© 1995 American Heart Association, Inc.
Articles |
Intravenous Injection of Rabbit Apolipoprotein A-I Inhibits the Progression of Atherosclerosis in Cholesterol-Fed Rabbits
From the Department of Biochemistry (A.M., M.S., H.H., Y.-I.S., S.H.), the Second Department of Pathology (M.N., Y.R., K.T.), and the Department of Pediatrics (T.O.), Kumamoto University School of Medicine, and the Chemo-Sero-Therapeutic Research Institute (S.S., W.M., T. Takiue, F.M., T. Terano), Kumamoto, Japan.
Correspondence to Seikoh Horiuchi, MD, PhD, Department of Biochemistry, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860, Japan.
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Abstract The effects of intravenous injection of purified rabbit apoA-I on the progression of aortic atherosclerosis in cholesterol-fed rabbits were examined. In experiment 1, 28 rabbits were equally divided into groups A and B and fed a 0.5% cholesterol diet for 90 days. For the last 30 days, group B received 40 mg apoA-I every week. The fatty streak lesions in group B (23.9±15.6%) were significantly suppressed compared with those in group A (46.0±24.9%) (P<.05). In experiment 2, 33 rabbits were divided into four groups (8 or 9 rabbits per group) and fed a 0.5% cholesterol diet. Group A was killed on day 105, while groups B, C, and D were maintained for an additional 60 days on a normal diet, during which time groups C and D received 1 mg apoA-I every other day or 40 mg apoA-I every week, respectively. The lesions in group C (70.2±15.4%) and group D (65.7±20.0%) were significantly suppressed compared with those in group B (86.2±13.7%) (P<.05) but were not reduced to the level of group A (50.0±22.9%). Although apparent regression was not observed under these conditions, the present study provided the first evidence for the antiatherogenic effect of homologous apoA-I on the progression of atherosclerosis in cholesterol-fed rabbits.
Key Words: apoA-I • HDL • diet-induced atherosclerosis • cholesterol-fed rabbits • experimental atherosclerosis
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Introduction |
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Several epidemiological studies have demonstrated that plasma levels of HDL and apoA-I are inversely correlated with the incidence of coronary heart disease.1 2 3 4 Patients with congenital apoA-I deficiency are reported to show low HDL-C levels with precocious atherosclerotic disease.5 6 In contrast, individuals with CE transfer protein deficiency show high levels of HDL-C due to the inhibition of CE transfer from HDL to apoB-containing lipoproteins.7 Inazu et al7 have suggested that individuals with CE transfer protein deficiency might be resistant to atherosclerotic diseases and have longer life spans than normal individuals. These observations suggest that HDL may play a protective role in atherogenesis.
The antiatherogenic properties of HDL or apoA-I have also been demonstrated in experimental animals. Transgenic mice overexpressing human apoA-I are much more resistant to diet-induced atherosclerosis than nontransgenic mice.8 Moreover, when human apoA-I gene is overexpressed in apoE knock-out mice, which exhibit severe hypercholesterolemia and atherosclerosis on a normal diet, the development of atherosclerotic lesions is markedly inhibited.9 10 Badimon et al11 12 first demonstrated that intravenous administration of exogenous HDL could suppress experimental atherosclerosis in cholesterol-fed rabbits. In their first report,11 rabbits were fed a 0.5% cholesterol diet for 8 weeks while receiving injections (50 mg/wk IV) of the homologous HDL-VHDL plasma fraction (d=1.063 to 1.25 g/mL). Atherosclerotic lesions covered 37.9% of the luminal surface area of the aortas in the control rabbits, which was significantly greater than the percentage in rabbits that were treated with the HDL-VHDL fraction (14.9%). In their second report,12 rabbits were divided into three groups and fed a 0.5% cholesterol diet. After 60 days, group 1 was killed, while groups 2 and 3 remained on the same diet until they were killed on day 90. For the last 30 days, group 3 was treated with the HDL-VHDL fraction (50 mg/wk IV). The aortic area occupied by atherosclerotic lesions in group 3 (17.8%) was significantly less than those in groups 1 (34%) and 2 (38.8%), which suggests the possibility that injection of the HDL-VHDL fraction might not only suppress the progression of atherosclerosis but may also induce its regression.
Since apoA-I is thought to play a key role in the antiatherogenic effects of HDL, we considered that purified apoA-I would be effective in suppressing atherosclerosis. To test this hypothesis, we examined whether purified rabbit apoA-I, instead of the rabbit HDL-VHDL fraction, might have a similar antiatherogenic effect on atherosclerosis in cholesterol-fed rabbits. We found that apoA-I inhibited the progression of atherosclerosis in these experimental animals, suggesting that apoA-I has therapeutic potential as an agent for controlling atherosclerosis.
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Methods |
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Materials
[1,2,6,7(n)-3H]Cholesteryl oleate (2530 GBq/mmol) and 125I-Na were purchased from DuPont, NEN. Tissue culture media and reagents were obtained from Life Technologies, Inc. Other chemicals were the best grade available from commercial sources.
Large-Scale Purification of Rabbit ApoA-I
Rabbit apoA-I was purified by using the methods of
Carson13 and Ross and Carson14 with some
modifications. They purified human apoA-I from human plasma by column
chromatography by using phenyl-Sepharose followed by
gel filtration with Sephacryl S-300. We modified this method for
large-scale purification of rabbit apoA-I as follows.
Plasma (20 L) obtained from 500 New Zealand White rabbits was applied to a phenyl-Sepharose CL-4B column (6x25 cm) that had been preequilibrated with 50 mmol/L Tris, 0.1 mol/L NaCl, and 5 mmol/L EDTA (pH 7.6) (buffer A). The bound fraction was eluted stepwise at a flow rate of 250 mL/min (500 mL/fraction). The column was first washed with 10 L buffer A containing 40% propylene glycol (buffer B) and then with 10 L buffer A containing 6 mol/L urea (buffer C). The peak fractions eluted with buffer C (4 L), which contained concentrated apoA-I, were precipitated with 15 L ethanol/ethyl ether (3:2, vol/vol) and delipidated twice with 3 L of the same solvent. The precipitates were further delipidated twice with 2 L ethyl ether and evaporated to dryness.
The precipitates were dissolved in 500 mL buffer A containing 3 mol/L guanidine and dialyzed against 10 mmol/L Tris, 10 mmol/L NaCl, and 1 mmol/L EDTA (pH 7.6) (buffer D). The solution (800 mL) was applied to a Q-Sepharose Fast Flow column (11x10 cm) that had been preequilibrated with buffer D. After washing with 5 L buffer D, the bound fraction was eluted with 5 L of 10 mmol/L Tris, 125 mmol/L NaCl, and 1 mmol/L EDTA (pH 7.6) at a flow rate of 50 mL/min. The peak fractions (1000 mL) were concentrated to 100 mL by using an Amicon membrane. Aliquots (15 mL) were further applied to a Sephacryl S-200 column (5x100 cm) that had been preequilibrated with 50 mmol/L Tris, 0.1 mol/L NaCl, 1 mmol/L EDTA, and 3 mol/L guanidine (pH 7.6) and were then eluted with the same buffer. The major peak was collected and dialyzed against phosphate-buffered saline. Finally, 4 g rabbit apoA-I was purified to homogeneity from 20 L rabbit plasma. The purity of rabbit apoA-I was >95% when determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by densitometric scanning. The amino acid sequence of the purified protein was identical to the reported sequence of rabbit apoA-I.15
Human Lipoproteins and Their Modifications
Human LDL (d=1.019 to 1.063 g/mL) and HDL
(d=1.063 to 1.21 g/mL) were isolated by sequential
ultracentrifugation from human
plasma.16 Ac-LDL was prepared by chemical modification of
LDL17 and labeled with [3H]cholesteryl
oleate as described18 except that
[3H]cholesteryl oleoyl ether was replaced by
[3H]cholesteryl oleate. The specific radioactivity of
[3H]cholesteryl oleate–labeled ac-LDL was 4700
dpm/µg protein. To purify human apoA-I, HDL was delipidated and HDL
apolipoproteins were subjected to Sephacryl S-300 gel-filtration
chromatography.19
Cholesterol Efflux From Macrophage Foam
Cells
The capacity of rabbit apoA-I to induce cholesterol
efflux from macrophage foam cells was determined by the method
of Hara and Yokoyama.20 Mouse resident peritoneal
macrophages were collected from nonstimulated male DDY mice (20
to 25 g) and suspended in 2x106 cells/mL in
Dulbecco's modified Eagle's medium containing 0.2% bovine serum
albumin, 10 mmol/L HEPES (pH 7.4), 0.1 mg/mL streptomycin, and
100 U/mL penicillin (medium A).21 Cell suspension (1 mL)
was added to each 35-mm dish, and the cells were incubated at 37°C in
5% CO2 for 2 hours. Cell monolayers thus formed were
washed three times with 1 mL medium A. Macrophages were first
converted to foam cells by incubation for 24 hours with 25 µg/mL
[3H]cholesteryl oleate–labeled ac-LDL. The cells
were washed three times with 1 mL medium A and incubated for an
additional 24 hours with 5, 10, or 20 µg/mL rabbit apoA-I or human
apoA-I. The medium was collected and centrifuged to remove
detached cells, and the radioactivity released into the medium from
cells was determined by liquid scintillation counting. The cells were
washed three times with 1.5 mL phosphate-buffered saline, and
cellular lipids were extracted and run on thin-layer
chromatography, followed by determination of the
radioactivities of [3H]cholesterol and
[3H]CE.22
Animals
Male New Zealand White rabbits (2.5 kg body weight, 12 weeks
old) were housed in the Animal Research Center of
Chemo-Sero-Therapeutic Institute, Kumamoto, Japan. The temperature and
humidity were controlled at 24±2°C and 55±15%, respectively, with
a 12-hour light/dark cycle. To induce experimental
atherosclerosis, the rabbits were fed a 0.5%
cholesterol diet at 150 g/d. A standard rabbit chow (LABO R
stock) and one containing 0.5% cholesterol were purchased
from Nihon Nosan Industrial Co Ltd. Both chows contain 17.3% protein,
17% diet fibers, and 3% fat with standard fatty acid composition.
Experimental protocols were approved by the Experimental Animal Care
Committee of Kumamoto University School of Medicine, and procedures
were in accordance with the animal care guidelines of the
committee.
Plasma Clearance of Intravenously Injected Rabbit
ApoA-I
Purified rabbit apoA-I (240 mg) was labeled with 37 MBq of
125I by using the method of McFarlane23 to a
specific radioactivity of 72 000 cpm/µg. 125I-Labeled
rabbit apoA-I (40 mg) in 5 mL phosphate-buffered saline was infused
into each of three normolipidemic rabbits by a bolus injection. Labeled
apoA-I was also injected into each of three
hyperlipidemic rabbits that had been fed a 0.5%
cholesterol diet for 90 days. Blood (5 mL) was sampled at
the indicated times, and the radioactivities remaining in the plasma
were determined.
Determination of ApoA-I (Experiment 1) and Combined Effects of
ApoA-I and Change to Normal Diet (Experiment 2) on the Progression of
Atherosclerosis in Cholesterol-Fed
Rabbits
For experiment 1, normolipidemic rabbits were randomly divided
into group A (n=14) and group B (n=14). All the rabbits in both groups
were fed an atherogenic diet containing 0.5% cholesterol
for 90 days (Fig 1
). For the last 30 days
(from days 60 through 90) of the experiment, group B received an
injection of purified rabbit apoA-I (40 mg IV) in 5 mL saline once a
week. Each rabbit in group A was injected with an equal volume of
saline.
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For experiment 2, 33 normolipidemic rabbits were randomly divided into
four groups (Fig 1
). Rabbits in group A (n=9) were fed a
0.5% cholesterol diet for 105 days and then killed to
evaluate the extent of atherosclerosis before
treatment. Groups B, C, and D (n=8 for each) were fed the same diet for
105 days and then normal chow for 60 days (Fig 1). From
day 105 through day 165, group C was given a bolus injection of 1.0 mg
purified rabbit apoA-I in 5 mL saline every other day. Group D was
injected with 40 mg apoA-I in 5 mL saline once a week. Group B was
injected with an equal volume of saline instead of apoA-I.
Morphometric Evaluation of Atherosclerotic Lesions
Animals were killed under deep anesthesia with
sodium pentobarbital 25 mg/kg IV. The entire aorta from the aortic
valve to the iliac bifurcation was removed from each rabbit and opened
longitudinally. The vessel was fixed with 10% buffered formaldehyde
(pH 7.4). Atheromatous lesions were measured without
staining.24 Each aorta was photographed (0.7-fold
magnification), and the luminal surface area that was covered with
atheromatous plaques was determined by
computer-assisted planimetry (IMAGE software, version
1.44) on a Macintosh computer. Atheromatous lesions
were manually traced in the photographs, and the percent area of the
atheromatous lesions was calculated by using the same
computer system. The results obtained by the two methods without
staining showed good accordance; the data represent those
obtained by computer-assisted planimetry. Moreover, in a
preliminary experiment, we found that the area of the lesions
determined after Sudan IV staining agreed well with the data obtained
without staining.
Measurement of Cholesterol Contents in Vascular Walls
and Plasma
After each aorta was photographed for morphometric examination,
its cholesterol content was determined. The intima and
media were carefully removed from the adventitia25 26 and
homogenized with 10 mL saline. Lipids were extracted twice
with 15 mL hexane/isopropanol (3:2, vol/vol) and dried under nitrogen,
and [3H]cholesteryl oleate was used as an internal
standard. The amounts of TC and FC were measured independently by using
a standard enzymatic method (cholesterol E-test, Wako).
Plasma levels of cholesterol (CEs and FC) and triglycerides were determined by using standard enzymatic methods with a Hitachi 7450 automatic analyzer.27 28 HDL-C was determined by measuring the cholesterol contents of rabbit plasma after precipitating apoB- and apoE-containing lipoproteins with dextran sulfate.29
Statistical Analysis
Data were evaluated by Student's t test;
P<.05 was judged as significant.
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Results |
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Capacity of Rabbit ApoA-I to Enhance Cholesterol Efflux From Macrophage Foam Cells
To examine the capacity of purified rabbit apoA-I to promote cholesterol efflux from macrophage foam cells, mouse macrophages were converted to foam cells with [3H]cholesteryl oleate–labeled ac-LDL and then exposed to rabbit apoA-I. Significant amounts of radioactivities were released into the medium in the presence of rabbit apoA-I (Fig 2
). Thin-layer
chromatographic analyses of the lipids extracted
from the medium showed that all the radioactivities were derived from
those of [3H]cholesterol. The capacity of the
rabbit apoA-I for cholesterol efflux was indistinguishable
from that of human apoA-I that had been purified by a conventional
method.18 Similarly, rabbit apoA-I and human apoA-I were
equally effective in reducing cellular [3H]CE (20%
reduction from the control). These results indicate that the
purification procedure for rabbit apoA-I employed in the present
study may not significantly affect its cholesterol efflux
capacity when compared with a widely used method for the purification
of human apoA-I.
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) or human (
) apoA-I.
The medium was collected, and radioactivities were determined by liquid
scintillation counting.
Plasma Clearance of Intravenously Injected Rabbit
ApoA-I
Plasma clearance of rabbit apoA-I was examined by bolus injection
of 40 mg labeled apoA-I since plasma clearance of a higher dose of
apoA-I in rabbit has not been reported. The time required for a 50%
reduction in plasma radioactivity in normolipidemic rabbits was
approximately 20 hours, which is close to that for the trace amount of
apoA-I reported by Badimon et al11 (Fig 3). When 40 mg labeled apoA-I was
injected into cholesterol-fed
hypercholesterolemic rabbits, its plasma clearance
rate was much faster than that in normolipidemic rabbits
(P<.05 at 24 and 44 hours) (Fig 3).
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Effect of ApoA-I on the Progression of
Atherosclerosis in Cholesterol-Fed Rabbits
(Experiment 1)
Both the control (A) and the apoA-I–treated (B) groups were fed a
0.5% cholesterol diet throughout the experiment (90 days).
For the last 30 days of the experiment, group B received an
intravenous injection of purified rabbit apoA-I, and the
effect on the progression of atherosclerosis was
examined. The plasma level of TC increased to 2500 mg/dL with the
atherogenic diet and remained constant throughout the remainder of the
experiment (Fig 4). The
cholesterol (Fig 3) and HDL-C (Table 1) levels in the two groups were
indistinguishable. However, aortic fatty streaks in apoA-I–treated
rabbits (23.9±15.6%) were significantly inhibited compared with
those in control rabbits (46.0±24.9%) (P<.05) (Fig 5).
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Combined Effects of ApoA-I and Change to Normal Diet on the
Progression of Atherosclerosis in
Cholesterol-Fed Rabbits (Experiment 2)
In a preliminary experiment similar to experiment 1, some of the
rabbits were killed on day 60 to examine whether apoA-I could induce
regression of atherosclerosis during apoA-I treatment
(from days 60 through 90). Atheroma formation in the
apoA-I–treated group killed on day 90 was significantly suppressed
compared with that in the 90-day control. However, it was not lower
than that in the 60-day control (data not shown), indicating that
apparent regression did not occur under the present experimental
conditions. Therefore, in experiment 2, we changed the experimental
protocol so that the effect of apoA-I on the regression of
atherosclerosis could be more easily observed with a
change from the cholesterol diet to a normal diet during
apoA-I treatment. In addition, since some rabbits showed negligible
aortic lesions even on day 60, the duration of cholesterol
feeding before apoA-I treatment was prolonged from 60 days in
experiment 1 to 105 days in experiment 2 to induce moderately extensive
aortic lesions (around 50%). Thus, in experiment 2, rabbits were fed
normal chow during apoA-I-treatment (days 105 through 165) after being
fed the atherogenic diet for 105 days.
Plasma cholesterol levels rose to 2000 mg/dL with the 0.5%
cholesterol diet and gradually decreased after the change
in diet on day 105 (Fig 6). Plasma
cholesterol (Fig 6) and HDL-C (Table 2) levels in the apoA-I–treated rabbits
did not significantly differ from those in the control groups during
the experiment.
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Cholesterol contents in vascular walls were also determined
(Table 3). The amount of TC increased
from 36.1 mg cholesterol/g wet wt on day 105 (group A) to
59.3 mg cholesterol/g wet wt on day 165 (group B)
(P<.01), indicating that cholesterol
accumulation in vascular walls progressed during this period even with
a normal diet. The amount of CE also increased during the last 60 days.
With a higher dose of apoA-I (group D), TC levels decreased from 59.3
(group B) to 41.9 (group D) mg/g wet wt (P<.05). CE content
also decreased from 37.7 (group B) to 23.5 (group D) mg/g wet wt
(P<.05). When rabbits were treated with an even lower dose
of apoA-I, CE content significantly decreased, from 37.7 (group B) to
26.8 (group C) mg/g wet wt (P<.05), whereas the reduction
in TC was not significant.
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Morphometric analysis of aortic lesions showed that the area of
atherosclerosis progressed from 50.0±22.9% on day 105
(group A) to 86.2±13.7% on day 165 (group B) (Fig 7), indicating that the progression of
atherosclerosis was not inhibited by changing the diet
alone. When the rabbits were injected with 1 mg apoA-I every other day
(group C) from day 105 through 165, the extent of aortic fatty streak
formation (70.2±15.4%) was significantly suppressed compared with
that in group B (P<.05) (Fig 7). Group D was
injected with a higher dose (40 mg) of apoA-I every week, and
atheroma formation (65.7±20.0%) was also significantly
suppressed compared with that in group B (P<.05) (Fig 7). However, the areas of atheromatous
plaques in the apoA-I–treated groups (C and D) were not lower than
those in group A, indicating that the regression of
atherosclerosis could not be induced even though the
rabbits were treated with apoA-I and a change to a normal diet.
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Discussion |
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The present study directly demonstrated the antiatherogenic effect of purified apoA-I on experimental atherosclerosis. Although Badimon et al11 12 have demonstrated that intravenous injection of the homologous HDL-VHDL fraction could suppress the progression of atherosclerosis in cholesterol-fed rabbits, the present study provides the first evidence that purified apoA-I has an antiatherogenic effect in experimental atherosclerosis.
One of the important findings in the present study was that apoA-I
did not affect plasma cholesterol levels but did
significantly reduce cholesterol contents in the vascular
walls (Table 3), thus inhibiting the progression of
atherosclerosis (Fig 7). A reasonable
interpretation of these results may be as follows. ApoA-I might enhance
cholesterol removal from vascular walls to plasma and
subsequent transfer to the liver, thereby increasing net
cholesterol transport from peripheral tissue to
liver (reverse cholesterol transport), whereas plasma
cholesterol levels might not be altered under dynamic
equilibrium. In this context, intravenous injection of apoE
into Watanabe heritable hyperlipidemic rabbits is also
effective in suppressing the progression of
atherosclerosis without affecting plasma
cholesterol levels.30 It is likely that apoE
as well as apoA-I enhances a reverse cholesterol transport
system and inhibits the progression of
atherosclerosis.
According to Badimon et al,11 the plasma clearance rate of
a trace amount of apoA-I in normal rabbits is similar to that in
cholesterol-fed rabbits. In the present study,
although the plasma clearance rate of a higher dose of rabbit apoA-I
(40 mg) in normal rabbits was similar to their values,11
the corresponding rate in cholesterol-fed rabbits was
significantly faster than that in normolipidemic rabbits (Fig 3). The reason for the discrepancy is unknown. The
hypercholesterolemic rabbits we used for this
experiment were studied after 90 days' feeding of a 0.5%
cholesterol diet, while the duration of
cholesterol feeding in the experiment of Badimon et
al11 is not clear. Since apoE is a ligand for the LDL
receptor and its plasma levels are known to increase under
hypercholesterolemic states, incorporation of apoE
into HDL particles might help to accelerate the plasma clearance of
apoA-I in cholesterol-fed rabbits.
It is generally assumed that intravenously injected apoA-I is incorporated into HDL.31 ApoA-I could also induce remodeling of HDL. For example, when apoA-I is incubated in vitro with VLDL in the presence of fatty acids, apoA-I readily forms discoidal complexes with VLDL lipids.32 Therefore, it is possible that discoidal HDL particles might be generated in hypercholesterolemic rabbits through an interaction with ß-VLDL. However, it is not known whether free apoA-I or apoA-I complexes with lipids could be translocated into subendothelial space to serve as a cholesterol acceptor in situ.
If injected apoA-I enhances reverse cholesterol transport,
one would expect that apoA-I might adsorb excess
cholesterol from peripheral tissues, thus
increasing plasma HDL-C levels. However, this was not observed in the
current study (Tables 1 and 2). A similar result was obtained in
cholesterol-fed rabbits that were treated with the
HDL-VHDL fraction.11 12 These observations are somewhat
inconsistent with the result obtained from transgenic mice
overexpressing human apoA-I, whose HDL-C level was twofold
higher.8 This could be because intravenous
injection of apoA-I in rabbits might have a relatively small effect on
total apoA-I level compared with the genetic expression of apoA-I in
mice, which dramatically increased their total apoA-I
level.8 The discrepancy could perhaps be ascribed to the
method of determining HDL-C.29 HDL particles that received
apoE from other plasma fractions might have been precipitated as
apoE-containing lipoproteins during HDL-C determination.
Intravenous injection of exogenous apoA-I inhibited the
progression of atherosclerosis in
cholesterol-fed rabbits (Figs 5 and 7). However, no
apparent regression of established lesions was achieved even when the
diet was changed to normal chow during treatment with apoA-I (Fig 7). There could be some explanations for these results.
Plasma cholesterol levels as well as atherogenic ß-VLDL
levels remain high in cholesterol-fed rabbits for more
than 10 weeks, even after cessation of cholesterol feeding,
during which time cholesterol accumulation in aortic walls
is still in progress.33 The present results were
consistent with this observation; the lesion area (Fig 7) as well as cholesterol contents in
vascular walls (Table 3) continued to increase after a
normal diet was instituted (from days 105 through 165). Such an
atherogenic predisposition of cholesterol-fed rabbits
might have counteracted the antiatherogenic effect of injected apoA-I
even after the change in diet. Alternatively, apoA-I treatment may have
been started too late to induce a regression of
atherosclerosis in the present protocol. We started
apoA-I injection 105 days after cholesterol feeding (Fig 1). In contrast, Badimon et al12 began HDL
injection 60 days after cholesterol loading and observed
significant regression even when the atherogenic diet was continued
during the therapeutic period (days 60 through 90). All these results
strongly suggest that it would be practically difficult for us to
achieve a significant regression of atherosclerotic lesions in
cholesterol-fed rabbits within the time period of this
study. Although the present study did not show any regression of
atherosclerosis, this does not necessarily imply that
the antiatherogenic function of purified apoA-I is weaker than that of
the HDL-VHDL fraction.11 12 Further studies are needed to
compare the antiatherogenic properties of purified apoA-I with those of
the HDL-VHDL fraction.
Another important issue that remains to be addressed is whether an apoA-I complex with phospholipids may have a stronger antiatherogenic effect than free apoA-I in vivo. An in vitro study has demonstrated that the capacity of apolipoproteins for cholesterol efflux is increased by forming complexes with phospholipids.34 Phospholipids are also known to reduce the atherogenicity of atherogenic lipoproteins. The plasma obtained from cholesterol-fed rabbits injected with phospholipid liposomes shows a much weaker ability to induce CE accumulation in macrophages in vitro than that from control rabbits.35 This is explained by the transfer of apoE from ß-VLDL to liposomes, which reduces the ligand activity of ß-VLDL. Moreover, the apoE liposomes thus formed could compete with ß-VLDL for its binding to macrophages and reduce cellular uptake of ß-VLDL. When modified LDLs such as ac-LDL and oxidized LDL are incubated with apoA-I complexed with dimyristoylphosphatidylcholine (DMPC), DMPC was transferred to modified LDLs, which resulted in a decrease in their net negative charge, thus reducing their ligand activity for the macrophage scavenger receptors.18 36
The significant antiatherogenic effect observed with a low dose of
apoA-I for group C (1 mgx30 times) in experiment 2 gave us an
important suggestion. When assessed by the lesion area (Fig 7) and the CE content in vascular walls (Table 3), the antiatherogenic effect of a lower dose of apoA-I
for group C was indistinguishable from that of a higher dose of apoA-I
for group D (40 mgx8 times). This suggests that the therapeutic
efficiency of apoA-I could be improved by modifying the treatment
regimen. It is possible that a lower dose of apoA-I, when injected
frequently, might be efficiently converted to an HDL subfraction such
as pre–ß-HDL,37 which serves as an effective
cholesterol acceptor from vascular walls. To further
elucidate this point, more specific studies are needed to examine the
effects of various doses of apoA-I on lipoprotein compositions and
atherosclerotic lesions in cholesterol-fed rabbits.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a grant-in-aid for scientific research (No. 07770108) from the Ministry of Education, Science and Culture of Japan, and a grant from Ono Medical Research Foundation. We thank Hajime Matsui, Shu-Ji Katajima, and Yumi Kouroki of the Chemo-Sero-Therapeutic Institute, Kumamoto, Japan, for their collaboration. We are also grateful to Drs Takeshi Biwa, Takashi Kawano, Ding Yi, and Hirofumi Matsuda in our laboratory for helpful discussions. Jeff W. McCall is also acknowledged for his editorial assistance with this manuscript.
Received February 12, 1995; accepted September 5, 1995.
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