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Originally published In Press as doi:10.1194/jlr.M500249-JLR200 on September 8, 2005

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Journal of Lipid Research, Vol. 46, 2681-2691, December 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a]

William J. Cain1,*, John S. Millar{dagger}, Adam S. Himebauch*,§, Uwe J. F. Tietge{dagger},**, Cyrille Maugeais{dagger},{dagger}{dagger}, David Usher* and Daniel J. Rader{dagger}

* Department of Biological Sciences, University of Delaware, Newark, DE
{dagger} Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA
§ Department of Pediatrics, Division of General Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA
** Center for Liver, Digestive, and Metabolic Diseases, University of Groningen Medical Center, Groningen, The Netherlands
{dagger}{dagger} Pharmaceutical Division, Hoffmann-La Roche, Ltd., Basel, Switzerland

Published, JLR Papers in Press, September 8, 2005. DOI 10.1194/jlr.M500249-JLR200

1 To whom correspondence should be addressed. e-mail: will{at}udel.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cellular and molecular mechanisms responsible for lipoprotein [a] (Lp[a]) catabolism are unknown. We examined the plasma clearance of Lp[a] and LDL in mice using lipoproteins isolated from human plasma coupled to radiolabeled tyramine cellobiose. Lipoproteins were injected into wild-type, LDL receptor-deficient (Ldlr–/–), and apolipoprotein E-deficient (Apoe–/–) mice. The fractional catabolic rate of LDL was greatly slowed in Ldlr–/– mice and greatly accelerated in Apoe–/– mice compared with wild-type mice. In contrast, the plasma clearance of Lp[a] in Ldlr–/– mice was similar to that in wild-type mice and was only slightly accelerated in Apoe–/– mice. Hepatic uptake of Lp[a] in wild-type mice was 34.6% of the injected dose over a 24 h period. The kidney accounted for only a small fraction of tissue uptake (1.3%). To test whether apolipoprotein [a] (apo[a]) mediates the clearance of Lp[a] from plasma, we coinjected excess apo[a] with labeled Lp[a]. Apo[a] acted as a potent inhibitor of Lp[a] plasma clearance. Asialofetuin, a ligand of the asialoglycoprotein receptor, did not inhibit Lp[a] clearance.

In summary, the liver is the major organ accounting for the clearance of Lp[a] in mice, with the LDL receptor and apolipoprotein E having no major roles. Our studies indicate that apo[a] is the primary ligand that mediates Lp[a] uptake and plasma clearance.

Supplementary key words kidney • tissue distribution • kinetics • LDL receptor • apolipoprotein E • asialoglycoprotein receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipoprotein [a] (Lp[a]) is a LDL-like lipoprotein that has been associated with increased risk of coronary heart disease, stroke, and restenosis (1). Lp[a], which closely resembles LDL in lipid composition, contains a single apolipoprotein B-100 (apoB-100) molecule and an additional apolipoprotein, called apolipoprotein [a] (apo[a]), which is connected via a disulfide linkage to apoB-100 (1). Apo[a] is a polymorphic glycoprotein that contains repeating domains of varying length that are homologous to kringle IV of plasminogen. More than 30 different isoforms of apo[a] have been described in humans, ranging in size from <300 kDa to >800 kDa (2).

Plasma levels of Lp[a] vary greatly between individuals, from <1 mg/dl to >100 mg/dl (2). The source of most of this variability in plasma concentrations is variability in the production rate of Lp[a] (3, 4), which, in turn, is controlled largely by the apo[a] gene locus (5). Apo[a] is synthesized by hepatocytes and rapidly associates with LDL after secretion to form Lp[a] in the sinusoids of the liver (2, 6). Although the steps involved in Lp[a] production are known, the major mechanisms involved in Lp[a] clearance from plasma are not currently known. It has been suggested that the kidney might play a major role in Lp[a] clearance, after several clinical studies reported increased plasma Lp[a] levels in patients with renal failure (7, 8). In addition, Kronenberg et al. (9) used measurements of renovascular arteriovenous differences in Lp[a] plasma concentrations in patients without renal insufficiency to demonstrate that the human kidney may play an active role in Lp[a] catabolism.

Several receptors that mediate the binding and uptake of lipoproteins containing apoB-100 have been proposed as receptors for Lp[a] catabolism. These include the LDL receptor (LDLR) (10–15), megalin/gp330 (16), the LDL receptor-related protein (LRP) (17), and the VLDL receptor (18). The latter two receptors can mediate binding to lipoproteins through apoE. According to the secretion-capture model, apoE that is secreted by the liver rapidly binds to remnants associated with heparan sulfate proteoglycans. These captured lipoproteins are then cleared by receptor-mediated endocytosis (19). ApoE has been proposed to play a similar role in Lp[a] catabolism (20).

The current studies were designed to test potential pathways responsible for Lp[a] uptake in mice through the use of a radioiodinated, intracellularly trapped ligand, tyramine cellobiose (TC), which was covalently attached to human Lp[a] and injected into mouse models in the C57BL/6 genetic background. Our results demonstrate that in this model, the liver is the major organ responsible for the plasma clearance of Lp[a] and that the kidney plays a very minor role under normal conditions. In addition, we examined the role of the LDLR, apoE, and the asialoglycoprotein receptor (ASGPR) in the clearance of Lp[a] in mice. These studies show that the LDLR, apoE, and the ASGPR do not have a major role in Lp[a] catabolism in mice. Lastly, we demonstrated that the administration of excess apo[a] can effectively block the plasma clearance of Lp[a], suggesting that it is the apo[a] moiety that mediates the plasma clearance of Lp[a].


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Wild-type C57BL/6, LDL receptor-deficient (Ldlr–/–), and apoE-deficient (Apoe–/–) mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were maintained on a chow diet under a 12 h light/dark cycle.

Isolation of lipoproteins and apo[a]
Lp[a] was isolated from single-donor plasma as described previously (3, 4, 21). Briefly, plasma was immediately brought to 0.01% NaN3, 0.01% Na2EDTA, and 1 mM benzamidine and adjusted to a density of 1.21 g/ml, and total lipoproteins were isolated by ultracentrifugation. The total lipoprotein fraction was dialyzed against phosphate buffer (0.1 M sodium phosphate, 0.01% NaN3, 0.01% Na2EDTA, and 1 mM benzamidine, pH 7.4) and then applied to a lysine-Sepharose column (Amersham Pharmacia) equilibrated with phosphate buffer. The column was then washed with phosphate buffer, and the unbound fraction, which contained LDL, was collected. The bound fraction, which is composed mostly of Lp[a], was eluted with phosphate buffer containing 100 mM {varepsilon}-aminocaproic acid (ACA). This fraction was brought to 7.5% (w/w) CsCl and centrifuged in a Beckman 60Ti rotor at 50,000 rpm and 15°C for 27 h. The self-forming density gradient was pumped from the centrifuge tubes. Those fractions containing purified Lp[a] were pooled and dialyzed against PBS (0.1 M NaCl, 0.01 M sodium phosphate, 0.01% NaN3, 0.01% Na2EDTA, and 1 mM benzamidine, pH 7.4). The unbound fraction from the lysine-Sepharose column was dialyzed against histidine buffer (0.2 M NaCl, 0.025 M histidine, and 0.01% Na2EDTA, pH 6.0) and applied to a column containing PBE94 (Bio-Rad) equilibrated with histidine buffer. The column was washed with histidine buffer, and the unbound fraction, which contained LDL, was collected and dialyzed against PBS. This fraction was concentrated using an Amicon stirred pressure cell, and the density was then adjusted to 1.35 g/ml with NaBr. Five milliliters of this solution was placed into 25 ml Beckman 60Ti centrifuge tubes and overlaid with a 0–25% NaBr gradient. The tubes were centrifuged for 90 min at 15°C and 50,000 rpm. The density gradient was pumped from the centrifuge tubes, and those fractions containing purified LDL were pooled and dialyzed against PBS.

Apo[a] was prepared by a modification of the procedure described by Edelstein et al. (22). Lp[a] was brought to a final concentration of 100 mM ACA and 2 mM dithioerythritol and incubated at room temperature for 1 h. The reaction mixture was dialyzed against PBS for ~1 h and adjusted to a density of 1.3 g/ml with NaBr. Approximately 5 ml of the density-adjusted reaction mix was placed into a Beckman 60Ti centrifuge tube and overlaid with a 10–25% NaBr gradient to a volume of 25 ml. This was centrifuged at 50,000 rpm and 15°C for 90 min to separate apo[a] from the LDL released by reduction and intact Lp[a]. The density gradient was pumped from the centrifuge tubes, and those fractions containing purified apo[a] were pooled, dialyzed against PBS, and concentrated using Vivaspin concentrators (Vivascience).

The BCA protein assay (Pierce) was used to quantify all lipoprotein and apolipoprotein preparations. When Lp[a] was coinjected with excess apo[a], we estimated the molar excess of apo[a] as follows. We determined that the apparent molecular weight of apo[a] used in this study was the same as that of apoB-100 and made the simplifying assumption that an equimolar amount of Lp[a] would have twice the amount of protein as apo[a] when measured by the BCA assay. The fold molar excess was then calculated as (micrograms of apo[a] injected)/(micrograms of Lp[a] injected x 0.5).

Radioactive labeling of proteins
Lipoproteins, apo[a], and asialofetuin (from fetal calf serum; Sigma) were directly iodinated with either Na131I or Na125I (New England Nuclear) using a modification of the iodine monochloride procedure (4); alternatively, TC was iodinated with either Na131I or Na125I and then chemically coupled to LDL or Lp[a] using the procedure described by Pittman and Taylor (23). Typically, 0.1 µmol of TC was iodinated using Iodobeads (Pierce) and then chemically coupled to ~5 mg of protein with 0.1 µmol of cyanuric chloride. The purity of the radiolabeled preparations was verified using SDS-PAGE and 1% agarose native gels (Ciba Corning).

Metabolic studies
Radioiodinated lipoproteins in saline were injected via a tail vein. Blood was drawn from the orbital sinus into heparin-coated tubes, and plasma was separated by low-speed centrifugation. Radioactivity in 10 µl aliquots was quantified in a {gamma} counter. The fractional catabolic rates (FCRs) were calculated using SAAM II (SAAM Institute, Seattle, WA).

Tissue uptake of proteins labeled with TC was examined in mice housed in metabolic cages (four mice per cage). One day after injection of radiotracer, the mice were anesthetized and bled from the inferior vena cava immediately followed by perfusion with saline. Unless stated otherwise, organs were removed whole, weighed, and counted. Tissue samples were taken from visceral adipose tissue, muscle from the hind leg, and skin from the abdomen. These samples were weighed and counted. The accumulations of radiotracer in skin, muscle, and adipose tissue were calculated by assuming that these tissues accounted for 16.5, 38.4, and 6.4% (w/w), respectively, of the mass of a mouse (24). Feces and urine were collected and counted. The injected dose was calculated using the plasma counts at 5 min as follows: (counts per minute per milliliter of plasma at 5 min) x (mouse weight in grams) x (0.035 ml of plasma per gram of mouse body weight). The counts remaining in the plasma at 24 h were calculated as follows: (counts per minute per milliliter of plasma at 24 h) x (mouse weight in grams) x (0.035 ml of plasma per gram of mouse body weight). The tissue distribution of radiotracer is expressed as a percentage of the injected dose.

Statistical analysis
All statistical analyses were performed using the unpaired two-tailed Student's t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of radiolabeled Lp[a] and LDL
Lipoproteins were either radiolabeled directly by the iodine monochloride method (4) or, alternatively, radiolabeled TC was chemically cross-linked to lipoproteins by the method of Pittman and Taylor (23). For each study, lipoprotein preparations were analyzed by SDS-PAGE under reducing and nonreducing conditions. A representative gel is shown in Fig. 1A . The Lp[a] used in this study had a single apo[a] isoform with the same apparent molecular weight as apoB-100, and this was verified by immunoblotting (data not shown). Nondenaturing agarose gel electrophoresis was also performed (Fig. 1B), and for all preparations, a single band with preß mobility was observed. Lipoproteins labeled by either of the two methods showed essentially identical migration patterns when examined using both electrophoretic methods. The 125I-TC-Lp[a] contained ~10% of the label on apo[a] and the remainder on apoB-100 (data not shown).



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  		Fig. 1. Analysis of radiolabeled lipoproteins. The lipoprotein [a] (Lp[a]) and LDL used for this study were isolated from a donor who possessed a single apolipoprotein [a] (apo[a]) isoform with the same apparent molecular weight as apolipoprotein B-100. A: Analysis by SDS-PAGE. The panel shows an autoradiograph of 125I-Lp[a], reduced (lane 1) and not reduced (lane 2); 125I-tyramine cellobiose (TC)-Lp[a], reduced (lane 3) and not reduced (lane 4); 131I-TC-LDL, reduced (lane 5). B: Autoradiograph of 125I-TC-Lp[a] and 131I-TC-LDL separated by nondenaturing agarose gel electrophoresis. C: Analysis by SDS-PAGE. The panel shows Coomassie staining of Lp[a], reduced (lane 1); and apo[a], not reduced (lane 2) and reduced (lane 3).

 
Tissue uptake of Lp[a] and LDL in wild-type mice
To assess which organs or tissues were responsible for Lp[a] uptake and catabolism, male C57BL/6 mice were injected simultaneously with both 125I-TC-Lp[a] and 131I-Lp[a]. For comparison, another set of male mice was injected with 125I-TC-LDL and 131I-LDL. The clearance of LDL was significantly faster than the clearance of Lp[a] for both 125I-TC-labeled lipoproteins (P = 0.02) and 131I-labeled lipoproteins (P = 0.0002) (Fig. 2) . Furthermore, the plasma clearance of 125I-TC-labeled lipoproteins was similar to that of the 131I-labeled forms. The plasma clearance of 125I-TC-LDL (3.0 ± 0.2 d–1) and 131I-LDL (3.2 ± 0.3 d–1) were not significantly different (P = 0.3), whereas the clearance of 125I-TC-Lp[a] (2.0 ± 0.2 d–1) was slightly slower than that of 131I-Lp[a] (2.5 ± 0.3 d–1) (P = 0.03).



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  		Fig. 2. Plasma clearance of human Lp[a] and LDL in wild-type mice. A: Four male mice were injected with both 125I-TC-labeled Lp[a] (TC-Lp[a]) and 131I-Lp[a] (Lp[a]). B: Four male mice were injected with both 125I-TC-labeled LDL (TC-LDL) and 131I-LDL (LDL). Error bars indicate SD.

 
After 24 h, the animals were euthanized and tissues counted. The results are shown in Fig. 3 , and values are expressed as percentages of the injected dose retained by each tissue. The tissue distribution of TC-Lp[a] and TC-LDL were quite similar. The liver was the major organ that accumulated the TC-labeled lipoproteins, 21.3 ± 3.1% of the injected Lp[a] and 20.5 ± 2.5% of the injected LDL. The kidney contained only 1.3 ± 0.3% of the Lp[a] and <1% of the LDL. Intestinal tissue contained 2.1 ± 0.3% and 1.9 ± 0.5% of the Lp[a] and LDL, respectively. Combined values for the stomach, spleen, adrenals, testes, heart, lungs, and brain contained <2% of either lipoprotein. We used tissue samples to estimate the amount of TC-labeled lipoproteins that accumulated in the skin, muscle, and adipose tissue (see Methods). Both the muscle and skin retained a considerable amount of lipoprotein. The muscle contained 11.7 ± 2.0% of the Lp[a] and 7.9 ± 4.4% of the LDL, whereas the skin retained 3.2 ± 1.1% of the Lp[a] and 4.0 ± 0.3% of the LDL. Adipose tissue retained only a small amount of label.



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  		Fig. 3. Tissue distribution of radiolabeled human Lp[a] and LDL in wild-type mice. Groups of four mice each were housed in metabolic cages. Twenty-four hours after injection of radiotracer, the animals were euthanized and their tissues were counted. The results are expressed as percentages of the injected dose. A: Four male mice were injected with both 125I-TC-Lp[a] and 131I-Lp[a]. B: Four male mice were injected with both 125I-TC-LDL and 131I-LDL. C: Four male mice were injected with 125I-TC-apo[a]. Error bars indicate SD.

 
As expected, the tissue distribution of 131I-LDL and 131I-Lp[a] that lacked the trapped TC ligand was considerably different from that of the TC-labeled lipoproteins (Fig. 3). The liver retained relatively little LDL and Lp[a] when these were not coupled to TC. Most of the labeled lipoprotein retained by tissues was found in the muscle and skin. The largest amount of radiolabel was recovered in the urine and accounted for 43% and 39% of the injected dose for LDL and Lp[a], respectively. This is an underestimation because there were some urine losses during some of the mouse bleeds. Indeed, in a similar study, when blood samples were not taken over the course of the urine collection, the radiolabel recovered in the urine accounted for almost all of the counts not recovered in the carcass when lipoproteins were labeled directly with iodine (data not shown).

To determine whether the 125I-TC-Lp[a] injected into mice was still intact in the plasma after 24 h, plasma samples were adjusted to 7.5% (w/w) CsCl and ultracentrifuged to separate Lp[a] from LDL. Under these conditions, LDL floats near the top of the self-forming density gradient, whereas Lp[a] is found toward to the bottom. Figure 4 clearly shows that almost all of the radiolabel in the plasma after 24 h was found as intact Lp[a]. Free apo[a], which would be found in the bottom of the tube, was not detected.



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  		Fig. 4. Distribution of 125I-TC between Lp[a] and LDL at 24 h after injection of 125I-TC-Lp[a]. Ten microliters of plasma, obtained form a mouse at 24 h after injection with 125I-TC-Lp[a], was added to a 7.5% CsCl solution, and the LDL was separated from Lp[a] by density gradient ultracentrifugation.

 
Metabolism of Lp[a] and LDL in Ldlr–/– and Apoe–/– mice
Wild-type, Ldlr–/–, and Apoe–/– male mice were injected with both 125I-TC-Lp[a] and 131I-TC-LDL. The plasma clearance of LDL was considerably slower in Ldlr–/– mice than in wild-type controls (1.47 vs. 2.96 d–1; P = 0.0002), whereas the clearance was accelerated considerably in Apoe–/– mice (4.69 vs. 2.96 d–1; P < 0.0001) (Fig. 5A) . In contrast, the plasma clearance rate of Lp[a] was not affected significantly by the absence of the LDLR and was modestly increased by 15% in the Apoe–/– mouse (Fig. 5B) compared with the wild-type mouse (2.95 vs. 2.43 d–1; P = 0.01).



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  		Fig. 5. Plasma clearance of TC-Lp[a] and TC-LDL. Mice were injected with both 125I-TC-Lp[a] and 131I-TC-LDL. A: Clearance of 131I-TC-LDL. B: Clearance of 125I-TC-Lp[a]. Wild-type mice, n = 7; LDL receptor-deficient (Ldlr–/–) mice, n = 4; apolipoprotein E-deficient (Apoe–/–) mice, n = 4. Error bars indicate SD.

 
The distribution of the trapped TC ligand among the tissues of the three mouse strains is shown in Table 1. As shown previously for wild-type mice, the uptake of radiolabel by different tissues was very similar for TC-Lp[a] and TC-LDL when injected into wild-type mice, with the liver accounting for 23.2% and 21.4% of the injected dose for TC-Lp[a] and TC-LDL, respectively. LDLR deficiency resulted in a markedly lower accumulation of TC-LDL in the liver (8.0%) but only a small decrease in the accumulation of TC-Lp[a] in the liver (19.5%). Apoe–/– mice had a marked increase in the accumulation of TC-LDL in the liver (41.8%) but only a small increase in the accumulation of TC-Lp[a] in the liver (27.7%).


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  		TABLE 1. Tissue distribution of 125I-TC-Lp[a] and 131I-TC-LDL

 
Hepatic uptake of Lp[a] and LDL
Pittman and Taylor (23) showed in rat and in rabbit that trapped radiotracer from TC-labeled plasma protein did not redistribute between tissues after initial uptake, within 24 h after injection. However, they did detect significant leakage of radiolabel from the liver, which appeared in the gut contents and the feces. Because the liver is a major site for Lp[a] and LDL uptake in mice, we believe that radiotracer appearing in the gut contents and feces is probably hepatic in origin. Therefore, a better estimate for the hepatic uptake and catabolism of LDL and Lp[a] in mice would be obtained by adding the amounts recovered in the liver, gut contents, and feces. The net hepatic contribution to plasma clearance was calculated and expressed as a percentage of the injected dose (Fig. 6) . In these studies, the intestinal tissue and contents were not separated. We estimated the net hepatic clearance as the sum of the label found in the liver, intestine with contents, and feces. Because intestinal tissue contained only ~2% of the injected dose for both TC-LDL and TC-Lp[a] in wild-type mice, the error caused by including the intestinal tissue in the net hepatic uptake should be relatively small. In wild-type mice, the net hepatic uptake of TC-labeled LDL after 24 h was 41.7 ± 2.3% of the injected dose. Hepatic uptake of TC-LDL was decreased by >2-fold in Ldlr–/– mice to 18.7 ± 0.7% (P < 0.0001), whereas hepatic uptake in Apoe–/– mice was increased nearly 2-fold to 70.25 ± 4.0% (P < 0.0001). In contrast, hepatic uptake of TC-Lp[a] showed a small decrease in Ldlr–/– mice to 27.9 ± 1.3% compared with 34.6 ± 3.1% in wild-type mice (P = 0.003), and it increased in Apoe–/– mice to 39.33 ± 2.1% (P = 0.04).



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  		Fig. 6. Hepatic clearance of TC-LDL (A) and TC-Lp[a] (B). Hepatic clearance was estimated by adding the tissue counts found in the liver, intestine with contents, and feces at 24 h after the injection of 125I-TC-Lp[a] and 131I-TC-LDL. The results are expressed as percentages of the injected dose. Wild-type mice, n = 7; Ldlr–/– mice, n = 4; Apoe–/– mice, n = 3. Error bars indicate SD.

 
Plasma clearance and tissue uptake of apo[a]
Apo[a] that was purified from Lp[a] had the same apparent molecular weight as apoB-100 when analyzed by SDS-PAGE under reducing conditions and, as expected, migrated considerably faster under nonreducing conditions (Fig. 1C). The plasma clearance of 125I-TC-apo[a] was monitored in four male mice, and after 24 h, tissues were harvested and counted. Plasma clearance of apo[a] was faster than that of Lp[a], with a FCR of 7.38 ± 0.53 d–1. The tissue distribution of 125I-TC-apo[a] was similar to that for Lp[a] (Fig. 3C). The liver was the predominant site of apo[a] catabolism and retained 34.7 ± 1.9% of the injected dose, whereas the kidney retained only 0.9 ± 0.2%. The hepatic contribution to plasma clearance (liver + intestinal contents + feces) was 52.7%.

Apo[a] mediates the plasma clearance of Lp[a]
Because we found that Lp[a] was not cleared to any significant extent by pathways involving the LDLR or apoE, we hypothesized that Lp[a] clearance could be mediated by the apo[a] moiety of the lipoprotein. To test this hypothesis, we measured the clearance of Lp[a] in the presence of a large (~30-fold) molar excess of apo[a] (Fig. 7A) . Lp[a] clearance was measured in mice injected with 72 µg of 125I-TC-Lp[a] protein and 1.1 mg of unlabeled apo[a] protein and compared with Lp[a] clearance in mice injected with label only. The plasma clearance of Lp[a] was greatly reduced by apo[a]. In mice coinjected with label and apo[a], 99.1 ± 6.2% of the labeled Lp[a] remained in the plasma after 30 min, whereas in mice injected with label only, 76.8 ± 1.2% of the 125I-TC-Lp[a] remained in the plasma (P = 0.007). By 1 h, this effect was still apparent, with 93.5 ± 5.6% of the label remaining in the plasma of mice coinjected with apo[a] compared with 65.7 ± 3.3% in mice receiving label only (P = 0.003).



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  		Fig. 7. Effect of apo[a] and asialofetuin on the plasma clearance of Lp[a]. A: Effect of apo[a]. Mice were injected with 125I-TC-Lp[a] either without or with 1.1 mg of apo[a] (+ competitor) (n = 4 for each group). B: Effect of asialofetuin (AF). Mice were injected with 125I-TC-Lp[a] and 131I-TC-asiaolfetuin either without or with 7.5 mg of asialofetuin (+ competitor) (n = 4 for each group). Error bars indicate SD.

 
To assess the contribution of the liver in the plasma clearance of Lp[a] during the first hour after injection, mice were injected with 125I-TC-Lp[a] and at 1 h the animals were euthanized and perfused. After 1 h, 39.6 ± 3.5% of the injected dose was cleared from the plasma and 10.5% of the injected dose was recovered in the liver (Fig. 8) . The spleen and kidney contained 0.6 ± 0.3% and 0.7 ± 0.1% of the injected dose, respectively.



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  		Fig. 8. Plasma clearance (A) and tissue distribution (B) of 125I-TC-Lp[a] at 1 h after injection (n = 4). Results are expressed as percentages of the injected dose. Error bars indicate SD.

 
Apo[a] is a highly glycosylated protein rich in sialic acid, and it has been suggested by Hrzenjak et al. (25) that the ASGPR found in the liver may play an important role in the plasma clearance of Lp[a]. We examined the clearance of 125I-TC-Lp[a] and 125I-TC-asialofetuin, an ASGPR ligand, in both the presence and absence of excess unlabeled asialofetuin (Fig. 7B). Mice were injected with 60 µg each of labeled Lp[a] and asialofetuin, either with or without 7.5 mg of asialofetuin. The plasma clearance of asialofetuin is very rapid, so that the initial plasma sample taken at 5 min occurs after most of the label has been cleared. To obtain meaningful data for the clearance of asialofetuin, we found it necessary to estimate the actual injected dose at time zero based upon the volume injected rather than use the 5 min time point. At 5 min, only 3.9 ± 0.4% of the label remained in the plasma. In contrast, in those mice also injected with excess unlabeled asialofetuin, 95.3 ± 2.8% of the injected label remained in the plasma after 5 min. The inhibitory effect is still clearly seen after 1 h. The plasma clearance of Lp[a] was essentially unaffected by the presence of asialofetuin over the 1 h study period.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mice do not possess Lp[a], which has been described only in certain primates and hedgehogs (26). Despite this limitation, studies using transgenic mice producing human Lp[a] have already contributed much to our understanding of Lp[a] synthesis (27–30) and its role in atherosclerosis (31–33). Furthermore, we believe that mice may also offer an attractive model for the study of Lp[a] catabolism, because they are able to clear the lipoprotein in a way that appears to be similar to that in humans. In this study, we examined the plasma clearance and tissue uptake of both human LDL and Lp[a] in mice. As we demonstrated previously in humans (3, 4, 21), in wild-type mice the clearance of LDL is faster than that of Lp[a], and the relative FCR of LDL to Lp[a] is similar to that in humans. In this study in mice, FCR values for LDL were ~20% higher than the values for Lp[a], whereas in humans, FCR values were ~40% higher for LDL than for Lp[a]. The plasma clearance of Lp[a] has been examined in human subjects with homozygous familial hypercholesterolemia who lack LDLRs (21). In the present study, the influence of LDLR deficiency in mice on Lp[a] turnover is remarkably similar to the results seen in humans (Table 2). In both species, LDLR deficiency reduces the LDL FCR to ~50% of normal, while only reducing the Lp[a] FCR by ~10% (a difference that in both the mouse and human studies was not statistically significant). Gabelli et al. (34) examined the plasma clearance of LDL in a human subject who was apoE-deficient and reported an FCR that was 100% greater than normal. Similarly, in Apoe–/– mice, the LDL FCR was increased by 58%. Thus, the plasma clearance of both human LDL and Lp[a] in mice resembles what occurs in humans, suggesting that the pathways of catabolism in these species are similar.


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  		TABLE 2. Comparison of FCR values for human Lp[a] and LDL in normal and Ldlr–/– individuals

 
We used radioiodinated TC to examine the tissue uptake of both human LDL and Lp[a] in mice. For both lipoproteins, the liver was by far the major organ responsible for clearance, whereas the kidney retained only ~1% of the injected dose. Furthermore, the spleen retained only ~1% of the injected label, indicating that the labeled proteins were not recognized as foreign or denatured. The hepatic uptake, as measured by the sum of the label found in the liver, intestines, and feces, accounted for 34.6 ± 3.1% of the injected dose. A better indicator of the liver's role in Lp[a] catabolism is obtained when uptake is expressed as a percentage of the cleared dose (injected dose minus the plasma counts at 24 h). When expressed in this manner, hepatic uptake of Lp[a] accounts for 42.3 ± 4.9% of the cleared dose. In rat, Ye et al. (35) found similar results when they examined the tissue uptake of Lp[a] that was labeled with [3H]cholesteryl linoleyl ether, a nonhydrolyzable lipid. After 24 h, the liver contained 21.4% of the injected dose, compared with 23.2% (Table 1) in the mice used in this study. They also found <1% of the injected dose retained by the kidney.

The kidney has been hypothesized to play a major role in Lp[a] metabolism. Patients with impaired renal function have increased plasma Lp[a] levels (7, 8, 36, 37). In addition, Kronenberg et al. (9) found arteriovenous differences in Lp[a] plasma concentrations, indicating that Lp[a] was removed from the renal circulation in these patients. Several studies have identified large apo[a] fragments in human urine (7, 38, 39), and evidence suggests that these fragments are formed extrarenally and excreted by the kidney (38, 40, 41). In our study, ~7% of the injected radiolabel from 125I-TC-Lp[a] was found in the urine after 24 h (Table 1), and only ~10% of the radiolabel was TCA-precipitable (data not shown). We did not detect any fragmentation of Lp[a] in the plasma. Mooser et al. (38) injected human Lp[a] into mice and detected only trace amounts in the urine by ELISA, and they detected no apo[a] fragments in either the urine or plasma by immunoblot analysis. Thus, in mice with normal renal function, the kidney does not play a major role in Lp[a] catabolism.

It has been proposed that Lp[a] is a ligand for the LDLR and that the liver plays a prominent role in plasma clearance of Lp[a] through the LDLR pathway. Although some in vitro studies support this proposal (11–13), others do not (42–44). In addition, several studies have reported that individuals with familial hypercholesterolemia, in which there is a genetic absence of functional LDLRs, have increased plasma Lp[a] levels, suggesting a role for the LDLR and the liver in the plasma clearance of Lp[a] (10, 14, 15). However, kinetic studies in humans injected with 125I-labeled Lp[a] have shown that Lp[a] plasma clearance is essentially the same in individuals lacking the LDLR compared with normal and heterozygous familial hypercholesterolemia family members (21), and only a minor role for the LDLR was suggested. Furthermore, Lp[a] isolated from patients with familial defective apoB-100 did not contain a higher proportion of defective apoB-100 than normal apoB-100, supporting the concept that the role of the LDLR in Lp[a] clearance is minimal (45).

Transgenic mice and rabbits have been used to further explore the potential role of the LDLR in Lp[a] clearance. Mice overexpressing the human LDLR were found to have accelerated clearance of human Lp[a], and the authors concluded that the LDLR has the potential to play a major role in the plasma clearance of Lp[a] in humans (46). Transgenic rabbits have been created that express human apo[a], and they form circulating Lp[a] that is composed of human apo[a] linked via a disulfide bound to rabbit LDL (47). When these rabbits were cross-bred with WHHL rabbits, progeny that produce Lp[a] but have defective LDLR function showed markedly increased plasma Lp[a] levels compared with apo[a] transgenic animals with normal receptor function (48). Presumably, this increase in Lp[a] was caused by a reduced plasma clearance rate, although no in vivo studies of Lp[a] turnover were performed in this model.

The tissue distributions in wild-type mice of TC-labeled LDL and Lp[a] were very similar. For both lipoproteins, the liver was the primary site of uptake and catabolism. When expressed as a percentage of the cleared dose, hepatic uptake accounted for 42.3% and 47.8% of the plasma clearance of Lp[a] and LDL, respectively, in wild-type mice. This suggests the possible role of the LDLR in Lp[a] clearance in mice. Indeed, in Ldlr–/– mice, the FCR for Lp[a] was ~9% less and hepatic uptake was reduced by ~20% during 24 h. On the other hand, for LDL, the FCR was reduced by ~50% and the hepatic uptake was less than half that for wild-type mice. Thus, it is possible that in wild-type mice the LDLR may make a minor contribution to the hepatic uptake of Lp[a] but that its role is much more limited than it is in the uptake of LDL.

ApoE can associate with apoB-containing lipoproteins and facilitate receptor-mediated endocytosis by several receptors in the LDLR family (19). In addition, van Barlingen et al. (20) showed that enrichment of Lp[a] with apoE resulted in increased lipoprotein lipase-enhanced binding to heparan sulfate proteoglygans, concluding that the secretion-capture process of apoE was a possible catabolic route for Lp[a]. We examined the possible role of apoE in the catabolism of Lp[a]. In mice deficient in apoE, the plasma clearance of Lp[a] was slightly accelerated compared with that in wild-type mice (Fig. 5B), and there was a small increase in hepatic uptake (Fig. 6A). This suggests that Lp[a] catabolism is not primarily facilitated through the acquisition of apoE. In these mice, plasma clearance and hepatic uptake of LDL were greatly accelerated (Figs. 5A, 6B). Woollett et al. (49) made similar observations when they examined the catabolism of both mouse and human LDL in Apoe–/– mice. Their studies revealed that the increased catabolism of LDL was not attributable to an increase in hepatic LDLRs but rather to the lack of apoE-containing lipoproteins to compete for binding to the receptor.

Other members of the LDLR family have also been suggested to participate in Lp[a] catabolism. It has been shown that the VLDL receptor mediates the uptake and catabolism of Lp[a] in fibroblasts expressing the VLDL receptor and that this uptake is mediated by apo[a] (18). Furthermore, the plasma clearance of Lp[a] was delayed in VLDL receptor knockout mice. However, the VLDL receptor is found primarily in the heart, muscle, and adipose tissue (50) and is expressed only at very low levels in the liver. Hence, the VLDL receptor may play an important role in the apo[a]-mediated clearance of Lp[a] in certain nonhepatic tissues. Another candidate receptor, megalin/gp330, can mediate the uptake and degradation of Lp[a] in in vitro studies (16), but unlike the VLDL receptor, the uptake is primarily mediated by apoB-100. However, megalin/gp330 is found only in certain resorptive epithelial cells, such as the proximal tubules of the kidney, and is not found in the liver (51). Hence, it is unlikely that the VLDL receptor or megalin/gp330 participates significantly in the hepatic uptake of Lp[a]. The LRP is highly expressed by the liver, and one in vitro study suggested that it may act as a receptor for high molecular weight isoforms of Lp[a] (17), but other in vitro studies do not support this notion (44, 52). It is well known that the LRP can mediate the hepatic catabolism of chylomicron and VLDL remnants containing apoE (24). However, because apoE deficiency in mice did not reduce the hepatic uptake of 125I-TC-Lp[a], it is doubtful that the LRP plays a similar role in the catabolism of Lp[a].

We demonstrate here for the first time that the plasma clearance of Lp[a] by the liver is greatly reduced when coinjected with a large molar excess of apo[a]. These results strongly suggest that the apo[a] moiety of Lp[a] is the major ligand that mediates plasma clearance by the liver. In separate studies, labeled apo[a] has a similar pattern of tissue uptake to Lp[a], with the liver being the predominant site, consistent with Lp[a] and apo[a] being ligands for a common receptor(s). Even if Lp[a] and apo[a] largely share the same receptor(s), they are still different molecules and might be expected to have different binding kinetics. In addition, the presence of the large LDL moiety found in Lp[a] could affect its access to putative receptors to which apo[a] also binds. This could explain why apo[a] is cleared more than twice as fast as Lp[a], and why uptake by the liver is enhanced compared with that of Lp[a].

Apo[a] is a heavily sialylated protein (53), and if desialylated it is a possible ligand for the ASGPR, which is highly expressed in hepatocytes (54). Asialofetuin is a well-characterized ligand of the ASGPR that is rapidly cleared from the plasma by the liver (55). We found that asialofetuin at concentrations that greatly reduced its own plasma clearance rate had no effect on Lp[a] clearance. Hrzenjak et al. (25) showed that asialo Lp[a] is rapidly cleared from the plasma of mice and hedgehogs. In addition, they showed that in ASGPR-deficient mice the plasma clearance of native Lp[a] is delayed. However, in the absence of the ASGPR, Lp[a] clearance was only slowed by a small percentage, leading the authors to speculate that other mechanisms are involved in Lp[a] clearance. Indeed, the plasma clearance of Lp[a] expressed as a percentage of the injected dose was not significantly slower in ASGPR-deficient mice until 4 h after injection. We also saw a small but nonsignificant slowing of Lp[a] clearance in mice injected with asialofetuin, which was only apparent after 4 h (data not shown).

Keesler et al. (56) identified an apo[a] receptor that mediates the binding and internalization of apo[a] and Lp[a] in cholesterol-loaded macrophages. They showed that the region around kringle IV6–7 of apo[a] was important in receptor binding. Recently, Devlin et al. (57) suggested the existence of one or more receptors in the liver that bind to Lp[a] through the kringle IV5–8 region of apo[a] and that also mediate the plasma clearance of remnant lipoproteins.

In this study, we used Lp[a] possessing a single isoform of apo[a] with the same apparent molecular weight as apoB-100. It is possible that the catabolism of other isoforms may be somewhat different. However, the FCR is independent of isoform size in humans (3), and presumably the sites of tissue uptake are independent of isoform size as well. We believe that this is probably also true for mice, although that issue was not addressed in this study.

In conclusion, we demonstrate that the liver is the primary organ responsible for the uptake and catabolism of Lp[a] in mice, whereas the kidneys are responsible for only a small fraction of the tissue uptake. Neither the LDLR nor apoE plays an important role in the hepatic uptake of Lp[a]. We show that a large molar excess of apo[a] dramatically reduces the plasma clearance of Lp[a], which suggests that the apo[a] moiety plays a major role in mediating the plasma clearance and tissue uptake of Lp[a]. The nature of the major receptor(s) that mediate the plasma clearance and tissue uptake of Lp[a] remains to be determined.


   ACKNOWLEDGMENTS
 
The authors thank Anthony Secreto for expert technical assistance. This work was supported by Grants HL-59407 and HL-57811 from the National Heart, Lung, and Blood Institute and by Grant M01 RR-00040 from the National Center for Research Resources supporting the University of Pennsylvania General Clinical Research Center. U.J.F.T. is a recipient of research fellowship grants from the Deutsche Forschungsgemeinschaft and the American Heart Association, Pennsylvania-Delaware Affiliate. D.J.R. is an Established Investigator of the American Heart Association and a recipient of the Burroughs Wellcome Foundation Clinical Scientist Award in Translational Research.

Manuscript received June 14, 2005 and in revised form August 17, 2005 and in re-revised form August 23, 2005.


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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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