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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1552-1558

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1552-1558.)
© 1996 American Heart Association, Inc.

Articles

Induction of Hepatic Uptake of Lipoprotein(a) by Cholesterol-Derivatized Cluster Galactosides

Erik A.L. Biessen; Helene Vietsch; Theo J.C. van Berkel

the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.

Correspondence to Dr E.A.L. Biessen, Division of Biopharmaceutics, Sylvius Laboratories, University of Leiden, PO Box 9503, 2300 RA Leiden, Netherlands.


*    Abstract
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 *Abstract
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 down arrowResults
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We have previously developed triantennary galactosides [TG(4Å)C and TG(20Å)C] that lower cholesterol levels by inducing liver uptake of lipoproteins via galactose-recognizing hepatic receptors. In this study, we have investigated whether this strategy could also be applied to reduce elevated serum levels of the atherogenic lipoprotein(a) [Lp(a)]. Both TG(4Å)C and TG(20Å)C could be incorporated into Lp(a). Incorporation of these glycolipids induced a rapid clearance of Lp(a). Concomitantly, the hepatic uptake of 125I-Lp(a) was enhanced from 4±1% to 80±4% of the injected dose for TG(4Å)C (P<.0001) and to 17±4% of the injected dose for TG(20Å)C (P<.006). TG(4Å)C was apparently more effective in accelerating the serum decay of 125I-Lp(a), which may be caused by the higher hydrophobicity of this glycolipid relative to TG(20Å)C. The TG(4Å)C- and TG(20Å)C-induced stimulation of the serum decay and liver uptake of 125I-Lp(a) could be significantly inhibited (>85%) by preinjection of N-acetyl-galactosamine (150 mg), indicating that galactose-recognizing receptors are involved in the liver uptake of the glycolipid/Lp(a) complexes. The TG(4Å)C-induced liver uptake of 125I-Lp(a) could be ascribed mainly to Kupffer cells (76±7%), whereas the parenchymal liver cell was the major site for liver uptake of TG(20Å)C-laden 125I-Lp(a) (55±12%). In conclusion, both TG(4Å)C and TG(20Å)C stimulate the catabolism of 125I-Lp(a) by enhancing hepatic uptake. Because endocytosis of the substrate via galactose-recognizing receptors on Kupffer and parenchymal liver cells is followed by lysosomal degradation, we anticipate that both approaches for Lp(a) targeting may prove valuable as therapeutic modalities for lowering atherogenic levels of Lp(a).


Key Words: apolipoprotein(a) • glycolipids • atherosclerosis • lipoproteins • hypercholesterolemia


*    Introduction
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 *Introduction
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Epidemiological studies have established that elevated serum levels of Lp(a) are an independent risk factor for coronary heart disease,1 2 premature myocardial infarction,3 and ischemic cerebrovascular disease.4 In addition, elevated levels of Lp(a) impart a risk of stenosis in vein grafts after coronary artery bypass surgery.5 The involvement of Lp(a) in atherogenesis and restenosis was further substantiated by a number of in vitro and animal studies (for a review, see Reference 6). Cushing et al7 and Rath et al8 have demonstrated Lp(a) to be present in the neointima of human vein grafts. Moreover, it has been reported that Lp(a) interferes with the thrombolytic system9 and stimulates proliferation of vascular smooth muscle cells.10 A direct correlation between Lp(a) and atherogenesis was recently found by Lawn et al,11 who showed that transgenic mice expressing apolipoprotein(a) are more susceptible to atherosclerosis than control mice.

Although our understanding of the putative role of Lp(a) in atherogenesis is rapidly growing, only marginal progress has been attained in the development of Lp(a)-lowering therapies. Clinical studies revealed that application of hypocholesterolemic agents that increase the catabolism of LDL by upregulating hepatic LDL receptors,12 such as ß-hydroxy-ß-methylglutaryl-CoA reductase inhibitors,13 14 15 cholestyramine,16 and fibrates,14 15 16 does not influence the Lp(a) level. Moreover, a low-cholesterol/low-fat diet does not reduce high Lp(a) levels.15 Only LDL apheresis15 and combined treatment with niacin and neomycin17 are effective in reducing high blood levels of Lp(a).

A promising approach to reduce high serum levels of Lp(a) may be the removal of Lp(a) via galactose-recognizing hepatic receptors.18 19 20 This approach has been elaborated on by our department21 22 23 24 25 26 27 and by others28 in the past. Cholesterylated triantennary cluster galactosides have been synthesized containing a lipophilic moiety to enable incorporation of the glycoside into lipoproteins and a terminal galactose residue to provide affinity for galactose-recognizing hepatic receptors.20 25 26 These glycolipids, TG(4Å)C and TG(20Å)C, are able to efficiently target lipoproteins to the liver. Furthermore, they exert a marked hypocholesterolemic activity in rats, establishing that it is possible to lower serum cholesterol levels by enhancing hepatic uptake via nonlipoprotein receptors.22 27

The above results clearly illustrate that the aforementioned strategy for lowering serum LDL levels is effective. Because Lp(a) closely resembles LDL in terms of lipid and apolipoprotein composition, a similar approach may also prove valid to enhance the catabolism of Lp(a). In the present study, we have therefore evaluated whether cholesterol-derivatized cluster galactosides interact with Lp(a) and whether this leads to an enhanced catabolism of the lipoprotein in rats.


*    Methods
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 *Methods
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Chemicals
Heparin sepharose, Superose 6 prep grade, and Sephadex G-50 were purchased from Pharmacia LKB, Biotechnology AB. Trypan Blue, collagenase (type IV), bovine serum albumin (fraction V), and N-acetyl-ß-D-galactosamine were purchased from Sigma Chemical Co. Kallikrein (Trasylol) was obtained from Bayer, and sodium pentobarbital (Nembutal) was purchased from Ceva. Na125I (carrier free) was obtained from Amersham International. TG(4Å)C21 and TG(20Å)C25 were synthesized as described previously (for structure, see Fig 1Down). All other chemicals were of analytical grade.



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  		Figure 1. Chemical structures of the cholesterol-derivatized galactosides TG(4Å)C and TG(20Å)C.

Isolation of Lp(a) From Human Serum
For isolation of human Lp(a), blood was collected from healthy individuals into sterile EDTA K3 evacuated tubes (Becton Dickinson Vacutainer Systems Europe), and kallikrein (100 international units per milliliter), proline (1 mmol/L), and PMSF (10 µmol/L) were added. Lp(a) (B isotype) was isolated from the serum of a single donor by density gradient ultracentrifugation in KBr/NaCl/HEPES/EDTA buffers, according to Redgrave et al, at 10°C.29 Lp(a) was recovered in the density area between LDL and HDL (1050<d<1120 kg·m-3). Further purification was established by size-exclusion chromatography over a Superose 6 column (volume: 100 mL) using NaPi buffer (0.1 mol/L, pH 7.4) containing 0.5 mol/L NaCl, 2 mmol/L EDTA, 10 µmol/L PMSF, and 1 mmol/L proline as eluent. Lp(a)-containing fractions were pooled and thoroughly dialyzed at 4°C under a N2 atmosphere. The purity and isotype of the Lp(a) pool was estimated by PAGE (5% acrylamide) under nonreducing conditions and by agarose gel electrophoresis (relative electrophoretic mobility 0.28±0.01 versus 0.19 for LDL).30 31 An additional purification step was performed in case the isolated Lp(a) was not completely free of LDL. Residual LDL was removed by affinity chromatography, with the use of heparin sepharose,32 by a NaCl gradient of 100 to 300 mmol/L in sodium barbital buffer (6 mmol/L, pH 7.4) that contained 10 µmol/L PMSF, 0.01% EDTA, and 0.01% NaN3. The particle size of the purified Lp(a) was measured by photon-correlation spectroscopy with the use of a submicron particle analyzer and was 26.8±1.8 nm (n=4).33 Purified Lp(a) was stored at 4°C under N2 and used within 4 weeks after isolation.

Radiolabeling of Lipoproteins
Lp(a) was radiolabeled with carrier-free 125I by the iodine monochloride method of McFarlane as modified by Bilheimer et al.34

Interaction of TG(4Å)C and TG(20Å)C With Lp(a)
TG(4Å)C or TG(20Å)C, dissolved in PBS (10 mmol/L NaPi, pH 7.4, 150 mmol/L NaCl, and 1 mmol/L EDTA) at a concentration of 10 and 11.2 mg/mL, was mixed with (radiolabeled) 125I-Lp(a) at a ratio of 1 mg glycolipid per milligram of apolipoprotein. After 30 minutes at room temperature, the mixtures were put on ice until use. The incubation mixtures were subjected to gradient density ultracentrifugation in KBr/NaCl buffer (1006<d<1220 kg·m-3; 150 000g; 22 hours). After the ultracentrifugation step, the gradient was subdivided into 0.3-mL fractions, and the fractions were assayed for total cholesterol and ß-D-galactose content as described below. The recovery of cholesterol amounted to 81%, 82%, and 87%, respectively, for Lp(a), Lp(a)/TG(4Å)C, and Lp(a)/TG(20Å)C, while the galactose recovery varied between 84% and 111% for the various samples.

In Vivo Serum Clearance and Liver Association
Male Wistar rats weighing {approx}250 to 300 g were anesthetized by intraperitoneal injection of 15 to 20 mg sodium pentobarbital. The abdomen was opened, and complexes of 125I-Lp(a) (50 µg apolipoprotein in 500 µL PBS) and TG(4Å)C or TG(20Å)C (prepared as described above) were injected into the inferior vena cava. At the indicated times, blood samples of 0.2 to 0.3 mL were taken from the inferior vena cava. The samples were centrifuged for 2 minutes at 16 000g, and the serum was counted for radioactivity. The total amount of radioactivity in serum was calculated by use of the equation: Serum Volume (mL)=[0.0219xBody Weight (g)]+2.66.35 At the indicated times, liver lobules were tied off, excised, weighed, and counted for radioactivity. The excised liver tissue amounted to <15% of the total liver mass. The liver uptake of the injected compound was corrected for the radioactivity in serum assumed to be entrapped in the tissue at the time of sampling (85 µL/g fresh weight).36

Isolation of Liver Cells
Rats were anesthetized, and complexes of 125I-Lp(a) (50 µg apolipoprotein in 500 µL PBS) and TG(4Å)C/TG(20Å)C were injected intravenously as described in the previous section. Ten minutes later, the portal vein was cannulated and the liver was perfused with Ca2+-free Hanks' balanced salt solution that contained 10 mmol/L HEPES, pH 7.4 (8°C) at a flow rate of 14 mL/min. After 8 minutes, a lobule was tied off for determination of the total liver uptake. Then, the liver was perfused with 0.01% (wt/vol) collagenase at 8°C in Hanks' solution containing 10 mmol/L HEPES, pH 7.4, and parenchymal and nonparenchymal cells were isolated as described previously.26 The nonparenchymal cell preparation was further fractionated into endothelial and Kupffer cells by centrifugal elutriation as described in detail previously.26 The contributions of the various cell types to the total liver uptake were calculated from the radioactivity recovered in the respective liver cell fractions after correction for the protein content and assuming that 92.5%, 2.5%, and 3.3% of the total liver protein content can be ascribed to parenchymal, Kupffer, and endothelial cells, respectively.37 As found previously with other substrates, no significant loss of radioactivity from the cells during the isolation procedure was observed.

Determination of Protein, Cholesterol, and Carbohydrate Content
Protein concentrations in cell suspensions and subcellular fractions were determined by the method of Lowry et al,38 with bovine serum albumin as the standard. The total cholesterol content was determined colorimetrically with the use of the CHOD-PAP assay (Boehringer Mannheim). The ß-D-galactose content was determined colorimetrically according to the ABTS assay.39 Two hundred microliters of ABTS reagent containing ABTS (1.14 g/L), peroxidase (1.2 mL/L, 4.8 U/L), and ß-D-galactose oxidase (2.5 U/L) in potassium phosphate buffer (0.1 mol/L, pH 7.0) was added to the 20-µL sample of the density gradient fractions. After incubation for 180 minutes at 37°C, the extinction at 405 nm was monitored. ß-D-Galactose, TG(4Å)C, and TG(20Å)C were used as a standard. The extinction coefficients of the three substrates were 0.181, 0.079±0.004, and 0.73±0.04 AU/µg, respectively (n=2). Uncomplexed Lp(a) did not stain significantly in the ABTS assay (<1.6x10-4 AU/µg).

Data Analysis
Differences in the contribution of various liver cell types to hepatic Lp(a) uptake between control and glycolipid-treated Lp(a) were analyzed statistically by the use of one-way ANOVA and subsequent Dunnett's posttesting. Other data were analyzed statistically by the use of unpaired two-tailed Student's t test.


*    Results
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 up arrowMethods
 *Results
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Interaction of TG(4Å)C and TG(20Å)C With Lp(a)
After incubation of Lp(a) with the cholesterol-derivatized cluster galactosides, we studied the association of TG(4Å)C and TG(20Å)C with Lp(a) by assaying the cholesterol and carbohydrate profile of glycolipid/lipoprotein mixtures after density gradient centrifugation in a NaCl/KBr buffer (1006<d<1220 kg·m-3) (Fig 2Down). In the absence of lipoprotein, TG(4Å)C and TG(20Å)C are mainly recovered in the bottom fractions at densities >1110 kg·m-3. However, in the presence of Lp(a), the carbohydrate and cholesterol profiles coincide. Aside from a small fraction of unassociated glycolipid at high density (d>1150 kg·m-3), the major fraction of TG(4Å)C (82%) and TG(20Å)C (73%) is recovered at intermediate densities (1110<d<1220 kg·m-3). Compared with untreated Lp(a), the cholesterol peak is shifted to higher densities in the presence of either of the glycolipids.



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  		Figure 2. Interaction of TG(4Å)C and TG(20Å)C with Lp(a). TG(4Å)C (200 µg in 1 mL PBS; {square}, {blacksquare}) or TG(20Å)C (200 µg in 1 mL PBS; {circ}, {bullet}) were incubated for 30 minutes at 20°C in the absence (top right) or presence of 200 µg Lp(a) (bottom right and left, respectively). Subsequently, the incubation mixtures were subjected to density gradient ultracentrifugation (1220>d>1006 kg/m3), and the density fractions were monitored for cholesterol ({circ}, {square}) and galactose content ({bullet}, {blacksquare}). As a control, the density gradient ultracentrifugation was also performed with Lp(a) (200 µg in 1 mL PBS; top left).

In Vivo Fate of TG(4Å)C- and TG(20Å)C-Laden Lp(a)
The effect of various amounts of cholesterol-derivatized cluster galactosides on the serum decay and liver association of 125I-Lp(a) was studied (Figs 3Down and 4Down, respectively). Ten minutes after injection of 125I-Lp(a) into rats, only 9.1±4.1% of the injected dose was cleared from the circulation. The serum clearance of 125I-Lp(a) was markedly and dose dependently enhanced on premixing of Lp(a) with TG(4Å)C; 10 minutes after injection, up to 93±2.7% of the injected dose can be removed from the bloodstream at a dose of 1.0 mg TG(4Å)C per milligram of protein (P<.0001). Even at a dose of 0.10 mg TG(4Å)C per milligram of protein, the serum decay of the complex was accelerated compared with native 125I-Lp(a) (29.1% of the injected dose was removed 10 minutes after injection). Interestingly, the serum level of TG(4Å)C/125I-Lp(a) complexes stabilized within 2 to 5 minutes after injection of the complex. In agreement with the slow serum decay (t½=115±11 minutes), liver uptake of untreated 125I-Lp(a) was low (3.9±1.4% of the injected dose at 10 minutes after injection). However, incubation of 125I-Lp(a) with TG(4Å)C markedly and dose dependently stimulated the hepatic uptake of the lipoprotein to 80.3±4% of the injected dose [P<.0001; 1.0 mg TG(4Å)C per milligram of 125I-Lp(a)].



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  		Figure 3. Effect of TG(4Å)C on the serum decay and liver association of 125I-Lp(a). TG(4Å)C was mixed with 125I-Lp(a) (50 µg apolipoprotein dissolved in 500 µL PBS) at a ratio of 0.0 ({circ}), 0.1({blacktriangledown}), 0.3 ({bullet}), and 1.0 mg ({blacksquare}) TG(4Å)C per milligram of apolipoprotein. After incubation for 30 minutes at room temperature, the mixtures were injected intravenously into rats. At the indicated times, radioactivities in serum (left) and the liver-associated radioactivities (right) were determined. Data points are mean±SD of a determination in triplicate. Where error bars are not visible, it indicates that they fall within the symbol size.



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  		Figure 4. Effect of TG(20Å)C on the serum decay and liver association of 125I-Lp(a). TG(20Å)C was mixed with 125I-Lp(a) (50 µg apolipoprotein dissolved in 500 µL PBS) at a ratio of 0.0 ({circ}), 0.1 ({blacktriangledown}), 0.3 ({bullet}), and 1.0 mg ({blacksquare}) TG(20Å)C per milligram of apolipoprotein. After incubation for 30 minutes at room temperature, the mixtures were injected intravenously into rats. At the indicated times, radioactivities in serum (left) and the liver-associated radioactivities (right) were determined. Data points are mean±SD of a determination in triplicate.

The effects of premixing 125I-Lp(a) with TG(20Å)C on the kinetics of serum removal and liver uptake of 125I-Lp(a) were similar, although less pronounced. Preincubation of 125I-Lp(a) with TG(20Å)C caused a dose-dependent acceleration of the clearance of 125I-Lp(a), which was accompanied by an increase in liver association (P<.002). The maximal liver uptake of 125I-LDL/TG(20Å)C complexes amounted to 16.7±3.9% of the injected dose at a ratio of 1 mg TG(20Å)C per milligram of 125I-Lp(a) (P<.006).

Effect of GalNAc on the TG(4Å)C- and TG(20Å)C-Induced Liver Uptake of Lp(a)
To verify whether hepatic galactose-recognizing receptors are involved in the increased uptake of Lp(a)/TG(4Å)C and Lp(a)/TG(20Å)C complexes, we studied the effect of preinjection of N-acetyl-galactosamine on the liver uptake of these complexes. It appeared that the hepatic uptake of both TG(4Å)C/125I-Lp(a) and TG(20Å)C/125I-Lp(a) was inhibited by 93% (P<.003) and 78% (P<.04), respectively, by preinjection of GalNAc (Fig 5Down). After blocking of the galactose-receptor mediated uptake, clearance and liver uptake of TG(4Å)C/125I-Lp(a) and TG(20Å)C/125I-Lp(a) were nearly identical to that of untreated Lp(a).



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  		Figure 5. Effect of preinjection of GalNAc on the liver uptake of TG(4Å)C-laden (left) or TG(20Å)C-laden 125I-Lp(a) (right). 125I-Lp(a) (50 µg apolipoprotein in 500 µL PBS) was mixed with TG(4Å)C or TG(20Å)C at a ratio of 1 mg per milligram of apolipoprotein, and the mixture was incubated for 30 minutes at room temperature. One minute before injection of the lipoprotein/galactoside complex, either solvent ({blacksquare}) or 150 mg GalNAc ({blacktriangleup}) was injected. Subsequently the mixtures were injected into rats, and the liver uptake was monitored for 40 minutes. As a control, the liver uptake of native 125I-Lp(a) is shown ({bullet}).

Contribution of the Various Liver Cell Types to the Hepatic Uptake of Complexes of Lp(a) and TG(4Å)C or TG(20Å)C
Two galactose-recognizing hepatic receptors have been identified in the liver: the asialoglycoprotein receptor on the parenchymal liver cell20 and the fucose/galactose receptor on Kupffer cells.18 19 To identify the sites of cellular uptake, we determined the contribution of the various liver cell types to the TG(4Å)C- and TG(20Å)C-stimulated hepatic uptake of Lp(a). The various cell types were isolated by collagenase perfusion at 8°C 10 minutes after injection of the glycolipid/Lp(a) complexes. In agreement with liver uptake studies (Fig 3Up), the recovery of Lp(a) in the liver 10 minutes after injection was increased 12-fold from 4.2±0.2% to 52±4% for TG(4Å)C (P<.001; n=3) and 15.2±3% for TG(20Å)C (P<.01; n=3) (Fig 6Down). Kupffer cell uptake increased almost 20-fold after complexing with TG(4Å)C (P<.001), whereas uptake of Lp(a) by endothelial and parenchymal liver cells was stimulated to a lesser extent (10-fold and 4-fold, respectively). Kupffer cells, accounting for 76±7% of the total liver uptake, were the main cell type responsible for TG(4Å)C-induced uptake of Lp(a). In contrast, TG(20Å)C-Lp(a) complexes were mainly recovered in parenchymal liver cells (51±13%), whereas 40±10% of the hepatic uptake could be attributed to Kupffer cells.



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  		Figure 6. Effect of incubation of 125I-Lp(a) with TG(4Å)C or TG(20Å)C on the contribution of various cell types to the liver uptake of Lp(a). 125I-Lp(a) (50 µg apolipoprotein) was added to a solution of PBS (500 µL; open bars) or PBS containing TG(4Å)C (50 µg; crosshatched bars) or TG(20Å)C (50 µg; hatched bars). After incubation for 30 minutes at room temperature, the mixtures were injected into rats. Ten minutes after injection, parenchymal (PC), endothelial (EC), and Kupffer cells (KC) were isolated from the liver and counted for radioactivity. Values are means of three experiments (±SD) and are expressed as percentage of the total injected dose.

In agreement with the liver uptake studies, the effect of TG(20Å)C appeared to be less pronounced. Total liver uptake was increased by a factor of four, at which parenchymal cell uptake was enhanced from 1.7±0.09% of the injected dose to 8.3±2.1% of the injected dose (P<.02; n=3) and Kupffer cell uptake was increased 2.8-fold. In contrast, endothelial uptake was not significantly enhanced compared with control Lp(a).


*    Discussion
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We have previously developed cholesterol-lowering glycolipids21 22 25 27 that induce galactose receptor–mediated liver uptake of lipoproteins like HDL and LDL.23 24 26 These glycolipids, TG(4Å)C and TG(20Å)C, consist of a lipophilic cholesterol group that provides anchoring to lipoproteins and a galactose-terminated glycoside. The glycoside confers recognition of the glycolipid by hepatic galactose receptors, ie, the asialoglycoprotein receptor on parenchymal liver cells20 and the fucose/galactose receptor on Kupffer cells.18 19 We have previously demonstrated that TG(20Å)C displays a 2000-fold higher affinity for the asialoglycoprotein receptor on parenchymal liver cells than TG(4Å)C.40 As a consequence, TG(20Å)C tends to target LDL to parenchymal liver cells, whereas TG(4Å)C induces hepatic uptake of LDL by Kupffer cells.23 However, because both compounds were able to stimulate hepatic uptake of LDL, and in view of the high similarity between Lp(a) and LDL, the above strategy may also prove effective for enhancing the catabolism of Lp(a).

Analysis of the density gradient fractions of a solution of TG(20Å)C, incubated in the absence and presence of Lp(a), showed that both glycolipids indeed incorporate into Lp(a). At least 82% of the added TG(4Å)C and 73% of TG(20Å)C were recovered in the cholesterol-containing fractions. This indicates that under these conditions (1 mg glycolipid per milligram of protein), {approx}730 molecules of TG(4Å)C and 420 molecules of TG(20Å)C are incorporated into a single molecule of Lp(a). Assuming that these glycolipids incorporate in the surface shell of the lipoprotein ({approx}2100 nm2), this corresponds to 0.34 and 0.2 molecules of glycolipid per square nanometer, respectively. In comparison to TG(4Å)C, the relative amount of incorporated TG(20Å)C is slightly lower, which may be caused by the higher hydrophilicity of the latter compound. On incubation with TG(4Å)C and TG(20Å)C, the particle density of Lp(a) is slightly increased from 1080 kg·m-3 to 1132 and 1135 kg·m-3 due to incorporation of TG(4Å)C and TG(20Å)C, respectively. The extent of the Lp(a) density shift corresponds with the theoretically calculated shift after incorporation of 0.82 mg TG(4Å)C (d=1134 kg·m-3) and 0.73 mg TG(20Å)C per milligram of Lp(a) (d=1139 kg·m-3).

Subsequently, the effect of both glycolipids on the in vivo behavior of Lp(a) was addressed in the rat. Because the serum half-life of Lp(a) in the rat is comparable to that in humans, the rat is an appropriate model system to study the effects of glycolipids on its clearance. Premixing of 125I-Lp(a) with TG(4Å)C strongly accelerates the serum decay of the lipoprotein. Accordingly, hepatic uptake of 125I-Lp(a) was markedly and dose dependently increased and reached a maximal value of 80% of the injected dose at 1.0 mg TG(4Å)C per milligram of 125I-Lp(a). This suggests that almost 100% liver uptake of the injected glycolipid/Lp(a) occurs within 5 minutes after injection. The serum decay of TG(4Å)C/Lp(a) complexes stabilizes at 2 minutes after intravenous injection, probably as a result of the rapid exchange of TG(4Å)C from Lp(a) to endogenous lipid compartments and lipoproteins, as has been described for complexes of TG(4Å)C and HDL or LDL.23 24 In agreement with previous findings of Van Berkel et al23 regarding the in vivo kinetics of TG(4Å)C/LDL in the liver, serum levels of complexes of TG(4Å)C and 125I-Lp(a) slightly increase after a sharp initial decrease.23 This is probably caused by dissociation of the glycolipid from Lp(a) and subsequent release of a small part of native Lp(a) from the liver during internalization of the complexes by the liver.

The TG(4Å)C-induced liver uptake of 125I-Lp(a) could be completely prevented by blockage of the galactose receptor–mediated uptake. After preinjection of GalNAc, liver uptake of glycolipid/Lp(a) complexes essentially coincides with that of untreated Lp(a), suggesting that the pronounced liver uptake of the complexes is not due to a specific glycolipid-induced disintegration of Lp(a). Rather, it establishes that galactose-recognizing receptors are responsible for uptake of TG(4Å)C/Lp(a) complexes. Combined with the finding that uptake is primarily mediated by Kupffer cells, we conclude that the hepatic fucose/galactose receptor18 19 is responsible for the TG(4Å)C-induced uptake of 125I-Lp(a).

Premixing of 125I-Lp(a) with TG(20Å)C also enhanced the serum clearance and liver uptake of 125I-Lp(a), although this was less pronounced than with TG(4Å)C. A minimal ratio of 0.1 mg TG(20Å)C per milligram of Lp(a) corresponding to 42 mol TG(20Å)C per mol of Lp(a) was required for stimulating the hepatic uptake and serum decay of 125I-Lp(a). Surprisingly, TG(4Å)C is more effective in inducing Lp(a) liver uptake. It should be noted, however, that the lower efficacy of TG(20Å)C may very well result from the higher hydrophilicity of this compound, which facilitates exchange of TG(20Å)C from the premixed complex to endogenous lipid compartments and thus the formation of glycolipid-deficient Lp(a) particles. Recently we established that parenchymal liver cells are able to internalize and process particles with a size of up to 23 nm (lactosylated LDL) at a normal rate.41 This makes it rather unlikely that the lower efficacy of TG(20Å)C to stimulate liver uptake of Lp(a) reflects a reduced capacity of the asialoglycoprotein receptor to process Lp(a) (25 nm).

As with 125I-Lp(a)/TG(4Å)C, liver uptake of 125I-Lp(a)/TG(20Å)C complexes proceeds via galactose-specific receptors and can be blocked by 78% by preinjection of 150 mg GalNAc. The liver contains at least two galactose-recognizing receptors, the asialoglycoprotein receptor on parenchymal liver cells20 and the fucose/galactose receptor on Kupffer cells.18 19 The binding characteristics of these receptors differ markedly. The asialoglycoprotein receptor prefers multiantennary glycosides at which the terminal galactose groups have to be properly spaced.20 The fucose/galactose receptor, by contrast, recognizes large particles (>= 12 nm) exposing high galactose or GalNAc densities.41 42 Unlike TG(4Å)C, TG(20Å)C stimulated parenchymal liver cell uptake of Lp(a) rather than uptake by Kupffer cells; the parenchymal liver cell uptake was enhanced by a factor of five after premixing 125I-Lp(a) with TG(20Å)C, whereas the Kupffer cell uptake was only increased by 2.8-fold. The observed tendency of TG(20Å)C to target Lp(a) to parenchymal liver cells is in agreement with previous findings26 and may be explained by the 2000-fold higher affinity of TG(20Å)C for the asialoglycoprotein receptor compared with TG(4Å)C.40 Apparently, uptake of large galactose-exposing particles like LDL and Lp(a) by the galactose/fucose receptor on hepatic Kupffer cells can be circumvented, provided a high-affinity ligand for the asialoglycoprotein receptor is utilized within the glycolipid.

The kinetics of glycolipid-induced liver uptake of Lp(a) strongly suggests that Lp(a) is degraded after uptake. At 40 minutes after injection of the complexes, the liver-associated radioactivity decreases from a maximal value of 80% to 15% for TG(4Å)C and from 17% to 7% of the injected dose for TG(20Å)C. This is not surprising, given the fact the galactose receptor–mediated uptakes by Kupffer and parenchymal liver cells are both coupled to lysosomal processing of glycolipid/lipoprotein complexes.23 24 Both uptake pathways will therefore effect proteolysis of the atherogenic apolipoprotein(a) and elimination of Lp(a) from the bloodstream. Previous studies have established that glycolipid-induced uptake of lipoproteins by parenchymal liver cells27 as well as Kupffer cells22 will also lead to irreversible biliary removal of lipoprotein-derived cholesterol(esters) from the body. Studies by Pieters et al43 further substantiated that Kupffer cells are able to channel intracellular cholesterol(esters) via parenchymal liver cells to the bile. Consequently, we anticipate that both pathways for eliminating serum Lp(a) by glycolipids are beneficial.

In conclusion, both glycolipids tested in the present study stimulate hepatic uptake of Lp(a) and thus accelerate Lp(a) catabolism. In previous studies, we have demonstrated that incorporation and subsequent hepatic targeting of lipoproteins also occur after intravenous injection of TG(4Å)C and TG(20Å)C into rats, leading to a dramatic lowering of serum cholesterol levels22 27 at doses that were tolerated well and did not lead to hemolysis or altered hematocrit levels.27 By analogy, it is anticipated that glycolipids will also incorporate into serum Lp(a) after in vivo administration. In vitro studies on the distribution of TG(4Å)C and TG(20Å)C over lipoprotein fractions from human serum revealed that more than 90% of the glycolipid is lipoprotein-associated, 43% and 36%, respectively, being associated with LDL and 57% and 64%, respectively, being associated with HDL.21 25 The lack of preference of both glycolipids to incorporate into atherogenic lipoproteins such as Lp(a) and LDL may still form a drawback of these compounds. To overcome this, new glycolipids are currently being devised and synthesized that are provided with anchors that are specific for the aforementioned lipoproteins. The present data prompted us to further elaborate the pursued strategy because they illustrate that the concept is therapeutically feasible, which may eventually allow specific removal of potentially atherogenic Lp(a) particles from the circulation.


*    Selected Abbreviations and Acronyms
 
AU = arbitrary units
GalNAc = N-acetyl-galactosamine
HDL = high-density lipoprotein
LDL = low-density lipoprotein
Lp(a) = lipoprotein(a)
PBS = phosphate-buffered saline
PMSF = phenylmethylsulfonyl fluoride
TG(4Å)C = N-[tris-O-ß-D-galactopyranosyl)methyl]ethyl-N{alpha}-[(5-cholesten-3ß-yloxy)succinyl]-glycinamide
TG(20Å)C = N-[tris-O-(3,6,9-trioxaundecanyl-ß-D-galactopyranosyl)methoxymethyl]methyl-N{alpha}-[1-(6-(5-cholesten-3ß-yloxy)glycyl)adipyl]-glycinamide


*    Acknowledgments
 
This work was supported by the Dutch Heart Foundation (grant M93.001).


*    Footnotes
 
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form (Circulation. 1994;90[pt 2]:I-461).

Received December 14, 1995; revision received April 10, 1996;
*    References
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 *References
 

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