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
total DNA.
2
.
8
. Statistical analyses Results are expressed as mean 9 SEM. The means of
VSMC viability by MTT assay or trypan blue exclu- sion test and DNA fragmentation by thymidine re-
lease assay were analyzed using analysis of variance ANOVA for multiple comparisons. Paired analysis
between control and an individual treatment group was performed using Student’s t-test, where ANOVA indi-
cated significance for the multiple comparisons. Two- tailed probability values less than 0.05 were considered
significant. Linear regression and all comparisons were done by
GRAPHPAD INSTAT
2.0 program GraphPad software, CA, USA. The dose of high-oxLDL or
lysoPC for causing 50 SMCs death IC
50
were calcu- lated by
GRAPHPAD INPLOT
4.0 program.
3. Results
Morphological changes of VSMCs following treat- ments of oxLDL or lysoPC were observed. It was
found that 50 mgml high-oxLDL or 25 mmoll lysoPC could induce retraction of VSMCs [39]. With higher
concentrations of oxLDL 200 mgml or lysoPC 75 mmoll, numerous rounded and floated cells were ob-
served data not shown. These parallel changes be- tween oxLDL and lysoPC were similar to previous
findings, including our own [28,39].
Fig. 2A shows the dependence of cytotoxicity on the oxidative level of oxLDL. No significant cytotoxicity
was demonstrable by nLDL and low-oxLDL even at protein concentrations up to 200 mgml. However, at
the same concentration, the percentages of dead cells were 19.8 9 6.5 P B 0.05 and 93.2 9 3.8 P B
0.001 for medium-oxLDL and high-oxLDL, respec- tively. Thus, as the oxidation level of the lipoprotein
preparation increased, the cytotoxic effect also in- creased. Furthermore, VSMC viability decreased persis-
tently as the concentration of high-oxLDL increased Fig. 2B. At 200 mgml, cell viability was only 4.1 9
2.0, similar to that in Fig. 2A. The cytotoxic effect of high-oxLDL on VSMCs was hence also dose depen-
dent. The effective dose of high-oxLDL for causing 50 VSMC death IC
50
using sigmoid curve regression was calculated to be 108 9 4.1 mgml correlation coeffi-
cient = 0.997; P B 0.001. This estimate was consistent with earlier findings [40], where a low molecular weight
fraction of oxLDL was shown to induce cell death at 150 mgml but not at 75 mgml. To compare the re-
sponses of VSMCs to the treatments of oxLDL or lysoPC, a calculation of equivalent lysoPC content in
oxLDL was estimated based on the report by Sakai et al. [41], where about 200 – 600 nmol lysoPCmg oxLDL
protein was determined. Accordingly, a range of 0 – 100 mmoll lysoPC concentration was chosen for investiga-
tion. Similarly, lysoPC also induced VSMC cytotoxicity in the absence of serum, and the effect became signifi-
cant P B 0.001 at concentration ] 30 mmoll Fig. 3A. The decrease of cell viability was also dose depen-
dent and the IC
50
of lysoPC was 36.6 9 1.5 mmoll. However, at concentrations 5 80 mmoll, phosphatidyl-
choline PC did not cause any significant cytotoxicity,
Fig. 2. Cytotoxic effect of oxLDL on VSMC. VSMCs were exposed to nLDL or oxLDL of various oxidative levels concentrations all at
200 mgml in the presence of 10 LPDS. TBARS level of oxLDL: low-oxLDL = 3.5, medium-oxLDL = 12.7, high-oxLDL = 20.4 nmol
MDA equivalentmg protein A. Results were obtained from two experiments each with duplicate determinations. VSMCs were also
exposed to increasing concentrations 0 – 200 mgml of high-oxLDL B. Results were obtained from two experiments each with duplicate
determinations. Compared with control 10 LPDS alone, P B 0.05, P B 0.001.
Near-complete necrosis of VSMCs was found at 200 mM, the highest concentration employed. Thus, VSMCs
were much less sensitive to the toxic effect of lysoPC in the presence of FCS.
Cell cycle distribution was examined by flow cytome- try analysis. Following high-oxLDL 200 mgml treat-
ment 24 h, cells accumulated principally in the G
1
Fig. 3. Cytotoxic effect of lysoPC on VSMCs. VSMCs were treated with increasing concentrations 0 – 100 mmoll of PC or lysoPC
for 24 h in the absence of serum A. VSMCs were also incubated in the presence of 10 FCS B, while treated with lysoPC
, 0 – 300 mmoll or further tested with trypan blue exclusion , 0 – 200 mmoll. P B 0.05, P B 0.001, compared with control
serum-free MCDB107 for A; 10 FCS only for B. Results were combined from three or four experiments each with triplicate determi-
nations.
a slight cytotoxic effect about 10 was observed only at concentrations of 90 and 100 mmoll; it was much
less than that caused by equal concentration of lysoPC where near-complete cytotoxicity was found P B
0.001. In the presence of 10 serum FCS, the cyto- toxic effects of lysoPC were reduced. Fig. 3B illustrates
that cytotoxicity was only 25 assayed by MTT test at 100 mM added lysoPC. The value of IC
50
was 3-fold of that found before Fig. 3B compared with Fig. 3A;
note the different scale. Near-complete cytotoxicity was not achieved until concentration of lysoPC reached
250 mM; again, almost a 3-fold increase as before cf. Fig. 3A. We also examined the effect of lysoPC on
membrane intactness by trypan blue exclusion assay in the presence of 10 FCS Fig. 3B, triangles. Signifi-
cant cell lysis was observed at a lysoPC concentration of 30 mM Fig. 3B and the effect was dose dependent.
Table 1 Cell cycle distribution of SMCs in the treatment of nLDL or oxLDL
VSMC Treatment
G
2
M S
G G
1
80.4 9.7
Control
a
9.9 11.1
9.8 nLDL 200 mgml
79.1 78.3
10.4 Low-oxLDL 200 mgml
11.3 88.2
1.9 High-oxLDL 200 mgml
9.9
a
Control cells were cultured in MCDB107+10 LPDS alone.
Table 2 Cell cycle distribution of SMCs in the treatment of PC or lysoPC
Treatment VSMC
S G
G
1
G
2
M 45.9
46.8 7.3
Control
a
52.3 LysoPC 10 mmoll
37.9 9.8
53.9 LysoPC 50 mmoll
37.1 9.0
28.3 58.7
13.0 LysoPC 150 mmoll
PC 150 mmoll 54.8
37.4 7.8
a
Control cells were cultured in MCDB107+10 FCS alone.
of PC only exerted an effect similar to that of 10 mmoll lysoPC.
To quantify the extent of apoptosis, the percentage of fragmented DNA by [
3
H]thymidine release assay fol- lowing the treatment of oxLDL or lysoPC has been
measured. Although exposure to nLDL or low-oxLDL for 24 h failed to induce apoptosis Fig. 4A, incubation
in medium- and high-oxLDL caused thymidine release significantly, reaching 14.2 9 0.9 and 16.6 9 2.1, re-
spectively. Furthermore, there was a significant dose- dependent
increase when
treated with
different concentrations of high-oxLDL Fig. 4B. To determine
whether lysoPC at concentrations parallel to effective oxLDL doses also induces apoptosis in VSMC, total
DNA was isolated and analyzed. Typical DNA ladder pattern was observed from cells treated for 24 h with 25
and 50 mmoll lysoPC Fig. 5, lanes 3 and 4. However, clear DNA laddering at lysoPC concentration of 10
mmoll Fig. 5, lane 2 was not observed, consistent with an earlier report [27]. In addition, Fig. 6 illustrates that
lysoPC also induced apoptosis of VSMCs in a dose-de- pendent manner. Similar to the DNA ladder pattern, it
was found that VSMC treated with low concentration of lysoPC 10 mmoll was of no significant effect but at
higher concentration of lysoPC ] 25 mmoll, signifi- cant DNA fragmentation was observed.
To further examine the nature of lysoPC-induced apoptosis in VSMC, phosphatidylserine PS exposure
was employed as an early marker of apoptosis [37]. In a typical experiment, double staining of PS exposure
FITC-annexin V or AV, FL1-H as well as membrane disruption propidium iodide or PI, FL2-H were per-
formed see Section 2.5.1 by flow cytometry, and the results showed that PS exposure was detectable as early
as 3 h following lysoPC 25 mM treatment Fig. 7B, AV
+
PI
−
cells of lower right panel. At 24 h, both apoptosis and necrosis AV
+
PI
+
cells of upper right panel increased significantly Fig. 7C. Statistical anal-
ysis of six determinations indicated that fractional apoptotic VSMCs was significantly higher in lysoPC-
treated cells at 3 h 4.3 9 0.2 versus 1.4 9 0.1 when compared with control VSMCs; the fraction reached
14 or 7-fold of untreated VSMCs 2.0 9 0.1 at 24 h. Two more identical experiments showed similar
results.
4. Discussion
