© 1995 American Heart Association, Inc.
Articles |
Reverse Cholesterol Transport in Plasma of Patients With Different Forms of Familial HDL Deficiency
From the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität (A. von E., H.F., G.A.), and the Institut für Arterioskleroseforschung an der Universität Münster (Y.H., S.W., G.A.), Münster, FRG, and the Ospedale Regionale della Beata Vergine, CH-Mendrisio, Switzerland (G.N.).
Correspondence to Arnold von Eckardstein, Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität, Albert-Schweitzer-Strasse 33, D-48129 Münster FRG.
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Abstract |
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Abstract HDLs encompass structurally heterogenous lipoproteins that fulfill specific functions in reverse cholesterol transport. Two-dimensional nondenaturing gradient gel electrophoresis (2D-PAGGE) of normoalphalipoproteinemic plasma and subsequent immunoblotting with anti–apoA-I-antibodies differentiates pre-ß1-LpA-I, pre-ß2-LpA-I, pre-ß3-LpA-I,
-LpA-I2, and
-LpA-I3. Immunodetection with anti-apoE antibodies
differentiates 
-LpE and -LpE. Pulse-chase incubations of plasma
with [3H]unesterified cholesterol
([3H]UC)–labeled fibroblasts and subsequent 2D-PAGGE
revealed that cell-derived [3H]UC is taken up by
pre-ß1-LpA-I and -LpE. From these initial acceptors,
[3H]UC is transferred to LDL via
pre-ß2-LpA-I
pre-ß3-LpA-I-LpA-I.
Some UC is esterified in pre-ß3-LpA-I, and some is
esterified in -LpA-I after its retransfer from LDL. In this study we
investigated the effect of various forms of familial HDL deficiency on
reverse cholesterol transport. Plasma samples of patients with various
forms of HDL deficiency are characterized by the lack of specific HDL
subclasses. ApoE-containing HDLs, including -LpE, are present in
all kinds of HDL deficiency. However, all forms of LpA-I are absent in
apoA-I–deficient plasma, pre-ß3-LpA-I and -LpA-I from
the plasma of patients with Tangier disease (TD), and
pre-ß3-LpA-I and large -LpA-I from the plasma of
patients with lecithin:cholesterol acyltransferase (LCAT) deficiency
and fish-eye disease (FED). After a 1-minute pulse with labeled
fibroblasts, efflux of [3H]UC into HDL-deficient plasmas
decreased, compared with normal plasma, by 49% (apoA-I deficiency),
36% (TD), 21% (LCAT deficiency), and 28% (FED). In apoA-I
deficiency, only -LpE takes up cell-derived [3H]UC. In
the three other HDL-deficiency states, cell-derived
[3H]UC is initially taken up by both
pre-ß1-LpA-I and -LpE. The four HDL deficiencies are
also characterized by differences in the esterification of cell-derived
[3H]UC. No esterification occurs in LCAT-deficient
plasma. In FED plasma, [3H]UC is esterified in LDL. In
apoA-I deficiency and TD, however, [3H]UC is esterified
in lipoproteins free of apoA-I and apoB. In the two latter cases, the
transfer of [3H]cholesteryl ester to LDL is enhanced
compared with normal plasma. The lack of specific HDL subclasses and
the consequent changes in reverse cholesterol transport pathways
differently affect net mass efflux of cholesterol from fibroblasts into
HDL-deficient plasma. Compared with normoalphalipoproteinemic plasma,
net cholesterol efflux from fibroblasts into plasma is reduced by 48%,
12%, 60%, and 34% in apoA-I deficiency, TD, LCAT deficiency, and
FED, respectively. Removal of apoB-containing lipoproteins from plasma
of patients with apoA-I deficiency, TD, LCAT deficiency, and FED
further decreased net cholesterol efflux rates by 77%, 84%, 72%, and
64%, respectively, compared with a reduction of 39% in
normoalphalipoproteinemic control plasma. In conclusion, various
quantitatively minor HDL subfractions and LDL also present in
HDL-deficient plasma effectively contribute to reverse cholesterol
transport.
Key Words: HDL subclasses • apoA-I deficiency • familial LCAT deficiency • fish-eye disease • Tangier disease
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Introduction |
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Several epidemiological and clinical studies have revealed an inverse correlation between the plasma concentration of HDL cholesterol and the risk of myocardial infarction (reviewed in Reference 11 ). The ability of HDL to protect the vessel wall from atherosclerosis has usually been explained by the reverse cholesterol transport model (reviewed in References 2 through 42 3 4 ), in which HDL mediates the flux of excess cholesterol from peripheral cells to the liver. HDL, however, includes structurally and functionally heterogenous lipoproteins that can be differentiated on the basis of density, size, charge, and apolipoprotein composition.5 6 7 8 Pulse-chase incubations of plasma with [3H]cholesterol-labeled fibroblasts and subsequent nondenaturing two-dimensional gradient gel electrophoresis (2D-PAGGE) have helped to assign distinct roles to the various HDL subclasses in reverse cholesterol transport. From cell membranes, cholesterol is initially taken up by a subgroup of HDL that contains apoA-I as its only apolipoprotein and is termed pre-ß1-LpA-I because of its electrophoretic pre-beta mobility9 and by another subclass of HDL that contains only apoE and is termed
-LpE because
of its electrophoretic gamma mobility.10 From
pre-ß1-LpA-I, cell-derived cholesterol is rapidly
transferred to other lipoproteins in the order
pre-ß2-LpA-Ipre-ß3-LpA-I-LpA-ILDL.11
Details of cholesterol transfer subsequent to its uptake by -LpE are
not fully understood, except that cholesterol ultimately accumulates in
LDL. In normolipoproteinemic human plasma, lecithin:cholesterol
acyltransferase (LCAT) directly esterifies a minor portion of
cell-derived cholesterol during its passage through
pre-ß3-LpA-I; most cholesterol, however, is esterified in
-LpA-I after recycling from LDL.11 12 13
Several inborn errors of metabolism interfere with the formation of
normal HDL.14 Some mutations in the apoA-I gene prevent
synthesis and secretion of apoA-I. Clinically, the patients may
present with xanthomatosis, atherosclerosis, and/or corneal
opacifications.15 16 17 18 19 20 Mutations in the LCAT gene lead to
the expression of two different clinical and biochemical phenotypes.
Familial LCAT deficiency results from the complete failure to esterify
cholesterol in the plasma compartment and is characterized by increases
in the ratio of unesterified cholesterol (UC) to cholesteryl ester
(CE).21 22 23 Affected patients suffer from corneal
opacifications and nephropathy with proteinuria and renal
insufficiency. By contrast, in fish-eye disease (FED), LCAT fails to
esterify cholesterol in HDL but not in the apoB-containing lipoproteins
(LpB). Clinically, this selective loss of -LCAT activity is
characterized by the presence of massive corneal opacifications, which
provide the name of the disease.24 25 26 In another form of
partial LCAT deficiency characterized by the presence of corneal
opacities and a normal UC-CE ratio, the ability of the patient's
plasma to esterify radiolabeled cholesterol in VLDL, LDL, and HDL was
reduced because of a decrease in LCAT mass.27 The
pathogenesis of Tangier disease (TD) is as yet unknown but is thought
to mainly involve a disturbance of intracellular lipid transfer
processes in macrophages and Schwann cells. Patients with TD
present with abnormal tonsils, neuropathy, and
hepatosplenomegaly.28 Despite the absence or a severe
reduction of HDL, most patients with these familial HDL deficiency
syndromes appear not to be at increased risk for coronary disease.
Therefore, we hypothesized that the maintenance of reverse cholesterol
transport in both HDL-deficient and normal plasma does not depend on
the major part of HDL but on the presence of subfractions that might
compensate for one another. To prove this hypothesis, we performed
pulse-chase incubations of plasma samples from HDL-deficient patients
by using [3H]cholesterol-labeled fibroblasts. After
separation of these plasma samples by nondenaturing 2D-PAGGE, we
monitored the occurrence of radioactive cholesterol and CEs in the
various HDL subclasses. This helped us to identify those lipoproteins
that are involved in the initial uptake of cell-derived cholesterol as
well as its subsequent transfer and esterification.
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Methods |
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Subjects
Three normolipidemic probands and four patients with different forms of primary HDL deficiency were included in this study. Characteristics of their lipid metabolism are summarized in Table 1
. The 32-year-old Italian woman with apoA-I deficiency
is homozygous for a nonsense mutation in codon 32 of the apoA-I
gene.29 The truncated protein could not be detected in her
plasma. She was not affected by premature atherosclerosis. The
60-year-old German patient with TD has been reported
previously.30 The patient with familial LCAT deficiency is
homozygous for a mutation in codon 321 of the LCAT gene, which leads to
replacement of a Thr by an Ile.21 FED was diagnosed in the
30-year-old German woman, who presented with typical corneal
opacifications and selective loss of -LCAT activity. Like most of
the German patients with FED who have been described,24 25
this woman was homozygous for a missense mutation in the LCAT gene,
which leads to a ThrIle substitution at residue 123.
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Blood Samples
Blood samples were taken after the subjects had fasted overnight
and immediately placed on ice. Plasma samples and sera were obtained by
centrifugation at 4°C (2000g, 15 minutes), divided into
aliquots, and frozen at -70°C. In former studies we found that
freezing and thawing did not affect the ability of normal plasma to
take up, esterify, and transfer cell-derived cholesterol. Serum was
used for the quantification of lipids. LCAT and CE transfer protein
activities were determined in EDTA-plasma. For experiments in which
plasma was incubated with cells, streptokinase (Sigma Chemical Co) was
used as the anticoagulant at a final concentration of 150 U/mL.
Quantification of Lipids, Apolipoproteins, and Lipid Transfer
Enzyme Activities
Serum concentrations of triglycerides and cholesterol were
quantified with an autoanalyzer (Hitachi/Boehringer). HDL cholesterol
concentrations were measured after precipitation of LpB with
phosphotungstic acid/MgCl2 (Boehringer). LDL cholesterol
was calculated with the Friedewald formula.31
Concentrations of apoA-I and apoB were determined with a modified
commercially available turbidimetric assay (Boehringer
Mannheim).32 LCAT activity was determined as the amount of
esterified [3H]cholesterol that was incorporated into
apoA-I–containing proteoliposomes.33 The plasma activity
of CE transfer protein was determined as the amount of
[14C]cholesteryl oleate transferred from artificial
apoA-I–containing proteoliposomes to LDL, as reported
previously.34 35
Preparation of Lipoproteins
LDL (d=1.019 to 1.063 g/mL) and HDL
(d=1.063 to 1.21 g/mL) were isolated from fresh normal human
plasma by standard preparative ultracentrifugation techniques and
dialyzed against 10 mmol/L sodium phosphate buffer (PBS, pH 7.4)
containing 0.15 mol/L NaCl.36 In some experiments as
indicated, apoB-free plasma was obtained by precipitation of LpB by
phosphotungstic acid/MgCl2 as recommended by the
manufacturer (Boehringer Mannheim). The apoB-free supernatant was
subsequently dialyzed against PBS (pH 7.4) and used in experiments for
determining net cholesterol efflux. Complete removal of LpB was
ascertained by immunoturbidimetry of apoB.32
Nondenaturing 2D-PAGGE
The distribution of apoA-I– and apoE-containing lipoproteins in
the plasma from normoalphalipoproteinemic and HDL-deficient probands
was determined by nondenaturing 2D electrophoresis, in which agarose
gel electrophoresis was followed by polyacrylamide gradient gel
electrophoresis (2D-PAGGE).9 10 Briefly, in the first
dimension, 20 µL of normal or HDL-deficient plasma was separated by
electrophoresis at 4°C on a 0.75% agarose gel with a 50 mmol/L
merbital buffer (pH 8.7, Serva). Agarose gel strips containing the
preseparated lipoproteins were then transferred to a 2% to 20%
polyacrylamide gradient gel. Separation in the second dimension was
performed at 40 mA for 4 to 5 hours at 4°C in a buffer system that
has been described by Altland and coworkers.37 After
separation, the proteins in 2D-PAGGE gels were electroblotted onto a
nitrocellulose membrane. ApoA-I– and apoE-containing lipoproteins were
detected by the use of sheep antibodies against human apoA-I and human
apoE, respectively (Boehringer Mannheim), which were biotinylated
according to the manufacturer's recommendations (Sigma). The
antigen-antibody complexes were visualized with a
streptavidin-biotinylated horseradish peroxidase complex (Amersham) at
a dilution of 1:1000. 4-Chloro-1-naphthol was used as the
chromogen.
Cell Culture
Normal human skin fibroblasts were cultured in Dulbecco's
modification of Eagle's minimum essential medium containing 10% fetal
calf serum as described previously.38 After 5 to 10
passages, cells were plated on 3.5-cm-diameter dishes for the
pulse-chase experiments. When they were nearly confluent, the cells of
some dishes were incubated for 72 hours at 37°C with 0.5 mCi
[1,2-3H]cholesterol (51.7 Ci/mmol, New England Nuclear),
which was complexed with fetal calf serum. Before the incubations with
plasma, fibroblasts were washed six times with PBS (pH 7.4). The final
specific radioactivity in the labeled cells then amounted to
5.2±1.4x108 cpm/mg cell protein, or
1.7±0.8x107 cpm/µg cell cholesterol (mean±SD).
Pulse-Chase Incubations With Fibroblasts
In pulse-chase experiments, 1 mL of complete plasma or apoB-free
plasma was first incubated with labeled fibroblasts (pulse). After
either 1 or 5 minutes of incubation, the plasma was removed and used
for the chase incubations, which were performed in the absence of
cells. Conditions and time intervals were varied as indicated in
"Results." In some instances, LDL (50 µg protein) or
5,5'-dithio-bis(2-nitrobenzoic acid) at a final
concentration of 1.5 mmol/L (DTNB, Sigma) was added to complete or
apoB-free plasma before starting the chase incubations.
After the chase incubation, the plasma samples were either delipidated
by the addition of chloroform/methanol (2:1, vol/vol)39 or
used for nondenaturing 2D electrophoresis. Typically, an unlabeled
sample from a normoalphalipoproteinemic control subject and a labeled
sample from a patient were run in parallel on one gel. One half of the
gel containing the 2D electrophoretic pattern of the patient was stored
at 4°C. The other half of the gel containing the pattern of the
normoalphalipoproteinemic control subject was electroblotted onto a
nitrocellulose membrane to immunolocalize the lipoproteins containing
apoA-I and apoE. The immunoblot was then used as a template to localize
the corresponding lipoproteins in the other half of the gel (Fig 1). These lipoproteins were cut out, and their lipids
were extracted with chloroform/methanol (2:1, vol/vol) for 72 hours. In
some experiments, the total radioactive cholesterol in various
lipoprotein fractions was determined. To separately count their
radioactivities, in other experiments UC and CE were first separated by
thin-layer chromatography using silica gel plates (Merck) as the
immobile phase and hexane/ether (6:4, vol/vol) as the mobile phase.
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Determination of Cholesterol Net Mass Transfer
Net cholesterol mass transfer from fibroblasts describes the
difference in the plasma concentrations of UC after a 2-hour incubation
of plasma with and without fibroblasts and was determined as described
previously.11 40 This test is based on the assumption that
esterification of cholesterol will decrease the concentration of UC in
both the presence and absence of fibroblasts. However, efflux of
cellular cholesterol into the medium causes less of a decrease in UC in
the sample that has been incubated with fibroblasts. In brief, after
they were washed four times with PBS, dishes with confluently growing
fibroblasts that had been preloaded with cholesterol for 48
hours41 and dishes without cells were incubated with 2 mL
of 5% plasma or apoB-free plasma in PBS. The media were removed after
a 2-hour incubation at 37°C. Lipids in 1 mL of total medium were
extracted with chloroform/methanol (2:1, vol/vol) three times for 6
hours each. Cholesterol mass in the extracts was quantified by the use
of a modified fluoroenzymatic assay as reported
previously.40
General Procedures
Total protein concentrations were measured according to the
method of Lowry et al42 using bovine serum albumin as the
standard. Every experiment was performed three times on plasma samples
from each proband. In some instances percent values are presented.
They represent the amount of [3H]UC in one
particle as a percentage of total [3H]UC in all
lipoproteins (ie, -LpE+pre-ß-LpA-I+-LpA-I+LDL). VLDL was not
included in this calculation, because this large lipoprotein does not
migrate into the polyacrylamide gradient gel.
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Results |
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Characterization of ApoA-I– and ApoE-Containing Lipoproteins in Normoalphalipoproteinemic and HDL-Deficient Plasma Samples
Fig 1
presents the distribution of apoA-I– and
apoE-containing lipoproteins after nondenaturing 2D-PAGGE of plasma
from a normoalphalipoproteinemic proband. As described
previously,9 11 apoA-I can be immunochemically detected by
2D-PAGGE in a major HDL subfraction with -mobility (-LpA-I) as
well as in three minor subfractions with pre-ß mobility that differ
by size (pre-ß1-LpA-I, pre-ß2-LpA-I, and
pre-ß3-LpA-I). According to Fielding and
colleagues,43 we differentiated -LpA-I into smaller
-LpA-I3 with a Stokes' diameter of 7.5 to 9.5 nm and
larger -LpA-I2 with a Stokes' diameter of 9.6 to 12 nm
(Fig 1c). LDL also reacted with anti–apoA-I antibodies. ApoE is
immunolocalized in two particles; the bulk of apoE is present in a
particle that mainly colocalizes with -LpA-I. A minor subfraction of
apoE, however, is present in a distinct lipoprotein with
-mobility and a Stokes' diameter of 12 to 14 nm, which we
previously described as -LpE.10 In some Western blots,
LDL immunoreacted with anti-apoE.
Fig 2 shows the distribution of apoA-I–containing
lipoproteins in the plasma of patients with various forms of familial
HDL deficiency. ApoA-I–containing lipoproteins were undetectable in
apoA-I deficiency (Fig 2a) but present in TD, FED, and familial
LCAT deficiency (Fig 2b through 2d). Plasma of the TD patient contained
pre-ß1-LpA-I and pre-ß2-LpA-I at apparently
normal concentrations but no pre-ß3-LpA-I or -LpA-I
(Fig 2b). Pre-ß1-LpA-I and pre-ß2-LpA-I
were also present in the plasma of patients with FED and familial
LCAT deficiency; in the latter group, these lipoprotein fractions were
present in higher concentration (Fig 2c and 2d).
Pre-ß3-LpA-I was undetectable in FED and LCAT deficiency.
In contrast to TD and apoA-I deficiency, in LCAT deficiency and FED
some small -LpA-I particles with a Stokes' diameter of 7.5 to 9.5
nm, ie, -LpA-I3, were visible. By contrast with
normal plasma, LDL in HDL-deficient plasma did not react with
anti–apoA-I antibodies.
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Fig 3 presents the distribution of apoE-containing
lipoproteins in the plasma of the HDL-deficient patients. -LpE was
present in all plasma samples. -LpE was very heterogenous in the
various HDL deficiency conditions. In apoA-I deficiency, -LpE
covered a considerably larger area of the gel than did -LpE in
normal plasma, indicating a greater heterogeneity in size (Fig 3a). TD
plasma contained an anti-apoE immunoreactive particle with ß-mobility
rather than -mobility on agarose gel electrophoresis (Fig 3b). By
contrast with normoalphalipoproteinemic plasma and despite its
heterogeneity in size and charge, -LpE never colocalized with
anti–apoA-I immunoreactive particles in HDL-deficient plasma.
Moreover, LDL was not detected by the use of anti-apoE antibodies.
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Uptake and Transfer of Cell-Derived Cholesterol in Normal and
HDL-Deficient Plasma
For practical reasons we performed all subsequently
described studies on plasma samples that had been frozen at -70°C,
because in a pilot study we had found that freezing and thawing of
plasma did not affect the distribution of apoE- and apo A-I–containing
subclasses.
To determine their ability to release cholesterol from cells, plasma
samples from normoalphalipoproteinemic probands as well as
HDL-deficient patients were incubated with [3H]UC-labeled
cells for 1 minute (Table 2). Compared with that from
normoalphalipoproteinemic plasma, the efflux of cell-derived
[3H]cholesterol from HDL-deficient patients was reduced
by 49%, 36%, 21%, and 28% in apoA-I deficiency, TD, FED, and LCAT
deficiency, respectively (Table 2).
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Anti–apoA-I and anti-apoE immunoblots of 2D electrophoretograms of
normal plasma were used as templates to localize apoA-I– and
apoE-containing lipoproteins in native 2D electrophoretograms of normal
and HDL-deficient plasma samples that had been pulsed with radiolabeled
cellular cholesterol. Because the plasma samples of patients with FED
and familial LCAT deficiency contained only small -LpA-I (Fig 2c and 2d), -LpA-I was divided into -LpA-I3 (Stokes'
diameter, 7.5 to 9.5 nm) and -LpA-I2 (Stokes' diameter,
9.6 to 12.0 nm). -LpE was not considered separately for two reasons.
First, in normoalphalipoproteinemic plasma, most -LpE colocalizes
with -LpA-I2. Second, -LpE is heterogenous in HDL
deficiency. Table 3 summarizes the recoveries of
radioactivity extracted from the gels compared with the radioactivity
in total plasma, supernatants, and infranatants after precipitation of
LpB with phosphotungstic acid/MgCl2. These recoveries
ranged from 80% to 90% and did not differ significantly between
plasma samples from HDL-deficient patients and
normoalphalipoproteinemic control subjects. Experiments on every sample
were performed in triplicate and in independent series. The interassay
coefficients of variation of the radioactivity recovered from plasma or
the various HDL subfractions were below 20%.
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Figs 4 and 5 show the presence of
[3H]cholesterol in the various lipoproteins of normal and
HDL-deficient plasma after a 1-minute pulse with radiolabeled
fibroblasts (open bars) and an additional 1-minute chase without cells
(hatched bars). Fig 5 represents the amount of
[3H]UC in one particle as a percentage of total
[3H]UC in all lipoproteins (ie,
-LpE+pre-ß-LpA-I+-LpA-I+LDL). VLDL was not included in this
calculation because this large lipoprotein does not migrate into the
polyacrylamide gradient gel. Fig 4 gives absolute numbers as counts per
minute in the various lipoproteins. Table 4 presents
the initial efflux of cholesterol into the various lipoproteins as a
percentage of [3H]cholesterol in the cells. After a
1-minute pulse of normal plasma (Fig 5a), the percentages of
[3H]cholesterol in -LpE, pre-ß1-LpA-I,
pre-ß2-LpA-I, pre-ß3-LpA-I,
-LpA-I3, -LpA-I2, and LDL
amounted to 20±3%, 9±2%, 4±1%, 5±1%, 32±4%, 15±2%, and
15±2%, respectively. After an additional 1-minute chase, the
radioactivity in -LpE and pre-ß1-LpA-I decreased to
7±1% and 5±1% and simultaneously increased in
-LpA-I3, -LpA-I2, and LDL
to 40±4%, 18±2%, and 26±3%, respectively. As reported
previously,9 10 11 the occurrence of
[3H]cholesterol in the cholesterol-poor
pre-ß1-LpA-I and -LpE during the pulse and its
disappearance from these particles and increase in -LpA-I during the
chase indicate that in normal plasma, considerable proportions of
cell-derived cholesterol are taken up first by both -LpE and
pre-ß1-LpA-I and then transferred to -LpA-I (mainly
-LpA-I3) and LDL.
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In all forms of HDL deficiency, -LpE took up cell-derived
[3H]cholesterol in normal amounts (Figs 4b through 4e and
5b through 5e and Table 4). Because of the reduced efflux of
[3H]cholesterol into HDL-deficient plasma, the
percentages of radioactivity in -LpE were higher in HDL-deficient
plasma than in normoalphalipoproteinemic plasma samples, namely,
38±5%, 32±3%, 36±5%, and 34±5% in apoA-I deficiency, TD, FED,
and LCAT deficiency, respectively. After a 1-minute chase, the
radioactivity in -LpE decreased to approximately 10% in all forms
of HDL deficiency. In contrast to the similar degree of uptake of
cell-derived [3H]cholesterol by -LpE in all
HDL-deficient plasma, efflux into LpA-I-subfractions varied widely in
the different forms of HDL deficiency.
After a 1-minute pulse with apoA-I–deficient plasma (Figs 4b and 5b and Table 4), radioactivity was detectable only in -LpE, LDL, and
a fraction with the mobility of -LpA-I3. The latter
particle, however, did not react with anti–apoA-I antiserum (cf Fig 2a). By contrast, no radioactivity was detected in the fraction with
-LpA-I2–like mobility, although this fraction included
an apoE-containing lipoprotein (cf Fig 3a). After a 1-minute chase,
[3H]cholesterol disappeared from -LpE and the
-mobile lipoprotein and accumulated in LDL.
A 1-minute pulse incubation with TD plasma (Figs 4c and 5c and
Table 4) led to the regular uptake of [3H]cholesterol by
pre-ß1-LpA-I and -LpE. No radioactivity was found in
pre-ß2-LpA-I, pre-ß3-LpA-I, or
-LpA-I2, although -LpA-I contained apoE. Like
apoA-I–deficient plasma but unlike normal plasma, pulse incubation
with TD plasma led to the occurrence of small amounts of
[3H]cholesterol in an apoA-I–free fraction with
-LpA-I3–like mobility (Figs 4c and 5c; cf Figs 2b and 3b). During chase incubations, [3H]cholesterol
disappeared from all of these initial acceptors and accumulated in LDL
(Figs 4c and 5c and Table 4).
Pulse incubations of plasma samples from patients with FED and
LCAT deficiency led to efflux of cell-derived
[3H]cholesterol into pre-ß1-LpA-I and
-LpE (Figs 4d, 4e, 5d, and 5e and Table 4) followed by a decrease in
radioactivity in this fraction during chase incubation. In contrast to
plasma from control subjects, apoA-I–deficient patients, and TD
patients, high amounts of radioactivity accumulated in the
pre-ß2-LpA-I of both FED and LCAT-deficient plasma
samples. As with the other two forms of HDL deficiency, only trace
amounts of radioactivity were detected in fractions with the mobility
of pre-ß3-LpA-I and -LpA-I2,
although the latter fraction was anti-apoE immunoreactive.
The percentage of radioactivity in LDL after a 1-minute chase was
increased in HDL-deficient compared with normal plasma (26±3%),
LCAT-deficient and FED plasma (45±5%), and apoA-I–deficient and TD
plasma (60±8%). However, because exogenous
[3H]cholesterol and endogenous unlabeled cholesterol
within plasma lipoproteins equilibrate by diffusion and 75% of UC in
normal plasma and more than 90% of UC in HDL-deficient plasma are
present in LDL, higher amounts of [3H]cholesterol in
LDL of patients with HDL deficiency may simply reflect the
disproportionate distribution of UC among the various lipoproteins in
normal versus HDL-deficient plasma. To investigate this possibility, we
prolonged the chase incubation periods to various time intervals (0 to
60 minutes) and subsequently precipitated LpB with phosphotungstic
acid/MgCl2. This procedure allowed us to separately
determine the specific radioactivity (counts per minute of
[3H]UC per microgram of UC) in the apoB-free supernatants
and the apoB-containing infranatants (Fig 6). Without
chase (time 0), the specific radioactivity of cell-derived
[3H]UC in the supernatants of all plasma exceeded that in
the infranatants (ie, LDL+VLDL) by a factor 2 to 3. During subsequent
chases the specific radioactivity decreased in the supernatants but
increased in the infranatants. In control plasma, specific
radioactivity in the infranatant exceeded that in the supernatant after
22 minutes (Fig 6a). By contrast, in HDL-deficient plasma a higher
specific radioactivity in the infranatant was already observed after 6
to 8 minutes (Fig 6b through 6e). This indicates that the transfer of
cell-derived [3H]UC from the initial acceptors to LDL is
enhanced in HDL-deficient compared with normal plasma.
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) and apoB-containing (
) lipoproteins of plasma
from normoalphalipoproteinemic probands (normal) and various
HDL-deficient (def.) patients. Pulse incubations with radiolabeled
fibroblasts for 1 minute were followed by chase incubations without
cells for the indicated times. ApoB-free and apoB-containing
lipoproteins were separated by precipitation with phosphotungstic
acid/MgCl2. Subsequent to lipid extraction, radioactive UC
and cholesteryl ester were separated by thin-layer chromatography.
Specific radioactivity is expressed as counts per minute of
[3H]UC per microgram of UC in apoB-free and
apoB-containing lipoproteins. Each point shows the mean value and
standard deviation of four experiments. TD indicates Tangier disease;
FED, fish-eye disease; and LCAT, lecithin:cholesterol
acyltransferase.
Esterification of Cell-Derived Cholesterol in Normal and
HDL-Deficient Plasma
In subsequent studies, we compared the esterification of
cell-derived UC and the transfer of CE to various lipoproteins of
normal and HDL-deficient plasma. To obtain amounts of labeled
cholesterol sufficient to differentiate between [3H]UC
and [3H]CE, pulse incubations of plasma with radiolabeled
fibroblasts were prolonged to 5 minutes and chase incubations to 15
minutes (Table 5). To inhibit LCAT, all chase
incubations were done in the presence of 1.5 mmol/L DTNB. After a
5-minute pulse incubation, normal plasma esterified 3.6% of the
[3H]cholesterol released from cells into plasma. This
fractional esterification was increased to 4.3% in apoA-I deficiency
and to 5.5% in TD. In FED we observed a slight decrease to 3.1%. As
expected, no detectable amounts of [3H]CE were formed in
LCAT-deficient plasma.
|
In control experiments, approximately 32% of [3H]CE was
recovered in pre-ß3-LpA-I, 57% in -LpA-I, and 11% in
LDL (5-minute pulse). A 15-minute chase in the presence of the LCAT
inhibitor DTNB resulted in a 32% to 12% decrease of
[3H]CE in pre-ß3-LpA-I, a 57% to 69%
increase in -LpA-I, and an 11% to 19% increase in LDL. In the
plasma of patients with FED, apoA-I deficiency, and TD,
[3H]CEs were detectable only in LDL but not in
pre-ß-LpA-I or -LpA-I (Table 5). After pulse incubation, the
amount of [3H]CE in LDL was twofold to threefold higher
in the plasma of patients with FED, TD, and apoA-I deficiency compared
with normal plasma. In contrast to normal plasma, chase incubation with
these HDL-deficient plasmas did not significantly increase the amount
of [3H]CE in LDL (Table 5).
The more rapid appearance of [3H]CE in LDL raises the
possibility that in these HDL-deficient plasmas, cell-derived
cholesterol is esterified in LDL or transferred to LDL after generation
in lipoproteins that contain neither apoA-I nor apoB. Therefore,
pulse-chase incubations were repeated with apoB-free plasma from
normoalphalipoproteinemic subjects and HDL-deficient probands (Fig 7). No [3H]CE was generated in apoB-free
FED and LCAT-deficient plasma, whereas the amount of
[3H]CE gradually increased with prolonged chase
incubation in apoB-free supernatants of control plasma, TD plasma, and
apoA-I–deficient plasma. Esterification of cell-derived
[3H]cholesterol in apoB-free plasma from patients with
apoA-I deficiency and TD was increased twofold and threefold,
respectively, compared with apoB-free plasma from normal control
subjects. Thus, with the exception of FED and LCAT-deficient plasma,
esterification of cell-derived [3H]cholesterol occurred
in the absence of LpB (Fig 7).
|
), the
normoalphalipoproteinemic probands (
). b and c, After 60 minutes' incubation with radiolabeled
fibroblasts, 200 µL apoB-free plasma was removed and incubated for a
further 15 minutes with 200 µg LDL. Subsequently, the apoB-free
fraction (b) and LDL (c) were separated by precipitation with
phosphotungstic acid/MgCl2. After lipid extraction,
radioactive UC and CE in the apo B-free supernatants (b) and
LDL-containing infranatants (c) were separated by thin-layer
chromatography. Open and closed bars represent
[3H]CE in lipoproteins before and after chase
incubations, respectively.
To further investigate the more rapid transfer of CE to LDL in
apoA-I–deficient and TD plasma, apoB-free plasma samples were first
pulsed with cell-derived [3H]cholesterol, then
supplemented with exogenous LDL, and finally used for 15-minute chase
incubations. Exogenous LDL was then precipitated by addition of
phosphotungstic acid/MgCl2 to separately determine
[3H]UC and [3H]CE in the apoB-free
supernatants (Fig 7b) and in the LDL-containing infranatants (Fig 7c).
More than one fourth (29±4%) of [3H]CE was found in the
LDL-containing infranatant of normal plasma, whereas 76±3% and
73±3% were detected in the LDL-containing infranatants of TD plasma
and apoA-I–deficient plasma, respectively.
Net Cholesterol Efflux From Fibroblasts Into Normal and
HDL-Deficient Plasma
We also determined the net mass transfer of UC from fibroblasts to
both native and apoB-free plasma during a 2-hour incubation (Table 6). During this period, a net amount of 7.46±1.14 nmol
UC per milligram of cell protein was released into 100 µL plasma of
normoalphalipoproteinemic probands. Compared with normal plasma, the
net transfer into plasma from apoA-I–deficient, TD, FED, and
LCAT-deficient patients was reduced to 53±7%, 88±11%, 66±11%, and
41±7%, respectively. Removal of LpB from normal plasma resulted in a
39% decrease of net released cellular cholesterol, from 7.46±1.14 to
4.55±0.73 nmol UC per milligram of cell protein. Removal of LpB from
the plasma of patients with apoA-I deficiency, TD, FED, and LCAT
deficiency, however, decreased the net cholesterol transport rates by
77%, 84%, 72%, and 64%, respectively. These findings indicate that
LpB plays an important role in net cholesterol efflux in HDL-deficient
plasmas of various origins.
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|
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The antiatherogenic effect of HDL has generally been attributed to its ability to mediate the flux of excess cholesterol from peripheral cells to the liver.2 3 4 In normoalphalipoproteinemic plasma, this reverse cholesterol transport involves (1) uptake of cell-derived UC by pre-ß1-LpA-I and
-LpE; (2)
subsequent transfer of UC to LDL via pre-ß2-LpA-I,
pre-ß3-LpA-I, and -LpA-I; (3) esterification of UC,
mostly in -LpA-I but to a lesser extent in
pre-ß3-LpA-I; and (4) transfer of CE to
LDL.9 10 11 12 13 43 Patients with various forms of familial HDL
deficiency do not lack HDL completely but rather are deficient in
distinct HDL subclasses.19 44 45 46 47 48 In our study,
nondenaturing 2D electrophoresis revealed the absence of
pre-ß3-LpA-I and -LpA-I2 and the presence
of -LpE in all four kinds of familial HDL deficiency. Furthermore,
pre-ß1-LpA-I and pre-ß2-LpA-I were
present in all forms of HDL deficiency except apoA-I deficiency;
-LpA-I3 was detectable in LCAT deficiency and FED but
not in TD and apoA-I deficiency. Uptake, transfer, and esterification
of cell-derived cholesterol in the plasma of patients with familial HDL
deficiency syndromes were significantly different from those of normal
plasma (Table 7).
|
Efflux of Cell-Derived Cholesterol
Cholesterol efflux from cells is the result of complex mechanisms
that involve the synthesis of cholesterol in the endoplasmic reticulum,
hydrolysis of CE in lysosomes and cytosolic lipid droplets,
translocation of cholesterol to the cell membrane, and finally
desorption of cholesterol from the cell membrane into the plasma
compartment.4 49 These processes are regulated differently
in different cell types and depend on different extracellular stimuli
and acceptors of cholesterol, such as the various apolipoproteins and
HDL subclasses.50 In apoA-I deficiency, cellular
cholesterol was taken up by -LpE but not by
pre-ß1-LpA-I. The initial efflux of
[3H]cholesterol and the net efflux of cholesterol into
plasma were decreased by 50% to 60% compared with
normoalphalipoproteinemic plasma. Similarly, Fielding and coworkers
(Fielding and Moser51 and Kawano et al52 )
observed a 55% reduction in cholesterol efflux stimulation by plasma
that had been depleted of LpA-I by immunoaffinity chromatography. These
authors concluded that pre-ß1-LpA-I contributed to more
than half of a plasma's ability to release cholesterol from cells and
attributed the residual activity to nonspecific effects of
albumin.9 51 52 Our data, however, show that -LpE takes
up to twofold more [3H]cholesterol than does
pre-ß1-LpA-I, suggesting that this lipoprotein is also a
major contributor to the cholesterol efflux–stimulating activity of
plasma. The importance of -LpE for the initial efflux of
cell-derived cholesterol into plasma is also underlined by our
observation that the initial efflux of cell-derived
[3H]UC into TD plasma is only slightly higher than that
by apoA-I–deficient plasma, although TD plasma contains
pre-ß1-LpA-I.
Transfer of UC to LDL
In normoalphalipoproteinemic plasma, cell-derived cholesterol is
taken up by a number of particles and then transferred to LDL. This
transfer involves various lipoproteins, including
pre-ß2-LpA-I, pre-ß3-LpA-I, and -LpA-I.
From LDL, UC is either retransferred to -LpA-I for esterification or
taken up by cells.11 13 These transfer mechanisms of
cell-derived cholesterol to LDL were found to be operative in the
various HDL deficiency syndromes, suggesting first that -LpE may
directly participate in the transfer of cell-derived cholesterol
to LDL and second, that the specific absence of
pre-ß2-LpA-I, pre-ß3-LpA-I, and -LpA-I
does not interfere with the transfer of UC from
pre-ß1-LpA-I to LDL.
Esterification of Cholesterol and Transfer of CE to LDL
In normoalphalipoproteinemic plasma, most of the cell-derived
cholesterol is esterified in -LpA-I after retransfer from LDL. A
smaller amount is esterified during passage through
pre-ß3-LpA-I.11 13 In contrast to LCAT
deficiency, in which plasma cholesterol esterification activity is
completely lost, FED is characterized by the selective failure of
plasma to esterify cholesterol in exogenous HDL or apoA-I–containing
proteoliposomes while retaining the ability to esterify cholesterol in
exogenous LpB.53 54 55 In our experiments with FED plasma,
CEs accumulated in LDL and were not found in other lipoproteins. These
findings further suggest that LDL cholesterol can be directly
esterified by the mutant LCAT in FED. By contrast, in apoA-I–deficient
and TD plasma, CEs accumulated in LDL after undergoing esterification
in lipoproteins containing neither apoA-I nor apoB. The nature of these
lipoproteins is unclear at present. Other authors have also
detected LCAT activity in the apoA-I– and apoB-free fractions of TD
plasma30 46 56 and in apoA-IV–containing
particles.45 Obviously, the newly formed CEs can be
effectively transferred from these abnormal particles to LDL.
Familial HDL deficiency is very rare, so we were able to perform only exemplary studies on single patients and could not analyze the effect of possible heterogeneity within a given syndrome. For example, heterogeneity within apoA-I deficiency, LCAT deficiency, FED, or TD arises from allelic variation of the underlying defects in the genes of apoA-I, LCAT, or the as yet unidentified TD gene.14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Further heterogeneity may originate from variations in the genetic background of an individual and other factors, such as gender, age, concomitant diseases, or genetic polymorphisms. Nevertheless, we believe that the phenomena described in this article can be extrapolated to other patients with the same HDL deficiency syndromes, because they generally cause qualitative rather than quantitative changes in the functioning of a given HDL subfraction.
In this study, we searched only for those lipoproteins that contained
apoA-I or apoE. We may, therefore, have overlooked two classes of HDL
particles that may play a role in reverse cholesterol transport. First,
there is some evidence for the involvement of lipoproteins containing
neither apoE nor apoA-I, which are present even in
normoalphalipoproteinemic plasma. Thus, lipoproteins that contain
apoA-IV but no apoA-I or apoE can release cholesterol from
cells.57 58 LpA-IV also contains LCAT
activity.57 Second, because apoA-I is the most abundant
apolipoprotein in all subclasses, a lack or severe decrease in apoA-I
gives rise to abnormal lipoproteins. Thus, -LpE particles in apoA-I
deficiency and TD are heterogenous in size and charge and do not
entirely colocalize with apoA-I– or apoE-containing particles in
normal plasma. These apoE-containing particles, however, do not appear
to contribute substantially to reverse cholesterol transport, as
neither pulse nor chase incubations led to the occurrence of radiolabel
in those proportions of abnormal -LpE that do colocalize with
-LpA-I of normal plasma (Figs 2 through 6). Abnormal HDL that
contains apoA-II but no apoA-I has also been identified in some
patients with apoA-I deficiency or TD.28 29 47 59 In one
case, this apoA-II–containing particle was shown to promote
cholesterol efflux from cells.47 Despite the uncertainty
surrounding the role of these minor HDL subfractions in reverse
cholesterol transport, they appear either to contribute little to the
efflux of [3H]cholesterol from cells or to be entirely
colocalized in those electrophoretic fractions that, in normal plasma,
contain apoA-I or apoE, as 80% to 90% of the total plasma
radioactivity was recovered in LpE, LpA-I, and LDL of both normal and
HDL-deficient plasmas.
In summary, our studies demonstrate that the two crucial steps in
reverse cholesterol transport—efflux of cellular cholesterol into the
plasma compartment and its transfer to LDL for final targeting to the
liver—are maintained in all forms of HDL deficiency, although
quantitatively and qualitatively modified (Table 7). This may explain
why many forms of familial HDL deficiency do not put homozygous
carriers at increased risk of coronary disease. Our data suggest that
quantitatively minor plasma subfractions, eg,
pre-ß1-LpA-I and -LpE, together with LDL, are
important contributors to reverse cholesterol transport. In particular,
apoA-I does not play an exclusive role in reverse cholesterol
transport, as shown by our in vitro findings in A-I deficiency and the
absence of premature atherosclerosis in affected individuals. This is
also highlighted by recent observations in transgenic animals that do
not express apoA-I but fail to develop
atherosclerosis.60
|
Acknowledgments |
|---|
This project was the topic of the Bennigsen-Foerder-Award from Ministerium für Forschung und Wissenschaft Nordrhein Westfalen to Dr von Eckardstein. Further support was provided by a fellowship from Boehringer Ingelheim Fonds to Dr Huang. We gratefully acknowledge the assistance of Dr Ali Chirazi in the determination of lipid transfer enzyme activities and the help of Dr Paul Cullen in editing the manuscript.
Received July 13, 1994; accepted February 2, 1995.
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