Atherosclerosis 154 2001 103 – 112
Essential role for the hepatic LDL receptor in macrophage apolipoprotein E-induced reduction in serum cholesterol levels and
atherosclerosis
Miranda Van Eck
a,
, Ko Willems Van Dijk
b
, Nicole Herijgers
a
, Marten H. Hofker
b
, Pieter H.E. Groot
c
, Theo J.C. Van Berkel
a
a
Di6ision of Biopharmaceutics, LeidenAmsterdam Center for Drug Research, Syl6ius Laboratories, Leiden Uni6ersity, P.O. Box
9503
,
2300
RA Leiden, The Netherlands
b
Department of Human Genetics, Syl6ius Laboratories, Leiden Uni6ersity, Leiden, The Netherlands
c
SmithKline Beecham Pharmaceuticals, Department of Vascular Biology, Harlow, Essex, United Kingdom Received 12 October 1999; received in revised form 2 March 2000; accepted 10 March 2000
Abstract
Apolipoprotein E apoE is a high affinity ligand for several receptor systems in the liver, including the low-density lipoprotein LDL receptor, and non-LDL receptor sites, like the LDL receptor-related protein LRP, the putative remnant receptor andor
proteoglycans. Although the liver is the major source of apoE synthesis, apoE is also produced by a wide variety of other cell types, including macrophages. In the present study, the role of the LDL receptor in the removal of lipoprotein remnants, enriched
with macrophage-derived apoE from the circulation, was determined using the technique of bone marrow transplantation BMT. Reconstitution of macrophage apoE production in apoE-deficient mice resulted in a serum apoE concentration of only 2 of the
concentration in wild-type C57Bl6 mice. This low level of apoE nevertheless reduced VLDL and LDL cholesterol 12-fold P B 0.001 and fourfold P B 0.001, respectively, thereby reducing serum cholesterol levels and the susceptibility to atherosclero-
sis. In contrast, reconstitution of macrophage apoE synthesis in mice lacking both apoE and the LDL receptor induced only a twofold P B 0.001 reduction in VLDL cholesterol and had no significant effect on atherosclerotic lesion development, although
serum apoE levels were 93 of the concentration in normal C57Bl6 mice. In conclusion, a functional hepatic LDL receptor is essential for the efficient removal of macrophage apoE-enriched lipoprotein remnants from the circulation and thus for
normalization of serum cholesterol levels and protection against atherosclerotic lesion development in apoE-deficient mice. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Apolipoprotein E; Bone marrow transplantation; mouse apoE ELISA www.elsevier.comlocateatherosclerosis
1. Introduction
Chylomicrons and
very-low-density lipoproteins
VLDL are processed by lipoprotein lipase after enter- ing the blood circulation, resulting in the formation of
cholesterol and apolipoprotein E apoE enriched lipo- protein remnants [1]. ApoE is a 34 kDa arginine-rich
lipoprotein, that serves as a high-affinity ligand for several receptor systems in the liver, including the low-
density lipoprotein LDL receptor, LDL receptor-re- lated protein LRP, proteoglycans, andor the putative
remnant receptor [2 – 7]. Targeted inactivation of the apoE gene in mice is associated with a prominent
increase in VLDL and LDL cholesterol levels, indicat- ing that apoE plays an important role in the removal of
these lipoproteins from the circulation [8 – 11]. Choi et al. found that an anti-LDL receptor antibody reduced
the liver uptake of lipoprotein remnants by 50, indi- cating that the LDL receptor plays a major role in the
clearance of remnant lipoproteins [12]. However, in humans, rabbits, and mice, lacking a functional LDL
receptor, mainly LDL accumulates and not remnant lipoproteins [13 – 15]. These data indicate that in vivo
back-up receptor systems are present that can compen-
Corresponding author. Tel.: + 31-71-5276238; fax: + 31-71- 5276032.
E-mail address
:
m.ecklacdr.leidenuniv.nl M. Van Eck. 0021-915001 - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0021-91500000471-8
sate for lipoprotein remnant clearance when the LDL receptor is absent. A likely other candidate for lipo-
protein remnant clearance is the LRP. LRP is a multi- functional receptor, that binds a broad range of
ligands, including apoE-enriched lipoproteins, lipo- protein lipase, and a
2
-macroglobulin-proteinase com- plexes [3,4]. A 39 k-Da receptor associated protein
RAP was also identified that binds with high affinity to the multiple binding sites of LRP, thereby blocking
the receptor-ligand interaction [16]. Recently, Willnow et al. demonstrated that transient inactivation of LRP
in vivo in wild-type mice by injection of an adenoviral vector containing RAP did not induce an increase in
lipoprotein remnants, whereas overexpression of RAP in the liver of LDL receptor-deficient mice resulted in a
massive accumulation of VLDL and LDL cholesterol [17]. Furthermore, liver-specific inactivation of LRP in
LDL receptor-deficient mice by conditional gene target- ing induced accumulation of mainly LDL cholesterol
and to a lesser extent VLDL cholesterol, whereas in wild-type animals this caused a compensatory up regu-
lation of the LDL receptor in the liver [18]. Herz et al. further elucidated that the initial hepatic removal of
remnant lipoproteins is largely independent of either the LDL receptor or LRP [19]. Endocytosis of surface
bound remnants in wild-type mice is predominantly mediated by the LDL receptor and in LDL receptor-
deficient animals by slow endocytosis via LRP. The current model for liver uptake of lipoproteins involves
two successive stages, including sequestration of lipo- protein remnants by proteoglycans on parenchymal
liver cells and subsequent internalization by either the LDL receptor or LRP. For uptake via LRP further
enrichment with exogenous apoE during the phase of sequestration is suggested to be required [20,21].
Although the liver is the major source of apoE synthesis, apoE is also produced by a wide variety of
other cell types, including macrophages [22 – 24]. Re- cently, by transplantation of wild-type bone marrow
into apoE-deficient mice we [25] and others [26 – 28] found that macrophage-derived apoE can reduce hyper-
cholesterolemia in apoE-deficient mice due to increased recognition and uptake of lipoprotein remnants by
parenchymal liver cells, leading to a decreased suscepti- bility to atherosclerosis.
The aim of the present study was to investigate the role of the hepatic LDL receptor in macrophage apoE
induced reduction in serum cholesterol levels and atherosclerosis. To address this question, mice solely
deficient for apoE and mice deficient for both apoE and LDL receptors are reconstituted with apoE positive
bone
marrow. Our
results demonstrate
that macrophage-derived apoE is unable to normalize hy-
percholesterolemia and
decrease susceptibility
to atherosclerosis in apoE-deficient mice, lacking the LDL
receptor, despite high levels of apoE in the circulation. Therefore, it is concluded that for efficient clearance of
remnant lipoproteins that are enriched with extrahep- atic apoE, a functional hepatic LDL receptor is
essential.
2. Materials and methods
