intima. The principal source of EC-SOD is the smooth muscle cells [23]. The strategic location sug-
gests that EC-SOD exerts an important protective role against pathologies induced by the superoxide radical
in the vascular wall.
Both inflammatory and immunological responses are integral parts of the atherosclerotic process, and
also occur in other diseases of the vascular wall. Infl- ammatory cytokines have previously been found to
influence the synthesis of EC-SOD by human dermal fibroblasts [28]. In the present study we examined the
effects of cytokines involved in inflammatory and im- munological responses on SOD isoenzyme expression
by human arterial smooth muscle cells. We found large effects on EC-SOD and Mn-SOD synthesis, sug-
gesting that cytokines formed in vascular diseases may significantly alter the response of the vascular wall to
superoxide radicals.
2. Methods
2
.
1
. Cell culture and regulation experiments The arterial smooth muscle cell SMC lines were
initiated from pieces of human uterine artery as de- scribed [29]. Cell lines from five different persons were
established Au-1 – 5 and were used between the 5th and 8th passages. Their identity as smooth muscle
cells was assessed by morphological appearance and by their expression of smooth muscle a-actin [30]
Boehringer Mannheim GmbH, Mannheim, Germany as analyzed by flow cytometry. Uterine arteries con-
tained around 33 mgg wet weight of EC-SOD, a value similar to those previously found in coronary
arteries and aorta [23]. The cells were maintained in Waymouth MB 7521 medium containing 15 fetal
calf serum FCS, 10
5
unitsl bensylpenicillin, 100 mgl streptomycin, 2 mmoll glutamine and 1 mmoll Na
pyruvate. For synthesis regulation experiments the cells were
seeded into 12-well culture plates, bottom area 3.80 cm
2
, and grown into near confluence. During the ex- periments, the culture media were supplemented with
either 15 FCS or 1 bovine serum albumin BSA, as indicated. When supplemented with 1 BSA, the
medium was exchanged twice to medium with 1 BSA about 20 h before the start of the experiments.
The experiments were started by exchange to 0.5 ml medium with either 1 BSA or 15 FCS containing
indicated concentrations of cytokines or only medium with 1 BSA or 15 FCS controls. Every 24 h the
media were collected and replaced with fresh media containing cytokines. At the end of the experiments,
after 4 days, the media were collected and the wells were washed three times with 0.15 moll NaCl. To
collect and homogenize the cells, 0.5 ml ice-cold 50 mmoll Na phosphate, pH 7.4, containing 0.3 moll
KBr, 10 mmoll diethylene-triamine pentaacetic acid, 0.5 mmoll phenylmethylsulfonyl fluoride and 100
KIUml aprotinin the latter three additions to inhibit proteases was added to the wells. After sonication in
the wells, with the plate bathed in ice water, the ho- mogenates were centrifuged 20 000 × g for 10 min
and the supernatants were collected for analysis. All samples were kept at − 80°C until assay.
2
.
2
. Analysis of SOD isoenzymes EC-SOD protein in culture media and cell ho-
mogenates was determined with an ELISA [31]. EC- SOD is throughout presented as amount of enzyme
protein in figures and tables. In the cell homogenates, the CuZn-SOD and Mn-
SOD enzymatic activities were also measured, using the direct spectrophotometric method employing KO
2
as previously described [32,33]. 3 mmoll cyanide was used for primary distinction between the cyanide-sen-
sitive isoenzymes CuZn-SOD and EC-SOD and the nearly resistant Mn-SOD. One unit in the assay corre-
sponds to 4.3 ng human CuZn-SOD [34], 8.6 ng hu- man EC-SOD [35], and 65 ng bovine Mn-SOD. For
final calculation of the activity of the SOD isoen- zymes in the cell homogenates, the EC-SOD activity
was calculated from the amount of protein as deter- mined by ELISA and the specific activity 8.6 ng
unit. The Mn-SOD activity was then taken as the cyanide resistant activity corrected for a minor in-
hibitory effect of the cyanide minus remaining mini- mal
activity of
CuZn-SOD and
EC-SOD. The
CuZn-SOD activity was finally calculated as total SOD activity minus the calculated EC-SOD activity
and the Mn-SOD activity.
2
.
3
. Turno6er of EC-SOD in cell cultures To assess the rate of uptake of EC-SOD by cul-
tured cells, conditioned media from SMCs containing EC-SOD were incubated with human cell lines not
producing EC-SOD. Thus, Waymouth medium sup- plemented with 1 BSA was incubated with confluent
SMCs for 4 days, after which it was diluted with unconditioned medium to around 3 ngml of EC-
SOD, a typical level in the SMC experiments, c.f. Fig. 1. 0.5 ml of the mixed medium was then added to
triplicate wells with human cell lines in 12-well culture plates. The cell lines were MG-251, a glioma cell line;
Hep-G2, a hepatoma cell line, PL-3, and DU-145, prostate cancer cell lines. The concentrations of EC-
SOD in the media were determined at intervals over 24 h.
2
.
4
. Protein and DNA analysis For protein analysis, Coomassie Brilliant Blue G-250
was employed [36], standardized with human serum albumin. The DNA concentration was determined with
fluorimetry as
a complex
with bisbenzimidazol
Hoechst 33 258 [37] using calf thymus DNA as a standard.
Fig. 1. Effects of IFN-g, TNF-a and IL-4 on smooth muscle cell SOD expression. Smooth muscle cell lines were cultured in 3.8 cm
2
wells, and culture media 0.5 ml containing the cytokines at indicated concentrations were exchanged daily for 4 days. At the end of the experiment the cells
were homogenized and analyses were made on culture media and cell homogenates as described in Section 2. The data presented are the means of results from two wells. A The effect of IFN-g on the Au-2 cell line, media supplemented with 1 BSA; B the effect of TNF-a on the Au-1
cell line, media supplemented with 15 FCS; and C the effect of IL-4 on the Au-1 cell line, media supplemented with 1 BSA.
Fig. 1. Continued
2
.
5
. Incorporation of
35
S-methionine into protein Smooth muscle cells were cultured in 12-well culture
plates 3.8 cm
2
for 4 days as described above. At the end of the experiment, the cells were incubated in
methionine free medium with
35
S-methionine added for 1 h, followed by homogenization, protein precipitation
with 10 trichloroacetic acid, and analysis of
35
S in- corporation into protein, as previously described [28].
Aliquots of the cell homogenates were also analyzed for EC-SOD, protein and DNA as described above.
2
.
6
. RNA extraction and Northern blot analysis Smooth muscle cells of the Au-1 line were cultured
in medium containing 1 BSA as described above. At the end of the experiment, RNA was isolated by TRI-
zol reagent Gibco BRL, Life Sciences Inc, Gaithers- burg, USA. Total RNA 10 mg per lane was
electrophoresed
in formaldehyde-containing
1.2 agarose gels, transferred to nylon filters Hybond N,
Amersham plc, Amersham, UK and immobilized by UV-linkage. The filters were then prehybridized for 15
min and hybridized for 1 h with Quickhyb solution Stratagene Inc, La Jolla, CA, USA at 65°C in a
hybridization oven. For EC-SOD mRNA detection, a DNA probe was used, which corresponded to nucle-
otides 1018 – 1211 in the cDNA sequence 22. For glyceraldehyde-3-phosphate dehydrogenase GAPDH
mRNA detection, a 1100 base pairs cDNA sequence Clontech, Palo Alto, CA, USA was used.
32
P-labeling was achieved by random priming Megaprime-DNA
labeling systems, Amersham, UK. To allow rehy- bridization, boiling 0.1 SDS was twice poured onto
the filters. Radiolabeled filters were exposed to imaging screens for 3 – 4 days and analyzed in a Molecular
Imager BioRad, Life Technologies, Hercules, CA, USA.
2
.
7
. Materials The following recombinant human cytokines were
obtained from Genzyme Corp, Boston, MA, USA: IFN-g,
TNF-a, IL-1a,
IL-3, IL-4,
granulocyte macrophage colony stimulating factor monocyte-
derived. Recombinant human IFN-a-2b was obtained from Schering Corp, Kenilworth, NJ, USA; recombi-
nant human IL-6 and IL-8 were obtained from RD, Minneapolis, MN, USA; recombinant human IL-2 was
obtained from Boehringer Mannheim GmbH, Ger- many, and recombinant human growth hormone was
obtained from Pharmacia – Upjohn Ltd., Stockholm, Sweden. Recombinant human TGF-b, E. coli lipo-
polysaccharide, Complement 5A, and platelet activat- ing factor were obtained from Sigma, St Louis, MO,
USA. Waymouth cell culture medium and FCS were purchased from Flow Ltd., Irvine, Scotland and
GIBCO BRL, Life Technologies Ltd., Gaithersburg, MD, USA.
3. Results
