Fig. 2. Selenium-dependent glutathione peroxidase activities of con- trol smooth muscle cells, SMC and phospholipid hydroperoxide
glutathione peroxidase PHGPx-transfected SMCPHGPx smooth muscle cells. Cells were grown for 4 days in DMEM, 10 FCS, with
and without selenium 100 nM sodium selenite supplementation. Then cells were harvested and PHGPx activity measured with phos-
phatidylcholine hydroperoxide PCOOH and total glutathione per- oxidase activity measured with H
2
O
2
as substrates. Activity is expressed in mUmg protein. Values are means from seven individual
cultures 9 S.D. P B 0.01, P B 0.001 vs selenium-supplemented SMC.
In contrast, the parental cell line, SMC, did not show any significant increase in GPx activities upon selenium
supplementation neither when measured with PCOOH nor with H
2
O
2
. This data implies that the parental SMC are practically devoid of both cGPx and PHGPx,
and the increase of PHGPx acitivity seen upon selenium supplementation in SMCPHGPx is almost exclusively
due to the overexpression of the transfected PHGPx gene. The stability of increased PHGPx activity in the
SMCPHGPx clone was verified over a period of 2 years.
In absolute terms, the overexpression achieved is modest, but comparable with overexpression obtained
by others [45 – 50]. When high expression rates of, e.g. cGPx were obtained [51,52], they proved to be unstable
over time. The modest but stable overexpression of PHGPx was therefore considered satisfactory to study
the impact of improved lipid hydroperoxide metabolism in SMC.
3
.
2
. Validation of in situ functionality of o6erexpressed PHGPx
3
.
2
.
1
. Peroxide-cytotoxicity As expected from previous studies [46,47,49] overex-
pressed PHGPx reduced the cytotoxicity of different hydroperoxides like PCOOH and linoleic acid hy-
droperoxide in SMCPHGPx compared to SMC not shown. Cytoprotection correlated with the PHGPx
activity and was highest in SMCPHGPx grown in selenium supplemented medium.
3
.
2
.
2
. Intracellular DHR oxidation To further prove the functionality of transfected
PHGPx, intracellular oxidation of dihydrorhodamine 123 DHR 123 after challenging cells with LOOH was
measured. DHR 123 belongs to the oxidant-sensing fluorescent probes detecting a broad range of oxidizing
reactions that may be increased during intracellular oxidative stress, e.g. induced by LOOH [53]. As shown
in Fig. 3, intracellular DHR 123 oxidation upon LOOH exposure increased in SMC in a concentration depen-
dent manner and was independent of selenium supple- mentation. In contrast, in SMCPHGPx, intracellular
oxidation was by far lower and could not be further reduced upon selenium supplementation. Thus, the low
increase in PHGPx activity in SMCPHGPx grown without selenium supplementation is already sufficient
to
withdraw LOOH
from reacting
with dihydrorhodamine.
3
.
2
.
3
. Hydroperoxide-induced acti6ation of NFkB The SMC line used in this study belongs to the few
examples in which NFkB can be activated by hydroper- oxides Fig. 4. In unsupplemented SMC, 1 h incuba-
tion with 40 and 80 mM LOOH, respectively, led to a PAGE of apolipoproteins according to Ref. [41]. For
oxidation, native LDL 1 mgml was incubated with 0.2 mM FeCl
3
at 37°C. After 3 h, iron was removed by gel filtration. Oxidative modification of LDL was esti-
mated according to [42]. A total of 7.7 9 0.7 nmol MDAmg protein was considered adequate native
LDL: 1.2 9 0.2 nmolmg protein.
In separate samples prepared the same way, hy- droperoxide and hydroxide levels. were analyzed as
cholesteryl hydroperoxy linoleate by HPLC [12,43] or by absorption at 234 nm of extracted lipids [44]. Both
methods proved a hydroperoxide content of 1 – 2
m molmg protein.
3. Results
3
.
1
. Success of transfection Twelve Geneticin-resistant clones of PHGPx-trans-
fected cells were expanded for further analysis. They all exhibited the transfected porcine PHGPx mRNA in
Northern blot data not shown. Three of them, show- ing the highest mRNA content, were tested for PHGPx
activity and the selenium dependency thereof. The finally selected clone, SMCPHGPx, still expressed low
PHGPx measured with PCOOH and total GPx mea- sured with H
2
O
2
activity under selenium limiting con- ditions, but responded with a 3 – 4-fold enhanced
PHGPx activity when cultivated in selenium supple- mented medium Fig. 2.
concentration-dependent activation of NFkB. Corre- sponding to the unchanged GPx activity, selenium sup-
plementation did not show any effect. This is in contrast to other studies in which selenium supplemen-
Fig. 5. Selenium-dependent initiation of apoptosis by linoleic acid hydroperoxide LOOH in control smooth muscle cells, SMC and
phospholipid hydroperoxide glutathione peroxidase PHGPx-trans- fected SMCPHGPx smooth muscle cells. A DNA fragmentation
expressed as absorbance at 450 nm in untreated control and cells exposed to 40 mM LOOH for 8 h. Values are means from three
individual experiments 9 S.D. P B 0.01; P B 0.005. B DNA fragmentation in SMCPHGPx 9 Se treated with different concentra-
tions of LOOH for 18 h. Values are means from three individual experiments 9 S.D. P B 0.0001. For details see Section 2.
Fig. 3. Selenium-dependent linoleic acid hydroperoxide LOOH-in- duced intracellular oxidation in control smooth muscle cells, SMC
and phospholipid hydroperoxide glutathione peroxidase PHGPx- transfected SMCPHGPx smooth muscle cells. Cells were grown for
4 days in DMEM, 10 FCS, with and without selenium 100 nM sodium selenite supplementation. Then cells were loaded with dihy-
drorhodamine 123 DHR 123 and treated with LOOH for 1 h at the concentrations indicated. Oxidation was measured by fluorescence.
For details see Section 2. Values are means from three individual experiments 9 S.D. P B 0.05, P B 0.01 vs SMC 9 Se, respec-
tively.
Fig. 4. Selenium-dependent activation of NFkB by linoleic acid hydroperoxide LOOH in control smooth muscle cells, SMC and
phospholipid hydroperoxide glutathione peroxidase PHGPx-trans- fected SMCPHGPx smooth muscle cells. Cells were grown for 4
days in DMEM, 10 FCS, with and without selenium 100 nM sodium selenite supplementation. 24 h prior to stimulation serum
was withdrawn, then cells were stimulated with LOOH for 60 min at the concentrations indicated. NFkB activation was analyzed as de-
scribed in Section 2.
tation led to an inhibition of NFKB activation due to an increased activity of endogenous glutathione peroxi-
dases [54 – 57]. In SMCPHGPx, the degree of NFkB activation was slightly decreased in selenium deficiency
and almost completely abrogated in selenium supple- mented cells. Supershift experiments with the respective
antibodies against the different NFkB subunits iden- tified the activated band as composed of p65 and p50
but not c-rel data not shown.
3
.
2
.
4
. Hydroperoxide-induced apoptosis LOOH 40 mM, 8 h led to apoptotic cell death in
SMC as measured by a quantitative sandwich-enzyme- immunoassay allowing the determination of mono- and
oligonucleosomes in the cytosol of apoptotic cells. The degree of apoptosis was independent of the selenium
status in control cells Fig. 5A. In SMCPHGPx, LOOH-induced apoptosis was clearly inhibited even
under selenium limiting conditions. This is in accor- dance with the decreased intracellular oxidation upon
LOOH treatment of SMCPHGPx see Fig. 3, indicat- ing that secondary effects exerted by LOOH over a
short period of time can be counteracted by the small increment of transfected PHGPx. Selenium-supplemen-
tation, thus, could lead only to a small additional inhibition of apoptosis.
In contrast, after 18 h of LOOH treatment apoptosis dramatically increased also in selenium-deficient SMC
PHGPx Fig. 5B. Under these conditions the low additional PHGPx activity obviously is not sufficient to
prevent apoptotic cell death. In selenium supplemented SMCPHGPx apoptosis was still completely abolished
in agreement with the high PHGPx activity.
3
.
2
.
5
. oxLDL-induced SMC proliferation The experiments described so far prove that overex-
pressed PHGPx consistently counteracts effects caused by added hydroperoxides. The test system can thus be
used to identify effects of hydroperoxides possibly present in more complex systems such as the prolifera-
tive response of SMC and SMCPHGPx to oxLDL. In untransfected SMC, the rate of thymidine incorpora-
tion remained unchanged over a period of 40 h, irre- spective of selenium supplementation Fig. 6A,B.
Native LDL did not significantly affect the prolifera- tion of SMC. oxLDL induced a significant and dose-de-
pendent increase of the proliferation. This proliferative response was not affected by selenium supplementation,
which is consistent with the lack of endogenous se- lenoperoxidases in SMC.
The PHGPx-transfected cells behave quite differ- ently. First of all, their thymidine incorporation was
Fig. 6. Proliferation of control smooth muscle cells, SMC and phospholipid hydroperoxide glutathione peroxidase PHGPx-transfected SMCPHGPx smooth muscle cells treated with native and oxidized LDL oxLDL. SMC 9 Se A and B and SMCPHGPx 9 Se C, D
were seeded at 3 × 10
3
cellsml each into 96 well plates, allowed to adhere and made quiescent by serum deprivation for 24 h. Then they were stimulated with either native LDL nLDL, 5 and 20 mgml or oxLDL 5 and 20 mgml all in the presence of 1 FCS 9 Se. For controls, only
1 FCS 9 Se was added. Cells were then incubated for 24 or 40 h, respectively. Proliferation was assayed by the incorporation of [
3
H]thymidine. Values are means from four individual experiments 9 S.D. For details see Section 2.
lower than in SMC from the beginning Fig. 6C,D. It further decreases with time, irrespective of selenium
supplementation and exposure to native LDL. In terms of absolute proliferation rates, the response to oxLDL
blunted in selenium-deprived SMCPHGPx, when com- pared to control SMC. The relative proliferation of
oxLDL-exposed versus unstimulated SMCPHGPx, however, tended to be increased. This trend was more
pronounced in selenium supplemented SMCPHGPx, i.e. under optimized PHGPx activity compare Fig. 6C
and D. After 24 h of oxLDL exposure of selenium- supplemented SMCPHGPx the proliferation was still
low compared to identically treated SMC. After 40 h, however, the proliferation of the SMCPHGPx is prac-
tically the same as that of pertinent controls compare Fig. 6D with B.
Clearly, thus, expression of functional PHGPx affects the oxLDL response of SMC in a complex manner in
lowering basic proliferation rates, but increasing the oxLDL proliferative response, after long term exposure,
to that of control cells.
4. Discussion
