Table 2 Effect of irradation with rhenium-188 to the cytoskeletal structures of smooth muscle-a-actin, vimentin, a-tubulin, and the von vWF in HCPSMC,
HCMSMC, and HCAEC
a
HCMSMC HCPSMC
HCAEC 22.5 Gy
Control Control
22.5 Gy Control
22.5 Gy 8119
– –
– –
vWF 8218
– 8911
928 –
928 SM-a-actin
928 1000
a-Tubulin 1000
1000 955
1000 1000
1000 Vimentin
1000 1000
1000 1000
1000
a
A total of 100 cells per structure were evaluated regarding damage values given as unaffecteddamaged.
dent manner after irradition with 7.5 – 37.5 Gy Table 1, Fig. 2. After irradiation with 7.5 Gy an inhibitory
effect of more than 40 was found P = 0.002; paired t-test, the maximal inhibition of almost 60 was seen
after irradiation with 37.5 Gy P B 0.001; paired t-test; Table 1. HCAEC were significantly P B 0.05 less
radiosensitive
in comparison
to HCPSMC
and HCMSMC after irradiation with 7.5, 15, and 22.5 Gy.
After irradiation with 30 and 37.5 Gy a significantly decreased effect P B 0.001 and P B 0.01, respectively
of irradiation on HCAEC was seen in comparison to HCPSMC, differences in comparison to HCMSMC
were not statistically significant.
3
.
2
. Alterations after irradiation of cytoskeleton and the 6on Willebrand factor
The HCAEC were identified by detection of vWF, the HCPSMC and HCSMSC by binding of the anti-
body against smooth muscle a-actin. Eighteen hours after Re-188-irradiation with 22.5 Gy the localisation of
smooth muscle a-actin, vimentin and a-tubulin did not show any alteration in comparison to non-irradiated
cells Fig. 3; Table 2.
4. Discussion
The earliest observations that blood vessels in irradi- ated tissues show specific changes were made 100 years
ago [32]. The radiation-induced effects in the various tissues and the multitude of changes of the blood
vessels in these tissues have been repeatedly reported and have been recently reviewed by Fajardo [33]. Dose
dependent gain and loss of tissue irradiation are issues of constant interest, especially since Bo¨ttcher et al. [34]
reported that irradiation with 12 Gy after angioplasty in peripheral arteries can reduce the risk of restenosis.
The response of mammalian cells to ionizing radia- tion has been extensively studied since the development
of techniques and growth media to sustain growth in vitro [19]. At high doses e.g. \ 10 Gy the dominant
cellular response is cell death. The predominant mecha- nism of radiation induced cell death results from chro-
mosomal damage by misrepair of pairs of double stand breaks either by a single electron track or by two
independent electron tracks. Following a dose of 8 Gy, the surviving cell fraction after 10 days is 1 which
means that only one in a 100 cells remains viable, in the sense that it can proliferate indefinitely [19]. This can be
demonstrated with a colony-forming assay [9]. How- ever, prerequisite for this assay is a homogeneous irra-
diation of the dishes which can be easily obtained by X-ray but not by irradiation from a balloon catheter
filled with a b-emitter. In this geometry, only a small field opposite to the catheter is irradiated with equal
dose and would be increasingly falsified by migrating cells during prolonged incubation time. However, if
radiation energy is delivered to a dividing cell, its effects are independent of the source used. That is, cell division
should be equally inhibited by X-ray, g and b energy as long as the energy is brought to the intended target. We
wanted to investigate the cellular effects of irradiation by the same technique of a radioactive filled balloon
catheter which is as well applied in vivo.
Radiation-induced apoptosis can occur at different timepoints following irradiation [35,36]. The earliest
radiation induced apoptosis happens during interphase without any requirement for cell division. In intestinal
crypts, cells undergo apoptosis in 3 – 6 h following ionizing radiation [35,36]. This process is very radiosen-
sitive but contributes very little to the radiation damage because only 2 of crypt cells become apoptotic. Most
of the cells destined to radiation induced damage com- plete at least one division and die after two or more
divisions [37], death mainly caused by chromosomal aberrations. Five days after incubation with diltiazem,
Voisard et al. [24] demonstrated a significant dose-de- pendent inhibition of cell proliferation for HCPSMC
and HCMSMC. Furthermore, the structure of the cy- toskeleton was damaged. We used an incubation time
of only 18 h. For this time window and the technique applied [23] no reference values were available. How-
ever, we could demonstrate a normal population dou- bling per day in controls of all cells used within this
time interval.
Fig. 3. Cytoskeletal architecture of HCAEC A – F, HCPSMC G – M and HCMSMC N – T: irradiation with rhenium-188 upper row of each set demonstrates no effect within 18 h to the structures of smooth muscle-a-actin G, N; a-tubulin C, I, P; vimentin E, L, S; and the von vWF
A, as compared with untreated controls.
While in our experiments, 18 h after irradiation the proliferation rate decreased dose dependently, the struc-
ture of smooth muscle a-actin, vimentin, and a-tubulin remained unchanged with no hints of destruction. As
mentioned above, early apoptotic death may occur 3 – 6 h following irradiation [35,36]. Cells undergoing apop-
tosis may completely disappear within 4 h [38]. Eigh- teen hours after irradiation, a few early reacting cells
might have already completed apoptosis. However, in our experiments this could not be proved definitely.
Moreover, most of the cells die in late apoptosis caused by chromosomal aberrations after two or more divi-
sions [37]. Cell doubling times of 3 and 5 days require investigations after 10 – 15 days following irradiation.
This corresponds to observations made by Han et al. [39] and Bochaton-Piallat et al. [40] that the number of
apoptotic smooth muscle cells becomes important be- tween 9 and 15 days after denudation of the rat aorta.
It has been reported that the proliferative activity of HCMSMC is higher than that of HCPSMC with popu-
lation doubling per day of 0.28 and 0.15, respectively [24]. The lower proliferative activity and the higher
amount of extracellular matrix and debris in cultures of HCPSMC seemed to be responsible for the higher
resistance of HCPSMC against diltiazem compared to HCMSMC [24]. However, in this study no difference in
radiosensitivity could be stated in both strains of smooth muscle cells. In addition, smooth muscle cells
were more radiosensitive than endothelial cells. These findings are supported by observations by Fischell et al.
[8] who found that proliferating bovine endothelial cells are more radioresistant after low-dose irradiation from
radioactive wires than HCSMC. Fischer-Dzoga et al. [7] observed some difference in radiosensitivity between
aortic medial and intimal cells as well. Nevertheless, Brenner et al. [19] could not demonstrate any difference
in radiosensitivity between endothelial and smooth muscle cells by survival data from a colony-forming
assay. It is always difficult to compare in vitro findings with the in vivo situation. However, if HCSMCs are
significantly more radiosensitive than endothelial cells, the radiation might inactivate all of the HCSMCs and
allow the endothelial cells to re-populate and re-line the artery after the trauma of angioplasty.
The very steep energy loss of the b-energy of Re-188 of 50 within 0.5 mm stresses the importance of identi-
cal irradiation conditions. Even a filling pressure of 3 atm instead of 6 atm would increase the distance from
the balloon surface to the cell layer by 0.07 mm or decrease the dose by approximately 10. However, g
camera imaging revealed a balloon filling of 131 9 14 ml corresponding to a mean balloon diameter of 2.96 mm.
Balloon volumes for irradiation of HCAEC were not different from irradiation of HCSMCs 134 9 8 and
130 9 17 ml, respectively.
5. Conclusion
