© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Human Plasma Phospholipid Transfer Protein Increases the Antiatherogenic Potential of High Density Lipoproteins in Transgenic Mice
From MGC–Department of Cell Biology and Genetics (R.v.H., F.G., A.v.d.K., R.d.C.) and the Department of Biochemistry, Cardiovascular Research Institute COEUR (A.v.T., P.V., T.v.G., P.v.d.B.), Erasmus University Rotterdam, Rotterdam, the Netherlands; the Department of Biochemistry, National Public Health Institute (M.J., S.E.), Helsinki, Finland; and the Department of Vascular Surgery, Academic Hospital "Dijkzigt" (R.d.C.), Rotterdam, the Netherlands.
Correspondence to Dr Arie van Tol, Department of Biochemistry, Erasmus University Rotterdam, Dr. Molenwaterplein 50, PO Box 1738, 3000 DR Rotterdam, Netherlands. E-mail VANTOL{at}BC1.FGG.EUR.NL
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Abstract |
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Abstract—Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress human PLTP were generated. Compared with wild-type mice, these mice show a 2.5- to 4.5-fold increase in PLTP activity in plasma. This results in a 30% to 40% decrease of plasma levels of HDL cholesterol. Incubation of plasma from transgenic animals at 37°C reveals a 2- to 3-fold increase in the formation of pre-ß-HDL compared with plasma from wild-type mice. Although pre-ß-HDL is normally a minor subfraction of HDL, it is known to be a very efficient acceptor of peripheral cell cholesterol and a key mediator in reverse cholesterol transport. Further experiments show that plasma from transgenic animals is much more efficient in preventing the accumulation of intracellular cholesterol in macrophages than plasma from wild-type mice, despite lower total HDL concentrations. It is concluded that PLTP can act as an antiatherogenic factor preventing cellular cholesterol overload by generation of pre-ß-HDL.
Key Words: atherosclerosis • pre-ß-HDL • transgenic mice • macrophages • phospholipid transfer protein
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Introduction |
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Plasma phospholipid transfer protein (PLTP) is able to promote the net transfer of phospholipids between plasma lipoproteins1 2 3 4 and mediate the conversion of HDL.5 6 Because plasma levels of HDL are among the best indicators for the risk of atherosclerosis in epidemiological studies,7 8 PLTP could play a role in the prevention of the development of coronary heart disease and stroke via its effect on HDL.
HDLs are antiatherogenic because they mediate efflux of cholesterol from peripheral cells and transport cholesterol to the liver, for excretion and degradation to bile acids. This process is known as reverse cholesterol transport.8 9 10 It has been postulated that the antiatherogenic effect of HDL can be attributed mainly to a quantitatively minor subclass of HDL, called pre-ß-HDL.11 12 This assumption is based on in vitro studies showing that pre-ß-HDL is a very efficient acceptor of cellular cholesterol.13 14 The origin of pre-ß-HDL is not well understood, but the available evidence suggests that PLTP participates in its generation, at least in vitro.15
After the cloning of a human PLTP (HuPLTP) cDNA,16 2 groups independently generated transgenic mice for HuPLTP.17 18 Unfortunately, these mice showed low levels of expression of the transgene, and as a result, only small effects on plasma lipoproteins were observed. Changes in HDL levels and subfractions could be demonstrated only in a compound transgenic background with human apoA-I.18 Recently, the generation of a PLTP knockout mouse was reported by Jiang et al.19 These mice show a decrease of plasma cholesterol in all lipoprotein classes.
In the present study, we report the generation of transgenic mice that overexpress HuPLTP 2.5- to 4.5-fold. This results in decreased plasma total HDL cholesterol levels, in increased formation of pre-ß-HDL, and in a high plasma capability to prevent cholesterol accumulation in macrophages.
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Methods |
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Generation of HuPLTP Transgenic Mice
A human cosmid library was constructed from high-molecular-weight DNA isolated from the blood of a healthy volunteer. This library was screened for cosmids containing the PLTP gene by using HuPLTP cDNA (kindly donated by Drs A-Y. Tu and J.J. Albers, Northwest Lipid Research Laboratories, University of Washington, Seattle) as a probe. Isolated cosmids were mapped by using restriction fragments from the cDNA, and a cosmid containing the complete PLTP gene20 was selected. Vector sequences were removed by restriction endonuclease digestion, and DNA was dissolved in microinjection buffer (10 mmol/L Tris-HCl, pH 7.5, and 0.1 mmol/L EDTA) at a concentration of 1 to 2 µg/mL. The DNA was microinjected into fertilized oocytes from FVB mice. These oocytes were transferred into the oviducts of pseudopregnant foster females. All animal experiments were performed in compliance with the Guidelines of the Ethical Committee on the Use of Laboratory Animals of the Erasmus University Rotterdam (No. 132.97.06) and with the European Committee Standards on the care and use of laboratory animals (Ministry of Welfare, Health, and Cultural Affairs, the Netherlands).
DNA Analysis
Genomic DNA was isolated from tail clips of 10-day-old
mice and analyzed for the presence of the HuPLTP transgene by
polymerase chain reaction (PCR) analysis: sense primer
5'-GCCACAGCAGGAGCTGATGC-3', antisense primer
5'-GCGGATGGACACACCCTCAGC-3'; 25 to 30 cycles (94°C for 1 minute,
65°C for 1 minute, and 72°C for 1 minute).
Breeding and Treatment of Transgenic Mice
Transgenic founder mice were bred with FVB mice to obtain
transgenic mice. FVB transgenic HuPLTP mice were backcrossed with
C57Bl/6 mice for 4 generations. These mice were intercrossed to obtain
wild-type, hemizygous, and homozygous HuPLTP transgenic mice. Animals
were kept on regular chow and fasted overnight before collection of
blood from the orbital plexus.
Gene Expression Analysis by RT-PCR
Total RNA was isolated from various tissues obtained
freshly from either wild-type, hemizygous, or homozygous HuPLTP
transgenic mice. cDNA was obtained by reverse transcription (RT) primed
by oligo(dT). This was used in PCR reactions in the presence of
[32P]ATP (20 cycles at 94°C for 1 minute,
62°C for 1 minute, and 72°C for 1 minute). Primers used were as
follows: for HuPLTP, sense 5'-CCTGCTGAGCCCAGCAGTG-3', antisense
5'-CTGGACCTCAGGCTGGTCTG-3'; for murine PLTP (MuPLTP), sense
5'-TTGACTCTGCCATGGAGAGC-3', antisense 5'-GCTCCACTTCGGGCAACATG-3'; and
for hypoxanthine-guanine phosphoribosyltransferase, sense
5'-CGAAGTGTT- GGATACAGGCC-3', antisense
5'-GGCAACATCAACAGG-ACTCC-3'. PCR products were run on
polyacrylamide gels and visualized by using a PhosphorImager
(Molecular Dynamics).
Assay of Plasma PLTP Activity
Plasma PLTP activity was assayed by using a phospholipid
vesicle–HDL system.1 21 EDTA-plasma samples (25 µL of
plasma diluted 1:75) were incubated with
[3H]dipalmitoylphosphatidylcholine-labeled
(Amersham) phosphatidylcholine vesicles and excess pooled normal HDL
for 45 minutes at 37°C. After incubation, the vesicles were
precipitated as described,21 and the radioactivity
transferred to HDL was counted in the supernatant. Standard curves
based on dilutions of human plasma were included in each run. The
measured activity is linear with time for 1 hour. All samples were
analyzed in duplicate, and blanks without plasma were
subtracted. Duplicates of pooled human plasma, stored at -70°C, were
also measured in each series (reference plasma). The between-assay
coefficient of variation of reference plasma was 4.1%. Activities are
expressed as micromoles of phosphatidylcholine transferred per
milliliter of plasma per hour (µmol ·
mL-1 · h-1).
Anti-HuPLTP Polyclonal Antibodies
A synthetic peptide was made containing amino acids 470 to 493
of HuPLTP (Chiron Mimotopes Peptide Systems). The peptide was chosen to
obtain an antibody that recognizes HuPLTP specifically, without
cross-reaction with the MuPLTP. Its sequence has a similarity of 69%
with the corresponding murine sequence (GenBank accession numbers of
the human and murine sequences are L26232 and U37226, respectively).
The peptide was coupled to keyhole limpet hemocyanin. New Zealand White
rabbits were immunized with the peptide-hemocyanin complex. Amounts of
0.15 mg of the complex were given in a volume of 0.3 mL each time at
intervals of 3 weeks. Blood samples were taken after each period of 3
weeks, and the generation of antibodies was followed by Western blot
analysis. Starting after the second booster injection of the
antigen complex, serum from immunized rabbits (diluted 1:1000)
displayed a strong positive reaction with human plasma and with
purified HuPLTP. Furthermore, the reaction with antibody was
monospecific, because in both samples only 1 band with an identical
molecular mass of 80 kDa was recognized. This molecular mass
corresponds to the HuPLTP band obtained with plasma from transgenic
animals shown in Figure 2
. The antibody also shows a strong
reaction with recombinant HuPLTP, which was used as an internal
standard in all Western blotting experiments but does not recognize any
protein in wild-type mouse plasma (see Figure 2).
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Anti-Mouse ApoA-I Polyclonal Antibodies
ApoA-I was purified from mouse plasma HDL (density range 1.063
to 1.21 g/mL) essentially as described22 and used to
immunize rabbits by subcutaneous injection with 100 µg of mouse
apoA-I; standard procedures were used.
Quantification of Plasma Lipids and ApoA-I
Total cholesterol was enzymatically determined with
the F-Chol kit (Boehringer-Mannheim) after hydrolysis of
cholesteryl esters with cholesterol esterase from
Candida cylindracea (Boehringer-Mannheim).
Phospholipids were measured enzymatically with a PAP150 kit
(BioMérieux).
Mouse apoA-I was quantified by a sandwich ELISA, with use of a polyclonal rabbit anti-mouse apoA-I IgG, performed in 96-well plates coated with this antibody. Purified mouse apoA-I was used as a primary standard. Plasma samples were diluted in PBS+Tween 20 (0.1%) with BSA (0.5%). Bound apoA-I was detected by the addition of polyclonal rabbit anti-mouse apoA-I, conjugated to horseradish peroxidase. The assay is linear in the range of 6.5 to 420 ng/mL.
Measurement of Plasma Lipoprotein Profiles by Gel
Filtration
Lipoprotein cholesterol profiles in mouse
EDTA-plasma were obtained by gel permeation
chromatography on HR10/30 FPLC columns in tandem that
were filled with Superose 6 and Superose 12 (preparation grade,
Pharmacia Biotechnology), respectively. Columns were equilibrated and
run in 0.9% NaCl containing 0.02% NaN3, 5
mmol/L EDTA, and 2 mmol/L sodium phosphate buffer, pH 7.4. Pooled
plasma samples obtained from 5 mice were filtered through 0.45-µm
filters (diameter 13 mm, Millipore), and 0.5 mL of plasma was
loaded onto the Superose 6 column. The separation was performed at
4°C with a flow rate of 0.1 mL/min. Fractions of 0.8 mL were
collected for measurement of cholesterol, phospholipids,
and PLTP activity.
Quantification of Pre-ß-HDL by Crossed Immunoelectrophoresis
Freshly isolated plasma samples from mice were either directly
frozen or incubated at 37°C in the presence of iodoacetate to measure
the formation of pre-ß-HDL in vitro. Iodoacetate (1 mmol/L) was
added for complete inhibition of lecithin:cholesterol acyl
transferase (LCAT). This prevents maturation of the formed pre-ß-HDL
into 
-HDL by LCAT activity. The LCAT inhibitor had no
effect on the plasma pre-ß-HDL concentration if added to freshly
isolated plasma (not shown).
The crossed immunoelectrophoresis consisted of agarose electrophoresis
in the first dimension for separation of lipoproteins with pre-ß and
mobility. Electrophoresis in the second dimension (ie, antigen
migration from the first gel into an anti–apoA-I–containing gel) was
used to quantitatively precipitate apoA-I. Lipoprotein electrophoresis
was carried out in 1% (wt/vol) agarose gels in barbital buffer
(50 mmol/L, pH 8.6) and run in an LKB 2117 system (4°C for 2
hours, 250 V). Plasma was applied at 5 µL per well. The track of the
first agarose gel was excised and annealed with melted agarose to a gel
containing 7.5% (vol/vol) rabbit anti-mouse apoA-I antiserum that was
cast on GelBond film (Pharmacia). The plate was run in an LKB 2117
system (4°C for 20 hours, 50 V) in barbital buffer. Unreacted
antibody was removed by extensive washing in PBS. The gel was stained
with Coomassie brilliant blue R250 and subsequently dried. Areas under
the pre-ß-HDL and -HDL peaks were calculated by multiplication of
peak height and width at half height. The pre-ß-HDL area is expressed
as a percentage of the sum of -HDL and pre-ß-HDL areas.
Pre-ß-HDL concentrations are also given in absolute amounts
(micrograms of apoA-I present in pre-ß-HDL per milliliter
plasma). These values were calculated from the percentage of apoA-I
present in pre-ß-HDL and the total plasma apoA-I
concentrations.
Cholesterol Flux Experiments With Mouse Peritoneal
Macrophages
The ability of mouse heparin-plasma to interfere with the
intracellular formation by acyl coenzyme A:cholesterol
acyltransferase (ACAT) of labeled cholesteryl oleate from
[3H]oleate and acetylated LDL
(AcLDL)-derived cholesterol was tested by using the assay
developed by Brown et al.23 The formation of labeled
cholesteryl esters from [3H]oleate by ACAT is
an estimate of intracellular cholesterol concentration.
BSA (fraction V grade) was delipidated by extraction of free fatty acids with activated carbon.24 [9,10(n)3H]Oleic acid (10.0 Ci/mmol, Amersham) was complexed to BSA after evaporating 0.1 mmol of oleic acid (48 Ci/mol) to dryness under a stream of nitrogen. Subsequently, 10 mL of 12% (wt/vol) fatty acid–free BSA in DMEM at 56°C was added to the dried oleate and sterilized by passage through a 0.22-µm filter.
AcLDL was prepared from LDL (density range 1.019 to 1.063 g/mL), isolated from human plasma by differential centrifugation, and subsequently acetylated by repeated additions of acetic anhydride.25 Increased electrophoretic mobility of the AcLDL was confirmed by agarose electrophoresis at pH 8.6.12
C57Bl/6 mice were elicited by intraperitoneal injection of 0.8 mL of eliciting agent, prepared from Baker’s thioglycollate (DIFCO) according to the manufacturer’s instructions. After 4 days, macrophages were obtained as described.26 The macrophage monolayers were washed with DMEM and incubated with 500 µL aliquots of DMEM that contained 3 µg/mL AcLDL, 0.1 mmol/L BSA-[3H]oleate (48 Ci/mol), and plasma that was diluted (12 times) from fasted mice. After 18 hours, medium was removed, and the cells were washed twice with PBS. Cholesteryl esters were extracted from the intact monolayers with 1 mL hexane-isopropanol (3:2 [vol/vol]) and purified by thin-layer chromatography as previously described.27 Labeled cholesteryl ester bands were excised from the silica, and radioactivity was determined. Protein was extracted from the cell remnants with 0.1 mol/L NaOH and quantified by the method of Lowry et al28 using BSA as a standard. Duplicate assays were performed for each plasma sample.
Statistics
Data are given as mean±SD. Differences between groups were
analyzed by 1-way ANOVA. The Bonferroni correction was used for
multiple pairwise comparisons when the ANOVA indicated a
significant effect.
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Results |
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Generation of Transgenic Mice
A cosmid clone containing the complete HuPLTP gene was isolated and analyzed by Southern blotting. Approximately 15 kb 5' to the first exon and
3.5 kb 3' to the last exon of the PLTP gene were
present in the cosmid as well. The 3' end, but not the 5' end, of
the lysosomal protective protein gene was included (Figure 1). The purified 35-kb cosmid was
microinjected in fertilized oocytes. This resulted in 27 newborn mice,
2 of which harbored the transgene, as determined by PCR
analysis and Southern blotting (data not shown). Both founder
mice were bred into 2 independent lines of HuPLTP transgenic mice.
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Expression of the Transgene
PLTP activity was measured in plasma samples of mice from 2 HuPLTP
transgenic lines. In line 1, PLTP activity in the plasma was increased
by 281% compared with the activity in the plasma from wild-type mice
(Table 1), whereas in line 2,
activity was increased by 262% (not shown). Subsequent
analyses were performed with line 1 only. The use of either
human HDL or mouse HDL as acceptor in the PLTP activity assay showed
the same differences between wild-type, hemizygous, and homozygous
transgenic animals, demonstrating that HuPLTP interacts with human HDL
and mouse HDL (not shown).
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The expression of the transgene was confirmed by Western blotting
(Figure 2
). An antibody that reacts
specifically with human PLTP was used. Compared with hemizygous
transgenic mice, the mice homozygous for the transgene showed a higher
plasma protein level of HuPLTP. No immunoreactive PLTP was detected in
wild-type mice, because the antibody used does not cross-react with
MuPLTP. Human recombinant PLTP was used as a positive control and gave
1 immunoreactive band at a relatively low molecular weight because of
the lower extent of glycosylation in the baculovirus expression
system.
The transgene was found to be expressed in all tissues analyzed
(Figure 3), with relatively high
mRNA levels in adrenal, testis, and (to a lesser extent) lung. Moderate
mRNA levels were present in liver, kidney, intestine, brain, and
spleen. The mRNA expression of the endogenous PLTP gene is
highest in adrenal, testis, and lung. The prominent expression in lung
tissue is found for MuPLTP but not for the (human) transgene. The
expression of endogenous PLTP was not affected by HuPLTP
expression in any of the tissues tested (Figure 3).
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Effects of HuPLTP Overexpression on Plasma Lipids and
Lipoproteins
The overexpression of PLTP resulted in a decrease in plasma
cholesterol levels in the HuPLTP hemizygous transgenic mice
and a further decrease in levels in the homozygous transgenic mice
(Table 1). Plasma levels of cholesterol,
phospholipids, and apoA-I were decreased by about the same extent,
indicating that the decrease reflects a lowering of HDL, which is the
major lipoprotein in mouse plasma. Separation of plasma lipoproteins by
gel filtration confirmed that the decrease in plasma lipids can be
largely attributed to a decreased level of HDL (fractions 15 to 18),
because only minor changes were found in VLDL (fraction 4 to 6) and LDL
(fraction 8 to 11), as seen in Figure 4.
The peak of PLTP activity was found in fractions 13 to 15 on filtration
of plasma from wild-type mice, as well as plasma from HuPLTP transgenic
mice (Figure 4).
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To investigate changes in HDL subclass distribution, mouse plasma
was analyzed by crossed immunoelectrophoresis (Figure 5). Plasma samples from wild-type,
hemizygous, and homozygous HuPLTP transgenic mice were collected and
incubated in the presence of an inhibitor of LCAT to
prevent maturation of the formed pre-ß-HDL into
-HDL.14 The formation of pre-ß-HDL particles is
clearly increased in plasma from transgenic mice compared with
wild-type mice. Table 2 gives the
pre-ß-HDL values, before and after incubation in the presence of the
LCAT inhibitor. No significant differences were found in
freshly frozen plasma between the different genotypes. Before
incubation, the percentage of pre-ß-HDL tends to be highest in the
homozygous HuPLTP transgenic animals, but the differences between
genotypes are not significant. However, in incubated samples,
clear differences arise with the highest relative and absolute
concentrations of pre-ß-HDL in the transgenic animals.
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Effect of Wild-Type and Transgenic Plasma on Accumulation of
AcLDL-Derived Cholesterol in Mouse Peritoneal
Macrophages
We investigated whether differences in the HDL-subfraction pattern
affect the ability of wild-type and transgenic plasma to interfere with
the esterification of intracellular cholesterol by ACAT.
ACAT activity determined in this manner is a measure of the
intracellular cholesterol concentration. Mouse peritoneal
macrophages were incubated in the presence of
[3H]oleate, AcLDL, and diluted mouse plasma
containing the acceptor HDL particles (see Methods). Figure 6 shows that the formation of labeled
cholesteryl oleate by ACAT was 25.7±9.7% lower in the presence of
hemizygous transgenic plasma compared with wild-type plasma, indicating
less accumulation of cellular cholesterol in the presence
of plasma from transgenic animals. Thus, in spite of a lower HDL
cholesterol concentration, transgenic mouse plasma has the
ability to prevent cholesterol accumulation to a greater
extent. With plasma from homozygous HuPLTP transgenic mice, a
42.4±13.2% decrease in ACAT activity (compared with wild-type plasma;
see Figure 6) is measured, showing even less accumulation,
indicative of a dose-response effect of PLTP on the prevention of
cholesterol accumulation.
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Discussion |
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We generated transgenic mice for HuPLTP that have up to a 4.5-fold elevation of plasma PLTP activity levels. The total plasma concentrations of cholesterol, phospholipids, and apoA-I are decreased because of a 30% to 40% reduction in total HDL. Gel filtration, which is used to measure the plasma lipoprotein profile, confirmed the decrease in HDL cholesterol and showed little variation in LDL and VLDL. No change in HDL size was detected.
Plasma pre-ß-HDL concentrations are unchanged in HuPLTP
transgenic mice (Table 2). This does not preclude the
possibility of increased turnover of pre-ß-HDL in the transgenic
animals. Pre-ß-HDL is the substrate of plasma LCAT that converts it
into mature -HDL, normally carrying most of the circulating plasma
HDL cholesterol.14 The formation of
pre-ß-HDL during plasma incubation at 37°C is therefore masked by
the LCAT reaction, and the maximal capacity for pre-ß-HDL formation
can be measured only when LCAT is inactive. For this reason, plasma was
incubated in the presence of an LCAT inhibitor. These
experiments revealed that the relative and absolute plasma levels of
pre-ß-HDL are clearly increased in plasma from transgenic animals
incubated under these conditions (Table 2). These data indicate
that rapid pre-ß-HDL conversion to -HDL, driven by LCAT, may
obscure the effects of the HuPLTP gene on plasma pre-ß-HDL levels in
vivo. Our data imply that HuPLTP transgenic plasma has a much greater
ability to generate pre-ß-HDL than does wild-type plasma, in spite of
only marginal differences in pre-ß-HDL levels.
HuPLTP transgenic mice have been described previously by 2 other groups, but these models did not show an appreciable overexpression of the transgene. The mice described by Albers et al17 showed little expression of the transgene and only small changes in plasma lipoproteins. Jiang et al18 reported a 29% increase in PLTP activity, but significant effects on total plasma lipids or lipoproteins were not observed. After their HuPLTP transgenic mice were crossbred with mice transgenic for human apoA-I, they detected minimal effects on plasma lipids and lipoproteins (including an increase in pre-ß-HDL levels), together with a 47% elevation in plasma PLTP activity. It must be noted that compared with wild-type mice, human apoA-I transgenic mice already have elevated levels of total and pre-ß-HDL cholesterol.18 The HuPLTP transgenic mice described in the present study have altered HDL metabolism, without the complication of the additional human apoA-I gene, which by itself has substantial effects on HDL. Moreover, our mice are not transgenic for lysosomal protective protein. Jiang et al18 used a DNA construct containing both genes that have a 3' overlap on opposite DNA strands. Although unlikely, it is difficult to exclude the possibility that this condition interferes with lipoprotein metabolism.
HuPLTP has also been overexpressed in mice via
adenovirus-mediated transfer.31 32 These mice showed a 13-
to 40-fold elevation of PLTP activity in plasma several days after
treatment. This resulted in a dramatic decrease (by 91%) in total HDL
cholesterol levels, whereas pre-ß-HDL levels were
substantially elevated.31 These data are in line with our
present observations: high PLTP activity results in a decrease in
total HDL, whereas pre-ß-HDL levels are increased. It is clear that
the effects are transient and greatly exaggerated in the
adenovirus-treated mice, because of the extremely high plasma levels of
PLTP. Four-fold–elevated PLTP activity levels, measured at late time
points after adenovirus transfection, did not result in altered plasma
HDL cholesterol concentrations, whereas in our homozygous
HuPLTP transgenic mice, a 4-fold elevation in PLTP activity resulted in
decreased HDL concentrations. Another notable difference is that in the
adenovirus-treated mice, PLTP expression is restricted to the liver.
The tissue pattern of expression of the HuPLTP gene in transgenic mice
more closely resembles the pattern of expression of the
endogenous PLTP gene (Figure 3).33
Compared with plasma of wild-type mice, the plasma of HuPLTP transgenic mice was found to be much more efficient in partially preventing the AcLDL-induced accumulation of intracellular cholesterol in cultured macrophages, in spite of lower levels of total HDL.
The most likely explanation for this observation is the increased plasma concentration of pre-ß-HDL, which has been identified previously as a very efficient cholesterol acceptor.13 14 It is well known that an operative cholesteryl ester cycle, as present in macrophages,34 35 is important for cholesterol efflux. Miyazaki et al36 show that dimyristoylphosphatidylcholine/apoA-I complexes, but not HDL, may modify AcLDL in a concentration-dependent manner, resulting in decreased cellular uptake. However, the plasma concentration of pre-ß-HDL is not increased in our HuPLTP transgenic mice. Therefore, it is unlikely that the prevention of cellular cholesterol accumulation by transgenic mouse plasma is due to decreased uptake of AcLDL by the macrophages. The present results imply that the distribution of HDL subclasses is of major importance in the efficacy of HDL-mediated reverse cholesterol transport; it is even more important than total plasma HDL levels. Compared with plasma from hemizygous mice, plasma from homozygous HuPLTP transgenic animals appeared to be more efficient in preventing cellular cholesterol accumulation, indicating a dose response for the effect of PLTP on cellular cholesterol. In addition to the proposed mechanism of action of PLTP on cellular cholesterol efflux via the generation of pre-ß-HDL, PLTP could also contribute directly to cellular cholesterol efflux via its proposed cholesterol transfer activity.37
Plasma levels of total HDL cholesterol are inversely
correlated with the incidence of coronary artery disease in
humans.7 8 However, only a few studies have looked at the
relation between total HDL and pre-ß-HDL in human plasma. Recently,
O’Connor et al14 analyzed the steady-state levels
of pre-ß-HDL in 136 normolipidemic individuals by using an
isotope-dilution technique. Their relative values for pre-ß-HDL in
human plasma are quite comparable to the values measured in mouse
plasma (see Table 2). The percentage of pre-ß-HDL (percentage
of total plasma apoA-I) was negatively correlated with total HDL
cholesterol concentrations, in line with our observations
in mice. Plasma PLTP activity was not measured in their study.
Reconstituted HDL particles enriched in triglycerides, which are model particles for HDL prevalent during alimentary lipemia, are more rapidly converted by PLTP to pre-ß-HDL than are triglyceride-poor HDL particles.38 This observation suggests that hypertriglyceridemia may be associated with increased generation of pre-ß-HDL by PLTP. In addition, Syvänne et al39 reported a positive correlation between PLTP activity and the capability of plasma from patients with diabetes mellitus and coronary heart disease to induce cholesterol efflux from Fu5AH rat hepatoma cells.
Taken together, the combination of increased PLTP activity, increased pre-ß-HDL formation, and less accumulation of cellular cholesterol (as seen in our mouse model) is likely to exist also in the human situation. The rate of formation of pre-ß-HDL may very well be more important than its steady-state concentration. The present work shows for the first time that PLTP is important in the ongoing generation of plasma pre-ß-HDL and that the ability of plasma to prevent cholesterol accumulation in macrophages is increased at high PLTP activity levels. Our findings show that PLTP is a potential antiatherogenic factor because of its ability to generate pre-ß-HDL. To test this hypothesis directly in vivo, we will perform a comparative study in which the susceptibility to diet-induced atherosclerosis will be evaluated. We are currently expanding the colony of HuPLTP transgenic mice to this end. Because the PLTP-expressing mice are expected to be even more resistant to diet-induced atherosclerosis than are wild-type mice, these experiments will be very time-consuming. Another approach will be to measure (diet-induced) atherogenesis in mouse models susceptible to the development of atherosclerosis (eg, that due to LDL receptor deficiency40 or apoE deficiency41 42 ), with and without overexpression of HuPLTP.
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Acknowledgments |
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This study was supported by the Netherlands Heart Foundation (grant 96.139 to Dr van Tol) and from the Finnish Foundation for Cardiovascular Research (Drs Jauhiainen and Ehnholm). Thanks are extended to J. Huuskonen, J. Metso, and L.M. Scheek for their expert technical assistance and M. Kuit for his photographic work. Dr Christian Ehnholm (National Public Health Institute, Helsinki, Finland) is acknowledged for critically reading the manuscript and many fruitful discussions.
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Footnotes |
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1 Dr van Tol and Dr van Haperen contributed equally to this study.
Received June 1, 1999; accepted October 14, 1999.
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