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 ngml and IFNg 20 ngml 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 ngml and IFNg 20 ngml 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-hydroxycholesterol. Cholesterol-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
mgml 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 mgml, 48 h was 40
[14]. The corresponding value for 27-hydroxycholes- terol was 15 9 6 mean 9 S.D.; n = 3, which in-
creased to 22 9 5 mean 9 SD; n = 3 when TNFa 10 ngml and IFNg 20 ngml were added together with
5 mgml of 27-hydroxycholesterol. At a lower concen- tration of 2.5 mgml, 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 ngml and IFNg 20 ngml for 72 h did not induce cell death. C After
treatments with 5 mgml of 25-hydroxycholesterol in combination with TNFa and IFNg for 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 1 mgml, 96 h induced chromatin degradation, but not chromatin condensation.
Fig. 2. Percentage of TUNEL stained cells after treatments with 7b-hydroxycholesterol 5 mgml with black bars or without white
bars TNFa 10 ngml and IFNg 20 ngml 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 values 9 S.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
P B 0.05. Fig. 3. Toxicity of 7b-hydroxycholesterol to SMCs as measured by
MTT assay. Subconfluent SMCs were treated with 7b-hydroxycholes- terol 9 TNFa and IFNg for 1 – 3 days. The lowest values indicate the
highest toxicity. Results represent mean values 9 S.D. n = 3. SMCs treated with 10 mgml 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
7
b -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 mgml 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 ngml and IFNg 20 ngml 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 3.3 9 0.8 mean 9 S.D.; n = 3 of the cells, as measured by TUNEL staining after incuba-
tion for 4 days. Addition of TNFa and IFNg to these treatments resulted in a twofold increase in TUNEL
staining 6.8 9 1.4. 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 mgml of 7b-hydroxycholesterol, the maximal percentage of TUNEL positive cells, 7.3 9
2.9 mean 9 S.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 mgml 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 ngml and
IFNg 20 ngml increased TUNEL staining to 17 9 4 mean 9 S.D.; n = 3 after incubations for 4 days, and
resulted in 40 9 3 mean 9 S.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’s t-test n = 3.
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 ngml TNFa for 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 10 mgml of cholesterol-5a,6a-epoxide + TNFa 10 ngml and IFNg 20 ngml for 72 h C or 10 mgml of 7b-hydroxycholesterol for 24 h D.
MTT reduction was observed at a 7b-hydroxycholes- terol concentration of 1 mgml 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 intracellular 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
.
7
b -Hydroxycholesterol induces Ca
2 +
oscillations Perturbation of cellular Ca
2 +
homeostasis can cause disruption of intracellular organization. To find out
whether oxysterol treatment affects the intracellular concentration of Ca
2 +
, we directly measured Ca
2 +
in single smooth muscle cells. Addition of 7b-hydroxyc-
holesterol 10 mgml induced Ca
2 +
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 Ca
2 +
channels. Nominally Ca
2 +
-free medium partially inhibited the oscillations. Removal of extracellular Ca
2 +
after induc- tion of oscillations with 7b-hydroxycholesterol immedi-
ately decreased both the amplitude and the frequency of spikes Fig. 7b, suggesting that the intracellular
Ca
2 +
pools which give rise to oscillations have to be replenished very rapidly or that Ca
2 +
entry through the plasma membrane is synchronized with Ca
2 +
release from the intracellular pools. Cholesterol 10 mgml did
not induce Ca
2 +
oscillations data not shown. Thapsigargin, an exceptionally specific, irreversible
inhibitor of the sarcoplasmic reticulum and endoplas- mic reticulum Ca
2 +
-dependent ATPase SERCA with an IC
50
value of 10 – 30 nM [35], can be used to empty the inositol trisphosphate InsP
3
-sensitive Ca
2 +
pools in the ER. In SMCs incubated in nominally Ca
2 +
-free medium, thapsigargin caused an initial elevation of
cytosolic Ca
2 +
within 15 s to 2 min, followed by a slow decrease to baseline levels within approximately 10 min
Fig. 8a. Addition of Ca
2 +
to the medium rapidly elevated intracellular Ca
2 +
, but did not induce Ca
2 +
oscillations. Rather, the Ca
2 +
increase was more sus- tained than the thapsigargin-induced release in the ab-
sence of extracellular Ca
2 +
, and the return to baseline levels occurred over a longer 30 min time period.
Thapsigargin treatment 10 nM – 5 mM 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 InsP
3
-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 Ca
2 +
pools, since thapsigargin 100 nM treatment for 24 h resulted in a 17 9 7
mean 9 S.D., n = 3 decrease in MTT reduction. In thapsigargin-treated SMCs, pretreatment with 7b-
hydroxycholesterol 10 mgml for 10 min did not influ- ence the subsequent response to addition of Ca
2 +
to the culture medium Fig. 8b. This suggests that the
oxysterol did not affect plasma membrane permeability in a direct physical manner. The Ca
2 +
response curve was identical to that in control cells, with no Ca
2 +
oscillations. Analogous experiments without thapsi- gargin where SMCs were incubated in nominally Ca
2 +
-free medium for 24 h prior to addition of Ca
2 +
yielded similar results. Ca
2 +
oscillations were not seen in cells pretreated with thapsigargin prior to addition of 7b-hy-
droxycholesterol. Taken together, these findings indi- cate that Ca
2 +
pools in the ER contribute to the oscillations seen in SMCs treated with 7b-hydroxyc-
holesterol. Furthermore, treatment of SMCs with 7b- hydroxycholesterol 10 mgml for 6 h abolished the
thapsigargin-induced Ca
2 +
release, suggesting that oxysterol treatment results in depletion of the ER Ca
2 +
pools within a few hours. Overexpression of the anti-apoptotic protein Bcl-2
has been reported to maintain Ca
2 +
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 Ca
2 +
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 2.5 mgml 27-hydroxycholesterol, 16 h. B Formation of tubular networks of membranes after treatment with
27-hydroxycholesterol 2.5 mgml + TNFa 10 ngml and IFNg 20 ngml for 16 h.
Fig. 7. 7b-Hydroxycholesterol induces Ca
2 +
oscillations. The arrows indicate the time of addition a: 72 s, b: 240 s of 10 mgml 7b-hydroxycholes- terol denoted by 7bOHC. The induced oscillations in intracellular Ca
2 +
had a frequency of 0.3 – 0.4 min
− 1
. a Verapamil 1 mM did not inhibit the oscillations. b Ca
2 +
-free medium decreased both the frequency and the amplitude of the oscillations. Arrows indicate the times when the Ca
2 +
content of the medium was changed.
Hydroxycholesterol 5 mgml 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
2 +
signals are involved in the activation of MAPKs [38]. Contribution of Ca
2 +
to oxysterol-induced MAPK activation was confirmed by
treatments in the presence of the Ca
2 +
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.
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 mgml, 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 91 9 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- [
35
S]methionine. At 5 mgml, 25-hydroxycholesterol and
Fig. 8. 7b-Hydroxycholesterol does not directly influence uptake of extracellular Ca
2 +
. a Addition of 1 mM thapsigargin TG to cells pre-incubated in Ca
2 +
-free medium for 24 h released intracellular Ca
2 +
. Subsequent addition of Ca
2 +
to the medium resulted in a rapid rise of intracellular Ca
2 +
followed by slow return towards basal levels. b 7b-Hydroxycholesterol does not influence Ca
2 +
uptake after treatment with thapsigargin. SMCs pre-incubated in Ca
2 +
-free medium for 4 h were treated with 1 mM thapsigargin TG for 340 s. 7b-Hydroxycholesterol 10 mgml was then added, and 450 s later the oxysterol treatment was continued in the presence of normal amounts of Ca
2 +
. No oscillations were seen, and the Ca
2 +
signals were similar to treatments without the oxysterol.
Fig. 9. ERK activation by 7b-hydroxycholesterol. SMCs were treated with 5 mgml 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 10 mM 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 47 9 3 P B 0.001, n = 6, 7b-hydroxycholesterol by 19 9 5 P B 0.001, n = 6,
and cholesterol was without effect Fig. 11.
4. Discussion
Kategori