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Introduction
Apoptosis is a key event in the progression of advanced
atherosclerosis. Apoptosis of foam cells leads to the
development of the necrotic core of an atherosclerotic plaque,
and reduction of the excretion of cell matrix in the plaque
region, which may contribute to plaque instability and the
majority of cardiovascular
complications[1_4]. Several lines of evidence strongly suggest that oxidized low density
lipoproteins (ox-LDL) cause apoptosis and
necrosis of macrophage
cells[5,6]. After they are taken up by scavenger receptor
A, ox-LDL inhibit the efflux of cholesterol from cells,
resulting in the accumulation of a large amount of cholesterol within
them[7_10]. Accumulating evidence suggests that elevation
of cellular cholesterol plays a critical role in the regulation of
ox-LDL-mediated cell apoptosis[11_14]. Thus, accelerating
cholesterol efflux or preventing the accumulation of excess
cholesterol in cells may inhibit ox-LDL-induced apoptosis, which
could provide new perspectives for the prevention and
reversal of atherosclerosis.
Having low levels of high density lipoprotein (HDL)
cholesterol is an important cardiovascular risk factor. The
atheroprotective activity of HDL is often attributed to its
unique ability to facilitate the efflux of cholesterol from
non-hepatic cells and subsequently deliver to the liver and
steroidogenic organs, a process known as reverse cholesterol
transport (RCT)[15,16]. Previous studies have revealed that
HDL suppresses tumor necrosis factor a (TNF-a)-mediated
apoptosis in cultured human endothelial
cells[17]. However, whether HDL has a protective effect against ox-LDL-induced
apoptosis of macrophages and whether this effect
correlates with RCT has not yet been investigated.
b-Cyclodextrins (b-CD) are cyclic oligomers of glucose that have the
capacity to sequester cholesterol in their hydrophobic core.
Recent studies have shown that b-CD is capable of stimulating
efficient cholesterol efflux from cultured human
fibroblasts[18]. In addition, brefeldin A (BFA), an antibiotic, has been shown to
inhibit HDL-mediated cholesterol efflux from
cholesterol-enriched cells[19]. The main aim of the present study was to
observe whether HDL3 antagonized ox-LDL-induced
apoptosis by promoting cholesterol efflux in RAW264.7 cells.
Materials and methods
Reagents Dulbecco¡¯s modified Eagle¡¯s medium (DMEM)
and fetal bovine serum were purchased from Gibco/BRL
(Grand Island, USA). A Hoechst 33258 staining kit was
purchased from the Beyotime Institute of Biotechnology
(Jiangsu, China). HDL3 was purchased from Huanan Company (Guangzhou, China). RAW264.7 cells (a murine
macrophage cell line) were purchased from the Institute of
Biochemistry and Cell Biology (Shanghai Institute for
Biological Science, the Chinese Academy of Sciences, Shanghai,
China). All reagents were of analytical grade.
Cell culture RAW264.7 cells were maintained in DMEM
containing 10% fetal bovine serum in a humidified
atmosphere of 5% CO2 and 95%
O2. Cells were incubated with 50 mg/L ox-LDL for 48 h and then treated with various
concentrations of HDL3 (50, 100, or 200 mg/L) or BFA (4 µmol/L)
or b-CD (10 mmol/L) for 24 h before analysis. Untreated cells
were used as controls.
LDL isolation and oxidization LDL was isolated from
nonfrozen human plasma according to the method described
in our previous report[20]. The remaining sample in the
low-density fraction after isolation of very low-density
lipoproteins was aliquoted into Quickseal tubes (Brown Caskets,
Inc Miami, USA). The volume and the density of solution in
each tube were adjusted to 35 mL and 1.063 kg/L with 38%
NaBr and 0.15 mol/L NaCl, respectively. Tubes were sealed
and centrifuged at 129 400×g at a temperature of 8 °C for 20 h.
A sample of approximately 5 µL was recovered from
superna-tant. The isolated LDL was oxidized with
CuSO4 (10 µmol/L) for 18 h at 37 °C. The degree of oxidation of ox-LDL was
assessed on the basis of increased mobility in
agarose gel (compared with native LDL) and an increased level of
thio-barbituric acid-reactive substances.
Flow cytometry Cultured RAW264.7 cells were collected
and washed twice with phosphate-buffered saline (PBS).
This was followed by fixing with cold 70% ethanol at 4 °C
overnight and resuspending the cells in PBS. Cells were
incubated with RNase A for 45 min and then stained with 50 mg/mL propidium iodide in the dark at 4 °C for 60 min. The
suspension was analyzed with a FACScan flow cytometer
(EPICS-XL, Beckman Coulter, Fullerton, USA). The apoptotic
rate was determined on the basis of the "sub-G1" peak.
Hoechst 33258 staining Cells were incubated with or
without ox-LDL and then harvested. After being fixed with
4% paraformaldehyde for 30 min at 25 °C, the preparations
were washed with cold PBS 3 times and exposed to 10 mg/L
Hoechst 33258 in the dark at room temperature for 10 min.
Stained cells were observed under a fluorescence microscope.
The cells with condensed chromatin and shrunken nuclei
were counted as apoptotic cells.
High performance liquid chromatography
analysis Cells were scraped from the culture flasks into 0.9% NaCl (1 mL
per 50 cm2 flask) and homogenized on ice by sonication for
10 s. After protein concentration was determined by using a
BCA kit (Pierce Biotechnology, Inc Rockford, USA), an equal
volume of freshly prepared cold (-20 °C) KOH in ethanol (150
g/L) was added to cell lysates, and the mixture was
repeatedly vortexed until clear. An equal volume of 3:2
hexane-isopropanol (v/v) was then added. The mixture was vortexed
for 5 min, followed by centrifugation at
800×g (15 °C) for 5 min. The extraction procedure was repeated twice. The
combined organic phase was transferred to clean tapered glass
tubes and thoroughly dried under nitrogen at 40 °C. The
tubes were allowed to cool to room temperature. One
hundred microliters of isopropanol-acetonitrile 20:80 (v/v) was
added. The sample was solubilized in an ultrasound water
bath at room temperature for 5 min. After centrifugation at
800×g for 5 min, the samples were introduced into the high
performance liquid chromatography (HPLC) device(Agilent
1100, Agilent Technologies, Inc, Palo Alto, USA).
Cholesterol was eluted with 1 mL/min of eluent consisting of 20:80
isopropanol-acetonitrile (v:v) and detected by ultraviolet
absorption at 206 nm[21]
Assessment of cholesterol efflux Cells were treated with
[3H]cholesterol and cholesterol-loaded ox-LDL (50 mg/L) for
48 h. After being labeled with [3H]cholesterol, cells were
washed and incubated for an additional 24 h in media
containing bovine serum albumin (2 g/L) to allow for
equilibration of [3H]cholesterol with intracellular cholesterol. Cells
were then incubated with various concentrations of
HDL3 (50, 100, and 200 mg/L), or BFA (4 µmol/L) or
b-CD (10 mmol/L) in serum-free media. Media were recovered 24 h later. Cells
were dissolved in
N-2-hydroxyethylpiperazine-N¡¯-2-ethane-
sulfonic acid (HEPES; 1 mmol/L, pH 7.5) containing 0.5%
Triton X-100. Media were briefly centrifuged to remove
nonadherent cells. Aliquots of both supernatants and cells
were then subjected to scintillation (FJ-2107P type liquid
scintillator, Xi¡¯an Nuclear Instrument Factory, Xi¡¯an, China)
for the determination of radioactivity. Cholesterol efflux was
calculated by using the following formula:
[3H]cholesterol in
medium/([3H] cholesterol in
medium+[3H]cholesterol in cells)×100%.
Statistical analysis Results are expressed as mean±SD.
The two-tailed Student¡¯s t-test was used for statistical
comparisons. P<0.05 was considered to be statistically
significant.
Results ox-LDL induced apoptosis of RAW264.7 cells Exposure
of RAW264.7 cells to 50 mg/L ox-LDL increased the apoptosis
rate of cells in a time-dependent manner. The apoptotic rates
after 24 h (23.1%) and 48 h (47.7%) treatment were both
significantly higher than that in the control
(5.14%; Figure 1).
Effect of HDL3 on ox-LDL-induced apoptosis in
RAW264.7 cells Typical apoptotic morphological changes, such as
condensed chromatin and shrunken nuclei, were seen in
RAW264.7 cells after the cells were incubated with ox-LDL
for 48 h (Figure 2B). Co-administration of
HDL3 inhibited these morphological changes (Figure 2C_2E) and decreased
the apoptotic rate in a concentration-dependent manner
(Figure 3).
Effect of HDL3 on cholesterol efflux and cholesterol level
in RAW264.7 cells Exposure of RAW264.7 cells to ox-LDL
for 48 h increased total cholesterol from 153.4 mg/g protein
to 505.1 mg/g protein without significantly affecting the rate
of cholesterol efflux (Table 1). Many lipid droplets were
seen after ox-LDL treatment (Figure 4B). Co-treatment with
HDL3 and ox-LDL increased the rate of cholesterol efflux
(Table 1), and decreased the total cholesterol (Table 1) and
lipid droplets in cells (Figure 4C_4E). Both alterations were
concentration-dependent (Table 1, Figure 4).
Effect of BFA and b-CD on apoptosis and cholesterol
efflux in RAW264.7 cells Further observations with a
blocker (BFA) and an agonist (b-CD) of cholesterol efflux
were performed to investigate the relationship between
HDL3 and reverse cholesterol transport. Flow cytometry analysis
showed that BFA alone had no effect on cell apoptosis (Table
2). However, both the inhibition of ox-LDL-induced apoptosis
(Table 2) and the facilitation of cholesterol efflux (Figure 5)
by 100 mg/L HDL3 were significantly attenuated by BFA.
Cholesterol acceptor b-CD decreased the ox-LDL-induced
apoptotic rate from 47.7% to 14.2% (Table 2) and increased
the rate of cholesterol efflux from 5.2% to 36.5% (Figure 5).
Discussion
Apoptosis is a prominent feature of atherosclerotic
lesions in humans and experimental animals. It occurs in
macrophages, T lymphocytes, and smooth muscle cells of
the lesions, and is implicated in the
development and progression of the disease. Increasing evidence
suggests that apoptosis of foam cells, mostly macrophages that
accumulate large quantities of free cholesterol, contribute to the
expansion of a lipid core, and the large eccentric lipid core
could lead to plaque instability by conferring a mechanical
disadvantage on the plaque through redistribution of
circumferential stress to the shoulder regions of the plaque,
where nearly 60% of plaque ruptures tend to
occur[1_4]. Therefore, inhibition of macrophage apoptosis may prevent
the development of atherosclerosis.
Previous studies have shown that large amounts of cholesterol accumulate in macrophages treated with ox-LDL.
Such a change in cholesterol leads to the failure of lipid
homeostasis[10]. Furthermore, cellular accumulation of
excess cholesterol may serve as a trigger for cell death, and
promote ongoing inflammation, calcification, thrombosis and
plaque rupture, which are the major sequelae of advanced
atherosclerosis. HDL has been shown to protect cultured
human endothelial cells induced by TNF-a through
inhibition of caspase 3 and caspase 9 activity, and cytochrome
c release[17,22]. However, whether HDL could protect
monocyte-derived macrophage cells against ox-LDL-induced
apoptosis has not yet been studied. Our data demonstrate
that after incubation with 50 mg/L ox-LDL for 48 h,
RAW264.7 cells display the morphological changes that are typically
associated with apoptosis, such as condensed chromatin
and shrunken nuclei, and up to 47.7% of cells are apoptotic.
Treatment with HDL3 after 48 h pre-incubation with ox-LDL,
decreased the apoptotic rate in a dose-dependent manner,
and the apoptotic morphological changes were not present.
Cholesterol accumulation plays a critical role in the
occurrence of ox-LDL-mediated cell apoptosis. Evidence
suggests that accumulation of intracellular free cholesterol can
increase Bax expression, increase cholesterol overloading of
mitochondria, increase cytochrome c release, activate
caspase-9, and lead to macrophage
apoptosis[11_14]. Studies using experimental models of atherosclerosis indicate that
the pro-apoptotic effect of ox-LDL can be reversed by lipid
lowering interventions[23]. It is thus possible to protect cells
from apoptosis by promoting intracellular cholesterol efflux.
Efflux of cholesterol from extrahepatic tissue facilitated by
HDL is considered to be an important mechanism by which
HDL exerts its protective effects in atherosclerosis
progres-sion. HDL enhances not only the translocation of
cholesterol from intracellular compartments to the plasma membrane,
but also cholesterol efflux from the plasma membrane to the
extracellular space[24].
Various HDL subclasses are characterized by difference
in shape, density, size, charge, and antigenicity. Mature
HDL3 and HDL2 are generated from their precursors,
lipid-free apoA-1 and lipid-poor pre-b1-HDL. Small
HDL3 particles have a greater capacity to facilitate the efflux of cholesterol
than large HDL2 because their small size makes it easier to
move to the plasma membrane and bind to receptors (SR-B1)
and cholesterol transporters (ABCA1). Furthermore,
HDL3 exerts more powerful anti-oxidative effects than
HDL2[25_29]. However, whether the protective effect of
HDL3 on ox-LDL-induced apoptosis is mediated via the promotion of
cholesterol efflux remains unclear. In line with this notion, we found
that HDL3 dose-dependently increased the rate of cholesterol efflux, and significantly decreased the apoptotic rate of
ox-LDL-treated RAW264.7 cells. Interestingly, when BFA,
an antibiotic drug, was present at a concentration of
4 µmol/L during incubation of the cells with
HDL3, the protective effect of
HDL3 on ox-LDL-induced apoptosis was significantly
attenuated. It has previously been shown that BFA inhibits
HDL-mediated cholesterol efflux from cholesterol-enriched
cells by disturbing the translocation of cholesterol from
intracellular compartments to the plasma
membrane[19]. Consistent with this finding, our data also revealed that BFA
treatment decreased the rate of cholesterol efflux, and
increased the level of total cholesterol, accompanied by an
increase in the apoptotic rate of cells. In contrast, treatment
with cholesterol acceptor b-CD, a cholesterol efflux stimulator,
suppressed ox-LDL-induced apoptosis. Overall, our results
suggest that promoting cholesterol efflux has great
potential in the inhibition of ox-LDL-induced apoptosis in
RAW264.7 cells.
In conclusion, cholesterol efflux mediates the protective
effects of HDL3 on ox-LDL-induced apoptosis in RAW264.7
cells. This finding provides new evidence that HDL has a
functionally protective effect on atherosclerosis and related
complications.
Acknowledgements
The authors are indebted to Dr De-liang CAO (South
Illinois University, USA) and Dr Ying-jun CAO (Johns
Hopkins University, USA) for their critical discussion,
suggestions, and valuable help in the preparation of the
manuscript.
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