rat carotid as well as rabbit external iliac arteries, and this apoptosis was proposed to account for the lack of
cell accumulation at the injury site during the period of observation [6]. In advanced lesions, both macrophages
and VSMCs can accumulate lipid and have been iden- tified as foam cells. Cell debris are more prominent in
lipid-rich
areas, with
death occurring
in both
macrophages and VSMCs [7 – 9]. A high rate of apop- totic VSMC death in atherosclerotic plaque may hence
contribute to the destabilization of the fibrous cap, and increase the risk of plaque rupture and thrombosis
[10,11]. The triggers inducing VSMC apoptosis in le- sions, however, are unknown at present.
There is evidence that oxidatively modified low-den- sity lipoprotein oxLDL exists in atherosclerotic le-
sions of rabbit and humans [12]. OxLDL exerts several potentially atherogenic properties including cytotoxicity
to vascular cells [13,14], impairment of endothelium-de- pendent relaxation of blood vessel [15,16], stimulation
of monocyte recruitment and their adhesion to en- dothelial cells [17,18]. To study oxLDL-induced cyto-
toxicity, the cell culture system provides a controlled environment to examine mechanism of actions and to
identify active components of oxLDL with limited in- terference. OxLDL, whether oxidized in a metal-ion
system or by cells, has been shown to injure endothelial cells [19], smooth muscle cells [20], fibroblasts [21,22],
and
macrophages [14].
However, the
toxic-dose threshold for the cytotoxicity of oxLDL in VSMCs has
not been determined. Furthermore, lysophosphatidyl- choline lysoPC, produced during LDL oxidation by
endogeneous phospholipase A
2
[23], is also found in atherosclerotic lesions at high levels [24,25]. Although
lysoPC has been proposed as one of the cytotoxins contained in oxLDL [26], quantitative studies are yet to
be performed. Moreover, whether lysoPC could induce apoptosis in cultured VSMC was not clear [27,28]. A
parallel study between the effects of oxLDL and lysoPC may provide additional information concerning the na-
ture of oxLDL action.
We thus chose to investigate the relative importance of the amount andor the oxidative level in determining
the threshold concentration of oxLDL for cytotoxicity as well as the dose – response pattern of lysoPC. We
also tested whether lysoPC, over the range of concen- trations employed in cytotoxicity studies, could induce
apoptosis in cultured VSMCs.
2. Materials and methods
2
.
1
. Cell culture VSMCs were isolated from thoracic aorta of 20-
week-old Wistar – Kyoto rats, identified and maintained in supplemented MCDB107 J.R.Scientific, CA, USA
as described previously [29,30]. Cells between the fourth and eighth passages were used.
2
.
2
. Preparation and oxidation of LDL Blood samples were obtained from the Blood Bank
of Chang Gung Memorial Hospital. LDL d = 1.019 – 1.063 gml and lipoprotein-deficient serum LPDS
d ] 1.21 gml were isolated by sequential ultra-cen- trifugation as described by Havel et al. [31]. Briefly,
saturated NaBr and phosphate-buffered saline PBS NaCl, 136.75 mmoll; KCl, 2.6 mmoll; KH
2
PO
4
, 1.5 mmoll; Na
2
HPO
4
·2H
2
O, 7.9 mmoll; pH 7.4 were used for density adjustments. Following centrifugation
50 000 rpm for 24 h; Beckman 55.2 Ti rotor, isolated LDL and LPDS fractions were extensively dialyzed at
4°C in a cold room against 200 × volume PBS in the presence of 0.1 ethylenediamine tetraacetic acid
EDTA to remove remaining NaBr. Samples were then dialyzed against PBS overnight to remove EDTA prior
to sterilization by filtration through 0.45 mm Gelman filters Ann Arbor, MI, USA. Samples were stored at
4°C and used within 6 weeks. LDL concentration was expressed in term of its protein content [32], i.e. micro-
grams of protein per millilitre of media. The LDL samples were also analyzed for contents of cholesterol
and triglycerides standard test of lipid profile, Chang Gung Memorial Hospital, and the weight ratio was
1.0:3.8:7.1 triglyceride:protein:cholesterol. Apolipo- proteins apo A
1
and apo B were also analyzed by the method of Behring Turbitimer.
2
.
3
. Oxidati6e modification of LDL The oxidative modification of LDL was carried out
by incubating freshly-prepared native LDL nLDL with ferrous sulfate 50 mmoll in 0.9 NaCl solution;
pH 7.4 for up to 24 h at 37°C [33]. The oxidation was terminated by first filtering the sample 0.45 mm and
then dialyzing oxLDL against phosphate buffer with 0.1 EDTA; pH 7.4, 4°C for three changes. OxLDL
was sterilized by passing through a 0.22 mm filter Millipore and further dialyzed against the PBS for at
least 48 h. The relative degree of oxidation for these preparations was measured by analyzing the presence
of thiobarbituric acid-reactive substances TBARS and expressed as malondialdehyde MDA content nmol
MDA equivalent per milligram protein as described elsewhere [34]. The levels of conjugated dienes CD of
oxLDL 100 mgml was also measured as the ab- sorbance at 234 nm using a spectrophotometer DU-64;
Beckman. These chemical changes that occur during oxidation of LDL preparations employed in this study
are summarized in Fig. 1. Both TBARS and CD in- creased linearly with incubation time and thus allowed
one to designate LDL with low-oxidative level 8 h,
low-oxLDL, medium-oxidative level 16 h, medium- oxLDL, and high-oxidative level 24 h, high-oxLDL,
respectively. Utilizing a similar assay, untreated nLDL showed a value of TBARS less than 0.1 nmol MDA
equivalent per milligram protein. Furthermore, the al- tered surface charge on the LDL protein was assessed
by
measuring the
electrophoretic mobility,
using Lipofilm kit Sebia, France. The Lipofilm is composed
of a gelified acrylic polymer divided into two zones of different concentration: 2 in the upper layer in which
the sample wells are moulded, and 3 in the lower layer. Native LDL and oxLDL were stained with sudan
black, and the electrophoresis was performed in 0.005 sodium azide buffer at 12 mA for 45 min. Following
electrophoresis, the gel was dried at 51°C. The distance each sample travelled was measured in millimetres from
the origin to the front of the band. The relative mobil- ity was expressed as folds of the migration distance of
the sample to that of LDL standard. All oxLDL prepa- rations exhibited higher relative mobility than nLDL
data not shown.
2
.
4
. Determination of cell 6iability The cytotoxic effects of oxLDL and lysoPC
L
-a- lysophosphatidylcholine, palmitoyl on VSMCs were
tested in a standard 3-4,5-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide MTT assay as described
previously [35]. Briefly, cells were dispensed in a 24-well flat-bottomed plate Corning, NY, USA at a density of
5 × 10
4
cellscm
2
overnight for adequate attachment. Cells were treated with nLDL, oxLDL, PC
D
-a-phos- phatidylcholine, dipalmitoyl or lysoPC for 24 h, and
then MTT solution 0.5 mgml was added into all wells. Plates were then further pre-incubated at 37°C
for at least 4 h. After pre-incubation, acid – isopropanol 0.04 N HCl – isopropanol was added to all wells and
throughly mixed to dissolve the dark blue crystals for 30 min. The samples were read on a Dynatech MR710
Microelisa reader and absorbances were measured at 570 and 630 nm. The net difference of absorbances
between 570 and 630 nm was used to express the viability of VSMCs as follow: relative viability = A
e
× 100A
c
, where A
e
is the absorbance of treated cells and A
c
is the absorbance of untreated controls.
2
.
4
.
1
. Trypan blue exclusion test The trypan blue exclusion test was performed to test
for the loss of plasma membrane integrity [36]. VSMCs 5 × 10
5
cells per 35 mm dish were cultured in 10 fetal
calf serum
FCS-supplemented MCDB107
overnight and then were treated with lysoPC. Follow- ing 24 h incubation, the supernatant and VSMCs were
obtained by trypsinization and centrifugation 1000 × g, 5 min. The cell pellet was resuspended in PBS and
then was mixed with equal volume of 0.5 trypan blue solution Serva Feinbiochemica, USA. Intact VSMCs
excluded, the dye and cell number were counted using a hemacytometer Cambridge Instruments, Inc., USA.
2
.
5
. Flow cytometric analysis VSMCs 5 × 10
5
cells were cultured in 10 LPDS or 10 FCS-supplemented MCDB107 for 48 h and then
nLDL, oxLDL, PC, or lysoPC was added. Following 24 h incubation, cells were isolated by trypsinization
and centrifugation at 1000 × g for 5 min. The cell pellet was washed once, resuspended in 200 ml PBS at 4°C,
and fixed in 2 ml ice-cold 70 ethanol for 30 min. The fixed cells were recovered by centrifugation for 5 min at
1000 × g, washed twice in cold PBS. Cells were then treated with 0.1 mgml RNase A and 50 mgml propid-
ium iodide dissolved in PBS. Flow cytometric analysis was performed on FACScan Becton Dickinson, USA
and tens of thousands of events were analyzed for each sample. The cell cycle distribution of each sample was
calculated by
MODIFIT
2.0 software package. This time point 24 h was selected because no significant apopto-
sis was observed with propidium iodide before 18 h.
2
.
5
.
1
. Annexin V and propidium iodide staining VSMCs 5 × 10
5
cells per 35 mm dish were cultured in 0.5 FCS-supplemented MCDB107 overnight and
then were treated with lysoPC 25 mmoll for 3 or 24 h. The supernatant and VSMCs were obtained following
trypsinization and centrifugation 1000 × g, 5 min. Cells were washed once with PBS and then incubated
with 0.5 mgml FITC-annexin V AV Molecular Probes, Inc., USA in 0.5 ml binding buffer NaCl, 150
mmoll; CaCl
2
, 2.5 mmoll; MgCl
2
, 5 mmoll; Hepes, 10 mmoll; 20 bovine serum albumin; pH 7.4 for 15 min
at room temperature. Following AV binding, cells were collected by centrifugation and resuspended in 0.5 ml
binding buffer, and propidium iodide PI was added at
Fig. 1. Effect of oxidation of LDL on levels of CD in absorbance units and TBARS in nmol MDA equivalentmg protein. LDL was
exposed to ferrous sulfate for different time intervals and analyzed for the presence of TBARS and CD .
the concentration of 0.6 mgml. FACS analysis was performed immediately after staining. The translocation
of phosphatidylserine PS from the inner leaflet of the membrane outward PS exposure, with cells remaining
physically intact, represents an early event of apoptosis [37]. Apoptotic cells therefore can be stained with AV,
which binds with high affinity to PS, resulting in a green fluorescence when excited at 450 – 480 nm. At the
same time, PI capable of passing the plasma membrane is excluded AV
+
PI
−
. Necrotic cells have lost the physical integrity of their plasma membrane and are
therefore stained with PI, which fluorescences in the red when excited at 510 – 550 nm AV
+
PI
+
or AV
−
PI
+
. Cells which are neither apoptotic nor necrotic did not
stain with either dye AV
−
PI
−
. The percentage of apoptotic or necrotic VSMCs was calculated by the
CELLQUEST
software package.
2
.
6
. Gel electrophoresis analysis of fragmented DNA VSMCs 1 × 10
6
cells were treated with increasing concentrations of lysoPC for 24 h. After treatment, the
supernatant of cell culture and VSMCs were collected, and then DNA was purified by QIAamp Tissue Kit
QIAGEN GmbH, Germany. DNA samples were sep- arated on 1.8 agarose gels 90 V, 2 – 3 h containing
ethidium bromide and visualized under ultraviolet light.
2
.
7
. Quantitation of fragmented DNA The percentage of DNA fragmentation was measured
with the [
3
H]thymidine release assay as Baumgartner- Parzer et al. described [38]. Subconfluent cultures of
VSMCs were labelled with [
3
H]thymidine 1 mCiml for 36 h and the cells were then treated with indicated
LDL, oxLDL, lysoPC. DNA fragmentation was deter- mined as follows: 0.5 ml lysis buffer Tris, 20 mmoll;,
EDTA, 4 mmoll; 0.4 Triton X-100; pH 7.4 was added to each culture well and mixed by pipetting, and
the cell suspension was transferred to an eppendorf tube, incubated on melting ice for 10 min and cen-
trifuged at 8000 × g for 5 min at 4°C. Subsequently, fragmented radiolabelled DNA was counted in the
supernatant by liquid scintillation counting. Radioac- tivity of cells treated with lysis buffer and ultrasound
homogenator was used as total activity. Results of fragmented DNA were expressed as a percentage of
