toxin in oxidized LDL [17,18]. 7b-Hydroxycholesterol can also be produced endogenously from cholesterol
[19]. Ca
2 +
mobilization can induce apoptosis [20 – 23] by activating Ca
2 +
-dependent proteases and endonucle- ases and by depleting intracellular Ca
2 +
stores [24,25], the maintenance of which is required for cell
survival. In the present study we show that 7b-hydroxy- cholesterol
induces intracellular
Ca
2 +
oscillations which lead to depletion of thapsigargin-releasable Ca
2 +
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 TNFa and 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, Ca
2 +
-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 RD 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 anti-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 Uml 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 DMEMF-12 1:1 with-
out phenol red, supplemented with 5 FCS unless stated otherwise, penicillin 100 Uml and strepto-
mycin 100 mgml. 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 DMEMF12 1:1 without phenol red at a concentration of 5 mgml. 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 vv 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
Uml terminal deoxytransferase, 300 mM Tris – HCl pH 7.2, 10 mM CoCl
2
, and 300 mgml 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 15 mgml 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 mgml p-phenylenediamine, 10 vv PBS, 90 vv 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,
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 25 mg 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 Ca
2 +
Subconfluent SMCs on glass cover slips were loaded with 3 mM fura-2-AM for 30 min in culture medium
DMEF-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 340380 fluores- cence ratios directly representative of changes in intra-
cellular Ca
2 +
. 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 vv for sterol concentrations of 5
mgml. 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 Cimmol, 10 mCiml, 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 wv SDS, 0.008 wv
Bromophenol blue,
0.01 vv
2-mercap- toethanol, and 0.05 vv 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, wv.
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’s t-test was used to determine the statistical signifi-
cance of
the differences
between treatments
as indicated.
3. Results
