Effect of Up-regulating Individual Steps in the Reverse
Cholesterol Transport Pathway on Reverse Cholesterol Transport in
Normolipidemic Mice*
Khairul
Alam,
Robert S.
Meidell, and
David K.
Spady
From the Department of Internal Medicine, the University
of Texas Southwestern Medical Center, Dallas, Texas 75235
Received for publication, November 9, 2000, and in revised form, February 8, 2001
 |
ABSTRACT |
Cholesterol acquired by extrahepatic tissues
(from de novo synthesis or lipoproteins) is returned to the
liver for excretion in a process called reverse cholesterol transport
(RCT). We undertook studies to determine if RCT could be enhanced by
up-regulating individual steps in the RCT pathway. Overexpression of
7
-hydroxylase, Scavenger receptor B1, lecithin:cholesterol
acyltransferase (LCAT), or apoA-I in the liver did not stimulate
cholesterol efflux from any extrahepatic tissue. In contrast, infusion
of apoA-I·phospholipid complexes (rHDL) that resemble nascent HDL
markedly stimulated cholesterol efflux from tissues into plasma.
Cholesterol effluxed to rHDL was initially unesterified but by 24 h this cholesterol was largely esterified and had shifted to normal HDL
(in mice lacking cholesteryl ester transfer protein) or to apoB
containing lipoproteins (in cholesteryl ester transfer protein
transgenic mice). Most of the cholesterol effluxed into plasma in
response to rHDL came from the liver. However, an even greater
proportion of effluxed cholesterol was cleared by the liver resulting
in a transient increase in liver cholesterol concentrations. Fecal sterol excretion was not increased by rHDL. Thus, although rHDL stimulated cholesterol efflux from most tissues and increased net
cholesterol movement from extrahepatic tissues to the liver, cholesterol flux through the entire RCT pathway was not increased.
 |
INTRODUCTION |
Cholesterol that has been acquired by extrahepatic tissues
(from de novo synthesis or lipoproteins) is returned to the
liver for excretion in a process called reverse cholesterol transport (RCT)1 (1-3). The first step
in the RCT pathway is efflux of cholesterol from cell membranes to
nascent HDL in interstitial fluid (2, 3). Nascent HDL are discoidal
pre-
-migrating complexes of phospholipid and apoA-I (other
amphipathic apoproteins such as apoE and apoA-IV may also be present).
These particles are secreted by the liver (4, 5) and small intestine
(6) and are also formed during the metabolism of triglyceride-rich
lipoproteins from excess surface material. In addition, lipid-free
apoA-I can mediate the efflux of cholesterol and phospholipid from
cells generating pre
-migrating nascent HDL (7, 8). ATP-binding cassette transporter 1 (ABCA1) appears to play a key role in this process although the exact mechanism is unclear (9-12). Cholesterol that is transferred to nascent HDL is esterified by
lecithin:cholesterol acyltransferase (LCAT) to cholesteryl esters,
which by virtue of their hydrophobicity move into the core of the HDL
particle resulting in the formation of
-migrating spherical HDL. HDL
cholesteryl esters are cleared from plasma mainly by the liver
(13-16). HDL cholesteryl ester uptake by the liver is mediated by the
scavenger receptor BI (SR-BI), which selectively transports HDL
cholesteryl esters resulting in an HDL particle of reduced size and
cholesteryl ester content (17, 18). In species with cholesteryl ester transfer protein, a portion of HDL cholesteryl ester is transferred to
lower density apoB-containing lipoproteins and ultimately cleared mainly by the liver via the LDL receptor pathway (19). The final step
in the RCT pathway is excretion of cholesterol from the liver into
bile, either directly or after conversion to bile acids.
Although individual steps in the RCT pathway have been studied in
detail, little is known about what regulates cholesterol flux through
the entire pathway. The main obstacle to such studies has been the
inability to directly quantify cholesterol flux in the RCT pathway
in vivo. One approach that has been used in animal models is
to quantify the rate of cholesterol acquisition in extrahepatic tissues
as a measure of cholesterol flux through the RCT pathway because in a
steady state the rate of cholesterol acquisition by extrahepatic
tissues (from de novo synthesis and lipoproteins) equals the
rate of cholesterol return to the liver for excretion (with the
exception of cholesterol that is converted to steroid hormones or lost
when cells are sloughed from the gastrointestinal tract or skin).
Studies using this approach have shown that plasma HDL cholesterol
concentrations can be varied over a wide range as a result of diet or
genetic manipulation with no change in the rate of reverse cholesterol
transport (20-22).
The purpose of the current studies was to determine if enhancing
individual steps in the RCT pathway increases cholesterol flux through
the entire pathway. The RCT pathway involves a series of steps
beginning with cholesterol efflux from cells in extrahepatic tissues
and ending with the excretion of this cholesterol by the liver and its
loss in the feces. If one of these steps is rate-limiting, then
up-regulation of this step may result in increased cholesterol flux
through the entire pathway. Conversely, if all steps are functioning at
maximal capacity or if cholesterol can be diverted out of the pathway
at one or more points then up-regulating individual steps may not
increase cholesterol flux through the RCT pathway.
 |
EXPERIMENTAL PROCEDURES |
Animals--
C57BL/6 mice were purchased from Taconic.
Mice with targeted disruption of the apoA-I gene (23) were purchased
from Jackson Laboratories. Homozygous CETP transgenic (24) and
hemizygous SR-BI transgenic (25) mice were generously provided by Dr.
Alan R. Tall (Columbia University) and have been previously described (24, 25). Animals were subjected to light cycling for at least 2 weeks
before specific experiments, which were performed during the mid-dark
phase of the light cycle. All experiments were performed in nonfasting
male mice that were within the age range of 3-6 months. All
experiments were approved by the Institutional Animal Care and Research
Advisory Committee of the University of Texas Southwestern Medical
Center at Dallas.
Recombinant Adenoviruses--
The recombinant adenovirus
AdCMV-LCAT, carrying a gene encoding mouse LCAT expressed from a human
cytomegalovirus promoter, was generated by homologous recombination in
293 cells as described previously (26). The cDNA encoding murine
LCAT was generously provided by Dr. Catherine Hedrick (University of
Virginia). Generation of the recombinant adenovirus AdCMV-Luc, carrying
a gene encoding firefly luciferase (27), AdCMV-7
, carrying a gene
encoding rat hepatic 7
-hydroxylase (26), and AdCMV-Apo AI, carrying a gene encoding human apoA-I (28) were described previously. Large
scale production of recombinant adenovirus was performed by infecting
confluent monolayers of 293 cells grown in 15-cm tissue culture plates
with primary stock at a multiplicity of infection of 0.1-1.0 (28).
Infected monolayers were lysed with Nonidet P-40 when >90% of the
cells showed cytopathic changes, and recombinant virus was purified by
precipitation with polyethylene glycol 8000, centrifugation on a
discontinuous CsCl density gradient, and desalting by chromatography on
Sepharose CL-4B. Purified virus eluting in the void volume was
collected, snap frozen in liquid N2, and stored at
80 °C until used. Virus titer was determined by plaque assay in
monolayer cultures of 293 cells.
Determination of Cholesterol Synthesis Rates--
Rates of
cholesterol synthesis were measured in vivo using
[3H]water. Mice were administered ~25 mCi of
[3H]water intraperitoneally and then returned to cages
under a fume hood as previously described (16). Two hours after the
injection of [3H]water, the animals were anesthetized and
exsanguinated through the inferior vena cava. Aliquots of plasma were
taken for the determination of body water specific activity, and
samples of liver and various extrahepatic tissues were taken for the
isolation of digitonin-precipitable sterols. The entire remaining
carcass was also taken for the isolation of digitonin-precipitable
sterols. Rates of sterol synthesis are expressed as the nanomoles of
[3H]water incorporated into digitonin-precipitable
sterols per h per g wet weight of tissue (nmol/h per g).
Determination of HDL Cholesteryl Ether Transport--
Mouse HDL
was isolated in the density range of 1.07-1.21 g/ml using sequential
preparative ultracentrifugation and standard techniques (29) and
labeled with either the intracellularly trapped
[1
,2
-3H]cholesteryl oleyl ether (18, 30, 31) or
[cholesteryl-4-14C]oleate by exchange from donor
liposomes as described (13, 32). Rates of HDL cholesteryl ether
transport were determined using a primed infusion protocol as
previously described (16). Animals were administered a priming dose of
[3H]cholesteryl ether-labeled HDL intravenously followed
by a continuous infusion of the same radiolabeled lipoprotein at a rate
determined in preliminary studies to maintain a constant plasma
specific activity. The primed infusions of
[3H]cholesteryl ether-labeled HDL were continued for
4 h at which time each animal was administered
[14C]cholesteryl ester-labeled HDL intravenously (as a
marker of the volume of plasma within each tissue) and sacrificed 10 min later. Plasma and tissue samples were assayed for their
3H and 14C content as previously described
(16). The tissue spaces achieved by the labeled HDL at 10 min
(14C dpm/g of tissue divided by the 14C
dpm/µl of plasma) and at 4 h and 10 min (3H dpm/g of
tissue divided by the steady-state 3H dpm/µl of plasma)
were then calculated and have the units of microliters/g. The increase
in tissue space over the 4-h experimental time period equals the rate
of radiolabeled HDL cholesteryl ether movement into each organ and is
expressed as the microliters of plasma cleared of its HDL cholesteryl
ether content/h/g of tissue (16).
Determination of Fecal Sterol Excretion--
Feces were
collected daily for 4 days beginning immediately after the intravenous
administration of rHDL. Feces were dried, weighed, and ground. A 1-g
aliquot of this material was used to determine total bile acid content
by an enzymatic method as previously described (33). A second 1-g
aliquot was subjected to alkaline hydrolysis at 120-130 °C for
12 h. After drying the sample, 10 ml of water and 10 ml of ethanol
were added. The sample was extracted in 15 ml of petroleum ether to
which 1.0 mg of 5-cholestene (Sigma) had been added as an internal
standard. The amount of cholesterol, coprostanol, epicoprostanol, and
cholestanone in the extracts was quantified by gas chromatography (33).
The daily excretion rates of both bile acid and neutral sterol are
expressed as micromole/day per 100 g of body weight.
Determination of mRNA Levels--
Tissue SR-BI, LDL
receptor, ABCA1, LCAT, apoA-I, and 7
-hydroxylase mRNA levels
were determined using a ribonuclease protection assay.
Glyceraldehyde-3-phosphate dehydrogenase or
-actin was used as an
internal control. Species-specific 32P-labeled riboprobes
were synthesized using MAXIscript in vitro transcription
kits (Ambion Inc., Austin, TX) in the presence of 3 µM
(SR-BI, LDL receptor, ABCA1, LCAT, and 7
-hydroxylase) or 100 µM (glyceraldehyde-3-phosphate dehydrogenase and
-actin) labeled nucleotide.
Samples of liver were homogenized in RNA STAT-60 (TEL-TEST, Inc.,
Friendswood, TX). Total RNA (40 µg) was hybridized with 32P-labeled riboprobes simultaneously at 68 °C using the
HybSpeed RPA protocol (Ambion Inc). Following RNase digestion, the
mRNA-protected 32P-labeled probes were separated on 8 M urea, 5% polyacrylamide gels together with
32P-labeled MspI-digested pBR322 size standards.
The radioactivity in each band, as well as background radioactivity,
was quantified using a phosphorimaging system (Molecular Dynamics Inc.,
Sunnyvale, CA).
Determination of the Concentration of ApoA-I and Lipids in Plasma
and Cholesterol in Liver--
Plasma lipoproteins were separated by
FPLC using a Superose 6 HR column (Sigma). Two-hundred-µl aliquots
were collected and used for apoA-I and lipid assays. The following
assays were performed on plasma and FPLC column fractions: total
cholesterol (Roche Molecular Biochemicals, catalogue number 1127771),
free cholesterol (Wako Chemicals, USA, catalogue number 274-47109),
phospholipid (Wako Chemicals, catalogue number 990-54009), and apoA-I
(Sigma, catalogue number 356-A). Liver cholesterol was quantified by
capillary gas-liquid chromatography.
Statistical Analysis--
The data are presented as mean ± 1 S.D. To test for differences among groups, one-way analysis of
variance was performed. Significant results were further analyzed using
the Tukey multiple comparison procedure.
 |
RESULTS |
These studies were undertaken to determine if cholesterol flux
through the RCT pathway can be increased by up-regulating individual steps in the pathway. The initial step in the reverse cholesterol transport pathway is efflux of cholesterol to circulating acceptor particles. Extrahepatic tissues acquire cholesterol mainly from de novo synthesis (21). The cholesterol biosynthetic pathway is tightly regulated by cholesterol availability (34, 35) and the rate
of cholesterol synthesis or the cholesterol content of a tissue will be
altered in response to changes in sterol influx or efflux. We therefore
measured cholesterol synthesis rates and cholesterol concentrations in
the extrahepatic tissues under conditions in which individual steps in
the reverse cholesterol transport pathway were accelerated. Working
backwards from the liver, the final step in the RCT pathway is the
conversion of cholesterol into bile acids and their excretion into
bile. We stimulated this step in the RCT pathway by overexpressing
hepatic 7
-hydroxylase and determined the effect on cholesterol
synthesis rates and concentrations in the extrahepatic tissues. Rates
of cholesterol synthesis were measured in vivo 3 days after
the intravenous injection of 109 pfu of recombinant
adenovirus expressing 7
-hydroxylase from the CMV promoter
(AdCMV-7
) or control virus (AdCMV-Luc) into C57BL/6 mice.
Administration of AdCMV-7
increased hepatic 7
-hydroxylase activity by ~15-fold as previously described (26, 36).
Overexpression of hepatic 7
-hydroxylase lowered plasma
VLDL/IDL/LDL cholesterol concentrations but had little effect on plasma
HDL cholesterol concentrations as shown in Fig.
1A and no effect on hepatic
HDL cholesteryl ether clearance (Fig. 1B). As shown in Fig.
1C, overexpression of 7
-hydroxylase increased hepatic
cholesterol synthesis by 4.5-fold but had no effect on rates of
cholesterol synthesis in the extrahepatic tissues. Although not shown,
overexpression of hepatic 7
-hydroxylase had no effect on the
cholesterol content of extrahepatic tissues.

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Fig. 1.
Effect of overexpressing
7 -hydroxylase on plasma lipoprotein
cholesterol concentrations (panel A), hepatic HDL
cholesteryl ether clearance (panel B), and tissue
cholesterol synthesis (panel C).
Adenovirus-mediated gene transfer was used to overexpress
7 -hydroxylase in the livers of C57BL/6 mice. Studies were performed
3 days after the administration of adenovirus expressing
7 -hydroxylase (AdCMV-7 ) or luciferase (AdCMV-Luc, used as a
control virus). A, distribution of cholesterol among plasma
lipoproteins. Plasma from the 2 groups was pooled and lipoproteins size
fractionated by FPLC using a Superose 6 HR column. The retention times
for mouse VLDL (d < 1.006 g/ml), IDL/LDL (d
1.02-1.055 g/ml), and HDL (1.07-1.21 g/ml) are indicated.
B, hepatic HDL cholesteryl ether clearance rates.
C, tissue cholesterol synthesis rates. Each value in
panels B and C represents the mean ± 1 S.D.
for data obtained in five animals. *, differs significantly from the
control value, p < 0.5. SB, small
bowel.
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The liver takes up HDL cholesteryl esters directly (via SR-BI) or after
CETP-mediated transfer to apoB-containing lipoproteins (via the LDL
receptor pathway). In the mouse, which lacks CETP, HDL cholesteryl
esters are delivered to the liver mainly through the SR-BI pathway.
SR-BI transgenic mice have been created that manifest marked
overexpression of SR-BI in the liver, rapid clearance of HDL
cholesteryl ester by the liver, and a marked reduction in plasma HDL
concentrations (25). We confirmed that liver-specific overexpression of
SR-BI markedly lowers plasma HDL cholesterol concentrations (Fig.
2A) as a result of enhanced
clearance of HDL cholesteryl ester by the liver (Fig. 2B).
The rate of hepatic HDL cholesteryl ether clearance in SR-BI transgenic
mice was 21-fold higher than that of SR-BI wild type mice. The marked
increase in HDL cholesteryl ether clearance by the liver was due in
part to the reduced concentration of HDL cholesteryl ester in plasma; however, clearance rates were still increased 9-fold in SR-BI transgenic mice in which plasma HDL concentrations were normalized by
an infusion of unlabeled mouse HDL. As shown in Fig. 2C,
rates of cholesterol synthesis were not increased in the extrahepatic tissues of SR-BI transgenic mice; indeed, rates of cholesterol synthesis were significantly suppressed in several tissues. Although not shown, the cholesterol concentration in the extrahepatic tissues shown in Fig. 2C were not altered in SR-BI transgenic
animals. These data indicate that liver-specific overexpression of
SR-BI does not enhance reverse cholesterol transport and may reduce the
rate of cholesterol efflux from some tissues into plasma. It should be
noted that we did not specifically look at the adrenal glands in these
studies. RCT from the adrenal glands may not be necessary as they
convert cholesterol to steroid hormones. Because mouse adrenal glands
derive the majority of their cholesterol from HDL, it is likely that
cholesterol synthesis is increased in the adrenal glands of SR-BI
transgenic mice to compensate for the decrease in HDL cholesteryl ester
uptake that results from the near absence of HDL in the plasma of these
mice (25).

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Fig. 2.
Effect of overexpressing SR-BI on plasma
lipoprotein cholesterol concentrations (panel A),
hepatic HDL cholesteryl ether clearance (panel B), and
tissue cholesterol synthesis (panel C). Studies
were performed in transgenic mice with liver-specific overexpression of
SR-BI (hemizygous) or sib controls. A, distribution of
cholesterol among plasma lipoproteins. Plasma from the 2 groups was
pooled and lipoproteins size fractionated by FPLC using a Superose 6 HR
column. B, hepatic HDL cholesteryl ether clearance rates in
SR-BI wild type (wt), SR-BI transgenic (tg), and
SR-BI transgenic mice in which plasma HDL concentrations have been
raised to normal values by the infusion of unlabeled mouse HDL.
C, tissue cholesterol synthesis rates. Each value in
panels B and C represents the mean ± 1 S.D.
for data obtained in five animals. *, differs significantly from the
control value, p < 0.5. SB, small
bowel.
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Free cholesterol that is transferred from tissues to nascent discoidal
HDL is esterified by LCAT leading to the generation of mature spherical
HDL. We accelerated this step in the RCT pathway by overexpressing
mouse LCAT in the liver using adenovirus-mediated gene transfer and
determined the effect on cholesterol synthesis rates and concentrations
in the extrahepatic tissues. Rates of cholesterol synthesis were
measured 3 days after the intravenous injection of 1 × 109 or 2 × 109 pfu of recombinant
adenovirus expressing mouse LCAT (AdCMV-LCAT) or control virus into
C57BL/6 mice. Administration of AdCMV-LCAT resulted in the accumulation
of large amounts of LCAT mRNA in the liver (not shown) and in the
accumulation of cholesteryl ester-rich HDL particles in plasma that
contained mainly apoA-I and apoE (Fig.
3A). However, overexpression
of LCAT in the liver had no significant effect on the rate of
cholesterol synthesis (Fig. 3, B and C) or the
cholesterol concentration (not shown) in extrahepatic tissues.

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Fig. 3.
Effect of overexpressing LCAT on plasma
lipoprotein cholesterol concentrations (panel A) and
tissue cholesterol synthesis rates (panels B and
C). Adenovirus-mediated gene transfer was used to
overexpress LCAT in the livers of C57BL/6 mice. Studies were performed
3 days after the administration of adenovirus expressing 1 × 109 or 2 × 109 pfu mouse LCAT
(AdCMV-LCAT) or luciferase (AdCMV-Luc, used as a control virus).
A, distribution of cholesterol among plasma lipoproteins.
Plasma from each group was pooled and lipoproteins size fractionated by
FPLC using a Superose 6 HR column. The FPLC profiles from the 2 luciferase groups were superimposible so mean values are shown. Equal
volumes of FPLC fractions corresponding to HDL (fractions 16-30,
26-40 and 31-42 for high dose AdCMV-LCAT, low dose AdCMV-LCAT, and
control, respectively) were pooled, delipidated, and equal amounts of
protein separated on 2-15% gradient polyacrylamide gels.
B, tissue cholesterol synthesis rates in animals
administered 1 × 109 pfu AdCMV-LCAT or AdCMV-Luc.
C, tissue cholesterol synthesis rates in animals
administered 2 × 109 pfu AdCMV-LCAT or AdCMV-Luc.
Each value in panels B and C represents the
mean ± 1 S.D. for data obtained in five animals. *, differs
significantly from the control value, p < 0.5. SB, small bowel.
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The initial step in RCT is cholesterol efflux from the plasma membranes
of cells in extrahepatic tissues to nascent HDL particles in the
interstitial space. We employed two protocols aimed at increasing the
availability of nascent HDL. In the first we increased plasma apoA-I
concentrations by overexpressing apoA-I in the liver using
adenovirus-mediated gene transfer. ApoA-I
/
mice were
administered 109 pfu of recombinant adenovirus expressing
human apoA-I from the CMV promoter (AdCMV-apoA-I) or control virus, and
the plasma concentration of apoA-I, cholesterol, and phospholipid was
determined at 24-h intervals for 3 days. As shown in Fig.
4A, administration of
AdCMV-apoA-I increased plasma apoA-I concentrations from 0 to 309 mg/dl; plasma HDL phospholipid and cholesterol concentrations increased
to 221 and 119 mg/dl, respectively, with 91% of HDL cholesterol being esterified. In the second protocol, animals were infused with discoidal
complexes of phospholipid and apoA-I. These particles (referred to as
rHDL) contained human apoA-I and phospholipid in a ratio of ~1:5
(w/w) (37) and were generously provided by Dr. Peter G. Lerch, ZLB
Central Laboratory, Swiss Red Cross, Bern, Switzerland.
ApoA-I
/
mice were administered a primed infusion of
rHDL at a rate of 1 mg of rHDL apoA-I/h for 3 days through an
in-dwelling internal jugular vein catheter that exited the body through
a tether and was attached to a fluid swivel. This system allowed the
animals free range of motion and access to food and water. As shown in Fig. 4B plasma apoA-I concentrations were similar to those
in animals administered AdCMV-apoA-I, whereas plasma phospholipid concentrations were much higher (~1,400 mg/dl). Plasma cholesterol concentrations increased during the 3-day infusion to nearly 1,000 mg/dl with 87% of the cholesterol that accumulated in plasma being unesterified.

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Fig. 4.
Effect of apoA-I overexpression or rHDL
infusion on the plasma concentration of apoA-I, phospholipid, and
cholesterol. ApoA-I / mice were administered
recombinant adenovirus expressing human apoA-I (panel A) or
a primed infusion of rHDL at the rate of 1 mg of apoA-I/h (panel
B). Blood was obtained from the retroorbital sinus at 24, 48, and
72 h. Each value represents the mean for data obtained in five
(AdCMV-ApoAI) or four (rHDL) animals.
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Fig. 5 shows rates of cholesterol
synthesis in the extrahepatic tissues of apoA-I
/
mice
after 3 days of overexpressing apoA-I or infusing rHDL. As shown in
Fig. 5A, overexpressing apoA-I had no significant effect on
cholesterol synthesis in any extrahepatic tissue. In contrast, infusion
of rHDL markedly increased rates of cholesterol synthesis in most of
the extrahepatic tissues that were examined as well as the remaining
carcass (Fig. 5B). The greatest relative increases in
cholesterol synthesis were seen in lung (6-fold), heart (9-fold), and
skeletal muscle (10-fold). Tissue cholesterol concentrations were
measured at 3 days and were not altered by the overexpression of apoA-I
(not shown) or the infusion of rHDL (Fig. 5C). These studies
show that for the same elevation in plasma apoA-I concentrations, an
infusion of apoA-I·phospholipid complexes is far more effective than
overexpressing apoA-I at stimulating cholesterol movement from tissues
to plasma.

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Fig. 5.
Effect of apoA-I overexpression or rHDL
infusion on tissue cholesterol synthesis rates and concentrations.
Apo-A-I / mice were administered recombinant adenovirus
expressing human apoA-I (panel A) or a primed infusion of
rHDL at the rate of 1 mg of apoA-I/h (panel B). Tissue
cholesterol synthesis rates and concentrations were measured at 72 h. A, tissue cholesterol synthesis rates in animals
administered an adenovirus expressing human apoA-I (AdCMV-apoA-I) or
control virus (AdCMV-Luc). B, tissue cholesterol synthesis
rates in animals infused with rHDL. C, tissue cholesterol
concentrations in the animals are shown in panel B. Each
value represents the mean ± 1 S.D. for data obtained in five
(AdCMV-apoA-I) or four (rHDL) animals. SB, small
bowel.
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Further studies were undertaken to characterize the effect of an
intravenous bolus of rHDL on plasma lipids and parameters of RCT. We
performed these studies in apoA-I
/
mice, which have
very low background levels of total and HDL cholesterol, and in CETP
transgenic mice, which more closely mimic the human situation. Fig.
6 shows changes in plasma lipid and human
apoA-I concentrations following the intravenous injection of 6 mg of
rHDL apoA-I to apoA-I
/
or CETP transgenic mice. In
apoA-I
/
mice, plasma concentrations of human apoA-I and
phospholipid equaled 1,662 and 429 mg/dl 1 h after administration
of rHDL and had returned to near normal levels by 24 h (Fig.
6A). As shown in Fig. 6C, plasma cholesterol
concentrations peaked at ~6 h at which time 88% was unesterified.
Free cholesterol concentrations returned to near normal values by
24 h although total cholesterol (and thus esterified cholesterol)
was still elevated. Basal plasma lipid levels were higher in CETP
transgenic than in apoA-I
/
mice; however, changes in
plasma lipid and human apoA-I concentrations after rHDL (Fig. 6,
B and D) were similar to those in
apoA-I
/
mice. Also shown in Fig. 6 are the changes in
liver cholesterol concentrations following the administration of rHDL.
In both animal models, liver cholesterol concentrations were
significantly reduced 1 h after the administration of rHDL. Liver
cholesterol concentrations returned to normal values by 6 h and
were significantly elevated at 24 and 48 h before returning again
to normal levels at 72 h. Cholesterol concentrations were
significantly reduced in many extrahepatic tissues 1-2 h after rHDL
administration but were subsequently normal at 6, 24, 48, and 72 h
(data not shown).

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Fig. 6.
Effect of rHDL on the concentration of
apoA-I, phospholipid, and cholesterol in plasma and cholesterol in
liver. ApoA-I / and CETP transgenic mice were
administered rHDL (6 mg of apoA-I) by intravenous injection. Animals
were killed at 1, 6, 12, 24, 48, and 72 h. Each value represents
the mean for data obtained in five animals. The concentration of human
apoA-I, phospholipid, and cholesterol in plasma and cholesterol in
liver were determined as described under "Experimental Procedures."
*, differs significantly from the 0 time value, p < 0.05.
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Fig. 7 shows the lipoprotein distribution
of plasma cholesterol after administration of 6 mg of rHDL apoA-I. Six
hours after the injection of rHDL, cholesterol (88% of which was
unesterified) was mainly present in particles having the size of VLDL
and IDL/LDL in both apoA-I
/
and CETP transgenic mice.
By 24 h, cholesterol had shifted into particles having the size of
HDL in apoA-I
/
mice (Fig. 7A), but remained
in VLDL- and IDL/LDL-sized particles in CETP transgenic mice (Fig.
7B). By 48 h, the lipoprotein distribution of plasma
cholesterol had returned to that of control animals in both animal
models (data not shown). These changes in plasma lipid concentrations
are similar to those reported in humans administered rHDL (37).

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Fig. 7.
Distribution of plasma cholesterol by
particle size after rHDL administration. Plasma was pooled from
each of the groups shown in Fig. 6 and lipoproteins size fractionated
by FPLC using a Superose 6 HR column. Data are shown for the control
(time 0), 6- and 24-h groups. Data for the 48- and 72-h groups were not
different from the control group and are not shown.
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Rates of tissue cholesterol synthesis were measured in parallel groups
of apoA-I
/
and CETP transgenic mice 6, 24, and 48 h after the injection of 6 mg of rHDL apoA-I. In both
apoA-I
/
mice (Fig.
8A) and CETP transgenic mice
(Fig. 8B), rates of hepatic cholesterol synthesis were
markedly elevated (2-3-fold) at 6 h, were suppressed (<50% of
control values) at 24 h, and had returned to normal levels by
48 h after rHDL administration. Rates of cholesterol synthesis in
the extrahepatic tissues generally showed a similar pattern of changes
in response to a bolus of rHDL. In most tissues rates of cholesterol
synthesis were markedly elevated at 6 h, were suppressed below
control values at 24 h, and had returned to normal levels by
48 h. Tissue cholesterol synthesis rates at 72 h did not
differ significantly from those at 48 h and time 0 (not shown).
LDL receptor, SR-BI, and ABCA1 mRNA levels were measured by RNase
protection in liver, lung, spleen, heart, and muscle of individual
animals (5 mice/group) at 6, 24, 48, and 72 h after rHDL
administration as described under "Experimental Procedures." There
were no significant changes in mRNA levels for the LDL receptor,
SR-BI, or ABCA1 at any of these time points (data not shown).

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Fig. 8.
Rates of tissue cholesterol synthesis after
rHDL administration. ApoA-I / and CETP transgenic
mice were administered rHDL (6 mg of apoA-I) by intravenous injection.
Rates of cholesterol synthesis were quantified in vivo at 0, 6, 24, and 48 h and are expressed as the nanomole of tritiated
water incorporated into cholesterol per h per g of tissue (nmol/h per
g). Each value represents the mean ± 1 S.D. from data obtained in
five animals. SB, small bowel.
|
|
The data in Fig. 8 indicate that rHDL stimulates cholesterol efflux
from most tissues of the body. However, when cholesterol synthesis
rates are expressed per whole organ, it becomes apparent that much of
the cholesterol entering plasma after rHDL administration is derived
from the liver. Fig. 9 shows the changes
in cholesterol synthesis rates in liver, extrahepatic tissues, and
whole body after rHDL administration. In apoA-I
/
mice
(Fig. 9A), total body cholesterol synthesis increased 48% (13.7-20.6 µmol/h per 100 g) and hepatic cholesterol synthesis increased 77% (5.7-10.1 µmol/h per 100 g) 6 h after the
administration of rHDL. It can therefore be calculated that the liver
accounted for 64% of the increase in total body cholesterol synthesis
in response to rHDL. A similar pattern was seen in the CETP transgenic mice (Fig. 9B) where the liver accounted for 50% of the
increase in total body cholesterol synthesis in response to rHDL.
Cholesterol synthesis was suppressed in many tissues 24 h after
rHDL administration as cholesterol that effluxed into plasma was
cleared from plasma. In apoA-I
/
mice (Fig.
9A), total body cholesterol synthesis was suppressed 33%
below control values (13.6 to 9.1 µmol/h per 100 g) and hepatic cholesterol synthesis was suppressed 61% (5.7 to 2.2 µmol/h per 100 g) 24 h after rHDL administration. Thus, the liver
accounted for 78% of the decrease in total body cholesterol synthesis
at 24 h. A similar pattern was seen in CETP transgenic mice where the liver accounted for 79% of the decrease in total body cholesterol synthesis at 24 h.

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[in this window]
[in a new window]
|
Fig. 9.
Rates of cholesterol synthesis in whole body,
liver and extrahepatic tissues after rHDL administration. Rates of
cholesterol synthesis were quantified in vivo at 0, 6, 24, and 48 h and are expressed as the micromole of tritiated water
incorporated into cholesterol per h per 100 g body weight
(µmol/h per 100 g). Each value represents the mean ± 1 S.D. from data obtained in five animals.
|
|
To determine if rHDL administration increased net cholesterol flux
through the entire RCT pathway we performed sterol balance studies in
apoA-I
/
and CETP transgenic mice after rHDL
administration. Feces were collected daily (for 4 days) from groups of
5 animals beginning immediately after the intravenous administration of
6 mg of rHDL apoA-I. As shown in Fig.
10, fecal neutral sterol and bile acid output was not affected by rHDL in either mouse model. In other studies
mice received daily intravenous injections of rHDL (6 mg of rHDL
apoA-I/dose) for 7 days. Feces were collected daily for the
determination of bile acids and neutral sterols beginning 2 days before
the first injection and ending 2 days after the last injection. These
studies showed no effect of rHDL on external sterol
balance.2 Hepatic cholesterol
7
-hydroxylase is the rate-limiting enzyme in the main bile acid
biosynthetic pathway. We measured 7
-hydroxylase mRNA levels by
RNase protection at times 0, 6, 24, 48, and 72 h after rHDL
administration and found no significant effect of rHDL at any time
point as shown in Table I.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 10.
Fecal bile acid and neutral sterol output
after rHDL administration. ApoA-I / and CETP
transgenic mice were administered rHDL (6 mg of apoA-I) by intravenous
injection. Feces were collected daily for 4 days from groups of mice
and fecal bile acids and neutral sterols quantified as described under
"Experimental Procedures." Each value for apoA-I knockout mice
represents the mean from 2 pools of five animals each. Each value for
CETP transgenic mice represents the mean ± 1 S.D. from data
obtained in 3 pools of five animals each.
|
|
 |
DISCUSSION |
The main objective of these studies was to determine if
up-regulating individual steps in the RCT pathway leads to increased cholesterol flux through the entire RCT pathway. The RCT pathway encompasses a series of steps beginning in extrahepatic tissues with
the efflux of cholesterol to nascent HDL and culminating in the liver
and gastrointestinal tract with the secretion of sterols into bile and
their elimination in feces. We show that RCT is not significantly
enhanced in mice under conditions in which each of the major steps in
the RCT pathway is markedly accelerated. These observations suggest
that it will be necessary to up-regulate multiple (possibly all) steps
in the RCT pathway if RCT is to be significantly increased.
The initial step in RCT can be markedly accelerated by the intravenous
administration of apoA-I·phospholipid complexes (rHDL) that resemble
nascent HDL. Administration of rHDL induced a rapid increase in the
plasma concentration of cholesterol (initially unesterified) that was
accompanied by a transient decrease in the cholesterol content of many
tissues. Enhanced efflux of cholesterol from tissues to plasma
triggered a marked increase in tissue cholesterol synthesis that was
apparently fully compensatory because tissue cholesterol concentrations
were restored to normal values and tissue LDL receptor and SR-BI
mRNA levels remained unchanged. The greatest relative increases in
cholesterol synthesis (and thus cholesterol efflux) in response to a
bolus of rHDL occurred in heart, skeletal muscle, lung, spleen, and
liver. Small intestine was relatively resistant to rHDL and preliminary
studies showed the brain to be completely unresponsive to rHDL.
Overall, total body cholesterol synthesis increased ~50% 6 h
after the administration of rHDL and the liver accounted for
approximately half (CETP transgenic mice) to two-thirds
(apoA-I
/
mice) of this increase. Thus, the liver was
the single most important source of cholesterol entering plasma after
the administration of rHDL.
While the tissue source of cholesterol entering plasma in response to
rHDL can be determined quantitatively, the precise destination of this
cholesterol as it is cleared from plasma is less certain. Plasma
cholesterol concentrations peaked ~6 h after a bolus of rHDL and at
this time most of the cholesterol was unesterified. By 24 h the
majority of plasma cholesterol was esterified and present in either HDL
(in mice lacking CETP) or LDL (CETP transgenic mice). It is likely that
most of this cholesterol was transported to the liver since the liver
is the major site for the clearance of cholesterol carried in both HDL
(16, 18) and LDL (38). However, cholesterol synthesis was suppressed in
many extrahepatic tissues at 24 h indicating that these tissues
contributed to the clearance of cholesterol that had entered plasma in
response to rHDL. Overall, total body cholesterol synthesis was
suppressed ~33% 24 h after rHDL administration and the liver
accounted for nearly 80% of this decrease. Thus, the majority of
cholesterol that effluxed into plasma in response to rHDL came from the
liver; however, an even greater proportion of this cholesterol may have returned to the liver and if so this would result in a net flux of
cholesterol from extrahepatic tissues to the liver. This is supported
by the finding that hepatic cholesterol concentrations increased
significantly in both apoA-I
/
and CETP transgenic mice
as effluxed cholesterol was cleared from plasma whereas the cholesterol
concentration in extrahepatic tissues remained constant during this
time. The current studies were performed in mice maintained on a low
cholesterol diet. Under these conditions, basal rates of hepatic
cholesterol synthesis are high and the liver can compensate for changes
in cholesterol flux by adjusting the rate of de novo
cholesterol synthesis (39). It will be important to determine the
effects of rHDL under conditions in which hepatic cholesterol synthesis
is suppressed by dietary cholesterol or inhibitors of cholesterol synthesis.
Although rHDL clearly mobilized cholesterol from extrahepatic tissues
and resulted in the net movement of cholesterol from extrahepatic
tissues to the liver, there was no induction of hepatic 7
-hydroxylase expression or increase in fecal sterol excretion in
either apoA-I
/
or CETP transgenic mice in response to
rHDL administration. These observations, which indicate that rHDL
administration did not increase cholesterol flux through the entire RCT
pathway, contrast with those of Eriksson et al. (40) who
reported that fecal sterol excretion was markedly increased in 4 patients with heterozygous FH after the administration of
pro-apoA-I·phospholipid complexes. At this point we have no adequate
explanation for these apparently conflicting observations. The relative
dose of rHDL used in our studies was higher than that used in the study
by Eriksson et al. (40) resulting in a greater movement of
cholesterol from tissues into plasma; however, this should increase the
likelihood of detecting an effect of rHDL on fecal sterol excretion if
such an effect exists. It is possible that there are inherent species differences in response to rHDL or significant differences in the rHDL
preparations (pro-apoA-I versus apoA-I) used in the two studies.
To investigate the role of phospholipid in cholesterol efflux in
vivo, we quantified rates of cholesterol acquisition in the extrahepatic tissues of animals overexpressing apoA-I or infused with
rHDL. Plasma apoA-I concentrations were similar in the two groups
whereas plasma phospholipid concentrations were much higher in those
infused with rHDL. Overexpression of apoA-I raised HDL cholesterol
concentrations but had no detectable effect on cholesterol efflux from
any tissue. In contrast, infusion of rHDL markedly increased
cholesterol efflux from most extrahepatic tissues. These results
emphasize the important role of phospholipid in promoting cholesterol
efflux in vivo. Raising plasma HDL concentrations by
overexpressing apoA-I has been shown to protect against atherosclerosis in mouse models (41, 42). Our results suggest that the protective effect of apoA-I overexpression is not related to enhanced RCT. It
should be noted that free apoA-I-mediated cholesterol efflux has been
demonstrated most consistently in cholesterol-loaded macrophages (3, 7,
8), a cell type that was not evaluated in our studies. It is possible
that overexpression of apoA-I results in efflux of cholesterol from
foam cells in the arterial wall without causing efflux from any other
tissue but this seems unlikely.
While the infusion of nascent HDL-like particles markedly increased
cholesterol efflux from most extrahepatic tissues, up-regulating other
steps in the RCT pathway did not. Thus, liver-specific overexpression of LCAT, SR-BI, or cholesterol 7
-hydroxylase did not induce
cholesterol efflux from any extrahepatic tissue. LCAT plays a key role
in the RCT pathway by catalyzing the esterification of free cholesterol that has been transferred from cell membranes to nascent HDL. The role
of LCAT in RCT has been studied in transgenic mice overexpressing human
LCAT. Francone et al. (43) showed that HDL from LCAT
transgenic mice promoted cholesterol efflux from cells more efficiently
than control HDL. Moreover, HDL cholesteryl ester flux to the liver was
increased in LCAT transgenic compared with control mice (43) suggesting
that LCAT overexpression enhanced RCT. In contrast, Berard et
al. (44) concluded that RCT was decreased in human LCAT transgenic
mice based on turnover studies that suggested the increased HDL
cholesteryl ester levels in these animals was the result of decreased
clearance by the liver. In the current work overexpression of mouse
LCAT increased HDL cholesteryl ester concentrations in a
dose-dependent fashion but did not induce cholesterol
efflux from any extrahepatic tissue indicating no significant effect on
RCT. We have previously shown that the HDL cholesteryl ester uptake
pathway in the liver is saturated at normal HDL concentrations in the
mouse (16) and hamster (15). Thus increasing plasma HDL cholesteryl
ester concentrations to supernormal levels (by infusing normal HDL or
LCAT overexpression) cannot increase HDL cholesteryl ester delivery to
the liver in these species unless SR-BI or CETP expression is increased
(45).
Liver-specific overexpression of SR-BI markedly increases HDL
cholesteryl ester clearance by the liver suggesting enhanced RCT (25).
If overexpression of SR-BI in the liver does increase the rate of RCT,
cholesterol synthesis will increase in extrahepatic tissues to balance
the increased flux of cholesterol from these tissues to the liver or
the cholesterol content of extrahepatic tissues will fall. However, we
found no change in the cholesterol content or increase in rates of
cholesterol synthesis in the extrahepatic tissues of SR-BI transgenic
mice; indeed, synthesis rates were reduced in several extrahepatic
tissues. These observations indicate that liver-specific overexpression
of SR-BI does not increase cholesterol flux through the RCT pathway and
may inhibit RCT from some tissues. There are several possible reasons
why hepatic overexpression of SR-BI failed to enhance RCT. First,
although liver-specific overexpression of SR-BI markedly increases HDL
cholesteryl ester clearance by the liver (25), this results in the
virtual elimination of HDL from plasma. Thus, the absolute rate of HDL
cholesteryl ester transported to the liver (clearance multiplied by the
plasma concentration) may be normal or even decreased in SR-BI
transgenic mice. In addition, the very low plasma HDL concentration in
SR-BI transgenic mice is associated with decreased LCAT activity, which may impair RCT (25). It is possible that more modest overexpression of
SR-BI in the liver may result in enhanced RCT; however, we have been
unable to demonstrate this using adenovirus-mediated gene
transfer.3 Liver-specific
overexpression of SR-BI (~10-fold) markedly decreased fatty streak
development in heterozygous LDL receptor-deficient mice fed an
atherogenic diet but this protective effect was likely due to a
reduction in the plasma concentration of apoB-containing lipoproteins
(46). This conclusion is supported by the observation that
liver-specific overexpression of SR-BI (~10-fold) did not decrease
the plasma concentration of apoB-containing lipoproteins or inhibit
fatty streak development in human apoB transgenic mice fed an
atherogenic diet (47). On the other hand, modest overexpression of
SR-BI (~2-fold) did inhibit fatty streak development without significantly reducing non-HDL cholesterol levels suggesting that SR-BI
can exert an anti-atherogenic effect that is independent of changes in
plasma non-HDL cholesterol concentrations and that this protective
effect is not directly proportional to the level of SR-BI expression
(47). Further studies will be required to sort out the complex
inter-relationship among hepatic SR-BI expression, plasma lipoprotein
concentrations, RCT, and atherogenesis.
In summary, we show that up-regulation of individual steps in the RCT
pathway does not increase cholesterol flux through the entire pathway.
These observations suggest that it will be necessary to up-regulate
multiple (possibly all) steps in the RCT pathway to significantly
increase cholesterol flux from extrahepatic tissues to the liver and
into the stool. Although cholesterol flux through the entire RCT
pathway is not increased, infusion of nascent HDL-like complexes
markedly stimulates cholesterol efflux from most tissues into plasma
and may increase net cholesterol flux from extrahepatic tissues to the
liver. These findings raise the possibility that rHDL could stimulate
cholesterol efflux from foam cells in the arterial wall and induce
regression of atherosclerotic plaque. Phospholipid-containing acceptor
particles can mobilize cholesteryl esters from cholesterol-loaded
macrophages in vitro despite an increase in de
novo cholesterol synthesis (48). Moreover, intravenous administration of phospholipid-containing complexes has been shown to
induce regression of pre-existing atherosclerosis in animal models
(49-51). Although many questions remain, enhancing cholesterol efflux
from the arterial wall is an attractive approach that would complement
current strategies that are directed primarily at reducing cholesterol
influx into the arterial wall.
 |
ACKNOWLEDGEMENTS |
We thank Thomas van Dinter, Ahn Pho,
and Brian Jefferson for excellent technical assistance and Dr. Stephen
Turley for help with the fecal sterol excretion studies. The
rHDL used in these studies was provided by Dr. Peter G. Lerch, Head
Biochemistry, ZLB Central Laboratory, Bern, Switzerland. CETP
transgenic and SR-BI transgenic mice were provided by Dr. Alan R. Tall,
Columbia University, New York. Mouse LCAT was provided by Dr. Katherine Hedrick, University of Virginia.
 |
FOOTNOTES |
*
This work was supported National Institutes of Health Grants
HL-38049 and HL-47551.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Internal
Medicine, 5323 Harry Hines Blvd., Dallas, TX 75390-8887. Tel.: 214-688-4545; Fax: 214-688-8290.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M010230200
2
A. Kafrouni, K. Alam, and D. K. Spady,
unpublished data.
3
K. Alam, R. S. Meidell, and D. K. Spady, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
RCT, reverse
cholesterol transport;
LCAT, lecithin:cholesterol acyltransferase;
CETP, cholesteryl ester transfer protein;
SR-BI, scavenger receptor B1;
ABCA1, ATP-binding cassette transporter-1;
FPLC, fast protein liquid
chromatography;
HDL, high density lipoprotein;
VLDL, very low density
lipoprotein;
LDL, low density lipoprotein.
 |
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