investigated. Earlier studies showed that the addition of TGF-ßl to ASMCs caused a lengthening of the glycosaminoglycan chains on the chondroitin sulfate and dermatan sulfate proteoglycans synthesized by ASMCs,^17,18 and these differences were confirmed in the present study (data not shown). The glycosaminoglycan chains released from proteoglycans were bound to LDL in a concentration-dependent manner (Figure 2). The binding affinity for glycosaminoglycan chains released from ASMC proteoglycans treated with TGF-ßl was higher for LDL compared with that for glycosaminoglycan chains from untreated cells (Figure 2, P=0.04).

View larger version (54K): [in this window] [in a new window]   Figure 2. Glycosaminoglycan chains liberated from proteoglycans produced by TGF-ß1–treated ASMCs show increased binding to LDL. Cells were untreated (A) or treated with TGF-ß1 (B) for 24 hours in the presence of [35S]SO4. Glycosaminoglycans were chemically released from the core proteins of the proteoglycans and analyzed for binding to LDL, as described in the legend to Figure 1. The percent bound was calculated as a ratio of radioactivity retained at the origin vs total radioactivity in the lane. Panel C shows that glycosaminoglycan chains synthesized in the presence of TGF-ß1 (solid circles, solid line) demonstrate higher binding affinity than do control glycosaminoglycan chains (open circles, solid line; P=0.04). The figure shown is representative of 2 independent experiments that gave similar results.

Because previous and present data have suggested that TGF-ß1–mediated elongation of glycosaminoglycan chains and increased binding affinity may be related, further experiments were undertaken to determine whether the effect of TGF-ß1 was due to the effects on glycosaminoglycan chain elongation. By use of ß-D-xyloside (an initiator of glycosaminoglycan synthesis), it is possible to examine glycosaminoglycan synthesis in the absence of core protein synthesis. Thus, xyloside stimulates glycosaminoglycan synthesis but reduces proteoglycan synthesis in ASMCs.²⁵ ß-D-Xyloside caused an 2-fold increase in the incorporation of radiolabeled sulfate into glycosaminoglycans during a 24-hour labeling period compared with control cells (Figure 3). TGF-ß1 treatment enhanced this increase, suggesting that TGF-ß1 can have an effect on the glycosaminoglycan synthetic machinery in the absence of proteoglycan core protein synthesis.

View larger version (27K): [in this window] [in a new window]   Figure 3. TGF-ß1 increases incorporation of [35S]sulfate into proteoglycans and xyloside (Xylo)-initiated glycosaminoglycans by ASMCs. Serum-deprived ASMCs were untreated or treated with TGF-ß1 (2 ng/mL) for 24 hours in the presence of [35S]SO4 and with Xylo (0.5 mmol/L) added to the media as indicated. Data show mean±SEM of 4 replicates from a single experiment that was repeated 3 times with similar results. Con indicates control. **P<0.001 vs respective Con values. ppt. indicates precipitable.

To further evaluate the xyloside–TGF-ß1 effect, newly synthesized proteoglycans and glycosaminoglycans were evaluated by SDS-PAGE and molecular sieve chromatography (Figure 4). In the controls, xyloside caused a reduction in the amount of proteoglycans that separate at the top of the SDS-polyacrylamide gel and the appearance of a broad band of radioactivity between 18 and 43 kDa (Figure 4A). Previous studies have identified the high molecular weight band as versican and the lower molecular weight broad band as a mixture of chondroitin sulfate and dermatan sulfate chains.^17,18,25 In the TGF-ß1–treated cultures, xyloside also reduced high molecular weight proteoglycans but shifted the broad band of radioactivity to a range of 29 to 80 kDa, suggesting an increase in the molecular weight of components found in this band (Figure 4A). To further assess the effect of TGF-ß1 on the xyloside-initiated glycosaminoglycan synthesis, the preparations were hydrodynamically sized by using Sepharose CL-6B columns. Three peaks of radioactivity were observed in the media of control and TGF-ß1–treated ASMCs. The control ASMC cultures contained peaks at K_av 0.00 (peak I), K_av 0.25 (peak II, shoulder), and K_av 0.51 (peak III) (Figure 4). Similar peaks of radioactivity were observed for the preparations from TGF-ß1–treated ASMCs, with the exception that peak III was shifted to an earlier elution position, indicating a larger sized population of molecules (K_av 0.43). Our previous studies have shown that peaks I and II represent intact proteoglycans and that peak III contains a mixture of chondroitin sulfate and dermatan sulfate chains.^17,18,25 The shift in peak III caused by TGF-ß1 treatment corresponds to an increase in average molecular mass from 19.2 to 29.0 kDa.²⁹ These data confirm that TGF-ß1 can increase the length of glycosaminoglycan chains in the absence of core protein synthesis.

View larger version (45K): [in this window] [in a new window]   Figure 4. TGF-ß1 treatment of ASMCs increases the size of glycosaminoglycan (GAG) chains initiated on Xylo. Cells were untreated or treated with TGF-ß1 (2 ng/mL) for 24 hours in the presence of [35S]SO4, with Xylo (0.5 mmol/L) present as indicated. Proteoglycans and (GAGs) were purified on DEAE-Sephacel ion-exchange columns and then subjected to SDS-PAGE (4% to 16% gradient) as described in Methods (A). Lanes are as follows: A, 14C standards; B, control; C, control+Xylo; D, blank; E, TGF-ß1; and F, TGF-ß1+Xylo. Panel B shows molecular sieve profiles (Sepharose CL-6B) of radiolabeled proteoglycans and GAGs from the media of untreated (open circles) and TGF-ß1–treated (solid circles) samples incubated with Xylo. Note that TGF-ß1 shifts peak III to the left, indicating larger size.

To determine whether the longer xyloside-initiated glycosaminoglycan chains produced in the presence of TGF-ß1 were bound more avidly to LDL, the gel mobility shift assay was performed by using radiolabeled samples from the xyloside-treated preparations. The xyloside-initiated chains are smaller than glycosaminoglycan chains on intact proteoglycans in ASMCs (data not shown).²⁵ Xyloside-initiated chains from control cultures were bound to LDL with a K_d value of 1350 µg/mL LDL (2.45x10^-6 mol/L apoB), whereas xyloside-initiated chains isolated from cultures treated with TGF-ß1 were bound to LDL with a K_d value of 880 µg/mL LDL (1.6x10^-6 mol/L apoB, P=0.03), again indicating that TGF-ßl results in the synthesis of glycosaminoglycan chains with increased binding affinity for LDL (Figure 5).

View larger version (59K): [in this window] [in a new window]   Figure 5. TGF-ß1 treatment of ASMCs increases the binding of glycosaminoglycan chains initiated on xyloside to LDL. Cells were untreated (A) or treated with TGF-ß1 (B) for 24 hours in the presence of [35S]SO4 and xyloside (0.5 mmol/L). The glycosaminoglycan chains were purified on DEAE-Sephacel columns and exchanged into HEPES buffer, and LDL binding was analyzed as described in the legend to Figure 1. The percent bound was calculated as a ratio of radioactivity retained at the origin vs total radioactivity in the lane. Note that the LDL concentration to achieve saturation was between 1 and 5 mg/mL. Panel C shows that glycosaminoglycan chains initiated on xyloside in the presence of TGF-ß1 (solid circles, solid line) demonstrate higher binding affinity than do control glycosaminoglycan chains (open circles, solid line; P=0.03). The figure shown is representative of 2 independent experiments that gave similar results.

Gel mobility shift assays were also performed on control and TGF-ß1–stimulated proteoglycans labeled with [³⁵S]methionine, which had been treated with chondroitin ABC lyase and heparinase I and II to remove the glycosaminoglycans chains. No binding was seen between LDL and core proteins synthesized under control conditions or in the presence of TGF-ß1 (data now shown).

	Discussion

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Transforming growth factor-ß1 is an active and central participant in several vascular processes. TGF-ß1 stimulates the synthesis of proteoglycans by ASMCs.^17,18 Those proteoglycans are components of the extracellular matrix that bind and entrap lipoproteins, leading to lipid accumulation and plaque formation.^4–8 However, the effect of TGF-ß1 treatment on the lipoprotein-binding properties of the proteoglycans produced by ASMCs has not been reported previously. The present study demonstrates that TGF-ß1 stimulates the synthesis of proteoglycans that exhibit an increased LDL-binding affinity over those proteoglycans synthesized by ASMCs in the absence of TGF-ß1. The effect of TGF-ß1 on lipoprotein binding appears to be mediated by the ability of this cytokine to cause elongation of the glycosaminoglycan chains attached to the proteoglycans. By the use of xyloside, it could be demonstrated that TGF-ß1 causes glycosaminoglycan chain elongation in the absence of core protein synthesis, suggesting that the effect of TGF-ß1 on lipoprotein binding is mediated through its effect on the glycosaminoglycan synthetic machinery of the smooth muscle cells. However, the K_d for binding of the glycosaminoglycans to the lipoproteins is lower than the K_d for the intact proteoglycans, suggesting that the core proteins are contributing in some way. The importance of glycosaminoglycan chain length to lipoprotein binding has been observed in other studies. For example, Cardoso and Mourao³² used an LDL affinity column to show a relationship between glycosaminoglycan chain length from atherosclerosis-susceptible regions of human aorta and binding to LDL. Chang et al³³ used similar methodology to show that oxidized LDL treatment of ASMCs results in glycosaminoglycan chain elongation and increased LDL binding. Similarly, Camejo et al³⁴ used a gel mobility shift assay to demonstrate a relationship of larger proteoglycans and more avid binding of LDL to proteoglycans from proliferative versus quiescent ASMCs. Furthermore, proliferating ASMCs showed increased activity of the chain-elongating enzyme, xylosyl transferase, and N-acetylgalactosaminyl transferase,³⁵ suggesting that posttranslational processing of proteoglycans may be a critical determinant in their binding to LDL. Furthermore, Srinivasan et al³⁵ found that proteoglycans isolated from injured arteries contained longer glycosaminoglycan chains than those isolated from control arteries and that these proteoglycans exhibited increased binding to LDL. The increased binding of lipoproteins to proteoglycans is thought to result from an ionic interaction between positively charged moieties on the apolipoprotein and the increased number of negatively charged carboxylate and sulfate residues on the elongated glycosaminoglycan chains of the proteoglycans synthesized in the presence of TGF-ß1. It is also possible that the composition of the glycosaminoglycan chains influenced binding as well. For example, Westergren-Thorsson et al³⁶ found that TGF-ß1 influenced the copolymeric structure of the dermatan sulfate chains synthesized by embryonic skin fibroblasts, whereas Schönherr and colleagues^17,18 found that TGF-ß1 had no effect on the 4- and 6-sulfation of glycosaminoglycan chains synthesized by ASMCs. Chain composition as a determinant of LDL binding requires further exploration.

Elucidating the mechanism by which TGF-ß1 causes an increase in glycosaminoglycan chain length is a potential therapeutic target for intervention in the pathological process of binding and retention of lipoproteins within the vascular wall. These studies support a proatherogenic role for TGF-ß1.

	Acknowledgments

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Funding was received (P.J.L.) from the National Heart, Lung, and Blood Institute and Baker Medical Research Institute Exchange program in vascular disease and remodeling. The study was supported in part by National Institutes of Health grants DK-02456 and HL-30086 (A.C.), HL-18645 (T.N.W.), and DK-07247 (L.T.). We thank Drs Michael Kinsella, Stephen Evanko, and Susan Potter-Perigo for generous advice and discussion; Shari Wang, Mohamed Omer, Thomas Johnson, and Christina Tsoi for expert technical assistance; and Julie Nigro and Ellen Briggs for assistance with the preparation of the manuscript. We also thank Dr P. Hugh R. Barrett, University of Western Australia, for undertaking the data analysis of the proteoglycan-lipoprotein binding experiments.

Received October 4, 2001; accepted October 8, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Haffner SM, d’Agostine R Jr, Mykkanen L, Tracy R, Howard B, Rewers M, Selby J, Savage PJ, Saad MF. Insulin sensitivity in subjects with type 2 diabetes: relationship to cardiovascular risk factors: the Insulin Resistance Atherosclerosis Study. Diabetes Care. 1999; 22: 562–568.[Abstract]
2. Braunwald E. Shattuck lecture: cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med. 1997; 337: 1360–1369.[Free Full Text]
3. Wei M, Haffner SM, Gaskill SP, Stern MP. Effects of diabetes and level of glycemia on all-cause and cardiovascular mortality. Diabetes Care. 1998; 21: 1167–1172.[Abstract]
4. Radhakrishnamurthy B, Srinivasan SR, Vijayagopal P, Berenson GS. Arterial wall proteoglycans: biological properties related to pathogenesis of atherosclerosis. Eur Heart J. 1990; 11 (suppl E): 148–157.[Medline] [Order article via Infotrieve]
5. Williams KJ, Tabas I. The response to retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995; 15: 551–561.[Free Full Text]
6. Hurt-Camejo E, Olsson U, Wiklund O, Bondjers G, Camejo G. Cellular consequences of the association of apoB lipoproteins with proteoglycans: potential contribution to atherogenesis. Arterioscler Thromb Vasc Biol. 1997; 17: 1011–1017.[Free Full Text]
7. Camejo G, Hurt-Camejo E, Wiklund O, Bondjers G. Association of apo B lipoproteins with arterial proteoglycans: pathological significance and molecular basis. Atherosclerosis. 1998; 139: 205–222.[CrossRef][Medline] [Order article via Infotrieve]
8. Chait A, Wight TN. Interaction of native and modified low-density lipoproteins with extracellular matrix. Curr Opin Lipidol. 2000; 11: 457–463.[CrossRef][Medline] [Order article via Infotrieve]
9. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor beta 1 during repair of arterial injury. J Clin Invest. 1991; 88: 904–910.[Medline] [Order article via Infotrieve]
10. Nabel EG, Shum L, Ponpili VJ, Yang ZY, San H, Shu HB, Liptay S, Gold L, Gordon D, Derynck R, et al. Direct gene transfer of transforming growth factor ß1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A. 1993; 90: 10759–10763.[Abstract/Free Full Text]
11. Agrotis A, Saltis J, Bobik A. Transforming growth factor-beta 1 gene activation and growth of smooth muscle from hypertensive rats. Hypertension. 1994; 23: 593–599.[Abstract/Free Full Text]
12. Bobik A, Agrotis A, Kanellakis P, Dilley R, Krushinsky A, Smirnov V, Tararak E, Condron M, Kostolias G. Distinct patterns of transforming growth factor-beta isoform and receptor expression in human atherosclerotic lesions: colocalization implicates TGF-beta in fibrofatty lesion development. Circulation. 1999; 99: 2883–2891.[Abstract/Free Full Text]
13. Evanko SP, Raines EW, Ross R, Gold LI, Wight TN. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and the proximity of platelet-derived growth factor and transforming growth factor-beta. Am J Pathol. 1998; 152: 533–546.[Abstract]
14. McCaffrey TA. TGF-betas and TGF-beta receptors in atherosclerosis. Cytokine Growth Factor Rev. 2000; 11: 103–114.[CrossRef][Medline] [Order article via Infotrieve]
15. Christen T, Bochaton-Piallat ML, Neuville P, Rensen S, Redard M, van Eys G, Gabbiani G. Cultured porcine coronary artery smooth muscle cells: a new model with advanced differentiation. Circ Res. 1999; 85: 99–107.[Abstract/Free Full Text]
16. Scott L, Kerr A, Haydock D, Merrilees M. Subendothelial proteoglycan synthesis and transforming growth factor beta distribution correlate with susceptibility to atherosclerosis. J Vasc Res. 1997; 34: 365–377.[Medline] [Order article via Infotrieve]
17. Schönherr E, Järveläinen HT, Sandell LJ, Wight TN. Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J Biol Chem. 1991; 266: 17640–17647.[Abstract/Free Full Text]
18. Schönherr E, Järveläinen HT, Kinsella MG, Sandell LJ, Wight TN. Platelet-derived growth factor and transforming growth factor-beta 1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells. Arterioscler Thromb. 1993; 13: 1026–1036.[Abstract/Free Full Text]
19. Lin H, Ignatescu M, Wilson JE, Roberts CR, Horley KJ, Winters GL, Costanzo MR, McManus BM. Prominence of apolipoproteins B, (a) and E in the intimae of coronary arteries in transplanted human hearts: geographic relationship to vessel wall proteoglycans. J Heart Lung Transplant. 1996; 15: 1223–1232.[Medline] [Order article via Infotrieve]
20. O’Brien KD, Olin KL, Alpers CE, Chiu W, Ferguson M, Hudkins K, Wight TN, Chait A. Comparison of apolipoprotein and proteoglycan deposits in human coronary atherosclerotic plaques: colocalization of biglycan with apolipoproteins. Circulation. 1998; 98: 519–527.[Abstract/Free Full Text]
21. Gutierrez P, O’Brien KD, Ferguson M, Nikkari ST, Alpers CE, Wight TN. Differences in the distribution of versican, decorin, and biglycan in atherosclerotic human coronary arteries. Cardiovasc Pathol. 1997; 6: 271–278.[CrossRef]
22. Halpert I, Sires U, Potter-Perigo S, Wight TN, Shapiro DS, Welgus HG, Wickline SA, Parks WC. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localized to areas of versican deposits. Proc Natl Acad Sci U S A. 1996; 93: 9748–9753.[Abstract/Free Full Text]
23. Riessen R, Isner JM, Blessing E, Loushin D, Nikol S, Wight TN. Regional differences in the distribution of the proteoglycans, biglycan, and decorin in the extracellular matrix of atherosclerotic and restenotic human coronary arteries. Am J Pathol. 1994; 144: 962–974.[Abstract]
24. Stout RW, Bierman EL, Ross R. Effect of insulin on the proliferation of cultured primate arterial smooth muscle cells. Circ Res. 1975; 36: 319–327.[Abstract/Free Full Text]
25. Potter-Perigo S, Braun KR, Schönherr E, Wight TN. Altered proteoglycan synthesis via the false acceptor pathway can be dissociated from beta-D-xyloside inhibition of proliferation. Arch Biochem Biophys. 1992; 297: 101–109.[CrossRef][Medline] [Order article via Infotrieve]
26. Wasteson A, Uthne K, Westermark B. A novel assay for the biosynthesis of sulfated polysaccharide and its application to studies on the effects of somatomedin on cultured cells. Biochem J. 1973; 136: 1069–1074.[Medline] [Order article via Infotrieve]
27. Yanagishita M, Midura RJ, Hascall VC. Proteoglycans: isolation and purification from tissue cultures. Methods Enzymol. 1987; 138: 279–289.[Medline] [Order article via Infotrieve]
28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227: 680–685.[CrossRef][Medline] [Order article via Infotrieve]
29. Wasteson A. A method for the determination of the molecular weight and molecular-weight distribution of chondroitin sulfate. J Chromatogr. 1971; 59: 87–97.[CrossRef][Medline] [Order article via Infotrieve]
30. Heinecke JW, Baker L, Rosen H, Chait A. Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells. J Clin Invest. 1986; 77: 757–761.[Medline] [Order article via Infotrieve]
31. Hurt-Camejo E, Camejo G, Sartipy P. Measurements of proteoglycan-lipoprotein interaction by gel mobility shift assay. Methods Mol Biol. 1998; 110: 267–279.[Medline] [Order article via Infotrieve]
32. Cardoso LE, Mourao PA. Glycosaminoglycan fractions from human arteries presenting diverse susceptibilities to atherosclerosis have different binding affinities to plasma LDL. Arterioscler Thromb. 1994; 14: 115–124.[Abstract/Free Full Text]
33. Chang MY, Potter-Perigo S, Tsoi C, Chait A, Wight TN. Oxidized low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties. J Biol Chem. 2000; 275: 4766–4773.[Abstract/Free Full Text]
34. Camejo G, Fager G, Rosengren B, Hurt-Camejo E, Bondjers G. Binding of low density lipoproteins by proteoglycans synthesized by proliferating and quiescent human arterial smooth muscle cells. J Biol Chem. 1993; 268: 14131–14137.[Abstract/Free Full Text]
35. Srinivasan SR, Xy JH, Vijayagopal P, Radhakrishnamurthy B, Berenson GS. Injury to the arterial wall of rabbits produces proteoglycan variants with enhanced low-density lipoprotein-binding property. Biochim Biophys Acta. 1993; 1168: 158–166.[Medline] [Order article via Infotrieve]
36. Westergren-Thorsson G, Schmidtchen A, Sarnstrand B, Fransson LA, Malmström A. Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts. Eur J Biochem. 1992; 205: 277–286.[Medline] [Order article via Infotrieve]

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