© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Increased Cholesterol Efflux Potential of Sera From ApoA-IMilano Carriers and Transgenic Mice
From the Center E. Grossi Paoletti and Institute of Pharmacological Sciences (G.F., L.C., G.C., C.P., C.R.S., M.C.), University of Milano, and the Institute of Pharmacology and Pharmacognosy (F.B.), University of Parma, Italy.
Correspondence to Guido Franceschini Center E. Grossi Paoletti, Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milano, Italy. E-mail Guido.Franceschini{at}unimi.it
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Abstract |
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Abstract—The ability of HDL to remove cholesterol from peripheral cells and drive it to the liver for excretion is believed to explain most of the strong inverse correlation between plasma HDL cholesterol levels and coronary heart disease. Carriers of the ApoA-IMilano (A-IM) mutant have a severe hypoalphalipoproteinemia but are not at increased risk for premature of coronary heart disease. To explain this apparent paradox, we compared the capacity of serum from A-IM and control subjects to extract cholesterol from Fu5AH cells. Because the A-IM carriers are all heterozygotes for the mutation, we also compared the cholesterol efflux capacity of serum from transgenic mice expressing A-IM or wild-type ApoA-I (A-IWT), in the absence of murine ApoA-I. In the whole series of human or mouse sera, cholesterol efflux was significantly correlated with several HDL-related parameters; after adjustment for concomitant variables, the only parameter that remained significantly correlated with cholesterol efflux was the serum ApoA-I concentration (r2=0.85 in humans and 0.84 in mice). The same was true when samples from control subjects, A-IM carriers, A-IWT or A-IM mice were analyzed separately. Cholesterol efflux to sera from the A-IM carriers was only reduced slightly compared with control sera (25.0±4.2% versus 30.4±3.3%), although there was a large reduction (-45%) in the serum ApoA-I concentration in the former. Cholesterol efflux was also lower to sera from A-IM than A-IWT mice (15.6±3.8% versus 30.1±7.1%), but less than expected from the 70% reduction in serum ApoA-I concentration. A relative efflux potential of serum was calculated in each group as the slope of the regression line fitting cholesterol efflux to ApoA-I concentrations. Therefore, the relative efflux potential reflects the relative efficiency of ApoA-I in determining cell cholesterol efflux. The relative efflux potential of mouse and human sera was in the following order: A-IM mice>A-IM carriers>A-IWT mice=control subjects, suggesting a gene–dosage effect of the A-IM mutation on the efficiency of serum to extract cholesterol from cells. The high efficiency of A-IM-containing HDL for cell cholesterol uptake would result in an improved reverse cholesterol transport in the A-IM carriers, possibly explaining the low susceptibility to atherosclerosis development.
Key Words: reverse cholesterol transport • ApoA-I • cholesterol efflux • HDL • transgenic mice
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Introduction |
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Numerous case–control and prospective studies have shown a strong inverse relation between plasma HDL cholesterol (HDL cholesterol) concentrations and the risk of coronary heart disease.1 The mechanism(s) underlying this negative association are not fully understood, and several biological explanations have been proposed.2 Most prominent seems the function of HDL as vehicle of cholesterol in the reverse cholesterol transport (RCT),3 4 the process by which excess cholesterol in peripheral tissues, including the arterial wall, is transported to the liver for excretion.
The first step in RCT is the efflux of unesterified cholesterol from peripheral cells to either free apolipoproteins5 or lipoprotein acceptors6 present in the extracellular space. Several serum factors, including the acceptor structure/composition, and the concentration of lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and other lipoproteins are known to modulate cell cholesterol efflux.7 Therefore, the use of whole serum instead of isolated lipoprotein fractions would provide an ideal tool to evaluate the individual capacity to promote cell cholesterol efflux.8 This approach has the advantage that all of the potential factors affecting efflux are present in the experimental setting, and therefore seems particularly suitable to correlate the efficiency of the earliest step in RCT to atherosclerosis susceptibility in subjects,9 10 or animal models at different risks of cardiovascular disease.11 12
ApoA-IMilano (A-IM) is the first described mutant of human apolipoproteins.13 Thirty-eight heterozygous carriers have been identified, up to now,14 who represent the largest group of individuals with low plasma HDL levels because of a single defect in the ApoA-I gene. The plasma concentration of major factors involved in RCT, ie, HDL, ApoA-I, LpA-I, LCAT, and CETP is remarkably reduced in A-IM carriers compared with controls.15 16 17 The severe hypoalphalipoproteinemia and the partial LCAT and CETP deficiencies suggest a defective RCT, but the carriers do not suffer from premature coronary heart disease.14 To investigate the mechanism(s) behind this apparent paradox, we compared in the present study the cholesterol efflux potential of sera from A-IM carriers and control subjects. Because of the limited number of carriers and the difficulty in discerning the impact of the mutation in these heterozygous subjects, we also evaluated the cholesterol efflux potential of sera from transgenic mice expressing the human A-IM variant or wild-type ApoA-I, in the absence of murine ApoA-I. A-IM transgenic mice share with human carriers low plasma HDL levels, increased triglycerides, defective cholesterol esterification, and structural abnormalities in HDL particles,18 19 and therefore are a suitable model for studying the impact of the mutation in a homozygous condition.
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Methods |
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Subjects
Nineteen adult heterozygous A-IM carriers (9 females and 10 males), belonging to the previously described A-IM kindred,14 volunteered for this study. Nineteen age- and sex-matched noncarriers were selected among close relatives in the same families. All participating subjects were healthy, were taking no medications, and consumed a typical Mediterranean diet. All subjects were fully informed of the methods and end points of the study, which were approved by the Internal Review Board.
After an overnight fast, blood was collected both into empty plastic tubes and into tubes containing Na2-EDTA (final concentration, 1 mg/mL). Serum and plasma were prepared by low-speed centrifugation at 4°C. Serum aliquots were added with Na2-EDTA (1 mg/mL) and solid NaBr (final concentration, 5.1 mol/L) and kept at 4°C for HDL subfraction analysis by rate-zonal ultracentrifugation.20 Serum aliquots for cholesterol efflux were immediately frozen and stored at -80°C until assayed.
Transgenic Mice
The generation of transgenic mice expressing human ApoA-I
(A-IWT) and the A-IM
variant has been previously described.18 These mice
express A-IWT or A-IM
together with human ApoA-II, and do not express murine ApoA-I because
of gene targeting.21 Twenty-one A-IM
mice and 21 A-IWT mice, of similar age (16 to 24
weeks) and of both sexes, were used for the cholesterol
efflux experiments.
Blood was collected after an overnight fast from the retroorbital plexus. Serum was prepared by low-speed centrifugation at 4°C and assayed on the same day for human apolipoprotein and lipid/lipoprotein levels. Serum aliquots for cholesterol efflux were immediately frozen and stored at -80°C until assayed.
Cell Cholesterol Efflux
The cholesterol efflux potential of serum was
assayed as described by de la Llera Moya et al,8 by
incubating diluted serum with
[3H]cholesterol-labeled Fu5AH rat
hepatoma cells for 4 hours at 37°C. Cells were seeded in Corning
24-well (15.5 mm/well) plates, using 20 000 cells per well, and
grown in DMEM with 5% FCS for 2 days. Lipids were radiolabeled by
adding 2 µCi/mL
[1,2-3H]cholesterol (Amersham) to
25% FCS in DMEM. Cells were grown in the presence of radiolabeled
cholesterol for 2 additional days to obtain confluent
monolayers. The labeling medium was replaced with DMEM containing 1%
essential fatty acid–free albumin for 18 to 20 hours to allow
equilibration of the label. Cells were then washed 2 times with PBS and
incubated for 4 hours with control medium or serum diluted in DMEM with
essential fatty acid–free albumin 1%. All efflux assays were
performed by using the same dilution (5%) for mouse and human sera. At
the end of this period, the medium was removed, collected into tubes,
and centrifuged for 5 minutes at 2000 rpm to remove any
floating cells. An aliquot of the medium was then counted for
[3H]cholesterol radioactivity
(Formula 989, Packard). Cellular lipids were extracted with 2-propanol
by overnight incubation at room temperature and radioactivity was
measured in an aliquot of the extract (Insta-Fluor, Packard).
Cholesterol efflux was calculated as the percentage of
total label in each well released to the medium. Individual efflux
values were calculated as averages of 3 determinations in different
wells, normalized to the cholesterol efflux obtained with a
pool of normolipidemic sera tested in each experiment.
Analyses
Serum total and unesterified cholesterol (TC and
UC), triglycerides (TG), and phospholipid (PL) levels were determined
with standard enzymatic techniques by using a Roche
diagnostics Cobas autoanalyzer. The CE mass was
calculated as (TC-UC)x1.68. The protein (P) content in lipoprotein
fractions was determined by the method of Lowry et al.22
The lipoprotein surface/core ratio was calculated as (FC+PL+P)/(TG+CE).
HDL cholesterol levels in human sera were measured after precipitation
of the ApoB-containing lipoproteins by dextran
sulfate–MgCl2.23 HDL cholesterol
was measured in mouse sera after precipitation of ApoB-containing
lipoproteins with polyethylene glycol (20%, wt/vol) in 0.2 mol/L
glycine (pH 10).24 ApoA-I, ApoA-II, and ApoB levels were
determined by immunoturbidimetry,25 using the Cobas
analyzer with commercially available polyclonal antibodies
(Boehringer Mannheim). The anti-A-I antibody recognizes all
forms of A-IM (monomer, homodimer, and
heterodimer) as wild-type ApoA-I26 ; therefore, the ApoA-I
concentration determined in the sera of A-IM
carriers, who are heterozygotes for the mutation, is the sum of mutant
and wild-type ApoA-I, and the ApoA-I concentration in the sera of
A-IM mice, who are homozygotes for the mutation,
is the concentration of mutant ApoA-I.
Human plasma lipoproteins were separated by sequential ultracentrifugation,27 using a Beckman TL 100 ultracentrifuge equipped with a TL 100.3 rotor (Beckman Instruments). HDL subfractions were isolated by rate-zonal ultracentrifugation in a swinging bucket rotor20 ; 2 fractions, designated as HDL2 and HDL3, were collected, and the total cholesterol content measured by enzyme methods.
Statistical Analyses
Quantitative variables are expressed as mean±SD values.
Differences among the groups were evaluated by 1-way ANOVA, with post
hoc evaluation by the Neuman–Keuls test. Statistical significance was
defined as P<0.05. Simple and multivariate
regression analyses were performed and the significance of the
correlations was determined by the F parameter.
In the forward stepwise regression, the independent
parameters were included 1 at a time starting with the
parameter that had the highest correlation with the
dependent variable, fractional cholesterol efflux.
Additional parameters were included only if a significant
increase in goodness of fit was achieved. Logarithmic transformation
was performed on individual data when values were not normally
distributed.
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Results |
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Cholesterol Efflux to Human Sera
The fasting serum lipid and lipoprotein concentrations in the examined A-IM carriers and control subjects are given in Table 1
. The
A-IM carriers had significantly lower serum total
and LDL cholesterol, and phospholipid concentrations than
controls; although there was a trend for A-IM
carriers to have greater serum triglycerides and VLDL
cholesterol levels, the difference in average values
between carriers and controls did not reach statistical significance
(P=0.15 and 0.16, respectively). The
unesterified/esterified cholesterol ratio was
significantly higher in the A-IM carriers
(0.45±0.09 versus 0.34±0.05), mostly because of the lower plasma LCAT
concentration.16 As expected,15 serum
HDL cholesterol, HDL2 cholesterol,
HDL3 cholesterol, HDL phospholipid, ApoA-I, and
ApoA-II levels were much lower in the A-IM
carriers than controls. The ApoA-I/ApoA-II ratio was significantly
higher in the A-IM carriers than in controls
(4.65±1.20 versus 3.70±0.65), consistent with the reported
preferential reduction of LpA-I:A-II particles in the
former.17
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HDL particles from A-IM carriers were enriched in
triglycerides and phospholipids, and depleted in cholesteryl esters
compared with control HDL (Table 2); the
surface/core ratio was significantly higher in
A-IM HDL than in control HDL (4.29±1.26 versus
3.25±0.49), indicative of a prevalence of small particles in the serum
of A-IM carriers.28
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Cholesterol efflux data, expressed as percent efflux from
Fu5AH cells during a 4-hour incubation, obtained with sera from
A-IM carriers and controls, are shown in Figure 1. The average efflux value was 18%
lower in A-IM carriers (25.0±4.2%) than in
controls (30.4±3.3; P<0.001). Both female and male
subjects participated in this study; a significantly higher
cholesterol efflux to sera from female than male subjects
was found among A-IM carriers (27.0±4.0% versus
23.1±3.6%) but not controls (30.6±3.2% versus 30.1±3.6%). The
trend toward higher efflux with A-IM female serum
was paralleled by a trend for higher HDL cholesterol levels
in female than male serum (20.6±9.9 and 15.9±10.9 mg/dL,
respectively).
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Cholesterol efflux in the whole series of sera correlated
with several serum parameters, most of which are related to
HDL (Table 3). Stepwise regression
analysis was then performed to identify which of the correlated
parameters best predicted cholesterol efflux to
serum. In the whole series, the serum ApoA-I concentration was the
strongest predictor of cholesterol efflux
(r2=0.85); HDL triglycerides, HDL
phospholipids, and HDL unesterified cholesterol made small additions to
this correlation, the 4 variables explaining 90% of the variation
in cholesterol efflux. A separate stepwise analysis
was also performed with data from control and
A-IM samples. Serum ApoA-I concentration was
again the strongest predictor of cholesterol efflux to
control sera (r2=0.63), with serum
esterified cholesterol adding significantly to this
prediction (r2=0.72). In the
A-IM group, serum ApoA-I was the strongest
univariate predictor of cholesterol efflux
(r2=0.86); HDL phospholipids and the
HDL unesterified/esterified cholesterol made small
additions to this correlation, the 3 variables explaining 93% of
the variation in cholesterol efflux.
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Because the serum ApoA-I concentration is the best predictor of
cholesterol efflux, a relative efflux potential (REP) was
calculated as the slope of the regression line fitting
cholesterol efflux to ApoA-I concentrations11
for A-IM and control subjects, separately.
Therefore, REP reflects the relative efficiency of ApoA-I in
determining cell cholesterol efflux. The REP was 50%
higher in A-IM carriers (0.116) than controls
(0.075) (Figure 2), indicating that the
same relative variation in ApoA-I concentration causes a higher
cholesterol efflux to the serum of
A-IM carriers than of control subjects. This
difference in REP explains the relatively mild reduction in
cholesterol efflux to A-IM than
control sera, despite the severe ApoA-I reduction in
A-IM sera.
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Cholesterol Efflux to Mouse Sera
One of the major problems we encountered in evaluating the impact
of the A-IM mutation on lipoprotein
metabolism in the carriers is that they are all
heterozygotes for the mutation, carrying both wild-type and mutant
ApoA-I.14 To resolve this issue, transgenic mice were
generated that express the A-IM mutant
(A-IM mice) in the absence of murine
ApoA-I.18 We then compared the capacity of the serum from
these mice to extract cholesterol from cells with that of
serum from mice expressing wild-type human ApoA-I
(A-IWT mice). The serum lipid/lipoprotein levels
in A-IM and A-IWT
transgenic mice are reported in Table 4.
The A-IM mice had significantly higher serum
triglycerides and lower total cholesterol, HDL cholesterol, HDL
phospholipids, and ApoA-I concentrations than
A-IWT mice.18 Therefore, the
expression of the A-IM mutation in a transgenic
mouse reproduces the major lipid/lipoprotein abnormalities observed in
human carriers, providing a unique model to investigate the
metabolic effects of the A-IM
mutation in a homozygous state.
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The cholesterol efflux to mouse serum was strongly
positively correlated with the serum ApoA-I concentration in the whole
series of samples (r=0.92), and both in the
A-IM and A-IWT groups
(r=0.77 and 0.83, respectively), as found in experiments
with human sera. Indeed, the serum ApoA-I concentration was the only
parameter that remained significantly correlated with
cholesterol efflux after adjustment for concomitant
variables. Cholesterol efflux to serum was
significantly lower in A-IM than
A-IWT mice (Figure 1
and Table 4).
The serum REP was calculated as the slope of the regression lines
fitting cholesterol efflux to ApoA-I concentrations, as
done for human samples. The REP was 2-fold higher in
A-IM than A-IWT mice (0.151
versus 0.074) (Figure 2), indicating that for the same relative
variation in serum ApoA-I concentration, the percent
cholesterol efflux is higher with
A-IM than A-IWT mice sera,
as found in human subjects.
It is noteworthy that the REP was similar for the sera from
A-IWT mice and from control subjects (Figure 2), indicating that the efficiency of human ApoA-I in
determining cholesterol efflux is the same when the protein
is expressed in either human or murine background. Indeed a strong
positive correlation (r=0.87) was found between
cholesterol efflux and serum ApoA-I concentration, when
data from control subjects and A-IWT mice were
analyzed together.
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Discussion |
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Cholesterol efflux from peripheral cells to HDL acceptors is the first step in RCT, the pathway believed to explain the protective role of HDL against the development of vascular lesions.7 To investigate whether the low susceptibility of the A-IM carriers to atherosclerotic vascular disease may be explained by improved cholesterol efflux capacity, we examined the ability of serum from these subjects and from transgenic mice expressing the A-IM mutant to extract cholesterol from cultured cells. The present results indicate that in a series of samples from humans and mice expressing wild-type or mutant ApoA-I, the concentration of ApoA-I is the largest predictor of the ability of serum to extract cholesterol from cells. They also provide indirect evidence of a similar efficiency of human ApoA-I in promoting cell cholesterol efflux when the protein is expressed in humans or mice, and of an improved efficiency of the A-IM mutant versus wild-type ApoA-I in determining cell cholesterol efflux both in humans and mice.
The correlation analyses on the whole series of human data and on data from separate groups indicate that cholesterol efflux to serum is positively correlated with several HDL-related parameters, confirming that HDL particles are the major factors responsible for cholesterol efflux from cells to serum.8 By multivariate correlation analysis, the ApoA-I concentration in serum was the largest predictor of cholesterol efflux. A strong positive correlation between serum ApoA-I levels and cholesterol efflux was also found when data from 2 animal species expressing human ApoA-I, as control subjects and A-IWT mice, were combined. Thus, it follows that human ApoA-I determines the cholesterol efflux potential of serum, even in species with a widely different genetic background.
Recent cholesterol efflux studies with human samples, and with sera from transgenic mice and rats expressing human ApoA-I, suggest that the major serum factor involved in the regulation of cell cholesterol efflux is the HDL phospholipids content.11 29 30 31 Phospholipids may facilitate the interaction of HDL acceptors with cells through the scavenger receptor BI,32 33 therefore improving cholesterol desorption from the cell membrane. This is particularly true for Fu5AH cells, which display a high level of scavenger receptor BI expression.32 Indeed, in the present study, HDL phospholipid was strongly correlated with efflux, and contributed to the variability in cholesterol efflux among the various serum samples. However, the serum ApoA-I concentration was the strongest predictor of cholesterol efflux to serum. The reasons for this discrepancy are not immediately clear, and may relate to the different level of ApoA-I expression in the various transgenic lines. In transgenic animals expressing high levels of human ApoA-I, as those used in previous studies,11 29 the cholesterol efflux to serum became less efficient as the concentration of ApoA-I increased, because of a marked decrease in the HDL phospholipid/ApoA-I ratio, and to the appearance of poorly effective lipid-free ApoA-I in serum.11 29 This is not the case in the present study. The A-IM and A-IWT mice have low-normal serum ApoA-I levels, no lipid-free ApoA-I in serum,18 31 and a similar HDL phospholipid/ApoA-I ratio. The distinct physicochemical properties of acceptor particles containing wild-type or mutant ApoA-I18 28 34 35 may also contribute to the discrepancy between the present and previous findings, by affecting the interaction of HDL with scavenger receptor BI.
A major observation in this study was that, although there was a large
reduction in serum ApoA-I and HDL concentrations, the
cholesterol efflux to sera from the
A-IM carriers was only slightly reduced compared
with control sera. The explanation for this apparent paradox came from
the estimation of the efficiency of A-IM and
A-IWT in determining cell cholesterol
efflux, obtained by calculating the serum REP as the slope of the
linear regression line between cholesterol efflux and serum
ApoA-I concentration. The REP, which provides an indirect estimation of
the relative efficiency of acceptor particles to extract
cholesterol from cells,11 was higher in
A-IM carriers than control subjects, suggesting a
relative abundance of more efficient acceptors in the sera from
A-IM carriers. Previous extensive studies on the
characterization of HDL particles in these subjects demonstrate that
A-IM HDL are smaller in size than control
HDL,28 34 a characteristic that has been associated with
improved efficiency for cell cholesterol uptake. Moreover,
efflux studies with reconstituted HDL show that particles containing a
recombinant form of the disulfide-linked A-IM
dimer are more efficient than A-IWT-containing
particles in removing cholesterol from cultured
cells,36 possibly because of the unique conformation of
the mutant ApoA-I on the surface of HDL.35 A direct effect
of the mutation on the efficiency of HDL acceptors in cell
cholesterol uptake is demonstrated by the control
subjects=A-IWT mice<A-IM
carriers<A-IM mice gradient in the REP (Figure 2). Indeed, after adjustment for concomitant variables, a
highly significant (P=0.009) gene–dosage effect of the
A-IM mutation on REP was found.
Cholesterol efflux from cells to serum acceptors is only the first step in RCT, a pathway that involves many other processes.3 4 Therefore, variations in the serum capacity to extract cholesterol from cells would only partially contribute to the efficiency of RCT in vivo, and to the individual cardiovascular risk. Nevertheless, it is noteworthy that the A-IM carriers, who are not at increased risk despite the severe hypoalphalipoproteinemia, display an improved serum capacity for cell cholesterol uptake. This finding enhances our understanding of the impact of the A-IM mutation on RCT, and provides an explanation for the apparent protection of A-IM carriers against atherosclerotic vascular disease. It also supports the concept that cholesterol efflux is a major determinant of RCT in vivo,37 and may contribute significantly to the determination of individual cardiovascular risk.
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Acknowledgments |
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This work was supported in part by the Istituto Superiore di Sanità, Roma, Italy (ISS project "Gene Therapy"). The authors are indebted to E.M. Rubin, University of California at Berkeley, for the substantial help in generating transgenic mice.
Received September 4, 1998; accepted September 29, 1998.
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