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7 b

-Hydroxycholesterol induces Ca

_{oscillations, MAP kinase}

activation and apoptosis in human aortic smooth muscle cells

Mikko P.S. Ares

_{*, M. Isabella Po¨rn-Ares}

_{, Sara Moses}

_{, Johan Thyberg}

_,

Lisa Juntti-Berggren

_{, Per-Olof Berggren}

_{, Anna Hultga˚rdh-Nilsson}

_{, Bengt Kallin}

_,

Jan Nilsson

a_{Wallenberg Laboratory}_,_Uni

6ersity Hospital MAS,Lund Uni6ersity,S-20502Malmo¨,Sweden

b_Di

6ision of Experimental Pathology,Uni6ersity Hospital MAS,Lund Uni6ersity,S-20502Malmo¨, Sweden

c_{Department of Cell and Molecular Biology}_,_{Section for Connecti}₆_{e Tissue Biology}_,_{Lund Uni}₆_ersity_,_P_._O_._Box₉₄_,_S_-_{221 00}_Lund_,_Sweden d_{Department of Cell and Molecular Biology}_,_{Karolinska Institutet}_,_S_-₁₇₁₇₇_Stockholm_,_Sweden

e_{Rolf Luft Center for Diabetes Research}_,_{Department of Endocrinology}_,_{Karolinska Hospital}_,_{Karolinska Institutet}_,_S_-₁₇₁₇₆_Stockholm_, _Sweden f_{Department of Medicine}_,_{Atherosclerosis Research Unit}_,_{King Gustaf Vth Research Institute}_,_{Karolinska Hospital}_,_{Karolinska Institutet}_,

S-17176Stockholm,Sweden

Received 23 June 1999; received in revised form 25 November 1999; accepted 7 January 2000

Abstract

In the present study, we characterize the early cytotoxic effects of 7b-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7b-hydroxycholesterol induced Ca2+_{oscillations with} a frequency of :0.3 – 0.4 min−1_{. A few hours later, thapsigargin-sensitive Ca}2+_{pools were depleted, indicating that 7}_b -hydrox-ycholesterol perturbs intracellular Ca2+_{homeostasis. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (but not} JNK) were activated within 5 min after addition of 7b-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxyc-holesterol and 27-hydroxyc25-hydroxyc-holesterol were more potent in inducing apoptosis than 7b-hydroxycholesterol and cholesterol-5a,6a -epoxide, as determined by TUNEL staining. Addition of TNFa (10 ng/ml) and IFNg(20 ng/ml) enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7b-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4 – 8 h as determined by [35_{S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the}

Keywords:7b-Hydroxycholesterol; Ca2+_{oscillations; MAP kinase activation}

www.elsevier.com/locate/atherosclerosis

1. Introduction

Death of smooth muscle cells (SMCs) in the fibrous cap region of the atherosclerotic plaque is believed to play an important role in plaque rupture [1]. A high incidence of apoptotic cell death among SMCs in this region has been demonstrated [2,3]. Similarly, human vascular SMCs derived from coronary plaques are characterized by increased frequency of apoptosis [4].

Moreover, electron microscopic studies suggest that additional cell death by necrosis occurs in parallel [5]. Atherosclerotic plaques have been shown to contain higher amounts of oxysterols (oxidized cholesterol derivatives) than normal arteries [6 – 10]. As oxysterols are generally cytotoxic [11 – 13], they may contribute to cell death in atherosclerotic lesions.

We have previously shown that 25-hydroxycholes-terol induces activation of the protease caspase-3 [14], a key mediator of apoptotic cell death in many systems [15,16]. In the present study, we have compared the ability of different oxysterols to induce cell death and characterize several early events during exposure of cells to 7b-hydroxycholesterol, reportedly a major cyto-* Corresponding author. Tel.: +46-40-337674; fax: +

46-40-332550.

E-mail address:mikko.ares@medforsk.mas.lu.se (M.P.S. Ares).

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toxin in oxidized LDL [17,18]. 7b-Hydroxycholesterol can also be produced endogenously from cholesterol [19].

Ca2+ _{mobilization can induce apoptosis [20 – 23] by} activating Ca2+_{-dependent proteases and} endonucle-ase(s) and by depleting intracellular Ca2+ _stores [24,25], the maintenance of which is required for cell survival. In the present study we show that 7b -hydroxy-cholesterol induces intracellular Ca2+ _oscillations which lead to depletion of thapsigargin-releasable Ca2+ stores within a few hours.

Mitogen-activated protein kinases (MAPKs) play im-portant roles in cell proliferation. Activation of the extracellular signal-regulated kinases (ERKs) generally inhibits cell death [26], but pro-apoptotic effects have been reported as well [27,28]. ERKs have been impli-cated in CD95-mediated apoptosis [29]. The results of the present study show that several different oxysterols induce apoptosis, which is potentiated by the inflamma-tory cytokines TNFaand IFNg, which are known to be expressed in atherosclerotic vessels [30,31].

2. Materials and methods 2.1. Chemicals

Biotin-16-dUTP and terminal deoxytransferase were from Boehringer-Mannheim (Mannheim, Germany). BAPTA-AM, Ca2+_{-free medium,} choles-terol, cholesterol-5a,6a-epoxide, ExtrAvidin-FITC, 25-hydroxycholesterol, 7b-hydroxycholesterol, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-mide), p-phenylenediamine, and verapamil were purchased from Sigma Chemical (St. Louis, MO). Na-cacodylate (dimethylarsinic acid sodium salt trihydrate) was obtained from Merck (Darmstadt, Germany). TNFa was purchased from R&D Systems (Min-neapolis, MN), and IFNg was kindly provided by Boehringer Ingelheim (Ingelheim, Germany). Fura-2-AM was obtained from Molecular Probes (Eugene, OR). The monoclonal mouse human Bcl-2 anti-body (clone 124) was obtained from DAKO (Copen-hagen, Denmark). 27-Hydroxycholesterol was a generous gift of Dr Ingemar Bjo¨rkhem (Karolinska Institute, Stockholm, Sweden).

2.2. Cell culture

Human aortic SMCs were purchased from Cytotech (Symbion Science Park, Copenhagen, Denmark). The cells were grown in DMEM supplemented with 13% FCS, penicillin (100 U/ml) and streptomycin (100 mg/

ml). Cells in the 7th through the 18th passages were used in the experiments. During treatments with oxys-terols, cells were incubated in DMEM/F-12 (1:1)

with-out phenol red, supplemented with 5% FCS (unless stated otherwise), penicillin (100 U/ml) and strepto-mycin (100mg/ml). Cells were subconfluent at the time of experiments.

2.3. Cytotoxicity assay

Metabolic activity was analyzed using the MTT as-say. MTT was dissolved in DMEM/F12 (1:1) without phenol red at a concentration of 5 mg/ml. An amount of this solution equal to 10% of the culture medium volume was added to cell cultures. After 1 h, cultures were removed from the incubator and the formazan crystals solubilized by adding solubilization solution (10% (v/v) Triton X-100 and 0.1 N HCl in isopropanol) equal to the original culture medium volume. MTT reduction was quantitated by measuring light ab-sorbance at 570 nm.

2.4. TUNEL (terminal deoxytransferase-mediated

dUTP nick end labeling) assay

Cells cultured on glass coverslips were fixed in 100% methanol at −20°C for 30 min. The coverslips were air-dried, rinsed twice with water, and transferred to cell culture dishes covered with wet tissue paper. Sev-enty-five microlitres of the following solution was added to each cover slip: 20 mM biotin-16-dUTP, 200 U/ml terminal deoxytransferase, 300 mM Tris – HCl (pH 7.2), 10 mM CoCl2, and 300 mg/ml freshly added cacodylate. The samples were incubated at 37°C for 60 min. The coverslips were transferred to TB buffer (300 mM NaCl, 30 mM Na-citrate) for 15 min, rinsed twice with PBS, incubated in 2% BSA for 10 min, and rinsed 2×5 min with PBS. One hundred microlitres of Ex-trAvidin-FITC diluted to 15mg/ml in PBS was added to each cover slip. After 30 min at 37°C, the samples were washed three times with PBS and once with PBS con-taining 0.1% Triton X-100. The coverslips were mounted on slides with anti-fading solution (1 mg/ml p-phenylenediamine, 10% (v/v) PBS, 90% (v/v) glyc-erol). At least 200 cells were counted for quantitative analyses. TUNEL positive cells had brightly stained nuclei, showing either homogeneous staining of intact nuclei, or condensation of chromatin into distinct, brightly stained fragments.

2.5. Electron microscopy

Primary fixative (2% glutaraldehyde, 2% formalde-hyde, 0.1 M cacodylate, 0.05 M sucrose, pH 7.3) was added directly to cell culture dishes. Post-fixation was done with 1% osmium tetroxide in cacodylate buffer with 0.5% potassium ferrocyanate (2 h at 4°C). The samples were dehydrated in graded ethanol (70 – 100%), stained with 2% uranyl acetate in ethanol for 30 min,

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and embedded in Spurr low viscosity epoxy resin. The sections were cut with a diamond knife using an LKB Ultratome IV, picked up on carbon-coated formvar films, stained with alkaline lead citrate (3 min), and finally examined in a JEOL EM 100 CX operated at 60 kV.

2.6. MAP kinase assays

SMCs cultured in 10 cm dishes were serum-starved in Ham F-12 medium for 24 h. Just before the oxysterol treatments, one half of the medium in cell culture dishes was transferred to sterile tubes and the oxysterol was rapidly injected into this medium using a Hamilton syringe. The remaining medium in the cell culture dishes was then replaced with the oxysterol-containing medium, thus eliminating any risk of MAPK activation due to addition of fresh culture medium. After the treatments, cells were rinsed twice with ice-cold PBS and lysed on ice by adding 300 ml of lysis buffer (1% Triton X-100, 270 mM sucrose, 50 mM NaF, 5 mM Na-pyrophosphate, 10 mM Na-glycerolphosphate, 1 mM Na-orthovanadate, 1 mM EGTA, 1 mM EDTA, 0.1% 2-mercaptoethanol, 1 mM benzamidine, 0.1 mM PMSF). After 5 min, the extracts were frozen instanta-neously in liquid nitrogen. Protein concentrations in the extracts were determined using Coomassie brilliant blue (Pierce, Rockford, IL) according to the manufacturer’s instructions. Equal amounts of protein (25mg for 10×

8 cm gels) were run in 10% SDS-polyacrylamide gels and electroblotted at 100 V (400 mA) for 2 h. Western blotting was performed using antibodies (New England Biolabs, Beverly, MA) against phosphorylated or non-phosphorylated ERKs or JNKs as primary antibodies (diluted 1:1000), and a peroxidase-conjugated sec-ondary antibody (anti-rabbit IgG; New England Bio-labs; diluted 1:2000) for detection by enhanced chemiluminescence.

2.7. Measurement of intracellular Ca2+

Subconfluent SMCs on glass cover slips were loaded with 3 mM fura-2-AM for 30 min in culture medium (DME/F-12 (1:1), 5% FCS, without Phenol red). The cover slips were transferred to a thermostatically con-trolled (37°C), open perifusion chamber placed in a holder on the stage of an inverted epifluorescence mi-croscope (Zeiss Axiovert 35 M). Fura-2 fluorescence in single cells was measured using a SPEX fluorolog-2 CM1T11I system for dual-wavelength excitation fluorimetry, essentially as previously described [32]. The excitation wavelengths were 340 and 380 nm and the emitted light, selected by a 510 nm filter, was monitored by a photomultiplier attached to the microscope. Data from all measurements are presented as 340/380 fluores-cence ratios directly representative of changes in

intra-cellular Ca2+_{. Results shown are representative of} multiple independent single cell recordings.

2.8. Determination of the rate of protein synthesis Before the treatment with oxysterols, SMCs were incubated in DMEM containing 5% FCS for 24 h. Oxysterol treatments were started in the same medium, in order to avoid the effects of fresh serum or altered serum concentrations. As for other experiments, oxys-terols were added to the culture medium by rapid injection of a stock solution in ethanol, using a Hamil-ton syringe. Final ethanol concentration in the culture medium was 0.25% (v/v) for sterol concentrations of 5 mg/ml. For the injection, culture medium was temporar-ily transferred to a sterile test tube without cells. Slightly subconfluent SMCs were incubated with oxys-terols for 4 h. The culture medium was then replaced by fresh DMEM (methionine-free, Life Technologies, Rockville, MD) containing 5% FCS, the indicated con-centrations of sterols and supplemented with 100 mCi/

ml L-[35_{S]methionine (}_\_{1000 Ci}_/_mmol, _{10 mCi}_/_ml, ICN Pharmaceuticals, Costa Mesa, CA). After 4 h, cells were rinsed twice with ice-cold PBS and harvested by scraping in 1 ml ice-cold PBS. For counting of cells, identical treatments were done, whereafter the cells were detached with trypsin and counted in an electronic cell counter (Medonic CA470). Radiolabeled cells were pelleted by centrifugation at 8000×g at 4°C, lysed in 300 ml of 50 mM Tris (pH 6.8), 1% (w/v) SDS, 0.008% (w/v) Bromophenol blue, 0.01% (v/v) 2-mercap-toethanol, and 0.05% (v/v) glycerol, triturated vigor-ously using a 0.8 mm, 80 mm needle to shear the DNA, and boiled for 5 min. Aliquots (10 ml) of each sample were transferred to microcentrifuge tubes containing 300 ml ice-cold trichloroacetic acid (TCA, 12%, w/v). Proteins were precipitated on ice for at least 10 min, and trapped in glass fiber filters (1 mm pore size, Gelman Sciences, Ann Arbor, MI) pre-wetted in 12% TCA by vacuum aspiration. The filters were washed three times with ice-cold 6% TCA and dried. They were then transferred to scintillation vials, and radioactivity was measured using a scintillation counter. The data shown was normalized to the number of cells in order to compensate for any eventual loss of cells. Statistical analysis was based on six separate samples per treat-ment, each of which was analyzed in triplicate. 2.9. Statistical analysis

Mean values and S.D. were calculated for triplicate determinations, unless stated otherwise. Unpaired Stu-dent’st-test was used to determine the statistical signifi-cance of the differences between treatments as indicated.

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3. Results

3.1. DNA degradation and chromatin condensation

The TUNEL assay detects apoptotic cells based on the presence of DNA breaks. Nuclei of normal non-apoptotic cells remain weakly stained (Fig. 1A), while apoptotic cells display brightly stained chromatin (Fig. 1C and D). TNFa(10 ng/ml) and IFNg (20 ng/ml) did not induce death of SMCs (Fig. 1B) but they potenti-ated oxysterol-induced apoptosis, as explained below. The TUNEL method does not label necrotic cells until in late stages of necrosis [33], when the cells are charac-terized by diffusely distributed chromatin or totally degenerated appearance. In cell cultures, such cells can be identified and have not been counted as TUNEL positive cells in our experiments.

25-Hydroxycholesterol and 27-hydroxycholesterol in-duced DNA degradation (positive TUNEL staining) and chromatin condensation (fragmented appearance of chromatin) (Fig. 1C), whereas treatment with choles-terol-5a,6a-epoxide only resulted in DNA degradation, even when TNFa and IFNg were added to the treat-ments. 7b-Hydroxycholesterol alone induced TUNEL staining without chromatin condensation (Fig. 1D), but in combination with TNFa (10 ng/ml) and IFNg (20 ng/ml) a diffuse pattern of chromatin condensation could be seen (data not shown).

As all of the oxysterols induced shrinkage of the cells, it is likely that they induce essentially the same type of cell death. The most pronounced difference was that chromatin condensation was less distinct in apop-tosis induced by 7b-hydroxycholesterol, as compared to the side-chain hydroxylated sterols 27-hydroxycholes-terol and 25-hydroxycholes27-hydroxycholes-terol. Choles27-hydroxycholes-terol-5a,6a -epoxide, the least toxic of the oxysterols tested, did not induce chromatin condensation, even though DNA fragmentation and positive TUNEL staining were ob-served. In control experiments, cholesterol (up to 20 mg/ml) was completely non-toxic to SMCs, even for treatments up to 6 days. TUNEL staining was not increased in cholesterol-treated cells, as compared to nontreated controls (data not shown).

We have previously quantitated 25-hydroxycholes-terol-induced apoptosis in the same SMCs used in the present study [14]. In order to find out whether struc-tural differencies between oxysterols could explain their different toxic properties, we compared the potency of structurally distinct oxysterols to induce apoptosis. The percentage of TUNEL positive cells after treatments with 25-hydroxycholesterol (5 mg/ml, 48 h) was 40% [14]. The corresponding value for 27-hydroxycholes-terol was 1596% (mean9S.D.; n=3), which in-creased to 2295% (mean9SD;n=3) when TNFa(10 ng/ml) and IFNg (20 ng/ml) were added together with 5 mg/ml of 27-hydroxycholesterol. At a lower concen-tration of 2.5 mg/ml, 27-hydroxycholesterol alone

in-Fig. 1. TUNEL staining of SMCs showing chromatin fragmentation after treatment with various oxysterols. (A) Untreated control cells only showed weak background staining. (B) Treatment with TNFa(10 ng/ml) and IFNg(20 ng/ml) for 72 h did not induce cell death. (C) After treatments with 5mg/ml of 25-hydroxycholesterol in combination with TNFaand IFNgfor 24 h, two kinds of positive staining patterns were seen. Labeled cells with homogeneously distributed chromatin may be in the initial stage of chromatin degradation, whereas cells with condensed chromatin represent a late stage. (D) 7b-Hydroxycholesterol (1mg/ml, 96 h) induced chromatin degradation, but not chromatin condensation.

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Fig. 2. Percentage of TUNEL stained cells after treatments with 7b-hydroxycholesterol (5 mg/ml) with (black bars) or without (white bars) TNFa(10 ng/ml) and IFNg(20 ng/ml) for various times. After 3 days, cell death was so extensive in dishes incubated with the oxysterol in combination with the cytokines that quantitation was not possible. Results represent mean values9S.D. (n=3). Student’s t -test was used to compare results for treatments with 7b-hydroxyc-holesterol alone to the treatments where cytokines were included (*PB0.05).

Fig. 3. Toxicity of 7b-hydroxycholesterol to SMCs as measured by MTT assay. Subconfluent SMCs were treated with 7b-hydroxycholes-terol9TNFaand IFNgfor 1 – 3 days. The lowest values indicate the highest toxicity. Results represent mean values9S.D. (n=3). SMCs treated with 10 mg/ml 7b-hydroxycholesterol for 2 or 3 days were completely dead. No MTT reduction could therefore be observed in these dishes (missing bars).

3.2. Cytotoxicity of 7b-hydroxycholesterol

The MTT assay provides a measure of metabolic activity that is closely correlated with the number of living cells. The toxicity of high concentrations (10 mg/ml) of 7b-hydroxycholesterol to SMCs (Fig. 3), as estimated by the MTT assay, exceeded the proportion of TUNEL positive cells. This is at least partly ex-plained by detachment of dead cells from the culture dishes, resulting in a lower percentage of apoptosis as estimated by the TUNEL assay. As many of the cells with labeled chromatin were found on top of other cells, it is likely that most dying cells detach, at least temporarily, and move a short distance before adhering to other cells nearby. TNFa (10 ng/ml) and IFNg (20 ng/ml) alone stimulated MTT reduction (Fig. 3) after treatments for 48 or 72 h.

Low concentrations of oxysterols resulted in in-creased formazan formation. Maximal stimulation of duced apoptosis in 3.390.8% (mean9S.D.; n=3) of

the cells, as measured by TUNEL staining after incuba-tion for 4 days. Addiincuba-tion of TNFa and IFNg to these treatments resulted in a twofold increase in TUNEL staining (6.891.4%). While these values are apparently low, it should be noted that even a low-level stimula-tion of apoptosis is likely to have profound effects on tissue homeostasis, both regarding cell content and the turnover of extracellular matrix. In control cells, the percentage of TUNEL positive cells always remained below 0.3%.

7b-Hydroxycholesterol and cholesterol-5a,6a-epoxide were less potent in inducing apoptosis than 27-hydroxy-cholesterol. At 5 mg/ml of 7b-hydroxycholesterol, the maximal percentage of TUNEL positive cells, 7.39 2.9% (mean9S.D.; n=3), was observed after 3 days (Fig. 2). At this stage a large number of cells were dead, and necrosis was widespread in the cell populations. TUNEL staining of cells treated with cholesterol-5a,6a -epoxide alone (10 mg/ml) never resulted in more than 5% labeled cells, and viability remained at 67% of control levels even after 4 days, as determined by MTT assay. However, addition of TNFa (10 ng/ml) and IFNg(20 ng/ml) increased TUNEL staining to 1794% (mean9S.D.; n=3) after incubations for 4 days, and resulted in 4093% (mean9S.D.;n=3) viability com-pared to the control.

In subsequent experiments, we chose to further char-acterize the effects of 7b-hydroxycholesterol, which has been reported to be a major cytotoxin of oxidized LDL [17,18].

Fig. 4. Stimulation of MTT reduction by low concentrations of 7b-hydroxycholesterol. Subconfluent SMCs were serum-starved for 24 h and incubated with the indicated concentrations of 7b-hydroxy-cholesterol for 4 days, in the presence of 5% FCS. Some cells were used for the MTT assay, others were counted (data not shown). Cell proliferation did not account for the differences, the statistical signifi-cance of which were assessed by Student’st-test (n=3).

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Fig. 5. Electron micrographs showing ultrastructural changes in SMCs treated with calphostin C, cholesterol-5a,6a-epoxide, or 7b-hydroxycholes-terol. Cells treated with 10 ng/ml TNFafor 24 h (A) had the same morphology as control SMCs in the synthetic phenotype. (B) Calphostin C (100 nM, 2 h) disrupted normal ER and Golgi membranes. Similar changes were seen in cells treated with 10mg/ml of cholesterol-5a,6a-epoxide+ TNFa(10 ng/ml) and IFNg(20 ng/ml) for 72 h (C) or 10mg/ml of 7b-hydroxycholesterol for 24 h (D).

MTT reduction was observed at a 7b -hydroxycholes-terol concentration of 1 mg/ml (Fig. 4). In the same experiment, we counted cells which had been treated in the same way as for the MTT assay, and excluded any increase in cell numbers (data not shown). These results suggest that non-lethal concentrations of oxysterols have a stimulatory effect on cellular metabolism. 3.3. Electron microscopy

SMCs can express a range of phenotypes [34]. At one end of this range is the cell whose function is mainly that of contraction (contractile state). At the opposite end of the range is the synthetic state, in which the muscle cell is engaged in proliferation and the produc-tion of extracellular matrix. SMCs used in the present study were in the synthetic state, characterized by abun-dant ER and Golgi membranes. Treatment with TNFa (Fig. 5A), IFNg, or both in ombination (data not shown) did not result in ultrastructural changes. The protein kinase C inhibitor calphostin C, which induces apoptosis in many types of cells, induced blebbing and cytoplasmic condensation in SMCs. Calphostin C-treated cells displayed general disorganization of the ER and Golgi membranes (Fig. 5B), formation of membranous whorls with multilamellar and multivesic-ular structures, and intracellmultivesic-ular vacuolization. This was also seen in cells treated with cholesterol-5a,6a

-epoxide (Fig. 5C) or 7b-hydroxycholesterol (Fig. 5D). 27-Hydroxycholesterol induced similar changes (Fig. 6A), but in addition, this oxysterol induced formation of tubular networks (Fig. 6B) which most likely con-sisted of ER and Golgi membranes. Swelling did not account for the early loss of ordered ER and Golgi membranes. Rather, these membranes were replaced by less organized tubular networks and multilamellar or multivesicular inclusions.

3.4. 7b-Hydroxycholesterol induces Ca2+

oscillations Perturbation of cellular Ca2+

homeostasis can cause disruption of intracellular organization. To find out whether oxysterol treatment affects the intracellular concentration of Ca2+_{, we directly measured Ca}2+ _in single smooth muscle cells. Addition of 7b -hydroxyc-holesterol (10 mg/ml) induced Ca2+ _{oscillations, which} continued for at least 60 – 120 min (Fig. 7a). The oscilla-tions had a frequency of approximately 0.3 – 0.4 min−1_. Verapamil did not attenuate the oscillations (Fig. 7b), suggesting that the signals were not due to opening of L-type voltage-operated Ca2+ _channels.

Nominally Ca2+_{-free medium partially inhibited the} oscillations. Removal of extracellular Ca2+ _after induc-tion of oscillainduc-tions with 7b-hydroxycholesterol immedi-ately decreased both the amplitude and the frequency of spikes (Fig. 7b), suggesting that the intracellular

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Ca2+ _{pools which give rise to oscillations have to be} replenished very rapidly or that Ca2+ _{entry through the} plasma membrane is synchronized with Ca2+ _release from the intracellular pools. Cholesterol (10mg/ml) did not induce Ca2+ _{oscillations (data not shown).}

Thapsigargin, an exceptionally specific, irreversible inhibitor of the sarcoplasmic reticulum and endoplas-mic reticulum Ca2+_{-dependent ATPase (SERCA) with} an IC50value of 10 – 30 nM [35], can be used to empty the inositol trisphosphate (InsP3)-sensitive Ca2+ pools in the ER. In SMCs incubated in nominally Ca2+_-free medium, thapsigargin caused an initial elevation of cytosolic Ca2+ _{within 15 s to 2 min, followed by a slow} decrease to baseline levels within approximately 10 min (Fig. 8a). Addition of Ca2+ _{to the medium rapidly} elevated intracellular Ca2+_{, but did not induce Ca}2+ oscillations. Rather, the Ca2+ _{increase was more} sus-tained than the thapsigargin-induced release in the ab-sence of extracellular Ca2+_{, and the return to baseline} levels occurred over a longer (30 min) time period. Thapsigargin treatment (10 nM – 5mM) was not toxic to SMCs during a period of 4 days. While initially surpris-ing to us, this has been confirmed in hamster SMCs which remained viable and retained normal morphol-ogy and mitochondrial function for 7 days despite total release of InsP3-sensitive Ca

2+ _{[36]. However, cell} divi-sion was completely blocked by thapsigargin, and protein synthesis was reduced by approximately 70% [36]. The observed increase in MTT reduction in cells treated with low concentrations of oxysterols was not due to emptying of ER Ca2+ _{pools, since thapsigargin} (100 nM) treatment for 24 h resulted in a 1797% (mean9S.D., n=3) decrease in MTT reduction.

In thapsigargin-treated SMCs, pretreatment with 7b -hydroxycholesterol (10mg/ml) for 10 min did not influ-ence the subsequent response to addition of Ca2+ _to the culture medium (Fig. 8b). This suggests that the oxysterol did not affect plasma membrane permeability in a direct physical manner. The Ca2+ _{response curve} was identical to that in control cells, with no Ca2+ oscillations. Analogous experiments (without thapsi-gargin) where SMCs were incubated in nominally Ca2+ -free medium for 24 h prior to addition of Ca2+ _yielded similar results. Ca2+ _{oscillations were not seen in cells} pretreated with thapsigargin prior to addition of 7b -hy-droxycholesterol. Taken together, these findings indi-cate that Ca2+

pools in the ER contribute to the oscillations seen in SMCs treated with 7b -hydroxyc-holesterol. Furthermore, treatment of SMCs with 7b -hydroxycholesterol (10 mg/ml) for 6 h abolished the thapsigargin-induced Ca2+ _{release, suggesting that} oxysterol treatment results in depletion of the ER Ca2+ pools within a few hours.

Overexpression of the anti-apoptotic protein Bcl-2 has been reported to maintain Ca2+ _{uptake in the ER} of thapsigargin-treated cells [37]. Downregulation of Bcl-2 could therefore contribute to oxysterol-induced cell death. However, as determined by immunoblotting, the human aortic SMCs used in the present study did not express Bcl-2 (data not shown), but we can not exclude the possibility that other members of the Bcl-2 family might exert comparable functions in these cells. 3.5. MAP kinase acti6ation

Since the oxysterols induced Ca2+ _{oscillations, we} tested whether they would activate MAP kinases. 7b

-Fig. 6. Electron micrographs showing early ultrastructural changes in SMCs treated with 27-hydroxycholesterol. (A) Disorganization of ER and Golgi membranes is apparent (2.5mg/ml 27-hydroxycholesterol, 16 h). (B) Formation of tubular networks of membranes after treatment with 27-hydroxycholesterol (2.5mg/ml)+TNFa(10 ng/ml) and IFNg(20 ng/ml) for 16 h.

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Fig. 7. 7b-Hydroxycholesterol induces Ca2+_{oscillations. The arrows indicate the time of addition (a: 72 s, b: 240 s) of 10}_mg_/_ml

7b-hydroxycholes-terol (denoted by 7bOHC). The induced oscillations in intracellular Ca2+_{had a frequency of 0.3 – 0.4 min}−1_{. (a) Verapamil (1}_{mM) did not inhibit}

the oscillations. (b) Ca2+_{-free medium decreased both the frequency and the amplitude of the oscillations. Arrows indicate the times when the}

Ca2+_{content of the medium was changed.}

Hydroxycholesterol (5 mg/ml) activated both ERK1 (p44mapk_{) and ERK2 (p42}mapk_{) within 5 min (Fig. 9).} Similar effects were seen with 25-hydroxycholesterol. Oxysterol-induced activation of ERKs is in agreement with the notion that Ca2+ _{signals are involved in the} activation of MAPKs [38]. Contribution of Ca2+ _to oxysterol-induced MAPK activation was confirmed by treatments in the presence of the Ca2+ _chelator

BAPTA, which inhibited ERK activation by 39% as determined by densitometric analysis of the bands shown in Fig. 9. ERKs are typically activated by growth factors, but nontoxic, low concentrations of oxysterols did not stimulate cell proliferation at any of the serum concentrations tested (1, 5, 14%). As shown in Fig. 10, c-Jun N-terminal kinases (JNKs) were not activated by 7b-hydroxycholesterol within 8 h.

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3.6. Inhibition of protein synthesis

It is currently not clear why inflammatory cytokines potentiated oxysterol-induced cell death. Inhibitors of protein synthesis are known to potentiate TNF-induced cell death, and restore the sensitivity of TNF-resistant cell lines to the cytocidal activity of TNF [39]. We therefore examined the effect of oxysterols on protein synthesis. At a concentration of 1mg/ml,

25-hydroxyc-holesterol (but not 7b-hydroxycholesterol or choles-terol-5a,6a-epoxide) consistently reduced total protein levels in SMCs. Measured daily over a 3-day period, the average protein level was 9195% (P=0.003,n=9) of control. At higher concentrations, the antiprolifera-tive effects of oxysterols make it difficult to assess their effects on protein synthesis. Therefore, we measured the rate of protein synthesis by labeling with L-[35_{S]methionine. At 5}_m_g_/_{ml, 25-hydroxycholesterol and}

Fig. 8. 7b-Hydroxycholesterol does not directly influence uptake of extracellular Ca2+_{. (a) Addition of 1} _{mM thapsigargin (TG) to cells}

pre-incubated in Ca2+_{-free medium for 24 h released intracellular Ca}2+_{. Subsequent addition of Ca}2+_{to the medium resulted in a rapid rise of}

intracellular Ca2+_{followed by slow return towards basal levels. (b) 7b-Hydroxycholesterol does not influence Ca}2+_{uptake after treatment with}

thapsigargin. SMCs pre-incubated in Ca2+_{-free medium for 4 h were treated with 1}_{mM thapsigargin (TG) for 340 s. 7b-Hydroxycholesterol (10}

mg/ml) was then added, and 450 s later the oxysterol treatment was continued in the presence of normal amounts of Ca2+_{. No oscillations were}

(10)

Fig. 9. ERK activation by 7b-hydroxycholesterol. SMCs were treated with 5mg/ml 7b-hydroxycholesterol (7bOHC) for 0 – 20 min as indicated. A purified, phosphorylated MAPK (phospho-MAPK) and treatment with 5% FCS for 5 min were used as positive controls. Some cells were pre-treated with 10mM BAPTA-AM for 30 min prior to addition of 7b-hydroxycholesterol. Active MAPKs were detected by immunoblotting with an antibody that recognizes the phosphorylated (active) forms of ERK1 and ERK2. An antibody against the non-phosphorylated MAPK was used as a control.

7b-hydroxycholesterol inhibited protein synthesis within 4 – 8 h (Fig. 11). At this time point, the number of cells had not changed substantially due to prolifera-tion or cell death, but we nevertheless normalized the [35

S]methionine incorporation results to the number of cells. Under these conditions, 25-hydroxycholesterol in-hibited protein synthesis by 4793% (PB0.001,n=6), 7b-hydroxycholesterol by 1995% (PB0.001, n=6), and cholesterol was without effect (Fig. 11).

4. Discussion

The present findings show that oxysterols differ in their potency to induce apoptosis. 25-Hydroxycholes-terol was the most potent inducer of cell death, fol-lowed by 27-hydroxycholesterol, 7b-hydroxycholesterol and cholesterol-5a,6a-epoxide. TNFaand IFNg poten-tiated oxysterol-induced cell death, but were not toxic alone. Addition of TNFa and IFNg to the oxysterol treatments resulted in a higher proportion of cells with condensed chromatin.

The TNF-resistant cell line L929r2 can be killed by TNF in the presence of cycloheximide [39]. Hence, it is conceivable that the observed inhibition of protein syn-thesis by oxysterols could make the cells more sensitive to TNF. Recently, TNF was shown to induce perinu-clear translocation of mitochondria in mouse L929 cells [39]. Clustering of mitochondria by itself was not harm-ful to the cells, but potentiated TNF-induced cell death [39]. Other mechanisms by which TNF might potentiate oxysterol-induced apoptosis include activation of cas-pase-8 [40] and generation of reactive oxygen intermedi-ates [41]. Finally, we have previously shown that

oxysterols inhibit activation of NF-kB [42], which would be expected to sensitize cells to TNF cytotoxicity [43,44].

The importance of Ca2+ _{signals for triggering} apop-tosis has been demonstrated in many experimental sys-tems [20 – 23,45]. A sustained elevation of Ca2+ _can activate degradative enzymes such as Ca2+_-dependent proteases (e.g. calpain) and endonuclease(s) responsible for DNA fragmentation [46]. In the present study we have shown that intracellular Ca2+ _{plays an important} role in cell death induced by 7b-hydroxycholesterol. Within a few minutes after addition, 7b -hydroxycholes-terol induced intracellular Ca2+ _{oscillations with a} frequency of approximately 0.3 – 0.4 min−1_{. Oxysterol} treatment led to a depletion of thapsigargin-releasable Ca2+ _{stores within a few hours. Interestingly,} glucocor-ticoid treatment has been shown to result in sustained depletion of the ER Ca2+ _{pool in mouse lymphoma} cells undergoing apoptosis [24].

The mechanisms underlying induction of apoptosis through Ca2+ _{mobilization remain partly elusive; at} present there are two models to explain how alterations in Ca2+ _{homeostasis might trigger apoptosis. In one,} depletion of intracellular stores and possibly influx of Ca2+_{across the plasma membrane promote a sustained} Ca2+ _{increase that acts as a signal for apoptosis. In the} second, it is not the Ca2+ _{increase but the emptying of} intracellular Ca2+ _{stores that triggers apoptosis [24,25],} perhaps by disrupting intracellular architecture and al-lowing key elements of the effector machinery to gain access to their substrates.

Balloon injury of rat carotid artery [47] as well as elevated intracellular Ca2+ _{[38] have been shown to} result in MAPK activation. It is therefore not

(11)

surpris-Fig. 10. 7b-Hydroxycholesterol does not activate JNKs. SMCs were treated with 5mg/ml 7b-hydroxycholesterol for up to 8 h as indicated below. Active JNKs were detected by immunoblotting, using an antibody that recognizes the phosphorylated (active) forms of all three JNKs. An antibody against the non-phosphorylated JNKs was used as a control. Data from four separate blots is shown. Lane 1: control; lane 2: 15 min; lane 3: 30 min; lane 4: 1 h; lane 5: 2 h; lane 6: 3 h; lane 7: control (the same as in lane 1); lane 8: 4 h; lane 9: 5 h; lane 10: 6 h; lane 11: 7 h; lane 12: 8 h; lane 13: positive control (extract from UV-treated 293 cells).

Fig. 11. Oxysterols inhibit protein synthesis. L-[35_S]Methionine

incor-poration was inhibited by 5 mg/ml of 25-hydroxycholesterol and 7b-hydroxycholesterol but not by cholesterol. SMCs were first treated with oxysterols for 4 h, whereafter fresh culture medium with L-[35_{S]methionine and oxysterols was added and the treatments were}

continued for 4 h before harvesting of cells.

homeostasis. The fact that inflammatory cytokines po-tentiated oxysterol cytotoxicity implies that the combi-nation of lipid oxidation and a local inflammatory reaction in atherosclerotic plaques can produce an envi-ronment promoting cell death, even if the levels of individual toxic substances alone would not be lethal to the vascular cells.

Acknowledgements

We are grateful to Ingemar Bjo¨rkhem and Ulf Dicz-falusy for generously providing us with 27-hydroxyc-holesterol. This study was supported by grants from the Wenner-Gren Foundation, Swedish Medical Research Council (grant numbers 8311, 2471, 6537, 09890, and 00034), the Swedish Heart and Lung Foundation, the Wallenberg Foundation, the Swedish Medical Society, King Gustaf V 80th Birthday Foundation, Prof. Nanna Svartz Foundation, the Loo and Hans Osterman Fund, the King Gustaf V and Queen Viktoria Foundation, and Barndiabetesfonden.

References

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ERK2. This effect was probably mediated by Ca2+_{. As} oxysterols inhibit protein synthesis and block the syn-thesis of isoprenoids required for cell proliferation [48 – 50], they may induce contradicting mitogenic and antiproliferative signals in cells, leading to a metabolic and/or signaling imbalance which culminates in cell death.

Based on oligonucleosomal DNA fragmentation and ultrastructural findings, it has been reported that oxi-dized LDL induces apoptosis in macrophages [51,52] and smooth muscle cells [53,54]. Oxysterols have been shown to account for most of the cytotoxicity of oxi-dized LDL [17,18], and the present study has character-ized the potency of different oxysterols to induce apoptotic cell death. The side-chain hydroxylated oxys-terols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent than 7b-hydroxycholesterol and cholesterol-5a,6a-epoxide, in which the ring structure of the sterols has been subjected to oxidation. Treatment with 7b-hydroxycholesterol induced intracellular Ca2+ oscillations and ERK activation within minutes, and depletion of thapsigargin-releasable Ca2+ _{pools within} a few hours, suggesting that oxysterol toxicity was primarily due to perturbation of intracellular Ca2+

(12)

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K, Wa˚hlander K, Nicotera P, Dypbukt J, Orrenius S, Hallberg A, Berggren P-O. Increased activity of L-type Ca2+ _channels

exposed to serum from patients with type I diabetes. Science 1993;261:86 – 90.

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[24] Lam M, Dubyak G, Distelhorst CW. Effect of glucocorticoid treatment on intracellular calcium homeostasis in mouse lymphoma cells. Mol Endocrinol 1993;7:686 – 93.

[25] Baffy G, Miyashita T, Williamson JR, Reed JC. Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-de-pendent hematopoietic cell line is associated with repartitioning

of intracellular calcium and is blocked by enforced Bcl-2 onco-protein production. J Biol Chem 1993;268:6511 – 9.

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[27] Watabe M, Kawazoe N, Masuda Y, Nakajo S, Nakaya K. Bcl-2 protein inhibits bufalin-induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells. Cancer Res 1997;57:3097 – 100.

[28] van den Brink MR, Kapeller R, Pratt JC, Chang JH, Burakoff SJ. The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells. J Biol Chem 1999;274:11178 – 85.

[29] Goillot E, Raingeaud J, Ranger A, Tepper RI, Davis RJ, Harlow E, Sanchez I. Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway. Proc Natl Acad Sci USA 1997;94:3302 – 7.

[30] Rus HG, Niculescu F, Vlaicu R. Tumor necrosis factor-alpha in human arterial wall with atherosclerosis. Atherosclerosis 1991;89:247 – 54.

[31] Szekanecz Z, Shah MR, Pearce WH, Koch AE. Human atherosclerotic abdominal aortic aneurysms produce interleukin (IL)-6 and interferon-gbut not IL-2 and IL-4: the possible role for IL-6 and interferon-gin vascular inflammation. Agents Ac-tions 1994;42:159 – 62.

[32] Kindmark H, Ko¨hler M, Efendic S, Rorsman P, Larsson O, Berggren P-O. Protein kinase C activity affects glucose-induced oscillations in cytoplasmic free Ca2+ _{in the pancreatic B-cell.}

FEBS Lett 1992;303:85 – 90.

[33] Gold R, Schmied M, Giegerich G, Breitshopf H, Hartung HP, Toyka KV, Lassmann H. Differentiation between cellular apop-tosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 1994;71:219 – 25.

[34] Chamley-Campbell J, Campbell GR, Ross R. The smooth mus-cle cell in culture. Physiol Rev 1979;59:1 – 61.

[35] Furuya Y, Lundmo P, Short AD, Gill DL, Isaacs JT. The role of calcium, pH, and cell proliferation in the programmed (apop-totic) death of androgen-independent prostatic cancer cells in-duced by thapsigargin. Cancer Res 1994;54:6167 – 75.

[36] Ghosh TK, Bian J, Short AD, Rybak SL, Gill DL. Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth. J Biol Chem 1991;266:24690 – 7. [37] He H, Lam M, McCormick TS, Distelhorst CW. Maintenance of

calcium homeostasis in the endoplasmic reticulum by Bcl-2. J Cell Biol 1997;138:1219 – 28.

[38] Enslen H, Tokumitsu H, Stork PJS, Davis RJ, Soderling TR. Regulation of mitogen-activated protein kinases by a calcium/ calmodulin-dependent protein kinase cascade. Proc Natl Acad Sci USA 1996;93:10803 – 8.

[39] De Vos K, Goossens V, Boone E, Vercammen D, Vancomper-nolle K, Vandenabeele P, Haegeman G, Fiers W, Grooten J. The 55-kDa tumor necrosis factor receptor induces clustering of mitochondria through its membrane-proximal region. J Biol Chem 1998;273:9673 – 80.

[40] Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involve-ment of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 1996;85:803 – 15.

[41] Goossens V, Grooten J, De Vos K, Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci USA 1995;92:8115 – 9.

[42] Ares MPS, Kallin B, Eriksson P, Nilsson J. Oxidized low-density lipoprotein induces transcription factor activator protein-1 but inhibits activation of nuclear factor-kB in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1995;15:1584 – 90.

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[43] Beg AA, Baltimore D. An essential role for NF-kB in preventing TNF-a-induced cell death. Science 1996;274:782 – 4.

[44] Xu Y, Bialik S, Jones BE, Iimuro Y, Kitsis RN, Srinivasan A, Brenner DA, Czaja MJ. NF-kB inactivation converts a hepato-cyte cell line TNF-aresponse from proliferation to apoptosis. Am J Physiol 1998;275:C1058 – 66.

[45] Lo´pez-Collazo E, Mateo J, Miras-Portugal MT, Bosca´ L. Re-quirement of nitric oxide and calcium mobilization for the induction of apoptosis in adrenal vascular endothelial cells. FEBS Lett 1997;413:124 – 8.

[46] Orrenius S, Ankarcrona M, Nicotera P. Mechanisms of calcium-related cell death. In: Siesjo¨ BK, Wieloch T, editors. Advances in Neurology (vol 71). Philadelphia: Lippincott-Raven, 1996:137 – 51.

[47] Lille S, Daum G, Clowes MM, Clowes AW. The regulation of p42 – p44 mitogen-activated protein kinases in the injured rat carotid artery. J Surg Res 1997;70:178 – 86.

[48] Saucier SE, Kandutsch AA, Taylor FR, Spencer TA, Phirwa S, Gayen AK. Identification of regulatory oxysterols, 24(S), 25-epoxycholesterol and 25-hydroxycholesterol, in cultured fibrob-lasts. J Biol Chem 1985;260:14571 – 9.

[49] Panini S, Sexton RC, Gupta AK, Parish EJ, Chitrakorn S, Rudney H. Regulation of 3-hydroxy-3-methylglutaryl coenzyme

A reductase activity and cholesterol biosynthesis by oxylanos-terols. J Lipid Res 1986;27:1190 – 204.

[50] Astruc M, Roussillon S, Defay R, Descomps B, Crastes de Paulet A. DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts. I. Temporal relationships between HMG-CoA reductase activity, sterol biosynthesis and thymidine incorporation into DNA. Biochim Biophys Acta 1983;763:1 – 10.

[51] Reid VC, Mitchinson MJ, Skepper JN. Cytotoxicity of oxidized low-density lipoprotein to mouse peritoneal macrophages: an ultrastructural study. J Pathol 1993;171:321 – 8.

[52] Reid VC, Hardwick SJ, Mitchinson MJ. Fragmentation of DNA in P388D1 macrophages exposed to oxidized low-density lipo-protein. FEBS Lett 1993;332:218 – 20.

[53] Nishio E, Arimura S, Watanabe Y. Oxidized LDL induces apoptosis in cultured smooth muscle cells: a possible role for 7-ketocholesterol. Biochem Biophys Res Commun 1996;223:413 – 8.

[54] Bjo¨rkerud B, Bjo¨rkerud S. Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibroblasts. Arterioscler Thromb Vasc Biol 1996;16:416 – 24.

(1)

Fig. 7. 7b-Hydroxycholesterol induces Ca2+_{oscillations. The arrows indicate the time of addition (a: 72 s, b: 240 s) of 10}_mg_/_ml

7b-hydroxycholes-terol (denoted by 7bOHC). The induced oscillations in intracellular Ca2+_{had a frequency of 0.3 – 0.4 min}−1_{. (a) Verapamil (1}_{mM) did not inhibit} the oscillations. (b) Ca2+_{-free medium decreased both the frequency and the amplitude of the oscillations. Arrows indicate the times when the}

Ca2+_{content of the medium was changed.}

Hydroxycholesterol (5

m

g

/

ml) activated both ERK1

(p44

mapk

_{) and ERK2 (p42}

mapk

_{) within 5 min (Fig. 9).}

Similar effects were seen with 25-hydroxycholesterol.

Oxysterol-induced activation of ERKs is in agreement

with the notion that Ca

_{signals are involved in the}

activation of MAPKs [38]. Contribution of Ca

_to

oxysterol-induced MAPK activation was confirmed by

treatments in the presence of the Ca

_chelator

BAPTA, which inhibited ERK activation by 39% as

determined by densitometric analysis of the bands

shown in Fig. 9. ERKs are typically activated by

growth factors, but nontoxic, low concentrations of

oxysterols did not stimulate cell proliferation at any of

the serum concentrations tested (1, 5, 14%). As shown

in Fig. 10, c-Jun N-terminal kinases (JNKs) were not

activated by 7

b

-hydroxycholesterol within 8 h.

(2)

3.6. Inhibition of protein synthesis

It is currently not clear why inflammatory cytokines

potentiated oxysterol-induced cell death. Inhibitors of

protein synthesis are known to potentiate TNF-induced

cell death, and restore the sensitivity of TNF-resistant

cell lines to the cytocidal activity of TNF [39]. We

therefore examined the effect of oxysterols on protein

synthesis. At a concentration of 1

m

g

/

ml,

25-hydroxyc-holesterol (but not 7

b

-hydroxycholesterol or

choles-terol-5

a

,6

a

-epoxide) consistently reduced total protein

levels in SMCs. Measured daily over a 3-day period,

the average protein level was 919

5% (

P

=

0.003,

n

=

9)

of control. At higher concentrations, the

antiprolifera-tive effects of oxysterols make it difficult to assess their

effects on protein synthesis. Therefore, we measured the

rate

of

protein

synthesis

by

labeling

with

L-[

_{S]methionine. At 5}

_m

_g

_/

_{ml, 25-hydroxycholesterol and}

Fig. 8. 7b-Hydroxycholesterol does not directly influence uptake of extracellular Ca2+_{. (a) Addition of 1} _{mM thapsigargin (TG) to cells}

pre-incubated in Ca2+_{-free medium for 24 h released intracellular Ca}2+_{. Subsequent addition of Ca}2+_{to the medium resulted in a rapid rise of}

intracellular Ca2+_{followed by slow return towards basal levels. (b) 7b-Hydroxycholesterol does not influence Ca}2+_{uptake after treatment with}

thapsigargin. SMCs pre-incubated in Ca2+_{-free medium for 4 h were treated with 1}_{mM thapsigargin (TG) for 340 s. 7b-Hydroxycholesterol (10}

mg/ml) was then added, and 450 s later the oxysterol treatment was continued in the presence of normal amounts of Ca2+_{. No oscillations were}

(3)

7 b

-hydroxycholesterol

inhibited

protein

synthesis

within 4 – 8 h (Fig. 11). At this time point, the number

of cells had not changed substantially due to

prolifera-tion or cell death, but we nevertheless normalized the

[

S]methionine incorporation results to the number of

cells. Under these conditions, 25-hydroxycholesterol

in-hibited protein synthesis by 479

3% (

P

B

0.001,

n

=

6),

7 b

-hydroxycholesterol by 1995% (

P

B

0.001,

n

=

6),

and cholesterol was without effect (Fig. 11).

4. Discussion

The present findings show that oxysterols differ in

their potency to induce apoptosis.

25-Hydroxycholes-terol was the most potent inducer of cell death,

fol-lowed by 27-hydroxycholesterol, 7

b

-hydroxycholesterol

and cholesterol-5

a

,6

a

-epoxide. TNF

a

and IFN

g

poten-tiated oxysterol-induced cell death, but were not toxic

alone. Addition of TNF

a

and IFN

g

to the oxysterol

treatments resulted in a higher proportion of cells with

condensed chromatin.

The TNF-resistant cell line L929r2 can be killed by

TNF in the presence of cycloheximide [39]. Hence, it is

conceivable that the observed inhibition of protein

syn-thesis by oxysterols could make the cells more sensitive

to TNF. Recently, TNF was shown to induce

perinu-clear translocation of mitochondria in mouse L929 cells

[39]. Clustering of mitochondria by itself was not

harm-ful to the cells, but potentiated TNF-induced cell death

[39]. Other mechanisms by which TNF might potentiate

oxysterol-induced apoptosis include activation of

cas-pase-8 [40] and generation of reactive oxygen

intermedi-ates [41]. Finally, we have previously shown that

oxysterols inhibit activation of NF-

k

B [42], which

would be expected to sensitize cells to TNF cytotoxicity

[43,44].

The importance of Ca

_{signals for triggering}

apop-tosis has been demonstrated in many experimental

sys-tems [20 – 23,45]. A sustained elevation of Ca

_can

activate degradative enzymes such as Ca

_-dependent

proteases (e.g. calpain) and endonuclease(s) responsible

for DNA fragmentation [46]. In the present study we

have shown that intracellular Ca

_{plays an important}

role in cell death induced by 7

b

-hydroxycholesterol.

Within a few minutes after addition, 7

b

-hydroxycholes-terol induced intracellular Ca

_{oscillations with a}

frequency of approximately 0.3 – 0.4 min

−1

_{. Oxysterol}

treatment led to a depletion of thapsigargin-releasable

Ca

_{stores within a few hours. Interestingly,}

glucocor-ticoid treatment has been shown to result in sustained

depletion of the ER Ca

_{pool in mouse lymphoma}

cells undergoing apoptosis [24].

The mechanisms underlying induction of apoptosis

through Ca

_{mobilization remain partly elusive; at}

present there are two models to explain how alterations

in Ca

_{homeostasis might trigger apoptosis. In one,}

depletion of intracellular stores and possibly influx of

Ca

_{across the plasma membrane promote a sustained}

Ca

_{increase that acts as a signal for apoptosis. In the}

second, it is not the Ca

_{increase but the emptying of}

intracellular Ca

_{stores that triggers apoptosis [24,25],}

perhaps by disrupting intracellular architecture and

al-lowing key elements of the effector machinery to gain

access to their substrates.

Balloon injury of rat carotid artery [47] as well as

elevated intracellular Ca

_{[38] have been shown to}

(4)

Fig. 11. Oxysterols inhibit protein synthesis. L-[35_S]Methionine incor-poration was inhibited by 5 mg/ml of 25-hydroxycholesterol and 7b-hydroxycholesterol but not by cholesterol. SMCs were first treated with oxysterols for 4 h, whereafter fresh culture medium with L-[35_{S]methionine and oxysterols was added and the treatments were} continued for 4 h before harvesting of cells.

homeostasis. The fact that inflammatory cytokines

po-tentiated oxysterol cytotoxicity implies that the

combi-nation of lipid oxidation and a local inflammatory

reaction in atherosclerotic plaques can produce an

envi-ronment promoting cell death, even if the levels of

individual toxic substances alone would not be lethal to

the vascular cells.

Acknowledgements

We are grateful to Ingemar Bjo¨rkhem and Ulf

Dicz-falusy for generously providing us with

27-hydroxyc-holesterol. This study was supported by grants from the

Wenner-Gren Foundation, Swedish Medical Research

Council (grant numbers 8311, 2471, 6537, 09890, and

00034), the Swedish Heart and Lung Foundation, the

Wallenberg Foundation, the Swedish Medical Society,

King Gustaf V 80th Birthday Foundation, Prof. Nanna

Svartz Foundation, the Loo and Hans Osterman Fund,

the King Gustaf V and Queen Viktoria Foundation,

and Barndiabetesfonden.

References

[7] Bjo¨rkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu RJ, Duan C, Lund E. Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol

ing that 7

b

-hydroxycholesterol activated ERK1 and

ERK2. This effect was probably mediated by Ca

_{. As}

oxysterols inhibit protein synthesis and block the

syn-thesis of isoprenoids required for cell proliferation [48 –

50], they may induce contradicting mitogenic and

antiproliferative signals in cells, leading to a metabolic

and

/

or signaling imbalance which culminates in cell

death.

Based on oligonucleosomal DNA fragmentation and

ultrastructural findings, it has been reported that

oxi-dized LDL induces apoptosis in macrophages [51,52]

and smooth muscle cells [53,54]. Oxysterols have been

shown to account for most of the cytotoxicity of

oxi-dized LDL [17,18], and the present study has

character-ized the potency of different oxysterols to induce

apoptotic cell death. The side-chain hydroxylated

oxys-terols 25-hydroxycholesterol and 27-hydroxycholesterol

were more potent than 7

b

-hydroxycholesterol and

cholesterol-5

a

,6

a

-epoxide, in which the ring structure of

the sterols has been subjected to oxidation. Treatment

with 7

b

-hydroxycholesterol induced intracellular Ca

oscillations and ERK activation within minutes, and

depletion of thapsigargin-releasable Ca

_{pools within}

a few hours, suggesting that oxysterol toxicity was

primarily due to perturbation of intracellular Ca