the media HCMSMC. A different entity is the smooth muscle cell in plaque material HCPSMC. Some inves-
tigators who have compared the sensitivity of endothe- lial cells with other cell types constituting the vessel wall
have concluded that the endothelial cells are the most radiosensitive [7,18]. However, other studies could not
demonstrate major differences in survival following ir- radiation [19]. In addition, the experience is limited and
restricted to a few tissues, and differences are known from animal and human cells as well as in different
individuals [18]. Moreover, data about the radiosensi- tivity of smooth muscle cells isolated from human
plaque tissue are very limited [20,21].
Different investigations have been published on the determination of endothelial cell survival in vitro in-
cluding different end-points e.g. fraction of growth control, endothelial colonies, apoptotic bodies or cy-
toskeletal alterations. A prescreening system for poten- tial antiproliferative agents was established by Voisard
et al. [22 – 24] which allows the estimation of the amount of proliferating cells and changes of cytoskele-
tal cell structure within 48 – 120 h after exposition to various drugs.
Therefore, we investigated whether the proliferation of the predominant cells in coronary artery disease
HCAEC, HCMSMC, HCPSMC can be reduced by irradiation from a b-emitter i.e. by liquid Rhenium-188
in a balloon catheter. Furthermore, we wanted to clarify whether this effect is dose dependent and com-
parable or different in the various cell types. To com- pare the antiproliferative effect with cell damage we
investigated cytoskeletal components 18 h after irradia- tion by immunofluorescence microscopy.
2. Material and methods
2
.
1
. Radionuclide and dosimetry Carrier-free Re-188 was obtained from the W-188
Re-188 generator by elution with saline generator is available from the Oak Ridge National Laboratory,
Oak Ridge, TN. The radiotracer concentration was increased using anion exchange columns [25]. The high-
energy b particles E
bmax
= 2.12 MeV, mean energy of
764 keV allow therapeutic uses cintigraphy can be performed from the gamma emission of 155 keV 15
intensity. Radiation absorbed dose from a balloon catheter was measured by means of thermoluminescent
dosimetry TLD and compared to calculations using the point kernel function of Re-188 [26]. Very good
correlation was demonstrated for the absolute radiation absorbed dose and the fast drop of to 50 within 0.5
mm [26]. Assuming a specific volume of 3.7 GBqml at the surface of a typical balloon catheter 3.0 × 20, 135
ml volume and at 0.5 mm distance i.e. 2 mm distance from the center of the balloon a dose of 7.8 and 3.9
Gymin could be achieved, respectively [26].
2
.
2
. Cell preparation and cell culture Smooth musle cells from human coronary plaque
material HCPSMC, plaque material of 52 patients, smooth muscle cells from the human coronary media
HCMSMC, Clonetics-Bio Whittaker, Verviers, Bel- gique and human coronary endothelial cells HCAEC,
Clonetics-Bio Whittaker were sucessfully isolated HCPSMC,
identified and
cultured HCPSMC,
HCMSMC, HCAEC. HCMSMC and HCAEC of pas- sage three were used in the study presented. For isola-
tion of
HCPSMC an
enzyme mixture
of collagenaseelastase Boehringer Mannheim, Germany
was used [27,28]. Due to the fact that only very few cells could be isolated from coronary plaque material,
cells of all 52 patients had to be pooled for the investi- gations. HCPSMC and HCMSMC were identified as
smooth muscle cells by positive reaction with antibodies against smooth muscle a-actin Progen, Heidelberg,
Germany as described elsewhere [27,28]. HCPSMC and HCMSMC of passages two and three were used for
the investigations. HCPSMC and HCMSMC were cul- tured in smooth muscle basal medium SmBM, Clonet-
ics-Bio Whittaker, supplemented with 5 fetal calf serum FCS, insulin 5 mgml, fibroblast growth factor
2 ngml, endothelial growth factor 5 ngml, gen- tamycin 50 mgml and amphotericin 50 ngml.
HCAEC were cultured in endothelial basal medium EBM, Clonetics-Bio Whittaker, supplemented with
bovine brain extract 12 mgml, epidermal growth fac- tor 10 ngml, FCS 5, gentamycin 50 mgml, am-
photericin 50 ngml, and hydrocortison 1 mgml. HCAEC were subcultured as described [29]; HCAEC of
passages three to five were used for the investigations. Identity of HCAEC was confirmed by the typical ‘cob-
blestone-growth pattern’ and by positive reaction with antibodies against the von Willebrand Faktor Dako-
patts, Hamburg, Germany. Cell doubling times of HCAEC, HCMSMC and HCPSMC were 3 and 5 days,
respectively.
All incubations were performed at 37°C in a humi- dified atmosphere of air containing 5 CO
2
. Tissues were extracted with the patient’s informed consent and
the study was approved by the ethical committee of the University of Ulm.
2
.
3
. Application of irradiation Before
irradiation HCAEC,
HCPSMC and
HCMSMC were seeded in a density of 2 – 5 × 10
3
cells cm
− 2
in rectangular four-well dishes Nunc, Cologne, Germany. Irradiation with rhenium-188 was per-
formed 24 h after cell seeding. The balloon catheter was
placed into a distance holder parallel to and above the cell sheet with a reproducible distance of 0.22 mm Fig.
1. The specific volume of Re-188 was 890 9 172 MBq ml causing a mean dose at balloon surface of 2.0 9 0.4
Gymin which dropped to 1.5 9 0.3 Gymin at the level of the cell sheet opposite the balloon. These radiation
doses rule out the possibility that repair of sublethal damage could occur during exposure. Predicted dose to
the cells increased from 7.5 to 37.5 Gy by prolonging irradiation time from 5.1 9 0.9 to 25.7 9 4.4 mins. Ev-
ery irradiation time was compared with its own control to avoid errors by changes in pH due to CO
2
deficiency during irradiation. The filling condition of the balloon
catheter was estimated at the end of each experiment by gamma camera imaging [26]. Each experiment was re-
peated three times.
2
.
4
. Cell proliferation 18 h before fixation bromodeoxyuridine and d-cy-
tidine BrdU and d-cyt, 20 mM, Serva, Heidelberg, Germany was added, immediately after irradiation, to
the cultures of HCPSMC, HCMSMC and HCAEC. After fixation with methanol − 20°C, the cells were
stained with the avidin – biotin method. Anti-BrdU-an- tibodies were used as primary antibodies Dakopatts,
Hamburg, Germany, biotinylated horse anti mouse secondary antibodies were purchased from Camon
Wiesbaden, Germany. 3-Amino-ethyl-carbacote was used as substrate. The number of BrdU-positive cells
Fig. 2 was analysed in a strictly limited field directly opposite to the Re-188 filled balloon catheter 500 cells
for each single assay. This cell number was compared with the cell number of untreated controls = 100.
2
.
5
. Cytoskeletal components and 6on Willebrand factor
6 WF
To characterize the effect of irradiation with rhe- nium-188 on cytoskeletal components of HCPSMC,
HCMSMC and HCAEC passage two and three these cells were seeded at a density of 3 – 5 × 10
3
cells cm
− 2
in four-well dishes. One day after seeding the culture
medium was exchanged and cells were treated with rhenium-188 30 Gy at balloon surface, respectively,
22.5 Gy as already described. After 18 h cells were fixed in methanol for 6 min at − 20°C and indirect
immunofluorescence was carried out as previously de- scribed [30,31]. The following primary antibodies were
used in a concentration of 10 mgml: 1 monoclonal anti-smooth muscle a-actin Progen Biotechnik, Heidel-
berg, Germany; 2 monoclonal anti-vimentin Camon, Wiesbaden, Germany; 3 monoclonal anti-a-tubulin
Amersham Buchler, Braunschweig, Germany; 4 polyclonal von Willebrand factor Dakopatts, Ham-
burg, Germany. Tetramethylrhodaminisothiocyanate TRITC-labelled secondary antibodies goat anti-
mouse IgG and fluorescein-isothiocyanate FITC goat anti-rabbit were purchased from Dianova Hamburg,
Germany.
All cells were mounted in Mowiol 4-88 [30,31] and examined with a Nikon Optiphot microscope Nikon,
Fig. 1. Culture dish with device for the fixation of the rhenium-188 filled balloon catheter right and irradiated area as illustrated by autoradiography left.
Fig. 2. BrdU-positive cell, indicating mitotic activity within the last 18 h magnification: 650 in irradiated smooth muscle cells 22.5 Gy A; and untreated control B. The different number of BrdU-positive cells is clearly demonstrated.
Du¨sseldorf, Germany equipped with appropriate filter sets. The destruction of cytoskeletal components was
quantified in percent in comparison with untreated controls. For each investigation 100 cells were exam-
ined according to the following criteria: cytoskeletal structure ‘intact’ if the localisaton of smooth muscle-
actin, vimentin, and a-tubulin was completely normal and ‘changed’, if the structures were little or severely
destroyed.
2
.
6
. Statistical e6aluation Results are expressed as mean 9 standard deviation.
