Atherosclerosis 151 2000 481 – 491
Lysophosphatidylcholine induces apoptotic and non-apoptotic death in vascular smooth muscle cells: in comparison with
oxidized LDL
Chien-Cheng Hsieh
a
, Mao-Hsiung Yen
b,1
, Hwan-Wun Liu
c
, Ying-Tung Lau
d, ,1
a
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
b
Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan
c
Department of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan
d
Department of Physiology, Chang Gung Uni6ersity College of Medicine,
259
Wen Hwa
1
Rd., Kwei-Shan, Tao-Yuan, Taiwan Received 25 February 1999; received in revised form 8 September 1999; accepted 2 March 2000
Abstract
Oxidized low-density lipoprotein oxLDL plays a key role in the development of atherogenesis, partly by causing injury to vascular cells. However, different preparations of LDL, methods of oxidation, andor active components often produce cellular
effects of various degrees. To explore the quantitative relationship between dose and level of oxidation of the oxLDL utilized, we employed combinations of different levels of oxidation and concentrations of oxLDL to induce cell death in cultured vascular
smooth muscle cells VSMC. We also examined the effect of lysophosphatidylcholine lysoPC, a putative active component of oxLDL, on VSMCs by determining, in parallel with a cytotoxicity test 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium
bromide MTT assay, DNA fragmentation [
3
H]thymidine release, and flow cytometric analyses. We found that oxLDL caused cytotoxicity in an oxidative level- and dose-dependent manner, lysoPC also caused dose-dependent cytotoxicity with or without
serum. Fragmentation of DNA was observed in both oxLDL- and lysoPC-treated VSMCs. Furthermore, lysoPC-induced DNA ladder was also demonstrated by gel electrophoresis at a concentration of 25 mmoll or higher. Flow cytometric analysis yielded
similar results for oxLDL- and lysoPC-treated VSMC; namely, an accumulation in the fraction of cells in G G
1
phase with a reciprocal change in S-phase fraction. Membrane phosphatidylserine exposure, detected by annexin V staining, provided
additional evidence that lysoPC induced significant apoptosis in VSMC. Taken together, the degree of oxLDL-induced cytotoxicityapoptosis of VSMC depended on combined effects of oxLDL concentration and oxidative level. Moreover, lysoPC
also elicited a dose-dependent apoptosis in addition to cytotoxicity. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Oxidized low-density lipoprotein; Lysophosphatidylcholine; Apoptosis; Vascular smooth muscle cell; Cytotoxicity www.elsevier.comlocateatherosclerosis
1. Introduction
Electron microscopic analyses revealed that vascular smooth muscle cell VSMC is the predominant cell
type in ruptured atherosclerotic plaques [1], and accu- mulated evidence also demonstrated that cell death
involves both oncosis and apoptosis of VSMCs during the progression of atherosclerosis [1 – 4]. Since vascular
remodelling during atherosclerosis could be mediated by many agents regulating both proliferation and apop-
tosis of VSMC simultaneously, and yet independently, in both early and late stages of the remodelling process,
apoptosis of VSMC is likely to play an important, albeit undefined, role for a reveiw, see Ref. [4]. In
early lesion of atherosclerosis, the proliferation of VSMCs and accumulation of matrix proteins synthe-
sized by VSMCs lead to the thickening of intima and formation of fibrous atheroma [5]. However, this in-
crease of arterial VSMC content was not observed at 12 weeks after injury [5]. Consequently, Walsh et al. re-
ported a high frequency of medial VSMCs apoptosis occurring as early as 30 min after the balloon injury of
Corresponding author. Tel.: + 886-03-328-3016; fax: + 886-03- 328-3031.
E-mail addresses
:
mhyenndmctsgh.edu.tw M.-H. Yen, yt- laumail.cgu.edu.tw Y.-T. Lau
1 1
Both authors are corresponding authors. Tel.: + 886-02-8792- 3100 M.H. Yen
0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 0 0 0 0 4 5 3 - 6
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
