Editor-in-Chief: Dr. Henry F. Hoff

Editor-in-Chief: Dr. Henry F. Hoff
ISSN:0098-6127

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Histiocytes trapped after 7 days in an artif icial vessel replacement in -the
thoracic
aorta oi sheep. Courtesy of M. Mateika, University of Vienna, A 1090 Vienna,
Austria.
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ARTERY 8(3): 288-293 (1980)
ALTERATTON OF CITROI"IATIN IN EARLY EXPERII\TENTAI
ARTERIOSCI,EROS]S
\-r)
Rolf Lehroann.*, Robert Denesl, Rozsa Nienhaus2,
Thomas Stej-nbachJ, Gabor Lusztig4, and Reza M. Sanatger5
fnstitute of Arteriosclerosi-s Research at the University of
.
*ünster, FRG
'2nd Department of Pathology, Senmelweis Universj-ty
Budapest, Ilungary
)-Conputer Center, Universitlr of Münster, FRG
2
/Departnent of Patholory, üniversity of llünster, FRG
4Institute of Pathology, County Hospital,
ecskenlet, Hungary
/Department of Surgery, University of Münster, FRG
ABSTRACT Our approach serves several purposes: [o denonstrate
the response of aortic ceI1 nuclei to a
\-r
changing environment (e.g. hypercholesterolemia,
experinental diabetes, drug treatrnent), to
applicate the acrj-dine orange (AO) ultracytochenical
method, and to deterrnine the euchromatln/
heterochronatin (EU/HE?) ratio by morrphonetric
procedures as suitable criteria, and to enphasize
the differential nuclear activity of adventitialce1Is.
Hypereholesterolemia in rabbits results in
an increase of AO positive nuclei'in the adventitia.
Additional treatnent with tinofedriner a new
cerebral vasod.ilator drug, reveals an even higher
number of AO positive nuclei in adventitial and
also in endothelial and plaque cel1s. In streptozotocin
induced iliabetes in rats the increased
number of A0 positi-ve ce1ls coincides with an
elevated EV/HET ratio.
+ Present address: Department of Dentistry, l/aldeyerstr.JO
Universit:r of I'Iünster, D-44O0 Münster,FRG

ARTERY
INTRODUCTlON
In recent years i-t has becone apparent that the cellnucleus
has to be eonsidered as a hiqhly dynamie structure.
rfnc nrrnlcrrs nlevq u srr an innnrtent frrPv rnlc i4 the cOntrol and
integratlon of ce11 behavior and naintaininq the differentiated
state. The function of a nucleus is moCi-fied by a
variety of extracellular factors eventually resulting in
alterations of protein svnthesis includinfl exbracellul,ar
rnatrix components. This general concept of ce11 biologv
should also apply to ce11s of the artery wafl in differential
models of experinental arteriosclerosis. Therefore it nay be
exnected that ce11 nuclei are responsive to alteratlons of
thei r environrnent nediate.i bv h:rpercholesterofemia, experimental
diabetes or hypertension, and drug treatment.
It was the ain of this paper to eive a brief survey on
our current approach to visual"ize and determine cluantitativelv
alterations of nuclear activity in experinental
arteriosclerosis. Guided by two experimental models (hyoernhn'l
cstern'l eni rr\4t e . d urqvv i ahetcs') tue wi I 1 cremonstrate that the
apoficabion of the ultrac.-/tochenical acridine oranEe method
(1,? rt r4) and specifying the euchromatin/heterochromatin ratio
are suitable nethods with which to gain inforraation on
al-terations of nuclear activity.
v
I'IATERIAL AND }IETHODS
The specimens for the first nocle1 (hypercholesterolernia,
tinofedri-ne) were taken from 1! rabbits divi-cled in 3
experimental groups: Grouos A and B were fed a high
cholesterol diet (tq/aa-r) for 12 weeks. Group 3 additionally
received a daily in;ection of tinofedrine i.v., a new
cerebral vasodilator

ARTERY
rabbits was dissected, fixed in 5% glutaraldehyde at 4oC for
2 h at p\ 7.2, washed i-n O.1li phosphate buffer and incubated
with 1O-'M acridine orange. Further details of ul-tracytochemical
and electron rnj-croscopic nethod.s have been publ-ished
elsewhere (=r7rB).
For quantitative evaluations 5 consecutive sections were
cut from the same region of each aortie specirnen on I different
1eve1s beginning just below the orlficium. Between each
of two 1eve1s about'lOO pn of aortic tissue weie discarded,
to make sure that the next 5 ultrathin sections always
contained a neur ce11 population. Ultrathin sections were
mounted on a copper grid. with a hole of 1 mm in di_anetet. For
ee11 counting and eval-uation of the euchromatin,/heterochromatin
ratio sections were randonly selected. First the percentaEe
of acridine orange positive ce1ls was deterrnined.
Set:ondly I acridine orange oositive and negative ce11 nuclei
of each cel1 tlroe and level were photographed and the euchromatin,/heterochromatin
nortion of individual nuclei deternined
bw Quantinet. The data were then processed bv eomputer
nethods
RESUI,TS
The first exanple compares a1terations of the pereentage
of acri-dine oranqe (AO) Dositive cell nuclei in the aorta
below the orifieium of the A.renalis of nornal, hvpercholesterolenic,
and in add,ition to cholesterol tinofedrinetreated
rabbits (table I). In controf aninals only in the
medi-a and adventitia a few AO positive cell- nuclei are found.
Ä distinct increase of the number of ÄO positive cell nuclei
is observed i-n the adventiti-a of cholesterol fed aninals.
The most remarkable increase occurs in the.group of
tinofedrine-treated animals in the endothelium as well as in
the plaques (fig. 1). But also the number of AO
positive nuclei in the adventitia is elevated whereas in the
nedia no

ARTERY
TABIE I
Alterations of the Percentage of Äcricline Orange Positive
Ce11s in the Aorta below the Orificiun of the A. renal-is.
E- Endotheliun, M - iledia, A - Adventitia
* Ieve1 of Significance c = O.O5
Control
Cholesterol
+
Cholesterol Tinofedrine
E
M
rl.aque
3.4
2.4
n
1.1
18.9
8.7
45.4
z.ö
zö.2
44.5
TABI,E II
Alterations of the Euchromatin,/Heterochronaiin (EU,/I{ET)
Ratio in Advential Cel1s of the -dorta below the Orificiun of
the A. renali-s after 2 lrleeks of Experinental Dj-abetes (Ex)
Conpared to Control (C) Rats and the Percentage of Acridine
Orange positive (96 LO) Nuclei of the same Region and Animals.
Ex
Ex
EUlIINT
%LO
1.26 1.5a 2J6 2.26
- 12.6 23.1
as the second example. Here alterations of the euchromatin,/
heterochromatin ratio are compared with the percentage of AO
positive nuclei of adventitial cells in the aorta below the
orificiurn of the A. renalis of normal and streptozotocin
treated rats (TABLE II). The results show an increase of the
euchromatin/heterochromatin ratio under experimental diabetic
conditions. Ce11 nuclei labeled with electron-Cense AO
chronatirr interaction products always have a higher ratio
than non-labeled nuclei regardless whether they are derived
from control or experimental aninals. However, the hi-ghest
ratio is found in Ao positive ce11 nuclei of diabetic rats.
v
291

ARTERY
FTGURE 1
Superficial foan cel1 of an aortie plaque below the orificium
of the A. renalis after 12 weeks cholesterol rich diet anti
additi-onal treatnent with tinofedrine beginning with the lth
week of the experiment. The nucleus contains a hieh nunber of
eleetron-dense acridine orange chronatin interaction products
in,ticatinE inereased nuclear activity.
CONC],USION
The results show that both applicatj_on of AO uLtra_
cvtochemistry and deternination of the euchrornatin/heterochromati-n
ratio are a sui.table approach to further evaluate
the response of vascular eel1s to a chanqi-nq environmenr on
the 1eve1 of chromatin. Thus it is possible to localize and
to determine the number of individual cell nucrei resnonding
to extracellular factors.
ACKNOhILEDGEI{ENTS
The authors are indebted to Mrs. E. Gräfin von
Plettenberg anil to Mrs. M. pflugbeil for phot.ographic aid.
Tinofedrine was kindly donated by cheniewerk Honburg, ZN der
Degussa, Frankfurt,/Mai.n, FRG through the courtesy of
nn I. T^+-
REFXRENCES
1. Frenster, J.H. 1971. Electron microseopic localization of
acridine oraneie binding to DNA within hunan leukenic bone
marrow cells. Cancer Res. 11: 11Za-1j13.
292

ARTERY
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f)
e
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v_
a
293