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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1414-1420.)
© 1997 American Heart Association, Inc.

Articles

Lipoprotein Lipase Can Function as a Monocyte Adhesion Protein

Joseph C. Obunike; Swarnalatha Paka; Sivaram Pillarisetti; ; Ira J. Goldberg

From the Division of Preventive Medicine and Nutrition, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY.

Correspondence to Joseph C. Obunike, PhD, Division of Preventive Medicine, BB 906, Department of Medicine, Columbia University, 630 W 168th St, New York, NY 10032.

	Abstract

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Abstract Lipoprotein lipase (LPL) is made by several cell types, including macrophages within the atherosclerotic lesion. LPL, a dimer of identical subunits, has high affinity for heparin and cell surface heparan sulfate proteoglycans (HSPGs). Several studies have shown that cell surface HSPGs can mediate cell binding to adhesion proteins. Here, we tested whether LPL, by virtue of its HSPG binding, could mediate monocyte adhesion to surfaces. Monocyte binding to LPL-coated (1-25 µg/mL) tissue culture plates was 1.4- to 7-fold higher than that of albumin-treated plastic. Up to 3-fold more monocytes bound to the subendothelial matrix that had been pretreated with LPL. LPL also doubled the number of monocytes that bound to endothelial cells (ECs). Heparinase and heparitinase treatment of monocytes or incubation of monocytes with heparin decreased monocyte binding to LPL. Heparinase/heparitinase treatment of the matrix also abolished the LPL-mediated increase in monocyte binding. These results suggest that LPL dimers mediate monocyte binding by forming a "bridge" between matrix and monocyte surface HSPGs. Inhibition of LPL activity with tetrahydrolipstatin, a lipase active-site inhibitor, did not affect the LPL-mediated monocyte binding. To assess whether specific oligosaccharide sequences in HSPGs mediated monocyte binding to LPL, competition experiments were performed by using known HSPG binding proteins. Neither antithrombin nor thrombin inhibited monocyte binding to LPL. Next, we tested whether integrins were involved in monocyte binding to LPL. Surprisingly, monocyte binding to LPL-coated plastic and matrix was inhibited by 35% via integrin-binding arginine-glycine–aspartic acid peptides. This result suggests that monocyte binding to LPL was mediated, in part, by monocyte cell surface integrins. In summary, our data show that LPL, which is present on ECs and in the subendothelial matrix, can augment monocyte adherence. This increase in monocyte-matrix interaction could promote macrophage accumulation within arteries.

Key Words: atherosclerosis • heparin • proteoglycans • integrins • artery

	Introduction

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Although the primary function of LPL is to hydrolyze the triglycerides in chylomicrons and VLDLs,^{1 2} recent results indicate that LPL may also have structural roles.³ LPL increases lipoprotein binding to the extracellular matrix and lipoprotein binding and uptake by cells, functions that do not appear to require catalytic activity.^{4 5 6 7 8 9 10} Because LPL has domains that interact with lipid and cell surface molecules, it has been postulated that LPL forms a "bridge" between lipoprotein lipid and cell surfaces.⁴ More recent results suggest that in addition to binding to lipid, LPL also interacts with the amino-terminal region of apoB, thereby facilitating binding and uptake of apoB-containing lipoproteins.^{11 12}

In vivo, LPL is thought to exist as a dimer of identical subunits.¹³ In models of LPL, the two subunits are aligned in a head-to-tail arrangement so that the carboxyl terminus of one subunit is near the amino terminus of the second subunit.^{2 14} The carboxyl-terminal region of LPL is considered a major determinant of LPL binding to cells. This domain is involved in LPL binding to the LRP.^{15 16} The ability of LPL to form a bridge between lipoproteins and the LRP appears to require that LPL be a dimer. This may be so because both lipid- and LRP-binding domains are in the carboxyl-terminal region of LPL. The carboxyl-terminal region of LPL also contains domains that bind to heparin,¹⁷ a property that mediates high-affinity LPL binding to cell surfaces and extracellular HSPGs.^{18 19 20 21} Specific oligosaccharide sequences in HSPGs and heparin then bind to LPL.^{22 23}

Cell surface HSPGs play a role in cell binding to extracellular matrix adhesion proteins.^{24 25 26} This action is due to the presence of heparin-binding domains in several adhesion proteins (eg, fibronectin, collagen, and laminin) that interact with cell surface HSPGs.²⁷ Most cell binding to these proteins, however, is mediated by cell surface integrins.²⁸ Monocyte binding to adhesion proteins on the endothelium and subendothelial matrix, an early event in atherosclerosis,²⁹ is mediated in part by specific integrins.

LPL is present on the endothelium and in the matrix, where it can interact with monocytes. LPL, in addition, is synthesized by monocytes/macrophages and is present on their surfaces. The possible role of LPL as an adhesion protein, however, has never been explored. LPL is ideally suited to perform this function, because it can bind to both cell surface and matrix HSPGs. Data from the study presented herein show that LPL can increase monocyte binding to ECs and matrix.

	Methods

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LPL
LPL was purified from unpasteurized bovine milk according to the method of Socorro et al³⁰ with modifications as described by Saxena et al.³¹ The average yield was 2.5 mg/L of milk. Purified enzyme (400 to 600 µg/mL) was stored in 1.5 mol/L NaCl buffer at -70°C. The purified LPL was found to be a dimer (molecular mass of 110 000 kDa) on the basis of its affinity for heparin (elution at 1.5 mol/L NaCl) and its elution from a Sephacryl S-200 gel filtration column. Enzyme activity was assayed with a glycerol-containing triolein emulsion as described previously.³¹ The purified enzyme had a specific activity of 40 to 50 mmol oleic acid released per hour per milligram of enzyme at 37°C.

For some experiments, purified LPL was radioiodinated enzymatically with glucose oxidase and lactoperoxidase as described previously.³² Radioiodinated LPL was repurified by heparin-agarose (Bio-Rad) affinity chromatography and stored at -70°C. Typical specific activity of the preparation was 1000 counts per minute per nanogram, and >90% of the radioactivity was precipitated with trichloroacetic acid. ¹²⁵I-LPL was purified by Sephadex G-50 gel filtration (PD-10, Pharmacia) prior to use to remove degradation products.

Monocytes
THP-1 cells were purchased from the American Type Culture Collection (Rockville, Md) and grown in RPMI 1640 (Life Technologies) supplemented with 1% (vol/vol) glutamine, 1% (vol/vol) penicillin and streptomycin, and 10% (vol/vol) fetal bovine serum (Gemini Bioproducts Inc). The cells in suspension were maintained at 37°C in an atmosphere of 5% CO₂ in air, and the medium was changed every 3 days.

ECs
Bovine aortic ECs were isolated and cultured as described.³² The cells (5 to 15 passages) were grown in DMEM containing 10% fetal bovine serum (Life Technologies).

Subendothelial Matrix
Confluent EC monolayers were grown in 24- or 48-well culture dishes (Falcon, Becton Dickinson). The matrix was prepared as described previously.³³ In brief, EC monolayers were washed three times with PBS and incubated for 5 minutes in a solution containing 20 mmol/L NH₄OH and 0.1% Triton X-100 at room temperature. Detached cells were removed by three washes in PBS followed by three washes in DMEM containing 3% BSA.

LPL Treatment
The 24- or 48-well plates were incubated with different concentrations of LPL in borate buffer containing 1% BSA for 18 to 20 hours at 37°C. Unbound LPL was removed and wells were washed three times with BSA before the monocytes were added. This protocol was used because the albumin stabilized LPL activity but did not appreciably interfere with LPL adherence to the plate. ECs or matrix was incubated with LPL in BSA for 2 hours at 37°C. Unbound LPL was removed and monocyte binding was assessed. In some experiments, the matrix was first treated with 1 U/mL each of heparinase and heparitinase (Sekagaku America Inc) before LPL binding.

For inhibition of LPL activity, THL was used (La Roche). THL inhibits LPL activity by >50% at the concentrations used.^{34 35} For the inhibition experiments, either LPL binding to matrix was carried out in the presence of THL or LPL was first bound to the matrix and then incubated with THL before the monocytes were added.

Monocyte Binding
Monocytes were incubated with Leu-deficient medium for 30 minutes before labeling. Approximately 100 µCi of [³H]Leu was added to 1x10⁷ cells and incubated for another 2 hours under cell culture conditions. Labeled cells were centrifuged at 800 rpm for 5 minutes to remove the unincorporated label. The cells were then washed four times with BSA and suspended in BSA. Suspended cells were then added to either monolayers of ECs or matrix in 24-well plates (2 to 4x10⁵ cells per well). Cells were allowed to bind for 1 hour at 37°C. The spontaneous release of radioactivity under these conditions was 5%. Unbound monocytes were removed by four washes with BSA, and bound radioactivity was extracted by incubation in 0.1N NaOH containing 0.1% SDS for 30 minutes at 37°C. Cells were also counted (Spotlight hemacytometer, American Scientific Products) in some experiments after trypsinization to determine whether increased radioactivity reflected an increase in the number of bound cells. In other experiments, monocytes were labeled with ⁵¹Cr and binding was assessed; the results (not shown) were similar to those obtained with [³H]Leu-labeled monocytes. All results described in this article are those that were obtained with [³H]Leu-labeled monocytes.

Albumin-coated plates bound 40 000 to 50 000 cells per well, and untreated matrix bound 100 000 to 125 000 cells per well (24-well plates). For enzyme treatments, labeled monocytes were incubated with heparinase and heparitinase (1 U/mL each) for 1 hour at 37°C. Cells were washed and binding to LPL-coated plates was assessed. For RGD inhibition experiments, labeled monocytes were incubated with 50 or 100 µmol/L RGD for 30 minutes at 4°C before they were added to the LPL-coated plates. For other competition experiments, labeled monocytes were mixed with the competing ligand (thrombin, antithrombin, or LPL, 50 µg/mL) before they were added to the LPL-coated plates.

	Results

Top Abstract Introduction Methods Results Discussion References

 
LPL Increases Monocyte Binding to Surfaces
We first examined whether LPL increased monocyte binding to plastic culture dishes. The 24-well plates were incubated with albumin or LPL-containing albumin for 16 hours at room temperature. Unbound protein was removed, and coated plates were incubated with labeled THP-1 monocytes for 1 hour at 37°C. Fig 1 shows that when plates were coated with 10 µg/mL LPL, 2.2-fold more monocytes were bound to the LPL-treated than the control plates. We next examined whether LPL would increase monocyte binding to the subendothelial matrix. Compared with untreated matrix, LPL (10 µg/mL) -treated matrix bound 2-fold more monocytes (Fig 1). This increased binding to LPL was completely abolished in the presence of a rabbit anti-bovine LPL antibody (not shown). These data suggest that LPL can function as a monocyte adhesion protein.

View larger version (64K): [in this window] [in a new window]   Figure 1. LPL increases monocyte binding to plastic and matrix. Plates (24 wells) were incubated with 10 µg/mL LPL in albumin-containing buffer for 18 hours at 37°C. Subendothelial matrix was prepared by incubating confluent monolayers of ECs in PBS containing 0.1% Triton X-100 and 20 mmol/L NH4OH. Matrix was then incubated with 10 µg/mL LPL for 2 hours at 37°C. LPL-coated plastic (P+L) and matrix (M+L) were incubated with labeled monocytes for 1 hour, and binding was assessed. Binding to plastic (P) and matrix (M) was increased in the presence of LPL. Values represent mean of triplicate experiments±SD. In controls, 40 000 to 50 000 monocytes bound per well to plastic and 100 000 to 125 000 cells per well to matrix.

Fig 2 shows how different concentrations of LPL affect monocyte binding to plastic, matrix, and ECs. When plates were coated with 1 to 25 µg/mL LPL, the number of bound monocytes increased from 1.4- to 7.8-fold (Fig 2A). Similarly, monocyte binding to LPL-coated matrix also increased in a dose-dependent fashion. With 10 µg/mL LPL, 170% more monocytes adhered to the matrix (Fig 2B). Monocyte binding to ECs was also increased by LPL (up to 200%).

View larger version (11K): [in this window] [in a new window]   Figure 2. Monocyte binding to plastic, matrix, and ECs (LPL dose curve). A, For monocyte binding to LPL-plastic, 24-well plates were incubated with indicated concentrations of LPL in albumin-containing buffer, and monocyte binding was assessed. Control represents monocyte binding to untreated plastic (no LPL). B, For monocyte binding to LPL-treated ECs and subendothelial matrix, confluent monolayers of ECs or subendothelial matrix in 24-well plates were incubated with indicated concentrations of LPL. Labeled monocytes were then added, and binding was performed at 37°C for 1 hour. Control represents monocyte binding to untreated matrix (no LPL). Values represent mean of triplicate experiments±SD.

Heparinase Treatment of Monocytes Prevents Monocyte Binding to LPL
We next examined whether monocyte adhesion to LPL was mediated by monocyte surface HSPGs. Labeled monocytes were incubated with 1 U/mL each of heparinase and heparitinase for 1 hour at 37°C. Cells were washed and added to LPL-coated plastic or matrix (Fig 3), and monocyte binding experiments were carried out at 4°C or 37°C. Experiments were performed at 4°C to minimize the possibility of de novo HSPG synthesis during the course of binding. LPL in this experiment increased monocyte binding by 195%, and approximately twice as many cells were bound to the LPL-coated plates at 37°C than at 4°C. Heparinase treatment of monocytes reduced the number of monocytes bound to LPL by 49±3% at 4°C and by 58±6% at 37°C (Fig 3A). Heparinase treatment similarly inhibited monocyte binding to LPL-coated matrix (Fig 3B). These results suggest that much of the LPL-mediated increase in monocyte binding was due to interactions with monocyte surface HSPGs.

View larger version (16K): [in this window] [in a new window]   Figure 3. Heparinase and heparitinase (H/H) treatment of monocytes decreases binding to LPL. A, For monocyte binding to LPL-coated plastic, labeled monocytes were incubated with 1 U/mL each of heparinase and heparitinase for 1 hour at 37°C, after which the cells were added to 24-well plates coated with albumin (with or without LPL [10 µg/mL]) and then incubated at either 4°C or 37°C for 1 hour. Binding of untreated (UT) and heparinase/heparitinase–treated (H/H) monocytes was assessed. B, For monocyte binding to LPL-coated matrix, untreated (UT) and H/H-treated radiolabeled monocytes were allowed to bind to LPL-coated subendothelial matrix for 1 hour at 37°C, and the number of monocytes bound was determined. Values represent mean±SD.

Heparin Treatment and Removal of Matrix HSPGs Inhibit Monocyte Binding to LPL
Because we hypothesized that LPL forms a bridge between cell surface HSPGs and matrix HSPGs, we next tested whether the removal of matrix HSPGs or the inclusion of heparin, which prevents LPL association with HSPGs, inhibited monocyte binding. LPL was allowed to bind for 1 hour at 37°C to untreated or heparinase/heparitinase–treated matrix. Labeled monocytes were then added, and binding was carried out for 1 hour (Fig 4). Heparinase treatment of control matrix increased monocyte binding to 126% of control values. This finding is in agreement with our previous observations,³³ which suggest that removal of HSPGs exposes other monocyte-binding proteins in the matrix (eg, fibronectin and collagen). LPL treatment of the matrix, as expected, increased monocyte binding: the number of cells bound to LPL-treated matrix was 180% of control in this experiment. LPL, however, did not increase the number of cells adhering to the heparinase-treated matrix. Addition of heparin (100 U/mL) inhibited monocyte binding to the LPL-treated matrix by 86±7%; approximately the same number of cells were bound to control and LPL-treated matrix in the presence of heparin. These results suggest that LPL association with matrix HSPGs is required for LPL to augment monocyte binding to the matrix.

View larger version (39K): [in this window] [in a new window]   Figure 4. Heparin treatment and removal of matrix HSPGs inhibit monocyte binding. Subendothelial matrix was treated with or without heparinase/heparitinase (H/H, 1 U/mL), after which LPL (10 µg/mL) was allowed to bind to untreated or H/H-treated matrix for 1 hour at 37°C. Labeled monocytes were then added, and binding was assessed at 37°C for 1 hour. Monocyte binding to LPL-coated matrix was also determined in the presence of heparin (100 U/mL, Matrix+LPL+H). Values represent mean of triplicate experiments±SD.

Effects of Other HSPG-Binding Proteins on Monocyte Binding to LPL
To further elucidate the role of monocyte HSPGs in binding to LPL, we tested whether other known HSPG-binding proteins competed for monocyte HSPGs and inhibited LPL-mediated binding. Labeled monocytes were mixed with the indicated concentrations of antithrombin, thrombin, and LPL and added to the LPL-coated matrix (Fig 5). LPL in solution, but not dissolved thrombin or antithrombin, inhibited the number of monocytes that bound to the LPL matrix. This result suggests that specific oligosaccharide sequences in monocyte surface HSPGs mediate binding to LPL.

View larger version (14K): [in this window] [in a new window]   Figure 5. Effects of HSPG binding proteins on monocyte binding to LPL. Labeled monocytes were mixed with antithrombin (AT, 50 µg/mL), thrombin (Th, 50 µg/mL), or LPL (50 µg/mL) before they were added to the LPL (10 µg/mL) -coated matrix. Monocyte binding (1 hour at 37°C) was assessed. Values represent mean of triplicate experiments±SD.

Lipase Activity Is Not Required for Monocyte Binding to LPL
We next tested whether the triglyceride hydrolase activity of LPL was required for LPL-mediated monocyte binding. THL has previously been used to inhibit LPL activity,^{34 35} and in our preliminary experiments, THL inhibited LPL activity by >70% at the concentrations used (not shown). Plates or matrix was coated with LPL in the presence or absence of the indicated concentrations of THL. In another experiment, we first coated the plastic or matrix with LPL and then incubated them with THL for 1 hour at 37°C before the monocytes were added. Although THL inhibited LPL activity, it did not affect the amount of LPL (as assessed by binding of ¹²⁵I-LPL) associated with the plastic or matrix (not shown). As shown in Fig 6, THL inhibited monocyte binding by <5%, suggesting that LPL activity is not necessary for monocyte binding.

View larger version (13K): [in this window] [in a new window]   Figure 6. Effects of THL, a lipase active-site inhibitor, on monocyte binding to LPL. Plates (24 wells) were coated with LPL (10 µg/mL) in the presence or absence of THL for 18 hours. In another experiment, LPL was first coated for 18 hours and then incubated with indicated concentrations of THL (THL post) for 2 hours at 37°C. Labeled monocytes were allowed to bind at 37°C for 1 hour. Control represents monocyte binding to untreated plastic (no LPL). LPL increased monocyte binding by 3-fold, and this binding was not affected by inhibition of lipase activity.

Effects of RGD-Containing Peptides on Monocyte Binding to LPL
Monocytes bind via their surface integrins to subendothelial adhesion proteins, such as collagen and fibronectin, that contain integrin-binding RGD sequences. We tested whether monocyte binding to LPL involved cell surface integrins. We used fibronectin as a control because monocyte binding to fibronectin is mediated by specific cell surface integrins and is inhibited by RGD peptides. Labeled monocytes were incubated in 50 µmol/L GRGDTP for 30 minutes at 4°C before they were added to LPL-coated plastic or matrix. RGD peptide inhibited monocyte binding to fibronectin by 58%. Surprisingly, RGD also inhibited monocyte binding to LPL-coated plastic by 39% and to LPL-coated matrix by 34%. These results suggest that part of the monocyte binding to LPL is mediated by monocyte surface integrins. We next tested whether removal of HSPGs and addition of RGD would further inhibit monocyte binding to LPL. Monocytes were first treated with heparinase and heparitinase for 1 hour at 37°C. Untreated and heparinase-treated monocytes were then incubated in the presence or absence of RGD as described above and then added to LPL-coated plates (Fig 7C). Heparinase treatment in this experiment decreased monocyte binding to LPL by 52%. Although RGD inhibited monocyte binding to LPL by 38%, it did not further increase the inhibition caused by heparinase treatment alone. This finding suggests that RGD effects require monocyte surface HSPGs.

View larger version (17K): [in this window] [in a new window]   Figure 7. A, Effects of RGD-containing peptides on monocyte binding to LPL-coated plastic. Plates (24 wells) were coated with 10 µg/mL LPL or 10 µg/mL fibronectin (FN) for 16 hours. Labeled monocytes were incubated with or without a peptide containing an RGD sequence (GRGDTP, 50 µmol/L) for 30 minutes at 4°C before they were added to the LPL- or FN-coated plastic. Binding was performed at 37°C for 1 hour. Control represents BSA-treated plastic. RGD peptide inhibited monocyte binding to FN, an integrin-binding protein, by 65%. In addition, RGD also inhibited monocyte binding to LPL-coated plastic by 39%. B, Effect of RGD on monocyte binding to LPL-treated matrix. Subendothelial matrix (M) was incubated with 10 µg/mL LPL in DMEM-BSA for 2 hours. Labeled monocytes were treated with RGD peptides as described in panel A and added to control or LPL-treated matrix. LPL in this experiment increased monocyte binding by 70% (M+LPL). RGD inhibited monocyte binding to matrix by 41% (M+RGD) and to LPL-matrix by 35% (M+LPL+RGD). C, Effects of RGD and heparinase/heparitinase (H/H) treatments on monocyte binding to LPL-treated plastic. Labeled monocytes were incubated in medium with or without H/H (1 U/mL each) for 1 hour at 37°C. Control and H/H-treated cells were then cooled, incubated in medium containing no or 50 µmol/L RGD peptides, and incubated further at 4°C for 30 minutes. The cells were then washed and added to control or LPL-coated wells, and binding was performed at 37°C for 1 hour. Values represent mean of triplicate experiments±SEM. H/H treatment of monocytes decreased binding to LPL by 52%, and this decrease was not affected further by addition of RGD peptides.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
LPL is synthesized primarily by cells on the abluminal (basolateral) side of the endothelium (eg, adipocytes and myocytes).^{2 3} Its main site of action, however, is the luminal side of the endothelium. Thus, LPL travels from its site of synthesis to the endothelium through the interstitial matrix. Immunohistochemical studies have identified LPL both on the luminal side of the endothelium and in the subendothelial (extracellular) matrix.^{36 37} LPL is also synthesized by macrophages in atherosclerotic lesions.^{38 39} Thus, LPL is present where it can interact with blood monocytes. The data presented in this article demonstrate for the first time that LPL can function as a monocyte-binding protein.

Monocyte binding to the endothelium and movement into the subendothelial space are early atherosclerotic events that have been noted in animals fed a high-fat, high-cholesterol diet.^{29 40 41} Although several steps in the process of monocyte interaction with ECs have been elucidated,^{40 41} most experiments designed to illustrate these processes were performed with cultured ECs, which do not contain cell surface LPL. This contrasts with the situation in vivo. Our data in Figs 1 and 2 show that LPL increases monocyte binding to both ECs and the matrix in a dose-dependent fashion. More (2- to 3-fold) monocytes were bound to LPL-coated cells and matrix than to untreated controls. This binding was inhibited by an LPL-specific antibody. The LPL effect was even more pronounced when non–matrix-containing tissue culture dishes were used. This event may have occurred because LPL binding to cells and matrix is limited by the amount of HSPGs present.

A major part of monocyte binding to LPL is mediated by monocyte surface HSPGs. Heparinase/heparitinase treatment of monocytes decreased binding to LPL by 59%. Previous studies have shown that cell surface HSPGs mediate cell binding to other adhesion proteins, fibronectin, and laminin.^{24 25 26} Saunders and Bernfield²⁵ showed that mammary epithelial cells have at least two distinct cell surface receptors for fibronectin. These cells have a trypsin-resistant molecule that binds to RGD sequences and a trypsin-labile HSPG that binds to the carboxyl-terminal heparin-binding domain of fibronectin. In addition to the matrix adhesion proteins, cell surface adhesion proteins also contain heparin and heparan sulfate–binding domains.⁴² These include L-selectin, P-selectin, and PECAM. For PECAM, binding to heparin is stronger than that between heparin and other PECAM molecules.⁴³

Molecules that form a bridge between matrix and cell surface molecules are ideally suited to act as cell adhesion proteins. Recently it has been reported that FGF-2 mediates cell binding to the matrix.⁴⁴ This property of FGF requires the presence of the high-affinity, non-HSPG receptor on the binding cell. A model has been proposed in which FGF forms a bridge between HSPG and the FGF receptor. In contrast, our studies suggest that most LPL-mediated monocyte binding requires both matrix and monocyte HSPGs. Because LPL has domains that exhibit stronger affinity for heparin than do many other heparin-binding proteins, it is not surprising that a major part of monocyte binding to LPL is mediated by cell surface HSPGs. Unlike FGF, which is a monomer, LPL can bridge HSPGs on two surfaces by utilizing each monomer. It should be noted, however, that although heparin treatment of the matrix almost completely abrogated LPL-mediated monocyte adhesion, we found a reduced but continued increase in monocyte binding to LPL-treated plates after elimination of monocyte surface HSPGs. Thus, in addition to HSPGs, LPL appears to interact with other monocyte surface proteins (see below).

Other HSPG-binding proteins did not significantly inhibit monocyte binding to LPL. The inability of thrombin and antithrombin to compete for monocyte binding to LPL was not surprising, because we had previously shown that neither of these ligands competed for ¹²⁵I-LPL binding to cells.⁴⁵ Although the amino acid sequences in heparin that bind to thrombin have not been identified, the oligosaccharide sequence in heparin that binds to antithrombin is quite different from that of LPL.⁴⁶ Thus, a specific set of monocyte surface HSPGs may bind to LPL and mediate monocyte adhesion.

A significant part of monocyte binding to LPL was inhibited by RGD-containing peptides. This result was surprising, because analysis of the bovine LPL sequence^{47 48} did not reveal any RGD sequences. There are, however, sequences that contain Arg-Gly but a different third amino acid. They are amino acids 189-191 (Arg-Gly-Ser) and 229-233 (Arg-Gly-Leu-Gly-Asp). It is possible that these sequences of LPL with the proper conformation may be able to interact with monocyte integrins. An alternative explanation for the RGD inhibitory effect is that RGD interferes with LPL-HSPG interactions. If this were the case, then an additional increase in RGD should eventually block LPL-mediated monocyte adhesion to the same extent as that found after heparin and heparinase treatment of the matrix. However, we found that (1) increasing the RGD concentration to 100 µmol/L (2-fold) did not further decrease the LPL-mediated monocyte binding and (2) RGD at 50 or 100 µmol/L did not affect ¹²⁵I-LPL association with the matrix (not shown). We therefore believe that the most likely explanation for our findings is that RGD blocks integrin-LPL interactions. One other protein that has been shown to mediate cell binding that involves both HSPGs and integrins is heparanase. Gilat et al⁴⁹ recently showed that heparanase can mediate CD4⁺ T-cell binding to matrix HSPGs by involving T-cell surface integrins. Thus, LPL appears to behave, in part, like heparanase in mediating monocyte binding.

We were also surprised that the effects of heparinase and RGD were not additive. One possible explanation for this is that integrin-mediated binding of monocytes to LPL requires an initial interaction between LPL and monocyte HSPGs. Without this initial interaction between monocyte HSPGs and LPL, monocyte integrins may not interact with the LPL "integrin binding sites." A similar situation also exists for monocyte binding to EC surface molecules. Although integrins interact with specific adhesion molecules on the EC surface, an initial interaction with carbohydrate ligands, such as selectins, appears to be necessary for monocyte binding to ECs.⁵⁰ Alternatively, removal of HSPGs may affect monocyte cell surface integrins, or RGD peptides may affect cell surface HSPGs, resulting in the lack of an additive effect.

In conclusion, our studies show that LPL can function as a monocyte adhesion protein. This function does not require LPL enzymatic activity but requires that LPL be present as a dimer. In addition, LPL-mediated monocyte binding to the matrix involves HSPGs in the matrix as well as HSPGs and integrins on the monocyte surface. Because atherosclerotic vessels have increased amounts of LPL, we hypothesize that LPL within the artery can contribute to increased monocyte retention.

	Selected Abbreviations and Acronyms

 

DMEM = Dulbecco's modified Eagle's medium EC = endothelial cell FGF = fibroblast growth factor HSPG = heparan sulfate proteoglycan LPL = lipoprotein lipase LRP = LDL receptor–related protein PECAM = platelet–endothelial cell adhesion molecule RGD = Arg-Gly-Asp THL = tetrahydrolipstatin

	Acknowledgments

 
This work was funded by grants HL-03323 (to J.C.O.) and HL-45095 and HL-21006 SCOR (to I.J.G.) from the National Heart, Lung, and Blood Institute. S.P. is an investigator of the American Heart Association, New York City Affiliate. We wish to thank Dr David Severson for providing us with the THL and Dr Salome Papaspyro-Rao for providing us with bovine milk.

Received April 25, 1996; accepted September 26, 1996.

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