© 1997 American Heart Association, Inc.
Articles |
Effect of Bone Marrow Transplantation on Lipoprotein Metabolism and Atherosclerosis in LDL Receptor–Knockout Mice
From the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Leiden University, Leiden, Netherlands (N.H., M. van E., Th.J.C. van B.); SmithKline Beecham Research and Development, Harlow, UK (P.H.E.G.); and the Department of Pediatrics, University Hospital, Leiden, Netherlands (P.M.H.).
Correspondence to Dr Nicole Herijgers, Division of Biopharmaceutics, Sylvius Laboratories, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, the Netherlands. E-mail Herijgers{at}LACDR.LeidenUniv.nl
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Abstract The LDL receptor (LDLR) plays an important role in the removal of LDL and its precursors, the intermediate and very low density lipoproteins, from the blood circulation. The receptor is expressed on various cell types. In this study the relative importance of the LDLR on macrophages for lipoprotein metabolism and atherogenesis was assessed. For this purpose, irradiated LDLR-knockout (-/-) mice were transplanted with bone marrow of normal C57BL/6J mice. DNA analysis showed that the transplanted mice were chimeric. The transplantation resulted in a slight decrease of total serum cholesterol when compared with LDLR-/- mice that were transplanted with LDLR-/- bone marrow. This modest decrease, however, did not reach statistical significance at all time points examined. This decrease can be almost completely attributed to a decrease in LDL cholesterol. The specific lowering of LDL cholesterol could clearly be observed at 4 weeks after transplantation, but the decrease was less at 12 weeks after transplantation. Quantification of atherosclerotic lesions of mice fed a 1% cholesterol diet for 6 months revealed that there were no differences in mean lesion area between mice transplanted with wild-type bone marrow or LDLR-/- bone marrow. We anticipate that in LDLR-/- mice transplanted with wild-type bone marrow, the LDLR is downregulated by the relatively high concentrations of circulating cholesterol. In vitro incubations of peritoneal macrophages with 125I-LDL indicated that the LDLR of these cells could be downregulated by 25-hydroxycholesterol. Peritoneal macrophages isolated from LDLR-/- mice transplanted with wild-type bone marrow, in contrast to those transplanted with LDLR-/- bone marrow, were able to degrade 125I-LDL, indicating that the capacity to express functional LDLR was achieved. In conclusion, introduction of the LDLR into LDLR -/- mice via bone marrow transplantation resulted in only a relatively modest decrease of LDL cholesterol that became less pronounced at later time points, possibly due to downregulation of the LDLR. To utilize the LDLR in macrophages for effective cholesterol lowering, either the sterol-regulatory elements have to be "silenced" or a high-expression LDLR construct has to be introduced into macrophages, eg, via transplantation of in vitro transfected hematopoietic stem cells.
Key Words: LDL receptor • atherosclerosis • lipoprotein metabolism • gene transfer • macrophages • Kupffer cells
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Introduction |
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The LDLR plays an important role in the regulation of plasma cholesterol level by determining the cellular uptake of LDL and its precursors, the intermediate density lipoproteins (IDLs) and VLDLs.1 The LDLR is expressed in a wide variety of tissues: the liver, aortic endothelium, muscle, adipose, steroid hormone–producing glands, and the skin. The majority of LDLR activity, however, is located in the liver, the only organ in which cholesterol can be irreversibly removed from the body via secretion into the bile.2 In rats, the Kupffer cell was found to be the main liver cell type responsible for the receptor-dependent catabolism of LDL.3 4 In the human liver, expression of the LDLR on Kupffer cells is relatively constant in contrast to its expression on parenchymal cells, which can be influenced by differences in genetic background, hormone status, and lipid content of the diet.5 6 Studies with isolated human liver parenchymal and Kupffer cells demonstrated that the association of LDL with Kupffer cells was more effectively coupled to catabolism rather than LDL's association with parenchymal cells.7 Thus, from these studies it can be concluded that at least in humans and rats, Kupffer cells can be important for the catabolism of LDL, especially under conditions when the LDLR on parenchymal cells is downregulated.
FH is a common hereditary disorder characterized by the absence (homozygous) or depleted numbers (heterozygous) of active LDLRs.8 9 The plasma levels of LDL in these patients are elevated, resulting in hypercholesterolemia and premature atherosclerosis. Recently, Herz and coworkers developed a mouse model for homozygous FH through targeted disruption of the LDLR gene: the LDLR-knockout (LDLR-/-) mouse.10 The total plasma cholesterol levels in these mice appeared to be twofold higher than those of wild-type littermates, mainly as a consequence of an increase in IDL and LDL, and the clearance of VLDL, IDL, and LDL from the plasma of these mice is delayed. On a cholesterol-rich diet, these mice exhibit massive xanthomatosis and atherosclerosis.11 Therefore, they form an excellent model to investigate the role of the LDLR in lipoprotein metabolism.
Uptake of LDL by Kupffer cells does not result in intracellular accumulation of cholesteryl esters, because it is coupled to transport to liver parenchymal cells and biliary secretion.12 13 Thus, these cells can form a potentially important target for the treatment of FH. BMT thereby allows specific study of the role of the LDLR on Kupffer cells and other macrophages in the clearance of cholesterol from the circulation. Introduction of wild-type hematopoietic stem cells into irradiated LDLR-/- mice may lead to the appearance of cells of hematopoietic origin with an intact LDLR gene. A long-standing controversy exists whether liver macrophages are derived from blood monocytes14 or whether they originate from precursor cells in the fetal liver and survive by local proliferation and self-renewal.15 16 17 18 Recently, however, Takezawa et al19 showed with fluorescence labeling that bone marrow cells in vivo can differentiate into Kupffer cells in the liver.
In our study, Kupffer cells and other macrophages of LDLR-/- mice were replaced by cells of WT mice by BMT. Our results indicate that transplantation can lead to a lowering of serum cholesterol, especially IDL/LDL cholesterol, although this lowering is insufficient to normalize cholesterol levels.
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Mice
Homozygous LDLR-knockout mice (LDLR-/-) were obtained from The Jackson Laboratory (Bar Harbor, Me) as mating pairs and bred in the Gaubius Laboratory, Leiden, the Netherlands. These mice were hybrids of the C57BL/6J and 129 Sv strains (F4 generation of backcrosses to C57BL/6J). C57BL/6J mice were obtained from the Gaubius Laboratory, Leiden, the Netherlands. The animals were housed in sterile filter-top cages. They were fed standard rat/mouse chow (SMR-A, Hope Farms). For analysis of atherosclerosis, the animals were fed a 1% cholesterol diet (Hope Farms) for up to 6 months. The sterile drinking water contained 67 mg/L polymyxin B, 84 mg/L ciprofloxacin, 5x10-4 mol/L HCl, and 6 g/L sugar. The experiments were approved by the ethics committee on animal experiments of the University of Leiden.
Irradiation and BMT
Female recipient mice (6 to 8 weeks old) were subjected to 13 Gy
of total-body irradiation (Roentgen source). Bone marrow was harvested
by flushing the femurs and tibias of 8- to 10-week-old male donor mice
with cold PBS. The cells were washed twice with PBS. Recipients
received 1x107 bone marrow cells by tail vein injection 24
hours after irradiation.
Serum Cholesterol and Lipoprotein Analyses
Blood samples (50 µL) were obtained by tail bleeding. Total
serum cholesterol was determined by enzymatic procedures
(Boehringer). Standardized serum (Precipath) was used as an
internal standard (Boehringer). Lipoproteins were fractionated
using the SMART system from Pharmacia. Serum (35 µL) was applied to
the column and eluted with a buffer containing 150 mmol/L
NaCl and 1 mmol/L EDTA. Fractions (38x 0.5 mL) were
collected and assayed for their total cholesterol
content.
Isolation of Spleen and Liver Cells
Mice were anesthetized by
intraperitoneal injection of sodium pentobarbital
(70 to 90 mg/kg body weight). The superior vena cava was
cannulated and the liver perfused at 37°C with 100 mL
oxygenated buffer containing 10 mmol/L HEPES,
142 mmol/L NaCl, and 16.7 mmol/L KCl at a rate
of 14 mL/min. After preperfusion, the liver was perfused with 50 mL
DMEM (GIBCO) containing 2% BSA and 0.05% collagenase
(type IV, Sigma). Liver cells were dissociated in Hanks' buffer
containing 0.2% BSA, filtered through a 250-µm filter, and
centrifuged (50g for 30 seconds). Parenchymal liver
cells were collected in the pellet, and the supernatant contained the
nonparenchymal liver cells. Spleen cells were obtained by forcing the
spleen through a 250-µm filter in PBS. The cells were washed three
times before analysis.
Isolation of DNA
Cells were lysed overnight at 55°C in a denaturing buffer
containing 10 mmol/L Tris HCl (pH 7.5), 150
mmol/L NaCl, 25 mmol/L EDTA (pH 7.5), 0.5% SDS, and
0.1 mg/mL proteinase K (Boehringer). DNA was extracted
twice with phenol/chloroform (1:1, vol/vol) and
precipitated with 0.3 mol/L sodium acetate and an equal
volume of isopropanol. After being washed in 70% ethanol, the DNA was
dissolved in 10 mmol/L Tris HCl (pH 7.5) and 0.1
mmol/L EDTA (pH 7.4) and quantified by spectrophotometry at 260
nm.
Southern Blot Analysis
Genomic DNA (10 µg) was digested with BamHI
(Biolabs) and fractionated on a 0.7% agarose gel. DNA was transferred
to a Hybond-N membrane (Amersham) by capillary blotting. Membranes was
prehybridized for 1 hour at 65°C in 15 mL hybridization mix
containing 0.5 mol/L NaPi, 7% SDS, and 1 mmol/L
EDTA. The LDLR probe (MLDL C90) has been previously described by Hoffer
et al.20 It consists of exons 2 to 4 of the murine LDLR
cDNA. Because of the presence of an extra BamHI site in the
neo expression vector, it is possible to distinguish WT LDLR
DNA from the disrupted LDLR DNA. The probe was labeled with
32P[dCTP] using the Multiprime DNA Labeling Kit
(Amersham). Hybridization was carried out at 65°C overnight.
Membranes were washed with a final stringency of 2x SSC buffer
containing 0.1% SDS. The membranes were exposed to Scientific Imaging
films (Kodak).
Peritoneal Macrophage Harvesting
Five days after peritoneal injection of 1 mL of 3% Brewer's
thioglycollate medium (DIFCO), peritoneal macrophages were
harvested by lavage of the peritoneum with 10 mL PBS/1
mmol/L EDTA. After three wash steps, the cells
(2.5x106) were plated out in 25-mm multiwell culture
dishes (Costar) with RPMI 1640 (DIFCO) containing 10% FCS, 2
mmol/L glutamine, 100 µg/mL streptomycin, and 100 IU/mL
penicillin. After 4 hours the nonadherent cells were removed by
washing, and the culture medium was replaced by RPMI 1640 containing
10% human lipoprotein–deficient serum, 2 mmol/L
glutamine, 100 µg/mL streptomycin, and 100 IU/mL penicillin.
To induce maximal expression of LDLRs, the cells were cultured for 2
days. When indicated, the cells were incubated with
25-hydroxycholesterol (Sigma) in medium for 20 hours at
37°C prior to the experiment.
LDL Isolation and Metabolism
LDLs were obtained from the sera of healthy volunteers. The sera
were centrifuged at 250 000g in a discontinuous KBr
gradient for 18 hours as reported by Redgrave et al.21 The
fraction of d=1.019 to 1.063 g/mL was
recentrifuged and dialyzed against PBS containing 1
mmol/L EDTA. LDL was labeled with 125I at pH 10.0
according to McFarlane22 as modified by Van Tol et
al.23 Free 125I was removed by Sephadex G50
gel filtration followed by dialysis against 1 mmol/L
PBS/EDTA.
LDL degradation studies were carried out with the indicated amounts of 125I-LDL for 3 hours at 37°C. Incubations of the cells were performed in RPMI 1640 (DIFCO) containing 2% (wt/vol) BSA in a total volume of 0.5 mL. After incubation the cells were washed three times with wash buffer (50 mmol/L Tris HCl, pH 7.4, containing 0.9% NaCl, 1 mmol/L EDTA, 5 mmol/L CaCl2, and 0.2% [wt/vol] BSA) followed by two washes with wash buffer without BSA. The cells were lysed in 0.1 mol/L NaOH and the cell protein content determined by the method of Lowry et al.24 Degradation of the radiolabeled LDL was determined as follows: to 0.5 mL of the medium, 0.4 mL of 35% trichloroacetic acid and 10 µL of 20% KI was added.25 The mixture was incubated for 30 minutes at 4°C and subsequently 0.25 mL of 0.7 mol/L AgNO3 was added. After centrifugation (5 minutes at 16 000g), the radioactivity in the supernatant was determined.
Analysis of Atherosclerosis
LDLR-knockout mice that were transplanted with either WT or
knockout bone marrow 5 months earlier, control C57BL/6J mice, and
LDLR-knockout mice were fed a 1% cholesterol diet for up
to 6 months. The mice were killed and the heart and vascular tree
perfused in situ with oxygenated Krebs'/Ringer's
bicarbonate buffer at 37°C under a pressure of 100 mm Hg for 30
minutes via a cannula in the left ventricle. The buffer was then
replaced by 3.7% neutral-buffered formalin (3.7% formaldehyde;
Formal-fix, Shandon Scientific Ltd), and the tissue was fixed during a
30-minute perfusion. Hearts and aortas were excised and stored in
formalin. The hearts were bisected just below the atria, and the base
of the heart and aortic root were taken for analysis. Cryostat
10-µm cross sections of the aortic root were taken and stained with
oil red O as described before.26 Atherosclerotic lesions
in the sections were quantified by using a light microscope connected
to a video camera and running optimas software version 5.1 (Bioscan
Inc). Mean lesion area was calculated from the first 10 sections in the
direction of the aortic arch from the point where all three aortic
valve leaflets first appeared, as previously
described.26
Statistical Analysis
Statistical analysis of the data was performed by ANOVA
and the Bonferroni t test.
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Results |
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Detection of Chimerism Following BMT
To assess chimerism following BMT, DNA of the spleen, bone marrow, and liver were isolated from the transplanted mice at different times (13 weeks to 6 months after transplantation) and analyzed by Southern blot analysis. In Fig 1
, examples of such
analyses are shown at 6 months after BMT for an LDLR-/- mouse
that was transplanted with WT bone
marrow. Hybridization of the MLDL C90
probe with spleen and bone marrow DNA resulted in a 16-kb band only,
showing the presence of WT donor LDLR DNA. Hybridization with liver
parenchymal cell DNA resulted in two bands (11 and 5.5 kb). These bands
are characteristic of recipient DNA, because the disrupted LDLR gene in
LDLR-/- mice contains an additional BamHI site.
Hybridization with liver nonparenchymal cell DNA resulted in both the
16-kb and the 11- and 5.5-kb bands, showing that cells from both donor
and recipient were present in this preparation. These results were
confirmed by polymerase chain reaction analysis (data not
shown). DNA analysis of the other mice in this study gave
similar results. The chimerism appeared to be steady in time (13 weeks
to 6 months). Taken the results from the Southern blots together, it
can be concluded that the spleen, bone marrow, and liver nonparenchymal
cells show a steady chimerism after transplantation.
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Effect of BMT on Total Serum Cholesterol Level
During the weeks after BMT, the total serum
cholesterol levels of the transplanted mice were repeatedly
determined. The results are shown in Fig 2. At 4 weeks after BMT, the
cholesterol level decreased in both the +/+ to -/-
transplanted mice and the -/- to -/- transplanted mice compared
with nontransplanted LDLR-/- mice. Because the decrease was
present in both transplanted groups, it suggested an effect of BMT
itself. The decrease in the -/- to -/- group, however, lasted only
until week 6 after BMT and was maximally 26% (P<.05)
compared with the untreated group. In the +/+ to -/– group, the
effect continued during the experiment (12 weeks) and reached a maximum
decrease of 42% (P<.001) in comparison with the control
group. When compared with the -/- to -/- group, the +/+ to -/-
group had lower total cholesterol values, which were
significant at 6 and 12 weeks after BMT. The decrease was maximally
27% (P<.001). These data thus show that introduction of
the LDLR in Kupffer cells and other macrophages in LDLR-/-
mice results in a decrease of total serum cholesterol,
although this is not sufficient to normalize cholesterol
levels.
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Effect of BMT on Distribution of Serum Lipoprotein
Cholesterol
The effect of BMT on the distribution of serum
cholesterol in the different lipoprotein classes was
analyzed by liquid chromatography. At 4 weeks
after BMT, the +/+ to -/- group showed a considerable decrease in LDL
cholesterol (Fig 3B); this
decrease was 46% (P<.001) compared with the
nontransplanted LDLR-/- mice (Fig 3A) and 37% (P<.01)
compared with the -/- to -/- group (Fig 3B). Although the decrease
in LDL cholesterol of the +/+ to -/- group was evident,
the level still remained higher than that of the control C57BL/6J mice
(Fig 3A). The amount of cholesterol in HDL remained
unchanged after BMT.
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;
n=4), closed squares represent C57BL/6J mice (
; n=2),
open circles represent LDLR-/- mice transplanted with
LDLR-/- bone marrow (
; n=6), and closed circles
represent LDLR-/- mice transplanted with WT bone marrow
(
; n=5). For clarity, only the SD of the fraction containing the
top of the IDL/LDL cholesterol peak is given. *Significant
difference vs -/- to -/- transplanted mice at P<.01
(Fig 4B
At 12 weeks after BMT, the +/+ to -/- group showed a decrease of 22%
(P<.05) in comparison with the control LDLR-/- mice and
19% (P<.05) in comparison with the -/- to -/- group
(Fig 3C
). When compared with 4 weeks after BMT, the +/+ to -/- group,
however, had a less pronounced decrease in LDL cholesterol.
Again, no significant difference in HDL cholesterol was
observed. These lipoprotein profiles thus show that introduction of the
LDLR into hematopoietic cells of LDLR-/- mice lowers LDL
cholesterol content and that this lowering was more
pronounced at 4 weeks than at 12 weeks after BMT.
Effect of BMT on 125I-LDL Metabolism by
Peritoneal Macrophages
Synthesis of the LDLR is susceptible to feedback inhibition by
intracellular cholesterol.27 Since LDLR-/-
mice have elevated serum cholesterol levels,10
it might be possible that the LDLR that has been introduced into
LDLR-/- mice by BMT is (partially) downregulated. It is therefore of
interest to investigate whether macrophages in LDLR-/- mice
that are transplanted with WT bone marrow have potentially obtained the
capacity to express functional LDLR and whether this LDLR can be
downregulated by cholesterol. To address this question,
peritoneal macrophages were isolated from mice at 20 to 25
weeks after BMT, and these cells were cultured for 2 days in medium
containing lipoprotein-deficient serum to induce maximal expression of
LDLR.
In Fig 4A, the degradation of
125I-LDL is shown. Degradation by macrophages of
the -/- to -/- group is comparable to that by macrophages
of the LDLR-/- mice. Macrophages of the +/+ to -/- group
showed a sixfold increase in degradation, though not as high as that of
macrophages of C57BL/6J mice (88%). When preincubated with
increasing concentrations of 25-hydroxycholesterol, the
degradation of 125I-LDL by macrophages of C57BL/6J
mice was decreased (Fig 4B), whereas degradation by macrophages
of LDLR-/- mice remained constantly low. This indicates that the LDLR
on peritoneal macrophages is susceptible to downregulation by
25-hydroxycholesterol. It thus appears that peritoneal
macrophages of +/+ to -/- transplanted mice, in contrast to
-/- to -/- transplanted mice, do have the capacity to express
functional LDLRs and that these LDLRs can be downregulated by
25-hydroxycholesterol.
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; n=4), and
(-/- to -/-) transplanted mice (
; n=2) were isolated and
cultured in lipoprotein-deficient medium for 2 days. A, Degradation of
125I-LDL by macrophages (3 hours at 37°C). B,
Degradation of 125I-LDL (25 µg/mL) by macrophages
of C57BL/6J (
Effect of BMT on Atherosclerosis
To investigate the effect of introducing the LDLR into LDLR-/-
mice via BMT on the development of atherosclerotic lesions,
transplanted mice, control LDLR-/- mice, and C57BL/6J mice were fed a
1% cholesterol diet for 6 months. The
cholesterol levels of the LDLR-/- mice, the -/- to
-/- transplanted mice, and the +/+ to -/- transplanted mice
increased about fourfold, whereas the cholesterol level of
C57BL/6J mice did not change significantly (Fig 5).
Tangirala et al28 found a
comparable increase of cholesterol levels in
LDLR-/- mice that were fed the same diet. No significant differences
in total serum cholesterol values between the LDLR-/-
control and transplanted mice were observed at the indicated time
points. After 6 months of the diet, the hearts and aortas were perfused
and fixed. Cross sections of the aortic root were examined, and
representative photomicrographs of the aortic valves
are shown in Fig 6. The mean lesion area
in the aortic root was calculated and is presented in Fig 7.
The cross sections of C57BL/6J mice hardly showed any lesions, whereas
extensive lipid-rich lesions could be demonstrated in LDLR-/- mice.
The increase in mean lesion area in LDLR-/- mice was approximately
sevenfold (P<.001). Cross sections of the transplanted
LDLR-/- mice, both -/- to -/- and +/+ to -/-, showed lesions
that were even more advanced than those of the LDLR-/- control mice
because of the presence of calcified areas in these sections. The mean
lesion area was increased approximately 1.5-fold (P<.05 in
case of LDLR-/- versus +/+ to -/-; P<.01 in the case of
LDLR-/- versus -/- to -/-). Thus, the BMT procedure itself
aggravated atherosclerosis. No statistically
significant differences in mean lesion area could be demonstrated
between the -/- to -/- and +/+ to -/- transplanted mice.
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BMT into knockout mice provides a unique method to study the role of an individual gene product from macrophages in various biological processes. Recently, this technique was successfully used by several groups to elucidate the role of apoE production by macrophages in lipoprotein metabolism and atherosclerosis29 30 . It appeared that the introduction of apoE-producing macrophages in apoE-knockout mice was sufficient to reduce the severe hypercholesterolemia and atherosclerosis which are characteristic features in these mice. In the present study we investigated the role of the LDLR on macrophages in lipoprotein metabolism and the development of atherosclerosis. The LDLR-positive hematopoietic cells were introduced into the LDLR-/- mice via transplantation of WT bone marrow. Transplantation resulted in a moderate decrease of total serum cholesterol, especially LDL cholesterol. The decrease, however, was not sufficient to normalize the serum cholesterol levels.
De Vries and Vos31 showed that 4 weeks after total-body
irradiation and subsequent BMT, the hematopoietic system is normalized.
In our study, bone marrow, liver nonparenchymal cells, and the spleen
of LDLR-/- mice that were transplanted with WT bone marrow did
contain the WT LDLR gene as shown by Southern blot analysis
(Fig 1). This chimerism appeared to be steady in time (13 weeks to 6
months). In the weeks after transplantation, total serum
cholesterol levels were determined frequently. At 4 weeks
after BMT, the cholesterol levels dropped in both the +/+
to -/- group and the -/- to -/- group. This drop in
cholesterol, independent of the type of bone marrow, is
probably caused by irradiation and/or the transplantation method
itself. During the repopulation of the hematopoietic system,
macrophages may become activated, which could result in
stimulation of the LDLR-independent uptake of cholesterol.
On the other hand, the influx of cholesterol into the
plasma may also be influenced by the irradiation and/or transplantation
procedure, as we recently observed in apoE-knockout mice. In these
animals, VLDL production by the liver decreased after BMT
(unpublished results, 1996). In the weeks after the initial drop in
cholesterol, the level in the (-/- to -/-) mice returned to
that of the control LDLR-/- mice, whereas the levels in the +/+ to
-/- group remained lower (Fig 2). Introduction of the LDLR into
LDLR-/- mice, therefore, can result in a decrease of total serum
cholesterol levels, although this is clearly not sufficient
to normalize serum cholesterol levels.
Using liquid chromatography we showed that the decrease
in total serum cholesterol in the +/+ to -/- group after
BMT can be almost entirely attributed to a lowering in LDL
cholesterol. The lipoprotein distribution pattern of the
LDLR-/- mice when compared with C57BL/6J mice showed an approximate
ninefold increase in LDL cholesterol (Fig 3A), as also
reported by Ishibashi et al.10 Transplantation of WT bone
marrow into LDLR-/- mice resulted in a 50% reduction of LDL
cholesterol after 4 weeks compared with control
-/- to -/- animals, whereas HDL cholesterol remained
unchanged (Fig 3B). At 12 weeks after BMT, the significant decrease in
LDL cholesterol was still present though less
pronounced than at 4 weeks after BMT. These data suggest that the
effect of introducing LDLR into LDLR-/- mice on
cholesterol metabolism is transient, although
these animals show a steady chimerism. One mechanism underlying this
observation might be the development of an immunological response to
the LDLR protein, which is a foreign-body protein in the LDLR-/-
mouse. Such antibodies might prevent proper functioning of the
introduced LDLR. On the other hand, the immune systems of donor mice,
those "familiar" with the LDLR protein, are also transferred during
transplantation, so one would not expect an immunological response
against the LDLR protein. On the receptor synthesis level, regulatory
mechanisms may also occur. Synthesis of the LDLR is subject to feedback
inhibition by intracellular cholesterol.27
From in vitro studies with cultured human fibroblasts, it is known that
LDLR downregulation by cholesterol can occur within 1
day.32 In vivo, however, a longer time is needed.
Srivastava et al33 described that in vivo regulation of
the LDLR by dietary cholesterol in mice can be observed 2
to 3 weeks after the start of the diet. Complete downregulation of the
receptor may take even a longer time. In our study we demonstrated that
the LDLR on murine peritoneal macrophages could be
downregulated by 25-hydroxycholesterol.
25-Hydroxycholesterol is an oxysterol and, like LDL itself,
a known potent inhibitor of LDLR synthesis in cultured
cells.34 Our studies on the catabolism of LDL by
peritoneal macrophages isolated from +/+ to -/- groups
indicated that the LDLR is functionally present in these
macrophages when cultured in lipoprotein-deficient serum, thus,
under conditions that the LDLR is upregulated (Fig 4A). This is in
contrast to peritoneal macrophages of the control -/- to
-/- transplanted group, which hardly showed any catabolism of LDL.
These results, therefore, support the suggestion that the LDLR in
transplanted LDLR-/- mice may be subject to downregulation in
vivo.
Transplantation of WT bone marrow into LDLR-/- mice will not only result in introduction of the LDLR into liver Kupffer cells but also lead to the presence of the LDLR gene in macrophages that are located in the arterial wall. The role of the LDLR on these arterial wall macrophages in mediating foam cell formation is unclear yet; controversy exists whether the expression of the LDLR in these arterial wall macrophages does mediate an increase in foam cell formation. According to Aviram and Rosenblat,35 binding of LDL to the LDLR is essential for macrophage-mediated oxidation of LDL. Tangirala et al,36 however, concluded from studies with peritoneal macrophages isolated from LDLR-/- mice that oxidative modification of LDL occurs independent of its binding to the LDLR. In our study, we quantified the aortic atherosclerotic lesions in control and transplanted mice. The mean lesion areas of the control LDLR-/- mice did largely correspond to those in the earlier publications of Tangirala et al.28 The -/- to -/- transplantation itself, however, already did cause a 1.5-fold increase in mean lesion area when compared with LDLR-/- control mice. This effect cannot be attributed to a change in serum cholesterol concentration because the cholesterol level of transplanted mice did not differ significantly from that of nontransplanted LDLR-/- mice. Numerous studies in humans, mice, and other animal models have shown that irradiation causes vascular damage and an accelerated development of atherosclerosis.31 37 38 It is suggested that the initial site of damage is the endothelial cell lining of the vessel wall.39 These studies are therefore in accordance with the observed aggravated atherosclerosis. No statistically significant differences in mean lesion area could be observed between the -/- to -/- and the +/+ to -/- transplanted group. Under the present conditions the absence or presence of the gene for the LDLR on macrophages thus does not influence the overall atherosclerotic process.
Very recently, Boisvert et al40 and Fazio et al41 reported comparable studies on BMT in LDLR-/- mice. Boisvert et al,40 but not Fazio et al,41 could detect a slight decrease in LDL cholesterol in +/+ to -/- transplanted mice. These results are generally in accordance with our data except that in addition, we found a much larger decrease in LDL cholesterol at an earlier time after BMT (4 weeks after BMT), suggesting that the decrease is temporary. Our quantitative studies of atherosclerosis were in agreement with the qualitative studies by Boisvert et al,40 namely, that the lesions in +/+ to -/- mice were not different from those in -/- to -/- mice. Fazio et al41 could not detect differences in binding of 125I-LDL by peritoneal macrophages of either +/+ to -/- mice or -/- to -/- mice, although the cells were cultured for 6 hours in lipoprotein-deficient medium. When measuring the degradation of 125I-LDL, we were able to clearly discriminate peritoneal macrophages of +/+ to -/- mice from those of -/- to -/- mice, proving the transplantation successful. Fazio et al41 also speculated on an induced downregulation of the LDLR in transplanted cells by the high plasma cholesterol concentration in LDLR-/- mice, which we could clearly indicate in establishing the effect of 25-hydroxycholesterol on expression of the LDLR in peritoneal macrophages.
In future studies we plan to overexpress the gene for the LDLR in macrophages to lower LDL-levels to a more beneficial extent. In the past, several studies have been performed in which the LDLR was overexpressed in parenchymal liver cells. In Watanabe heritable hyperlipidemic rabbits,42 43 FH patients,44 and LDLR-/- mice,10 this gene transfer resulted in correction of the LDLR deficiency. Also in normal mice, adenovirus-mediated transfer of LDLR accelerated cholesterol clearance,45 demonstrating the important role of the LDLR for LDL turnover. So far, no reports have been published in which the effects of overexpression of the LDLR gene in macrophages are described. In rats, the LDLR on Kupffer cells plays a significant role in the catabolism of LDL.3 4 Whether murine Kupffer cells play a similar important role is not yet known. Srivastava et al33 46 demonstrated that the LDLR is expressed in the mouse liver, although no distinction between parenchymal liver cells and Kupffer cells was made.
In conclusion we have shown that transplantation of WT bone marrow into LDLR-/- mice does result in a lowering of serum cholesterol, especially IDL/LDL cholesterol. This effect is not sufficient to normalize the cholesterol levels and appears to be temporary. No differences in atherosclerosis were observed in mice transplanted with WT bone marrow and LDLR-/- bone marrow. We suggest that the LDLR that is introduced into LDLR-/- mice via BMT is downregulated by the sustained increased serum cholesterol levels. Further studies with macrophages overexpressing the LDLR, or expressing the LDLR in a cholesterol-insensitive way, may lead to a more beneficial effect on cholesterol levels and possibly atherosclerosis.
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Acknowledgments |
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This research was supported by the Netherlands Heart Foundation (project 93188). We are grateful to Dr M.H. Hofker for providing the MLDL C90 plasmid. We would like to thank M. Vidgeon-Hart for expert technical help with the sectioning, J. Yates for technical support with the SMART system, and Dr G.M. Benson for helpful assistance with the image analysis of atherosclerotic lesions.
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Footnotes |
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This work was presented at the 69th Scientific Sessions of the American Heart Association, November 11-13, 1996, New Orleans, La, and published in abstract form in Circulation. 1995;94(suppl I):I-697.
Received March 24, 1997; accepted May 12, 1997.
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