© 1997 American Heart Association, Inc.
Articles |
Role of Free Apolipoprotein A-I in Cholesterol Efflux
Formation of Pre–
-Migrating High-Density Lipoprotein Particles
From the Division of Lipoprotein Metabolism and Pathophysiology, Department of Physiology, Louisiana State University Medical Center, New Orleans, La.
Correspondence to Laurence Wong, Division of Lipoprotein Metabolism and Pathophysiology, Department of Physiology, Louisiana State University Medical Center, New Orleans, LA 70112.
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Abstract |
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Abstract This article characterizes products formed by the interaction of purified apolipoprotein (apo) A-I and human fibroblasts. Fibroblasts were incubated with different concentrations of purified apoA-I (1 to 30 µg/mL) in tissue culture medium for different periods of time (0 to 24 hours). The medium was then characterized by one- (agarose) and two-dimensional (agarose : polyacrylamide nondenaturing gradient gel) electrophoresis. At any given concentration of apoA-I, the rate of cellular cholesterol efflux appeared linear over 24 hours. Incubating purified apoA-I with fibroblasts for 4 hours, we detected five pre-
lipoproteins with particle sizes between 114 and 684 kDa.
Formation of pre- lipoproteins was concentration-dependent. At low
concentrations (below 5 µg/mL apoA-I), all purified apoA-I (with
pre-ß mobility) was converted to pre- lipoproteins. At higher
concentrations (greater than 5 µg/mL apoA-I), more apoA-I remained
with pre-ß mobility. The pre- lipoproteins were characterized by
colocalization of apoA-I particles with
14C-cholesterol and
32P-phospholipids. Results showed that the pre- particle
of lowest molecular weight contained phospholipid and apoA-I but no
cholesterol. The remaining pre- particles contained all
three substances. When pre- particles were subjected to
ultracentrifugation, all particles floated at d<1.21
g/mL with some of the smallest phospholipid apoA-I only particles being
present in the d>1.21 g/mL fraction. Based on these results, we
postulated that in the first stages of reverse cholesterol
transport, pre- lipoproteins are formed by the interaction of lipid
free apoA-I and peripheral cells.
Key Words: high-density lipoproteins • pre- lipoproteins • pre-ß lipoproteins • cholesterol efflux • fibroblasts • cholesterol • two-dimensional electrophoresis • apolipoprotein A-I • phospholipids
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Introduction |
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Peripheral cells cannot break down and metabolize cholesterol. To maintain cell homeostasis, peripheral cells transfer excess cholesterol and transport it to hepatocytes to be metabolized.1 Numerous studies have examined the effect of cholesterol acceptors mediating cholesterol efflux in vitro from peripheral cells. The role of apolipoprotein (apo),2 HDL,3 recombinant HDL,4 and phospholipid vesicles5 on cellular cholesterol efflux has been studied. In most of these studies, cells were labeled with radioactive cholesterol, incubated with medium containing one or another of the above particles, and radioactivity of transferred cholesterol examined as a function of time. In other studies, total cholesterol mass in the medium was determined after introduction of acceptors. The results of these complementary studies can generally be summarized as follows: (1) most apoprotein appear to be adept in acting as cholesterol acceptors2,6; (2) in the presence of unlimited acceptors, the rate of cellular cholesterol efflux is limited by diffusion of cholesterol from the cell membrane3; (3) apoproteins may not be necessary for cholesterol efflux, and phospholipid vesicles are also effective2; and (4) artificial cholesterol acceptors, such as cyclodextrans,7 can also promote cholesterol efflux.
Currently, two major processes are generally accepted as being responsible for cholesterol efflux (reverse cholesterol transport). The first, a specific process of rapid movement of cholesterol from plasma membrane involves lipoproteins/apoprotein. The second, a nonspecific process, involves diffusion of a slowly exchanging pool of cholesterol from cell membrane.1,8,9
Recently, we developed a two-dimensional agarose nondenaturing polyacrylamide gradient gel electrophoresis system (2DE) to analyze plasma HDL.10 Using this procedure, we have observed "free apoA-I–like" particles in plasma.11 The purposes of the study presented here were to determine if these free apoA-I particles could accept cholesterol from peripheral cells and what type of a cholesterol-containing particle would result.
The study presented here differs from previous ones in its
methodology of characterization of the newly formed
cholesterol-apoprotein complex. To model this newly formed
complex in vitro, we incubated fibroblasts with purified apoA-I and
characterized the product of apoA-I cell interaction. Using the
method of 2DE,12 we demonstrated the presence of five
different lipoprotein particles (containing apoA-I), all with pre-
mobility but differing in size from 114 to 684 kDa. Particles with
other than pre- mobility were not recovered.
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Methods |
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ApoA-I Purification
ApoA-I was purified from normolipemic human plasma (Southeast Blood Center) HDL (1.080<d<1.21 g/mL) by modification of a procedure described by Nichols et al13 and Forte et al.14 Solid guanidine HCl (ICN) was added to 3 mg/mL HDL to give a final concentration of 2 mol/L. This solution was incubated at 37°C for 3 hours. After incubation, the mixture was dialyzed four times against a solution containing 150 mmol/L NaCl, 10 mmol/L Tris-HCl, and 0.01% EDTA at pH 8.0 to remove the guanidine HCl. All chemicals were from Sigma unless otherwise noted. After dialysis, density of the solution was adjusted to 1.21 g/mL using solid KBr. The HDL solution was then transferred into an ultracentrifuge tube (Beckman) and overlaid with an equal volume of KBr solution of the same density. The sample was then subjected to ultracentrifugation at 50 000 revolutions per minute for 24 hours in a 50.3 Ti rotor (Beckman) at 10°C. The bottom 1.5 mL was collected by tube slicing and dialyzed extensively against 0.01 mol/L NH4HCO3. The apoA-I was stored sterile in solution for experimental use. No resolubilization was necessary. Purity of the isolated apoA-I was determined by SDS polyacrylamide gradient gel electrophoresis followed by Coomassie Blue and silver staining. A single band corresponding to apoA-I was detected in all cases. In addition, apoA-I did not cross-react to antibodies to any known apoprotein except to anti–apoA-I by double immunodiffusion.
Cell Culture and Lipid Labeling
Normal human fibroblasts GM 3468A (Coriel Institute) from
passages 10 to 20 were grown in T75 plastic flasks in DMEM containing
10% fetal bovine serum (Life Science Technologies) at 37°C in a
humidified incubator equilibrated with 5% CO2 and 95%
room air. Before the experiments, one million cells were trypsinized
and seeded into 35-mm Petri dishes. The mean cell protein per dish was
160.1±14.7 µg.
Radioactive cholesterol [4-14C] (Amersham) was purified by thin-layer chromatography. After purification, cholesterol (161 µg) was dissolved in chloroform and dried in a stream of nitrogen. Thereafter, cholesterol was resuspended by addition of Molecusol HPB-20, 50 µL of 45% hydroxy propyl-cyclodextran (Molecusol HPB-20, Pharmatec Inc) in medium (DMEM, Gibco).15 Under these conditions, the cyclodextran solution is saturated with cholesterol and, therefore, cannot act as a net cholesterol acceptor.7 After stirring overnight, the Molecusol-cholesterol solution was sterile-filtered before addition to fibroblasts. Twenty-two microcuries of 14C-cholesterol was mixed with DMEM containing 10% fetal bovine serum, added to each 35-mm Petri dish of subconfluent fibroblasts, and incubated for 48 hours. After incubation, the labeled cells were washed four times with D-PBS and incubated with BioRich medium (ICN) for 24 hours before incubation with apoA-I. On the day of the experiment, the cells were washed three times with D-PBS, apoA-I (approximately 500 µg/mL) was diluted to 10 µg/mL with BioRich medium, and 1 mL was incubated with cells.
For phospholipid labeling, subconfluent fibroblasts in each 35-mm Petri dish were incubated for 72 hours with 100 µCi of 32P-orthophosphate mixed with DMEM containing 10% fetal bovine serum. Labeled cells were washed four times with D-PBS before incubation with apoA-I.
Other Methods
Separation of lipoprotein subpopulations by the technique of
2DE, agarose electrophoresis (1D) in the first dimension, and
nondenaturing PAGE in the second dimension (2D) has been previously
described.10 It should be noted that the agarose did not
contain albumin, permitting the separation of and
pre-–migrating particles.10,12 After transfer of
lipoproteins to nitrocellulose membranes, apoA-I–containing
lipoproteins were immunolocalized with monospecific goat anti-human
apoA-I. Goat antibody was then immunodetected with
125I-labeled anti-goat 
globulin. The radioactivity was
quantified on the phosphorimaging device (Molecular Dynamics). For both
the 1D and 2D analyses, 15 µL of unconcentrated medium was
used. We defined the apoA-I–containing lipoproteins by their
electrophoretic mobility on agarose gels as pre-ß, , and
pre-.10 The size of the lipoproteins was then defined by
their mobility by 2DE, compared with known molecular weight standards
(Pharmacia HMW standard consisting of: thyroglobulin, 669 kDa;
ferritin, 440 kDa; catalase, 232 kDa; lactate dehydrogenase 140 kDa;
and albumin, 67 kDa) that were run simultaneously
in the same gel. In some experiments designated herein as 2DE (3% to
16%), the 2DE was run with a 3% to 16% linear gradient gel.
Otherwise, the gels have a 3% to 35% concave gradient, designated 2DE
(3% to 35%). Molecular weight standards were not used in the 2DE (3%
to 16%) because electrophoresis was not carried to completion, and
migration of the proteins did not represent true molecular
weight. As a control, purified apoA-I and human plasma pre-ß segment
obtained from 1D were used as comparative standards.
Sequential colocalization of lipid label and apoA-I was carried out in a two-step process. After incubation of apoA-I with lipid-labeled cells, the lipoproteins in the medium were separated by either 1DE or 2DE. The lipoproteins were then transferred to nitrocellulose membrane and imaged by the phosphorimaging device. After imaging, the nitrocellulose membrane was washed with PBS (0.01 mol/L sodium phosphate, pH 7.4, 0.145 mol/L NaCl) containing 0.05% Tween 20. This effectively stripped the membrane of any lipids associated with the lipoproteins. The nitrocellulose membrane was then processed for immunolocalization as described above.
Medium content of free cholesterol was determined by gas-liquid chromatography using a Hewlett-Packard Model 5890 Gas Chromatography with automatic on-column sample injection (Hewlett-Packard) equipped with a DB17 capillary column (J&B Scientific). Injector temperature was 100°C. Temperature programming was as follows: 100°C for 5 minutes, then 30°C per minute until 280°C, and holding at 280°C for 9 minutes. Helium carrier pressure was 21.5 pounds per square inch at a constant flow of 40 mL/min. A known amount of internal standard (ß-stigmasterol) was added to the medium before the lipid was extracted for cholesterol determination. We assumed that the cholesterol specific radioactivity was constant over 24 hours. At times earlier than 24 hours, medium was collected, lipid extracted, and counted by liquid-scintillation counting. At the end of the experiment (24 hours), 0.5 mL of medium was extracted for cholesterol determination by GLC. An aliquot was also counted by liquid-scintillation counting to determine specific radioactivity at 24 hours. The concentration of cholesterol was then calculated from the radioactivity at each time point divided by the specific radioactivity at 24 hours.
Protein determination was completed according to the method of Lowry et al.16 Iodination of secondary antibodies was as previously described.17 For some experiments, iodinated apoA-I was used. The iodinated apoA-I was prepared from iodinated HDL using the method of MacFarlane,18 and purified apoA-I was prepared as described in the apoA-I purification section. HDL was chosen to protect from denaturation of apoA-I during iodination. Ultracentrifugation was carried out by the method of Havel et al.19
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Results |
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To study the rate of cellular cholesterol efflux, fibroblasts were labeled with radioactive cholesterol and then 10 µg/mL purified apoA-I was added to the medium. Medium was incubated with fibroblasts for various times. Cholesterol appearance in the medium as a function of time was determined by liquid-scintillation counting divided by the cholesterol specific radioactivity at the end of the experiment. The result, summarized in Fig 1
, suggested that the rate of
cholesterol efflux was linear over the 24-hour incubation
period (r=.99, P<.0001).
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We also determined the distribution of effluxed cholesterol
on 1D agarose gel. In this experiment, 10 µg/mL apoA-I was
used and 14C-cholesterol was substituted for
3H-cholesterol to allow detection by the
phosphorimaging device. The result, presented in Fig 2, showed a steady increase of
radioactivity from 4 to 24 hours in the pre- region. It should be
noted that there was not any radioactivity in the pre-ß region. We
also immunolocalized apoA-I on 1D gel (Fig 2). Incubation of apoA-I
with medium, without cells, resulted in pre-ß mobility in 1D. When 10
µg apoA-I was incubated with fibroblasts, both pre- and pre-ß
(free) apoA-I were found in the medium. The appearance of apoA-I with
pre- mobility increased with the time of incubation from 4 to 24
hours.
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To determine if the formation of pre- particles was dependent on
apoA-I concentration, 1 to 20 µg/mL of
125I-labeled apoA-I was used and analyzed by 1D.
The result is summarized in Fig 3. At
concentrations of 1 to 8 µg/mL, nearly all the purified apoA-I
was converted to pre-–migrating particles. Above this
concentration, the pre- formation approaches saturation. At 10 and
20 µg/mL, 80% and 50% of the pre-ß apoA-I was converted to
pre-, respectively. The formation of pre- particles appeared to
be dependent on the concentration of apoA-I present.
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Products of purified apoA-I incubations with fibroblasts were also
analyzed on 2DE (3% to 16%) gels. The result of the 24-hour
incubation of 10 µg/mL apoA-I in medium is shown in Fig 4. When apoA-I was incubated in medium
without fibroblasts (Fig 4a), only a pre-ß mobility particle was
found both in the 1DE (top insert) and 2DE (3% to 16%). Mobility of
this particle in the second dimension was similar to that of purified
apoA-I (standard 2) as well as that of free apoA-I–like particle of
human plasma (standard 1) obtained from the pre-ß region of the 1D
gel. When apoA-I was incubated in medium containing fibroblasts (Fig 4b), pre-ß and pre- mobility particles appeared in 1D and 2D as
well. The mobility of pre-ß was similar to the "free apoA-I." In
2D, five distinct pre- particles of different sizes were detected.
When no apoA-I was present in the medium, cholesterol
was effluxed from the cells at a lower rate (Fig 1
), but there were no
distinct spots when the medium was analyzed by 2DE. Also, there
were no distinct bands when the medium was analyzed by 1DE
(data not shown).
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To demonstrate that pre- particles contained lipid, we incubated
purified apoA-I with cells that were labeled with radioactive
cholesterol (14C). To test whether labeled
cholesterol would appear both in the pre-ß and pre-
region, we incubated 30 µg/mL apoA-I with fibroblasts. The
results are shown in Fig 5 (middle
panel). Immunorecognition with anti–apoA-I demonstrated the presence
of five particles with pre- mobility (Fig 5, left panel). Labeled
cholesterol was found in four of the pre- particles but
not in the smallest-sized particle. Importantly, there was no
radioactivity in the pre-ß region.
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To examine the possibility that this small particle that did not
contain cholesterol might contain phospholipid, fibroblasts
were labeled with 32P-orthophosphate. The smallest particle
in the pre- region was detected with 32P labeling in
addition to four higher-molecular-weight particles (Fig 5, right
panel). Immunorecognition by anti–apoA-I showed the presence of
pre-ß particles in the medium (Fig 5, left panel). To further confirm
that the radioactive counts were those of phospholipids, the region of
pre- was extracted with chloroform:methanol. Radioactive counts were
lipid-extractable and co-migrated on thin-layer
chromatography with plasma phospholipids.
To understand the nature of the pre- particles, their molecular
masses were determined by 2DE (3% to 35%). The results are summarized
in the Table. The molecular weight of the five pre-
particles ranged from 114 to 684 kDa. All particles contained
cholesterol, phospholipid, and apoA-I except the smallest
particle, which contained only phospholipid and apoA-I (Fig 5). The
phospholipid-apoA-I particle had a molecular weight of 114 kDa. The
higher-molecular-weight pre- particles appeared to be polymers of
the lowest-molecular-weight phospholipid-apoA-I particle.
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To determine if the pre- particles were lipoproteins, we incubated 5
µg/mL 125I-labeled apoA-I with fibroblasts. This
concentration was chosen to maximize formation of pre- particles and
minimize the presence of pre-ß particles. At the end of incubation,
medium density was adjusted to d=1.25 g/mL and transferred to an
ultracentrifuge tube. A potassium bromide solution of d=1.21
g/mL was overlaid on the medium, and the solution
ultracentrifuged. The resultant d<1.21 g/mL top and
d>1.21 g/mL bottom fractions were collected and
analyzed by 2DE (3% to 35%). The results showed that 85% of
the radioactivity was in the d<1.21 g/mL fraction, and 15% was
in the infranatant. The top 85% contained all five of pre-
particles (Fig 6), whereas the bottom
15% contained some of the phospholipid-apoA-I–only particle. SDS PAGE
of the pre- lipoproteins showed only apoA-I (data not shown).
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Discussion |
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Cellular cholesterol efflux occurs in the interstitial space. Lipoproteins and apoproteins must penetrate the interstitial space before reverse cholesterol transport can occur. Capillary endothelia sieve plasma proteins, allowing particles to penetrate the interstitial space in varying degrees, depending on their size. Lymph particles derived from plasma proteins are proportionally enriched in smaller particles.20 For this reason, interstitial fluid contains fewer LDL particles than plasma (about 8%). In the prenodal peripheral lymph (an accepted model of interstitial fluid), apoA-I concentration is only 10% to 15% that of plasma.20 ApoA-I–containing particles are heterogeneous, having several subpopulations that differ in size and charge both in plasma and lymph.9,10,12 Fielding9 postulated that a specific subpopulation of HDL, the pre-ß1 particles, participates in the initial stages of reverse cholesterol transport. Based on the properties of the interstitial space, we would expect that the lower-molecular-weight particles (pre-ß1 particles) would cross the interstitial space much more readily than the larger particles.
The concentration of pre- particles in lymph greatly exceeded the
pre- particles of plasma.10,21 Because of the larger
size of pre- particles, we expected a much lower concentration in
the lymph than we observed. The higher-than-expected pre-
concentration in lymph suggests generation of pre- particles in the
interstitial space. The higher concentration of
pre-ß1 particles in lymph than in plasma10,21
suggests that at least some of lymph pre-ß1 particles are
generated outside the plasma compartment.
Recently, we have demonstrated the presence of free apoA-I–like particles with pre-ß mobility, which differ from pre-ß1 particles,11 in plasma. Pre-ß particles may be generated either through lipolysis22 or through the action of cholesterol ester transfer protein on HDL.23 It should also be noted that although free apoA-I has pre-ß mobility on 1D, not all pre-ß–migrating particles are "free apoA-I."11 For example, the majority of dog lymph pre-ß particles have a molecular weight of 45 kDa21 and contain lipid.
The estimated molecular weight of free apoA-I–like particles is
similar to purified apoA-I (Fig 4). The relatively small molecular size
of these particles would increase their proportion passing into the
interstitial space as well as their rate of diffusion
within the interstitial space. We postulated that the
interaction of free apoA-I with peripheral cells could
result in cellular cholesterol efflux with the formation of
pre- and pre-ß particles. In an attempt to understand this
phenomenon, we studied lipoprotein formation in a tissue culture model
in which all the components could be defined. We used our 1DE and 2DE
methods, which allow clear differentiation between purified or free
apoA-I–like particles and pre-ß1 particles. Using this
approach, we investigated the effect of purified apoA-I particles on
cellular cholesterol efflux in a tissue culture model. In
this article, we have shown that during incubation of apoA-I with
fibroblasts, lipoproteins are formed with pre- mobilities.
It is estimated that the concentration of free apoA-I like particles in
plasma is between 10 to 20 µg/mL. As shown in Fig 3, apoA-I
concentration in the incubation medium at 8 µg/mL is almost
maximal for pre- particle formation. Our observation is
consistent with those of other investigators who found maximal
cholesterol efflux by macrophages,24
CHO cells,14 and fibroblasts25 to be about 10
µg/mL apoA-I; thus, it is conceivable that some pre-
particles found in lymph could be formed from "free apoA-I"
pre-ß–migrating particles through their interaction with
peripheral cells such as fibroblasts. Recently, Clay and
Barter26 have shown that lipoproteins could also be formed
in vitro through the interaction of free apoA-I and lipolysis
products.
The most important observation reported in this article is that the
newly formed cholesterol–apoA-I complex has pre-
mobilities (Figs 2 and 4-6). We have shown that this pre- particle
can be separated on 2DE into five apoA-I–containing particles with
distinctly different sizes. Cholesterol, phospholipid, and
apoA-I colocalized with one another in the four larger particles.
However, the smallest (114 kDa) particle contained only phospholipid
and apoA-I. We have shown that these particles are lipoproteins (Fig 6). After ultracentrifugation, the four larger
particles and a fraction of the smallest particles were recovered in
the d<1.21 g/mL fraction. Some of the smallest pre-
particles were also recovered in the d>1.21 g/mL fraction.
The presence of a phospholipid–apoA-I particle with pre- mobility
that is devoid of cholesterol is of considerable interest
(Fig 5). The phospholipid–apoA-I particle is the major pre-
particle. This particle may be similar to that of the 7.9-nm particle
reported by Forte et al.14 Assuming that the particle
contained one molecule of apoA-I, the particle would have 76% lipid
and 24% protein. If the particle contained two molecules of apoA-I, it
would still contain 53% lipid. If we assume an average molecular
weight of 645 for a phospholipid, the particle will have between 60 to
100 molecules of phospholipid. This amount of lipid is sufficient for
it to behave as a lipoprotein. Indeed, our experiment showed that the
smallest pre- particles were recovered both in the d<1.21
g/mL and the d>1.21 g/mL fractions (Fig 6). That this
particle could be recovered in the d>1.21 g/mL fraction may be
caused by ultracentrifugation. We have previously shown
that some pre- and particles could be recovered from the d>1.21
g/mL bottom.10 The molecular weight of the other
pre- particles suggests that they may be polymers of the 114 000
molecular weight particle. It is possible that this particle may be a
precursor particle for accepting cholesterol. Forte et
al14 reported a 7.9-nm particle that was not a good
cholesterol acceptor, and it is possible that our
phospholipid–apoA-I–only particle could be the 7.9-nm particle. Thus,
the exact function of this particle requires further study.
Although we have provided evidence that pre- particles could be
formed from free apoA-I, we have not clarified whether different
pre- particles are precursors of one another. Two hypotheses are
possible: (1) pre- particles are formed spontaneously and are
independent of lower-molecular-weight pre- particles; or (2)
lower-molecular-weight pre- particles are precursors of the
higher-molecular-weight pre- particles. Evidence in support of the
first hypothesis is our observation that at the earliest time when
pre- particles are observed, all pre- particles have already
formed.
The observation that the interaction of purified apoA-I with effluxed
cholesterol and phospholipids led to the formation of
lipoproteins with pre- mobilities may provide an explanation for our
previous observation of increased pre- lipoproteins in
lymph.12 We postulate that as a result of passage of plasma
apoA-I–containing particles through the capillary
endothelium, the particles will be exposed to
lipoprotein lipases and phospholipases. Interaction will increase the
number of free apoA-I particles at the site of filtration. When these
free apoA-I–like particles cross the endothelium and
interact with peripheral interstitial cells,
the effluxed cholesterol and phospholipid will transform
the free apoA-I particles into pre- particles. It is postulated that
this process is responsible for the increase in pre- particles in
lymph. To understand the subsequent fate of these newly formed
interstitial fluid pre- particles after they have
returned to the plasma compartment would be an important avenue of
future research.
In variance to other investigators, we were unable to find significant
amounts of lipid associated with apoA-I in the pre-ß region. Castro
and Fielding27 and von Eckardstein et al28 have
both shown radioactive cholesterol associated with
pre-ß1–migrating lipoproteins when plasma was incubated
with radioactive cholesterol–labeled cells for 1 to 2
minutes. We could not rule out the possibility that the amount of lipid
incorporated into pre-ß1 particles at 1- to 2-minute
incubations was too low to be detected. Indeed, the first detectable
amount of pre- particles appears at 4 hours, ample time to transfer
radioactivity from pre-ß1 to pre- particles. It should
also be emphasized that both Castro and Fielding27 and von
Eckardstein et al28 used whole plasma in their experiments.
Under their conditions, approximately 10% of total medium
cholesterol radioactivity was in the pre-ß1
region at the end of 1 minute; the majority of the remaining 90%
occurred in the region.28
Hara and Yokoyama24 used purified apoA-I on radioactive
cholesterol–labeled macrophages and found
radioactive cholesterol in pre-ß lipoproteins. In
contrast, Bielicki et al25 have found lipid labeled in near
-migrating apoA-I particles. The difference may be attributed either
to use of different cell lines (macrophages that secrete apoE
versus fibroblasts that do not) or to differences in apoA-I
purification.25 Our observation is more consistent
with that of Bielicki et al.25 Difference in
electrophoretic mobility could also be attributed to the method of 1D.
Bielicki et al25 used albumin in their agarose
electrophoresis. We have shown previously10 that using the
method of Noble29 for agarose gel electrophoresis caused
movement of pre-–migrating particles to the region.
In conclusion, we have shown that incubation of purified apoA-I with
fibroblasts resulted in formation of lipoproteins with pre-
mobility. These newly formed lipoproteins consist of five distinct
particles ranging in molecular weight between 114 and 684 kDa. The
particle of smallest molecular weight was an apoA-I–only phospholipid
particle. The other four particles contained apoA-I,
cholesterol, and phospholipids.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We wish to thank our colleagues Drs Howard Eder, C.H. Sloop, and J.J. Thompson for their many suggestions and discussions; Dr Theda Foster for her editorial assistance; and Katalin Horvath and Colleen Tierney for their expert technical assistance. Wenwu Zhang is supported by a student fellowship from the American Heart Association Louisiana Affiliate. This research was supported by NIH program project grant HL-25596 and NIH grant HL56160.
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Footnotes |
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Drs Asztalos and Zhang contributed equally to the study.
Received October 17, 1996; accepted January 9, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Johnson WJ, Mahlberg FH, Rothblat GH, Phillips MC. Cholesterol transport between cells and high-density lipoproteins. Biochim Biophys Acta. 1991;1085:273-298.[Medline] [Order article via Infotrieve]
2. Stein O, Stein Y, Lefevre M, Roheim PS. The role of apolipoprotein A-IV in reverse cholesterol transport studied with cultured cells and liposomes derived from an ether analog of phosphatidylcholine. Biochim Biophys Acta. 1986;878:7-13.[Medline] [Order article via Infotrieve]
3.
Bernard DW, Rodriguez A, Rothblat GH, Glick JM.
Influence of high density lipoprotein on esterified
cholesterol stores in macrophages and hepatoma
cells. Arterioscler Thromb. 1990;10:135-144.
4.
Davidson WS, Rogrigueza WV, Lund-Katz S, Johnson WJ,
Rothblat GH, Phillips MC. Effects of acceptor particle size on the
efflux of cellular free cholesterol. J Biol
Chem. 1995;270:17106-17113.
5.
Rothblat GH, Phillips MC. Mechanism of
cholesterol efflux from cells. Effects of acceptor
structure and concentration. J Biol Chem. 1982;257:4775-4782.
6. DeLamatre J, Wolfbauer G, Phillips MC, Rothblat GH. Role of apolipoproteins in cellular cholesterol efflux. Biochim Biophys Acta. 1986;875:419-428.[Medline] [Order article via Infotrieve]
7.
Kilsdonk EPC, Yancey PG, Stoudt GW, Bangerter FW,
Johnson WJ, Phillips MC, Rothblat GH. Cellular cholesterol
efflux mediated by cyclodextrins. J Biol Chem. 1995;270:17250-17256.
8. Barter PJ, Rye K-A. Molecular mechanisms of reverse cholesterol transport. Curr Opin Lipidol. 1996;7:69-76.[Medline] [Order article via Infotrieve]
9. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res. 1995;36:211-228.[Abstract]
10. Asztalos BF, Sloop CH, Wong L, Roheim PS. Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apoA-I containing subpopulations. Biochim Biophys Acta. 1993;1169:291-300.[Medline] [Order article via Infotrieve]
11.
Asztalos B, Roheim PS. Presence and formation of `Free
Apolipoprotein A-I-like' particles in human plasma. Arterioscler
Thromb. 1995;15:1419-1423.
12. Asztalos BF, Sloop CH, Wong L, Roheim PS. Comparison of apoA-I-containing subpopulations of dog plasma and prenodal peripheral lymph: evidence for alteration in subpopulations in the interstitial space. Biochim Biophys Acta. 1993;1169:301-304.[Medline] [Order article via Infotrieve]
13. Nichols AV, Gong EL, Blanche PJ, Forte TM, Anderson DW. Effects of guanidine hydrochloride on human plasma high density lipoproteins. Biochim Biophys Acta. 1976;446:226-239.[Medline] [Order article via Infotrieve]
14. Forte TM, Bielicki JK, Goth-Goldstein R, Selmek J, McCall MR. Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT. J Lipid Res. 1995;36:148-157.[Abstract]
15. Caprio JD, Yun J, Javitt NB. Bile acid and sterol solubilization in 2-hydroxypropyl-ß-cyclodextrin. J Lipid Res. 1992;33:441-443.[Abstract]
16.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
17. Wong L, Torbati A. Differentiation of intrahepatic membrane-bound and secretory apolipoprotein B by monoclonal antibodies-membrane-bound apolipoprotein B is more glycosylated. Biochemistry. 1994;33:1923-1929.[Medline] [Order article via Infotrieve]
18. McFarlane AS. Efficient trace-labeling of proteins with iodine. Nature. 1958;182:53.[Medline] [Order article via Infotrieve]
19. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.
20. Sloop CH, Dory L, Roheim PS. Interstitial fluid lipoproteins. J Lipid Res. 1987;28:225-237.[Abstract]
21.
Wong L, Sivok B, Kurucz E, Sloop CH, Roheim PS,
Asztalos B. Lipid composition of HDL subfractions in dog plasma and
lymph. Arterioscler Thromb. 1995;15:1875-1881.
22.
Barrans A, Collet X, Barbaras R, Jaspard B, Manent J,
Vieu C, Chap H, Perret B. Hepatic lipase induces the formation of
pre-ß1 high density lipoprotein (HDL) from
triacylglycerol-rich HDL2. J Biol
Chem. 1994;269:11572-11577.
23. Liang HQ, Rye K-A, Parter PJ. Dissociation of lipid-free apolipoprotein A-I from high density lipoproteins. J Lipid Res. 1994;35:1187-1199.[Abstract]
24.
Hara H, Yokoyama S. Interaction of free apolipoproteins
with macrophages—formation of high density lipoprotein-like
lipoproteins and reduction of cellular cholesterol.
J Biol Chem. 1991;266:3080-3086.
25. Bielicki JK, Johnson WJ, Weinberg RB, Glick JM, Rothblat GH. Efflux of lipid from fibroblasts to apolipoproteins-dependence on elevated levels of cellular unesterified cholesterol. J Lipid Res. 1992;33:1699-1709.[Abstract]
26. Clay MA, Barter PJ. Formation of new HDL particles from lipid-free apolipoprotein A-I. J Lipid Res. 1996;37:1722-1731.[Abstract]
27. Castro GR, Fielding CJ. Early incorporation of cell derived cholesterol into pre-ß-migrating HDL. Biochemistry. 1988;27:25-29.[Medline] [Order article via Infotrieve]
28.
von Eckardstein A, Huang Y, Wu S, Sarmadi AS, Schwarz
S, Steinmetz A, Assmann G. Lipoproteins containing apolipoprotein A-IV
but not apolipoprotein A-I take up and esterify cell-derived
cholesterol in plasma. Arterioscler Thromb. 1995;15:1755-1763.
29. Noble RP. Electrophoretic separation of plasma lipoproteins in agarose gel. J Lipid Res. 1968;9:693-700.[Abstract]
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