the reduction products of the corresponding hydroper- oxy fatty acids, proved to be even stronger inducers of
the endothelial cell adhesion molecule ICAM-1 [9]. They also induce the scavenger receptor CD36 respon-
sible for oxLDL uptake in monocytesmacrophages [11]. In chemically oxidized LDL, oxidized fatty acids
were primarily found esterified to cholesterol [12]. They can also be produced by the action of 15-lipoxygenases
in cultured endothelial cells [13], macrophages [14], 15-lipoxygenase overexpressing endothelial cells [10]
and fibroblasts [15], and after treatment of LDL with purified rabbit reticulocyte 15-lipoxygenase [16]. How-
ever, these findings are not undebated [17].
The selenium-containing glutathione peroxidases [18] inhibit the activation of lipoxygenases in vitro [19 – 21]
and efficiently reduce products thereof [18]. Their role in the prevention of atherogenesis in vivo is widely
discussed, since epidemiological studies revealed a cor- relation of cardiovascular diseases and selenium defi-
ciency [22,23]. Therefore, the manipulation of the peroxide metabolism was considered in SMC as a
promising approach to analyze the relevance of lipid hydroperoxides present in oxLDL to SMC biology.
While all glutathione peroxidases reduce H
2
O
2
or soluble alkyl hydroperoxides [24], only the phospho-
lipid hydroperoxide glutathione peroxidase PHGPx efficiently reduces hydroperoxy groups of complex
lipids including those of phospholipids [18,25] and cholesterolesters [26] even when present in lipoproteins
[27]. Overexpression of PHGPx was therefore consid- ered the optimum tool to define the effects of hydroper-
oxy and hydroxy lipids of oxLDL on cellular targets. With this in mind PHGPx was overexpressed in SMC,
verified its function in situ, and tested the proliferative response to oxLDL in rabbit aortic SMC.
2. Methods
2
.
1
. Cell culture Rabbit abdominal aortic SMC RAASMC [10,28]
and 8CRE and 8CRIP cells both modified from NIH 3T3 [29] were grown in DMEM Seromed supple-
mented with 5 or 10 FCS, 100 Uml penicillin, 100 m
gml streptomycin and 4 mM
L
-glutamine. If men- tioned, cells were supplemented with sodium selenite at
the concentrations indicated. A 1000-fold stock solu- tion of selenite Sigma in water was prepared, filtered
sterile and diluted by addition to the cell culture media. Selenium deficiency was obtained by growing the cells
in an FCS batch Seromed, batch 698E which con- tained 8.7 mg seleniuml, resulting in a selenium concen-
tration of 5.5 nM when 5 was used in the growth medium. Despite not knowing the bioavailability and
the form in which selenium was present in the FCS, this batch led to the lowest GPx activities obtainable with-
out selenium supplementation.
2
.
2
. Construction of the retro6iral 6ector All cloning work was done according to conventional
procedures [30].
2
.
2
.
1
. Construction of pLPHGPxRNL The cDNA of the pig PHGPx [31] was first trans-
ferred into the retroviral vector pLRNL [32]. The full PHGPx cDNA was taken from pMM3 [33] as NdeI ×
BamHI fragment and ligated into pBluescript II KS
+
Stratagene. To facilitate subsequent cloning proce- dures, an oligonucleotide containing an additional
BamHI restriction-site was inserted between the KpnI and NdeI site of the vector pBluescriptPHGPx result-
ing in pBluescriptPHGPxb Fig. 1A. Bluescript PHGPxb and pLRNL were digested with BamHI,
fragments PHGPx, 0.8 kbp; pLNRL, 6.4 kbp isolated via electroelution, ligated, and transformed into Es-
cherichia coli XL1-Blue Stratagene. The resulting pLPHGPxRNL Fig. 1B was analyzed by restriction
for the right orientation of PHGPx gene.
2
.
2
.
2
. Production of PHGPx 6ectors pLPHGPxRNL was first packaged in an ecotrophic
packaging cell line, 8CRE, and subsequently in an
Fig. 1. A Map of pBluescriptPHGPxb; B map of pLPHG- PxRNL. For explanations see Section 2 and text.
amphotropic packaging cell line, 8CRIP. Three days after infection of 8CRIP, cells were split and put under
selection Geneticin, 400 mgml. First clones 8CRIP PHGPx were visible after 14 – 16 days of the selection.
More than 70 clones were expanded and analyzed for the retroviral transcript by Northern blots and by
activity of PHGPx. Five clones with the highest PHGPx activity were chosen for the transfection of
target cells.
2
.
2
.
3
. Infection of target cells SMC were infected with supernatants from selected
8 CRIPPHGPx clones. On day 4 after infection, cells
were put under selection 400 mgml Geneticin and clones were isolated 12 – 16 days later.
2
.
3
. Acti6ity of glutathione peroxidases Glutathione peroxidase activity was measured with
the glutathione reductase-coupled test adapted for cul- tured cells as described [34].
2
.
3
.
1
. Hydroperoxides H
2
O
2
was purchased from Sigma. Phosphatidyl- choline hydroperoxide PCOOH was prepared by oxi-
dizing phosphatidylcholine
Sigma with
soybean lipoxygenase type IV Sigma in the presence of 3
deoxycholate, freed from detergent and concentrated by solid phase extraction with SEP PACK 18 reverse
phase cartridges Waters-Millipore, and quantified ac- cording to Ref. [35]. Linoleic acid hydroperoxide
LOOH was prepared according to Ref. [36], by oxi- dizing linoleic acid Sigma with soybean lipoxygenase
type IV Sigma.
2
.
4
. Assays for in situ PHGPx function Cytotoxicity of different hydroperoxides was mea-
sured by estimation of MTT reduction [37].
2
.
4
.
1
. Intracellular oxidation The amount of intracellular oxidation upon incuba-
tion with linoleic acid hydroperoxide LOOH was measured fluorographically via oxidation of dihy-
drorhodamine DHR 123 according to Ref. [38]. The 48-well plates with a confluent monolayer of cells
grown with or without sodium-selenite in DMEM with 5 FCS for 4 days were loaded with 20 mM DHR 123
Molecular Probes, Netherlands for 45 min at 37°C in Hank’s balanced salt solution HBSS, washed and
treated with 30 – 60 mM LOOH in serum-free DMEM for 60 min at 37°C. After washing with HBSS, cells
were scanned in a fluorescence reader Cytofluor II, Perceptives Biosystems, Germany with l
ex
485 nm and l
em
530 nm.
2
.
4
.
2
. Electrophoretic mobility shift assay Cells were grown without or with selenium 100 nM
sodium-selenite for 3 days in DMEM with 10 FCS. Then medium was changed to serum-free DMEM for
24 h before stimulation. Linoleic acid hydroperoxide was added at the concentrations indicated for 60 min at
37°C. Nucleic protein extracts were prepared according to Ref. [39].
A DNA fragment representing the consensus binding motif
of the
KB site
5-AATTCA CAAAGA
GGGACT TTCCCC TACATC CATTG-3 was used for gel shift analyses. Binding reaction was performed
at room temperature with 5 mg protein of the nuclear extract, 10 fmol of labelled DNA and 3 mg polydIdC
Pharmacia Biotech in 15 mM Hepes, 1 mM EDTA, 1 mM DTT, 10 wv glycerol in 10 ml. After 30 min
samples were immediately subjected to gel electrophore- sis on a native 4 polyacrylamide gel in 0.25 × TBE at
200 V.
2
.
4
.
3
. Apoptosis Cells grown in DMEM, 5 FCS, with or without
100 nM sodium-selenite for 3 – 4 days were seeded at 2 × 10
4
cellswell in 24-well plates and left to adhere overnight. Then media were changed to DMEM 5
FCS containing the peroxides indicated and cells incu- bated for 8 h. Thereafter cells were lysed and superna-
tants assayed immediately by the Cell Death Detection ELISA
plus
Boehringer Mannheim according to manu- factors’ instructions.
2
.
4
.
4
. Proliferation assay Cells were grown in DMEM medium, 10 FCS, in
the presence or absence of 100 nM sodium selenite. They were seeded at 3 × 10
3
cellsml into 96-well plates and allowed to adhere for 24 h. Then serum was
deprived for 30 h to obtain quiescence. Thereafter, cells were incubated with 1 FCS control, 1 FCS plus 5
or 20 mg nLDLml, 1 FCS plus 5 or 20 mg oxLDLml for 24 or 40 h. At the end of a stimulation period 10 ml
[
3
H]thymidine 0.5 mCiwell was added and incubations were continued for additional 2 h. The radioactive
medium was aspirated, cells were washed twice in ice- cold PBS buffer 200 ml followed by 5 TCA exposure
for 2 h at room temperature. The TCA solution was removed by aspiration and 75 ml of 1 N NaOH was
added to solubilize the precipitated material. After fur- ther 2 h 75 ml of 1 N HCl was added to each well for
neutralization.
Incorporated radioactivity
was quantified by liquid scintillation counting.
2
.
4
.
5
. Oxidation of LDL LDL were isolated from the plasma by density gradi-
ent ultracentrifugation according to Ref. [40], desalted in a Sephadex G-25 column equilibrated with PBS and
filtered sterile. Puritly of LDL was checked by SDS-
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
