Lysophosphatidylcholine induces apoptotic and non-apoptotic death in vascular smooth muscle cells: in comparison with oxidized LDL

Atherosclerosis 151 (2000) 481 – 491 www.elsevier.com/locate/atherosclerosis

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 1Rd., 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, and/or 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 ([3H]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 mmol/l or higher. Flow cytometric analysis yielded similar results for oxLDL- and lysoPC-treated VSMC; namely, an accumulation in the fraction of cells in G0/G1 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 cytotoxicity/apoptosis 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

1. Introduction Electron microscopic analyses revealed that vascular smooth muscle cell (VSMC) is the predominant cell type in ruptured atherosclerotic plaques [1], and accumulated evidence also demonstrated that cell death involves both oncosis and apoptosis of VSMCs during the progression of atherosclerosis [1 – 4]. Since vascular * Corresponding author. Tel.: +886-03-328-3016; fax: +886-03328-3031. E-mail addresses: [email protected] (M.-H. Yen), [email protected] (Y.-T. Lau1) 1 Both authors are corresponding authors. Tel.: + 886-02-87923100 (M.H. Yen)

remodelling during atherosclerosis could be mediated by many agents regulating both proliferation and apoptosis 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 synthesized by VSMCs lead to the thickening of intima and formation of fibrous atheroma [5]. However, this increase of arterial VSMC content was not observed at 12 weeks after injury [5]. Consequently, Walsh et al. reported a high frequency of medial VSMCs apoptosis occurring as early as 30 min after the balloon injury of

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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 identified 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 apoptotic 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 lesions, however, are unknown at present. There is evidence that oxidatively modified low-density lipoprotein (oxLDL) exists in atherosclerotic lesions of rabbit and humans [12]. OxLDL exerts several potentially atherogenic properties including cytotoxicity to vascular cells [13,14], impairment of endothelium-dependent relaxation of blood vessel [15,16], stimulation of monocyte recruitment and their adhesion to endothelial cells [17,18]. To study oxLDL-induced cytotoxicity, the cell culture system provides a controlled environment to examine mechanism of actions and to identify active components of oxLDL with limited interference. 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, lysophosphatidylcholine (lysoPC), produced during LDL oxidation by endogeneous phospholipase A2 [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 nature of oxLDL action. We thus chose to investigate the relative importance of the amount and/or 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 concentrations 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 20week-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 g/ml) and lipoprotein-deficient serum (LPDS) (d] 1.21 g/ml) were isolated by sequential ultra-centrifugation as described by Havel et al. [31]. Briefly, saturated NaBr and phosphate-buffered saline (PBS) (NaCl, 136.75 mmol/l; KCl, 2.6 mmol/l; KH2PO4, 1.5 mmol/l; Na2HPO4·2H2O, 7.9 mmol/l; 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. micrograms 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). Apolipoproteins (apo A1 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 mmol/l 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 mg/ml) was also measured as the absorbance 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 increased linearly with incubation time and thus allowed one to designate LDL with low-oxidative level (8 h,

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low-oxLDL), medium-oxidative level (16 h, mediumoxLDL), 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 altered 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 mobility was expressed as folds of the migration distance of the sample to that of LDL standard. All oxLDL preparations exhibited higher relative mobility than nLDL (data not shown).

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= (Ae × 100)/Ac, where Ae is the absorbance of treated cells and Ac is the absorbance of untreated controls.

2.4. Determination of cell 6iability

2.5. Flow cytometric analysis

The cytotoxic effects of oxLDL and lysoPC (L-alysophosphatidylcholine, palmitoyl) on VSMCs were tested in a standard 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium 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× 104 cells/cm2 overnight for adequate attachment. Cells were treated with nLDL, oxLDL, PC (D-a-phosphatidylcholine, dipalmitoyl) or lysoPC for 24 h, and then MTT solution (0.5 mg/ml) 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

VSMCs (5× 105 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 mg/ml RNase A and 50 mg/ml propidium 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 apoptosis was observed with propidium iodide before 18 h.

Fig. 1. Effect of oxidation of LDL on levels of CD (in absorbance units) and TBARS (in nmol MDA equivalent/mg protein). LDL was exposed to ferrous sulfate for different time intervals and analyzed for the presence of TBARS () and CD ( ).

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× 105 cells per 35 mm dish) were cultured in 10% fetal calf serum (FCS)-supplemented MCDB107 overnight and then were treated with lysoPC. Following 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).

2.5.1. Annexin V and propidium iodide staining VSMCs (5× 105 cells per 35 mm dish) were cultured in 0.5% FCS-supplemented MCDB107 overnight and then were treated with lysoPC (25 mmol/l) 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 mg/ml FITC-annexin V (AV) (Molecular Probes, Inc., USA) in 0.5 ml binding buffer (NaCl, 150 mmol/l; CaCl2, 2.5 mmol/l; MgCl2, 5 mmol/l; Hepes, 10 mmol/l; 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

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the concentration of 0.6 mg/ml. 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× 106 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 separated 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 [3H]thymidine release assay as BaumgartnerParzer et al. described [38]. Subconfluent cultures of VSMCs were labelled with [3H]thymidine (1 mCi/ml) for 36 h and the cells were then treated with indicated LDL, oxLDL, lysoPC. DNA fragmentation was determined as follows: 0.5 ml lysis buffer (Tris, 20 mmol/l;, EDTA, 4 mmol/l; 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 centrifuged at 8000× g for 5 min at 4°C. Subsequently, fragmented radiolabelled DNA was counted in the supernatant by liquid scintillation counting. Radioactivity 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 mean9SEM. The means of VSMC viability (by MTT assay or trypan blue exclusion test) and DNA fragmentation (by thymidine release 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 indicated significance for the multiple comparisons. Twotailed 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 (IC50) were calculated by GRAPHPAD INPLOT 4.0 program.

3. Results Morphological changes of VSMCs following treatments of oxLDL or lysoPC were observed. It was found that 50 mg/ml high-oxLDL or 25 mmol/l lysoPC could induce retraction of VSMCs [39]. With higher concentrations of oxLDL (200 mg/ml) or lysoPC (75 mmol/l), numerous rounded and floated cells were observed (data not shown). These parallel changes between 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 mg/ml. However, at the same concentration, the percentages of dead cells were 19.89 6.5% (PB 0.05) and 93.293.8% (PB 0.001) for medium-oxLDL and high-oxLDL, respectively. Thus, as the oxidation level of the lipoprotein preparation increased, the cytotoxic effect also increased. Furthermore, VSMC viability decreased persistently as the concentration of high-oxLDL increased (Fig. 2B). At 200 mg/ml, 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 dependent. The effective dose of high-oxLDL for causing 50% VSMC death (IC50) using sigmoid curve regression was calculated to be 108 9 4.1 mg/ml (correlation coefficient= 0.997; PB 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 mg/ml but not at 75 mg/ml. To compare the responses 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 lysoPC/mg oxLDL protein was determined. Accordingly, a range of 0–100 mmol/l lysoPC concentration was chosen for investigation. Similarly, lysoPC also induced VSMC cytotoxicity in the absence of serum, and the effect became significant (P B0.001) at concentration ]30 mmol/l (Fig. 3A). The decrease of cell viability was also dose dependent and the IC50 of lysoPC was 36.69 1.5 mmol/l. However, at concentrations 5 80 mmol/l, phosphatidylcholine (PC) did not cause any significant cytotoxicity,

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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 cytometry analysis. Following high-oxLDL (200 mg/ml) treatment (24 h), cells accumulated principally in the G1

Fig. 2. Cytotoxic effect of oxLDL on VSMC. VSMCs were exposed to nLDL or oxLDL of various oxidative levels (concentrations all at 200 mg/ml) in the presence of 10% LPDS. TBARS level of oxLDL: low-oxLDL= 3.5, medium-oxLDL = 12.7, high-oxLDL = 20.4 nmol MDA equivalent/mg protein (A). Results were obtained from two experiments each with duplicate determinations. VSMCs were also exposed to increasing concentrations (0–200 mg/ml) 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.

a slight cytotoxic effect (about 10%) was observed only at concentrations of 90 and 100 mmol/l; it was much less than that caused by equal concentration of lysoPC where near-complete cytotoxicity was found (PB 0.001). In the presence of 10% serum (FCS), the cytotoxic 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 IC50 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). Significant cell lysis was observed at a lysoPC concentration of 30 mM (Fig. 3B) and the effect was dose dependent.

Fig. 3. Cytotoxic effect of lysoPC on VSMCs. VSMCs were treated with increasing concentrations (0 – 100 mmol/l) 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 mmol/l) or further tested with trypan blue exclusion (, 0 – 200 mmol/l). * P B0.05, ** PB 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 determinations.

Table 1 Cell cycle distribution of SMCs in the treatment of nLDL or oxLDL Treatment

Controla nLDL (200 mg/ml) Low-oxLDL (200 mg/ml) High-oxLDL (200 mg/ml) a

VSMC (%) G0/G1

S

G2/M

80.4 79.1 78.3 88.2

9.7 9.8 10.4 1.9

9.9 11.1 11.3 9.9

Control cells were cultured in MCDB107+10% LPDS alone.

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Table 2 Cell cycle distribution of SMCs in the treatment of PC or lysoPC Treatment

Controla LysoPC (10 mmol/l) LysoPC (50 mmol/l) LysoPC (150 mmol/l) PC (150 mmol/l) a

VSMC (%) G0/G1

S

G2/M

46.8 52.3 53.9 58.7 54.8

45.9 37.9 37.1 28.3 37.4

7.3 9.8 9.0 13.0 7.8

Control cells were cultured in MCDB107+10% FCS alone.

Fig. 4. Induction of apoptosis in VSMCs by oxLDL. VSMCs were exposed to LDL (200 mg/ml) or oxLDL of various oxidative levels (200 mg/ml) for 24 h (A). TBARS levels of different oxLDL preparations are given in Section 2 (Fig. 1). Results obtained from two experiments each with triplicate determinations. VSMCs were also exposed to increasing concentrations of high-oxLDL (B). Results were obtained from two experiments each with triplicate determinations. * P B0.001, compared with control (10% LPDS alone).

phase of the cell cycle with a concomitant reduction (7.8%) in the proportion of cells in the S phase. In contrast, no change in the proportion of cells in the S phase was evident in VSMC after low-oxLDL or nLDL treatment (Table 1). Similarly, at concentration of 10 or 50 mmol/l, lysoPC led to a decrease of about 8% in the proportion of VSMCs in the S phase than that of control group. We also found a 17% reduction in the proportion of VSMCs in the S phase and 12% more cells accumulated in the G1 phase with 150 mmol/l lysoPC (Table 2). The same concentration (150 mmol/l)

of PC only exerted an effect similar to that of 10 mmol/l lysoPC. To quantify the extent of apoptosis, the percentage of fragmented DNA by [3H]thymidine release assay following 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 14.290.9 and 16.69 2.1%, respectively. Furthermore, there was a significant dosedependent 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 mmol/l lysoPC (Fig. 5, lanes 3 and 4). However, clear DNA laddering at lysoPC concentration of 10 mmol/l (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-dependent manner. Similar to the DNA ladder pattern, it was found that VSMC treated with low concentration of lysoPC (10 mmol/l) was of no significant effect but at higher concentration of lysoPC (] 25 mmol/l), significant 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 performed (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 analysis of six determinations indicated that fractional apoptotic VSMCs was significantly higher in lysoPCtreated cells at 3 h (4.390.2% versus 1.49 0.1%) when compared with control VSMCs; the fraction reached 14% or 7-fold of untreated VSMCs (2.090.1%) at 24 h. Two more identical experiments showed similar results.

4. Discussion Some of the chemical changes that occur during iron oxidation of LDL have been characterized, including increased negative charge (gel mobility), increased content of CD, and TBARS. Highly significant correlation could be derived (e.g. from Fig. 1) between the level of TBARS of oxLDL samples with relative electrophoretic mobility (r= 0.982; PB 0.001, n= 6) or with contents

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high-oxLDL (middle column, Fig. 2B). The TBARS contents of these preparations were also similar (2.0– 2.5 nmol MDA equivalents/ml). This ‘oxidative equivalency’ of induced cytotoxicity was further confirmed by considering the dose–response pattern of the highoxLDL-induced cytotoxic effect (Fig. 2B). If we convert cell viability to cytotoxicity, i.e. 1-viability, a linear response between cytotoxicity and the calculated

Fig. 5. Electrophoresis of DNA isolated from VSMCs cultured under serum-free conditions with 0, 10, 25 and 50 mmol/l lysoPC for 24 h (lanes 1 – 4, respectively). A 100 bp DNA molecular weight marker was loaded on lane M.

Fig. 6. Dose-dependent induction of apoptosis in VSMCs by lysoPC. Serum-deprived VSMCs were treated with increasing concentrations of lysoPC (0 – 50 mmol/l) for 24 h. Results were obtained from two experiments each with triplicate determinations. * PB 0.005, ** PB 0.001, compared with control (in the absence of lysoPC).

of CD (r= 0.985; PB 0.001, n =6). Therefore, the extent of lipid peroxidation, as measured by the concentration of TBARS of iron-oxidized LDL, could be used as a marker for oxidative modifications of lipoprotein similar to those of the copper-oxidized LDL preparations [42], lipoxygenase-modified LDL [43], and electronegatively charged LDL (LDL−) isolated from hyper-cholesterolemic plasma [19]. The TBARS content of oxLDL preparations was thus employed for quantitative analysis of relative cytotoxicity. A strong correlation between the cytotoxicity of the oxLDL and the respective extent of TBARS in various preparations of oxLDL was found (r =0.984; PB 0.001, n=32). When the content of TBARS was less than 1.0 nmol MDA equivalents/ml (amount contained in about 50 mg/ml high-oxLDL), there was no significant cytotoxicity even at high concentration (e.g. 200 mg/ml low-oxLDL in Fig. 2A). However, preparations of oxLDL with stronger oxidation killed VSMC and 200 mg/ml medium-oxLDL caused an extent of cytotoxicity (Fig. 2A) similar to that caused by 100 mg/ml

Fig. 7. Apoptotic and necrotic death of VSMCs were distinguished using FITC-annexin V (AV) label and propidium iodide (PI) stain. VSMCs were treated as indicated above each panel and then analyzed by flow cytometry. The lower left quadrants of each panel show the viable cells, which exclude PI and are negative for AV binding (AV−/PI−). The lower right quadrants represent the apoptotic cell, positive for AV binding and negative for PI uptake (AV+/PI−). The upper quadrants (left and right) represent the necrotic cells, positive for PI uptake with or without AV fluorescence. FL1-H, Fluorescence height of AV; FL2-H, fluorescence height of PI. Data was taken from one out of six determinations in a typical experiment. Three such experiments were performed. Panels A, B, C were typical results for untreated control, lysoPC-treated for 3 h, and lysoPC-treated for 24 h, respectively.

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TBARS content can be obtained (correlation coefficient = 0.993). It thus appeared that the toxic-dose threshold of oxLDL for VSMCs was approximate 1.0 nmol MDA equivalents/ml in the absence of serum, irrespective of the concentration of oxLDL preparations per se. Several lines of evidence suggest that lysoPC, a major component of oxidatively modified LDL, exerts vascular effects similar to those induced by oxLDL. Both oxLDL and lysoPC can induce cytotoxicity of vascular cells [13,26], impairment of endothelium-dependent relaxation [16,44], as well as activation of endothelial adhesiveness [17,45]. It was found that similar to oxLDL, lysoPC exhibited a dose-dependent (with IC50 =36.691.5 mmol/l; r= 0.999), and near-complete (at a concentration of 50 mmol/l) cytotoxic effect in the absence of serum (Fig. 3A). Even in the presence of 10% FCS, lysoPC exerted a dose-dependent cytotoxicity (Fig. 3B). These observed dose dependency of lysoPC-induced cytotoxicity are consistent with the view that lysoPC content in oxLDL preparations increases not only as amount of oxLDL increases [41], but also as level of oxidation increases [46]. The similarity between oxLDL- and lysoPC-induced cytotoxicity of VSMCs extends beyond the observations that both exhibited dose-dependent cytotoxicity in VSMCs. Serum proteins may bind oxLDL or lysoPC and thus hinder their transfer to vascular cell [47]; however, specific protective effects of serum on cell survival may contribute also. In fact, low concentrations of oxLDL and lysoPC (with 0.25% serum) both stimulate VSMC to enter cell cycle partially via an autocrine or paracrine action to release endogenous basic fibroblast growth factor [48], which is also responsible for the observed lysoPC-induced migration of VSMC [49]. Furthermore, it has been demonstrated that both oxLDL and lysoPC exerted cytostatic effect on VSMCs using flow cytometric analyses (Tables 1 and 2). Cytotoxicity of oxLDL and lysoPC could be due to selectivity of the proportion of VSMCs in the S phase, consistent with the findings of Hodis et al. where subconfluent proliferating-endothelial cells were more sensitive to the cytotoxic effects of oxLDL [19], and/or cells were inhibited from entering the S phase. Although the mechanism responsible for the cytotoxicity of lysoPC is not well understood, Kume et al. had reported that lysoPC is a polar phospholipid that can exhibit detergent-like properties [50]. Amphiphiles in an aqueous solution can cause cell lysis above their critical micellar concentrations (CMC). Previous studies have shown that CMC of lysoPC (C16:0) is 40 – 50 mmol/l in protein-free physiological salt solutions [51]. Above CMC, lysoPC may directly perturb membrane structure and impair the function of macromolecules embedded in the membrane. We found significant cytotoxicity of

lysoPC over a broad range of concentrations in the absence (Fig. 3A) or presence of serum (Fig. 3B) and that cell damage was observed at sub-CMC, suggesting that a non-detergent action of lysoPC could not be excluded. However, direct test of membrane intactness by trypan blue exclusion indicated significant damage occurred at sub-CMC concentrations (Fig. 3B), consistent with a detergent action of lysoPC. In addition, Ohara et al. had reported that lysoPC can activate protein kinase C in intact vessels [52], leading to an − increase in O− 2 production. O2 also provides a source of other oxygen-centred radicals, such as H2O2 and.OH, which may cause membrane damage. It is also well recognized that lipoproteins, especially LDL and oxLDL, raises intracellular Ca2 + level probably through the activation of calcium channel (for a review, see Ref. [53]). Recent report suggests that oxLDL but not nLDL activates the L-type Ca2 + channel in smooth muscle-derived cell line [54], and it is interesting to note that lysoPC may also activate such a Ca2 + channel [55]. Therefore, lysoPC could injure VSMCs via multiple mechanisms. Our results also have shown that oxLDL or lysoPC could lead to distinct types of VSMCs death: apoptosis and necrosis. In the presence of 200 mg/ml oxLDL, 95.99 2.0% of VSMC died (Fig. 2B) while the mean fractional fragmented-DNA content was 17.490.5% (Fig. 4B). Moreover, 50 mmol/l lysoPC showed similar effects on VSMCs: the cytotoxicity of VSMCs was 82.19 1.1% (Fig. 3) and the mean fractional fragmented-DNA content was 26.49 2.3% (Fig. 6). These observations were consistent with the findings of Crisby et al.. They demonstrated that the vast majority of injured VSMCs in the plaque undergoing cell death by necrosis and less by apoptosis [1]. Furthermore, lysoPC-induced apoptosis, as indexed by PS exposure, could be detected as early as 3 h and increased with time to 14% at 24 h following lysoPC treatment (Fig. 7B,C). Therefore, oxLDL and its active component, lysoPC (]25 mmol/l), both cause VSMC apoptosis. Watanabe et al. had also demonstrated that oxLDL could induced apoptosis in cultured VSMCs [27]. They further found that oxLDL induced apoptosis through activation of a CPP32-like protease and bcl-2 protein downregulation [56]. The signal transduction pathway for the induction of apoptosis by lysoPC is still unknown. Since lysoPC can bind to a receptor in the plasma membrane such as fatty acid binding protein or to the plasma membrane in a receptor-independent manner [57], the capacity to activate various signal pathways may exist. However, the oxidation of LDL leads to the formation of several cytotoxins, including lipid hydroperoxides, alkenals, oxysterols and lysoPC [58]. Therefore, the cytotoxic effect of oxLDL to VSMCs may derive from a dominant toxin among these, but also may be

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due to the combined actions of multiple agents. Several studies had demonstrated that the cytotoxicity of oxLDL could be mimicked by a lipid extract of the LDL [59,60]. Hughes et al. had identified the most toxic fraction of copper-oxidized LDL to be 7-ketocholesterol for cultured procine VSMCs [61]. In addition, Chisolm et al. reported that 7b-hydroperoxycholesterol was the principal cytotoxin of oxLDL when examined using human and rabbit VSMC culture [62]. The current results showed that lysoPC was highly cytotoxic on VSMC as well. Apparently, methods for preparing oxLDL samples (e.g. LDL source and oxidation procedure), conditions for cytotoxicity test (e.g. incubation time and types of test), and the culturing cell systems (e.g. medium serum content and origin of cells) employed may all influence the outcome of cytotoxicity test; it is hence useful to compare the actions of oxLDL with its putative component by quantitative analysis and by determining more than one parameter, such as necrosis versus apoptosis. In conclusion, oxLDL and its active component, lysoPC, both induce VSMC death in a dose-dependent manner. However, a certain threshold oxidative level (ca. 1.0 nmol MDA/ml) could be detected such that the dose –response pattern actually reflected an ‘oxidative equivalency’. Both oxLDL and lysoPC elicited dose-dependent cytotoxicity and apoptosis, suggesting that lysoPC, being a major lipid component of oxidized LDL, could account for not only the cytotoxic effect, but also some of the apoptosis induced by oxLDL.

Acknowledgements This study was supported by the National Science Council (NSC87-2314-B-182-088), Department of Health, Executive Yuan (Research Center DOH86-HR610) and Chang Gung University (CMRP 736) to Y.T.L. C.C.H. also thanks the scholarship support from the Foundation of Biomedical Sciences of National Defense Medical Center. The editorial assistance of Cynthia Huang and the excellent assistance of L.Y. Chen in the preparation of the manuscript is gratefully acknowledged.

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