Proline and Thymidine Uptake in
Rabbit Ear Artery Segments In Vitro
Increased by Chronic Tangential Load
WILLIAM R.
HUME, P H . D .
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SUMMARY Indices of structural change were examined in blood vessel walls subjected to increased tangential load in a new, in vitro model system. Ring segments of rabbit ear artery were maintained in organ culture
medium for times up to 9 days. Tangential load was chronically applied with small, intraluminal springs made
of 0.010 in. diameter stainless steel wire. The applied load was considered to produce lerels of circumferential
wall tension corresponding to those induced by a range of levels of blood pressure. The indices of stnicturai
change examined were the uptake of radioactively-labelled proline and thymidine, which indicate protein synthesis and cell division respectively. Increased uptake of both proline and thymidine was noted in artery
segments under elevated mechanical tension after a latency of 3 to 4 days. The degree of uptake was related to
the degree of calculated wall tension elevation. The work indicated that cell division and protein synthesis can
be induced in the blood vessel wall by increased wall tension alone, in vitro.
(Hypertension 2: 738-743, 1980)
KEY WORDS
• proline uptake
• thymidine uptake
• tangential load
T
HE blood vessels of the arterial tree thicken
during the development of hypertension. The
observed changes differ in degree among
different vessels and among various experimental
hypertensive animal models, but generally involve
both hypertrophy and hyperplasia of medial smooth
muscle cells, and increased synthesis and content of
mural scleroproteins.1"7 Similar structural changes occur in hypertensive human subjects;*' • the effects of
the change in structure on flow resistance have been
studied both in man and the spontaneously hypertensive rat.1*-" A hypothesis of the etiology of essential
hypertension was based on the observed thickening,
and on observed and theoretical changes in the
resistance to flow. Folkow and co-workers'- "•1B
proposed that adaptive, structural changes following
intermittent episodes of hypertension may cause increased peripheral resistance and therefore the establishment of maintained hypertension, in susceptible
individuals. These workers showed that an increase in
• in vitro • artery segments
the wall thickness/lumen ratio alone could account for
the observed increase in flow resistance at maximal
dilation.
Flow resistance increase due to enhanced medial
contractility was also proposed. The large body of
evidence favoring this hypothesis has recently been
reviewed by Folkow." The observed change in structure has implications beyond this proposed
mechanism. There is potential for alteration in a wide
range of nerve-, hormone- and drug-mediated effects
on vascular diameter. The transfer of nutrients and
metabolites between the blood and other tissues may
change with the increase in wall thickness of small
blood vessels. It is also possible that change in arterial
structure favors the later development of mural
pathology.19
The various animal models of hypertension have
provided the basis of our present understanding of
hypertensive structural change. However, complex
variations in both autonomic motor nerve activity and
in the levels of several humoral factors very probably
occur during the phase of pressure increase, and such
variations may influence the development of the
change in structure. The onset time and the degree of
pressure rise are also difficult both to predict and to
measure in animal models. For these reasons, I have
developed a simplified in vitro model that avoids the
influence of nerve and hormone activity, and in which
both the mechanical and the humoral conditions in-
From the Section of Oral Biology and Department of Pharmacology, Center for the Health Sciences, Los Angeles, California.
Supported by American Heart Association, Greater Los Angeles
Affiliate Grant 597, and by National Heart, Lung and Blood
Institute Grant HL20581.
Address for reprints: Dr. W. R. Hume, Section of Oral Biology,
UCLA School of Dentistry, The Center of the Health Sciences, Los
Angeles, California 90024.
Received October 27, 1979; revision accepted May 16, 1980.
738
LOADED ARTERY SEGMENTS IN WJKO/Hume
739
fluencing blood vessel segments are precisely controlled and may be rapidly and readily changed. While
there are obvious limitations which must be considered when interpreting data derived from studies
using in vitro models, such data when viewed with that
generated by the study of animal models and man can
contribute greatly to the understanding of biological
mechanisms.
Mechanical Considerations
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The following is not intended to imply quantitative
equivalence between mechanical conditions in the
vessel wall in vitro and in vivo but rather to outline the
qualitative analogy on which the present model is
based.
For a blood vessel segment of length (1) and radius
(r), the tangential load (W), which will produce circumferential wall tension (Tc) corresponding to that
at a particular level of blood pressure (P B ), can be expressed as follows. The relationship between blood
pressure, circumferential wall tension, and radius in a
blood vessel can be expressed as:
Tc = PBr, - P o r o
where P o is the outside pressure and r! and r0 are the
inside and outside radii respectively. If P o is negligible,
then20
Tc = P B r, . . .
(1)
But in a blood vessel ring segment held under load, as
shown in figure 1, wall tension may be expressed as:
Tc =
W
(2)
"IT
From equations (1) and (2),
FIGURE 1. Top and center: two spring types; the base
spring with a ring segment of artery tissue on its tips and below, and an auxiliary spring. Bottom: Oblique view showing the relation between the artery segment, the base spring,
and the auxiliary spring.
W
2-1
i.e.,
W = 2 PB-1T,
...
(3)
For example, in segments 0.1 cm in length and 0.1 cm
in diameter, the spring load giving circumferential
wall tension equivalent to that generated by a mean
pulse pressure of 100 mm Hg (1.3 X 105 dynes/em 2 )
can be calculated from equation (3),
W =
2.PB-1T,
= 1.3 X 103 dynes.
Spring Fabrication and Design
Springs were handmade from 0.010 in. (0.25 mm)
diameter stainless steel wire (Unitek Corporation) according to the designs shown in figure 1. The springs
were made to deliver the required loads at an opening
distance of half the circumference of the vessel
segments. The load delivered by each spring or pair of
springs was measured with a force transducer. One
spring type (the "base" spring) was designed to accept
a ring segment of blood vessel at its tips, as shown,
and was made to deliver a load that would generate
wall tension analogous to normal blood pressure. The
spring was made so that a second ("auxiliary") spring
could be added, placing additional, known load on the
vessel segment analogous to a higher level of pressure.
With one spring the segment was under mechanical
conditions analogous to normal blood pressure; with
two it was under conditions analogous to some degree
of high blood pressure. Several recognizably different
designs of a second spring were made to deliver
different loads. The auxiliary spring mechanism was
included in the experimental design so that the in-
HYPERTENSION
740
creased load could be applied at a time well removed
from that of dissection, transfer to culture medium,
and other manipulation.
Methods
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Seventeen young adult New Zealand white rabbits
of either sex, 1.8 to 2.3 kg in weight, were stunned and
killed by exsanguination. The central artery of each
external ear was removed using aseptic surgical
technique, the segment removed being that extending
from the artery's emergence from the muscular layer
at the base of the ear to a point approximately 5 mm
distal to the major side-branch of the ear vein. The
artery segments were placed in tissue culture medium
(Medium RPMI 1640, with 20% fetal calf serum and
gentamycin 50 Mg/ml> a ' l fr°m Microbiological
Associates, Inc., Los Angeles). All tissue-handling
procedures subsequently described until weighing of
tissue samples were carried out using aseptic technique and a limited access, glass-covered, working
area.
The artery tissue was divided into ring segments 2-3
mm in length, and each segment was placed onto the
tips of a spring as shown in figure 1 (bottom). Fourteen to 16 segments of ear artery were obtained from
each rabbit, and were subsequently treated identically
except for the degree of wall tension induced. The
segments and springs were kept in the culture medium
described above in air containing 5% CO a at 95%
humidity and a temperature of 37.5°C. After 1 day or
more, auxiliary springs were added to approximately
three-quarters of the base springs, inducing a range of
wall tension levels in the group of segments. The
culture medium was completely changed every 2 days.
After periods ranging from 1 to 8 days following
placement of the auxiliary springs, the culture medium
was replaced with the same medium containing both
tritium-labelled proline ('H-proline) 0.5 /*Ci/ml (proline 2,3'H, 20 Ci/mmole, New England Nuclear) and
"carbon-labelled thymidine ( u C-thymidine) 0.02
jtCi/ml (thymidine 2- u C, 51.6 Ci/mmole, New
England Nuclear). After 24 hours under the same incubation conditions, the springs and segments were
rinsed in three changes of the medium without label
over a further 24 hours. The segment lengths were
then measured, and each segment was weighed,
digested in 0.2 ml of Soluene (Packard) overnight at
60°C in a glass scintillation vial, and 'H and U C determined using a Beckman LS 100c liquid scintillation
spectrometer. The scintillant contained Spectrafluor
PPO-POPOP (Amersham/Searle) 4.2% and 15%
methanol in toluene. Counting efficiency was 58% for
S
H and 72% for M C. The uptake of labelled proline and
thymidine into each segment was calculated and expressed as moles incorporated per milligram of wet
weight of tissue per hour.
For each group of segments the relationship
between'the uptake of each labelled compound and the
induced wall tension was examined by a plot-scatter
diagram of paired parameters. Pearson's productmoment correlation coefficient (r) was calculated for
VOL 2, No 6, NOVEMBER-DECEMBER
1980
each group, and the p value for testing the statistical
significance of the correlation coefficient was determined from table A-30a of Dixon and Massey.21
Results
The load delivered by each spring or pair of springs
was measured both before and after each experiment.
No differences were detected in the load delivered
between the beginning and end of individual experiments, i.e., no fatigue of the springs was apparent.
The rabbit ear artery segments took up *H-proline
from the incubation medium over a range from
1 X 1 0 " to 1.2 X 10-" moles/mg wet weight of
tissue/hr, and 14 C-thymidine from 7 X 10"" to
5.2 X 1 0 " mole/mg wet weight of tissue/hr. Segments pre-treated with 0.1 M potassium ferricyanide
for 1 day before such exposure took up less than 0.5%
of the lower of the values for each labelled compound,
the absolute counts being at or slightly above (less
than two times) background.
The results of an experiment where a group of
segments from one rabbit were maintained under load
for 5 days before the addition of label are shown in
figure 2 (proline uptake vs circumferential wall tension) and figure 3 (thymidine uptake vs circumferential wall tension). It was apparent that, as wall tension
increased, so did the uptake of proline and thymidine.
The values for r and p are shown.
Table 1 summarizes the results of all experiments,
arranged in sequence by the number of days the
segments were under load. There was no statistically
significant increase (95% confidence level) in the uptake of proline or thymidine in groups of segments
maintained under load for 1, 2, or 3 days. Such increase was apparent in all segment groups maintained
for 5 days or longer. No differences related to the sex
of the donor animal were observed.
Discussion
The studies described showed first, and most
simply, that segments of rabbit ear artery remained
viable under the conditions described for up to 9 days.
That the uptake of the labelled compounds used was a
valid index of tissue life was confirmed by treatment of
a number of segments under load with 0.1 M
potassium ferricyanide, a metabolic poison, for 24
hours in the standard medium before the addition of
label. The amount of *H and MC remaining in these
segments after rinsing was less than 0.5% of the usual
values, and was not influenced by circumferential wall
tension. While it was found in the present work that
the uptake of label fell slightly with increasing total incubation time over the time range examined, i.e., that
the rate of the metabolic processes declined, comparisons such as were made between segments within a
group were valid. An examination of conventionally
prepared hematoxylin-and-eosin-stained sections of
several segments of rabbit ear artery treated for 7 days
on springs in the culture system described showed
light-microscopic cellular morphology similar to that
in tissue treated for less than 1 hour on springs (fig. 4).
LOADED ARTERY SEGMENTS IN VITRO/Hume
741
5
\
I
w
6_
I
1"
s
r-.6O5
p<.025
5
a
a
fasH
2I
15
20
10
Circumferential Wall Tension
(Dynes/cm x 103)
1^
o
r-.769
p<.00S
IS
10
20
Circumferential Wall Tension
(Dyn«s/fcm x 103)
25
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FIGURE 2. Relationship between the circumferential wall
tension and the incorporation of tritium-labelled proline into
segments of rabbit ear artery maintained with spring load in
culture medium for 5 days.
FIGURE 3. Relationship between the circumferential wall
tension and the incorporation ofucarbon-labelled thymidine
into segments of rabbit ear artery maintained with spring
load in culture medium for 5 days.
In segments loaded for more than 4 days, variation
in the level of circumferential wall tension influenced
the uptake of both proline and thymidine directly. We
demonstrated previously7 that the in vitro uptake of
proline into blood vessel segments was directly related
to the incorporation of the amino acid into tissue pro1
tein, and was therefore a valid index of new protein
synthesis. It may therefore be concluded that the present study demonstrated an increase in protein synthesis rate in artery segments subjected to increased
wall tension for 5 days or more. Similarly, the increased incorporation of labelled thymidine into the
same segments indicated an increase in rate of cell
division, since thymidine is incorporated into cells synthesising DNA, prior to mitosis." While not identifying which cells were involved, the study showed that
increase of circumferential wall tension alone induced
increased cell division and protein synthesis in a model
system totally removed from the donor animal. The
humoral environment was identical for all segments of
a group, and all segment were functionally denervated.
Some previous studies using hypertensive animal
models have indicated that increased local blood
pressure, rather than humoral or nerve-mediated factors, induced structural change. In the rabbit, with
part of its circulation hypertensive and part normotensive, structural change occurred in arterial vessels
subject to increased pressure but not in those in the
normotensive circulation or in veins,8' '• "• " indicating that the change was in response to a primary drive
of local pressure increase. Antihypertensive drug
treatment was shown to prevent structural change induction in the SHR;S>" collagen biosynthesis, a sensitive index of structural change, increased in the
arteries but not in the veins of both the spontaneous
and the DOCA-salt hypertensive rat.2" The possibility
that some systemic factor, in addition to local pressure
rise, is necessary to induce structural change was not
excluded by the above studies. There is recent evidence
for structural change independent of pressure rise in
the normotensive vessels of both the SHR and the rat
with coarctation hypertension, indicating that
humoral factors alone can induce structural
TABLE 1. Incorporation of Labelled Proline or Thymidine
versus Induced Circumferential Wall Tension for Group* of
Segments of Rabbit Ear Artery
Proline
Days'
rt
nt
1
1
2
2
3
3
3
4
4
-0.123
0.096
-0.051
-0.196
0.169
0.383
0.392
0.118
0.232
0.497
0.652
0.605
0.590
0.570
0.795
0.781
0.728
13
13
15
15
11
16
15
14
14
13
14
14
14
5
5
5
5
6
7
7
8
15
15
12
12
Thymidine
Pi
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
<0.05
<0.01
<0.025
<0.05
<0.05
<0.005
<0.005
<0.005
r
n
V
0.399
0.032
0.432
0.420
0.349
0.148
0.637
0.513
0.569
0.769
15
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
<0.01
<0.05
<0.025
<0.005
0.828
0.715
0.564
0.527
15
11
16
15
14
14
13
14
14
<0.005
15 < 0.005
12 <0.05
12 <0.05
15
*Number of days for which the group of segments was
maintained -with increased load before the addition of label.
•[Correlation coefficient.
JNumber of segments, each from one rabbit.
§Probability that the slope of the regression line differs from
742
HYPERTENSION
VOL 2, No
6, NOVEMBER-DECEMBER
1980
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FIGURE 4. Transverse sections of rabbit ear artery segments treated on springs as described in the text and
stained with hematoxylin and eosin. Intima (I), media (M), and adventitia (A). a) Segment treated for 7 days
before removal and fixation. The arrows show the tissue adjacent to the loading springs. Scale, 100 fi. b)
Higher-power view of the same section, in a region away from the springs. Scale, 50 p. c) Segment treated
for 30 min before fixation. Scale, 50 p.
change.27' u The present results show conclusively,
however, that some degree of structural change in
hypertensive blood vessels can be attributed solely to
the local effects of increased pressure.
The study also gave an indication of the latent
period between increase of wall tension and increase of
the metabolic indices of structural change. Folkow et
al.18 noted signs of vascular structural adaptation in
the renal artery-clipped rat 1 week after surgery.
Bevan8 reported an increase in tritium-labelled
thymidine incorporation in histological sections of
arteries from the hypertensive circulation of the
PAAC rabbit 6 days after surgery, at a time closely
following the first observed pressure rise. In the SHR,
and in man, the onset of hypertension is gradual, but it
appears likely in light of the observations above that
change in structure follows pressure rise relatively
closely in time. In the present work, statistically
significant increase in proline and thymidine uptake
was seen only after 4 days of increased load. The duration of load increase, and not the time in culture
medium, was the determining factor, since maintaining groups of segments for 4 days before placement of
the auxiliary springs did not alter the threshold for
observation of the increased parameters. It is likely
that quantitative differences would exist between the
latent period seen in the present study and the latent
period obtaining in vivo. However, it can be stated
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that there is a strong likelihood that structural change
induction does not occur until a period of time
measurable in days has elapsed following an acute and
sustained rise in blood pressure. The effects of intermittent, acute increases in circumferential wall tension
on induction of structural change have yet to be determined.
The possibility exists that the increased uptake was
a response to injury in the segments which varied with
spring load, particularly since histological examination of segments treated for 7 days showed evidence of
cell death in the spring contact areas. Experiments
were therefore made in which the segments were
treated as described above for 6 days under a range of
load levels, and after treatment with the labelled compounds were cut longitudinally to discard that tissue
adjacent to the spring. The pattern of uptake in the remaining tissue was the same as that seen in the wholesegment experiments, indicating that the springrelated damage was unlikely to be the determining
factor of the increase in uptake.
Leung et al.Wi *° demonstrated that cyclic stretching
of smooth muscle cells grown in tissue culture on an
elastin membrane increased the synthesis of collagen
and other matrix components by those cells. The present model has some similarities to that of Leung et al.
in that cells are maintained in vitro and are subject to
mechanical load. The principal difference is, however,
that in the present model the relationship between
cells in the vessel wall resembles the in vivo situation
more closely. The model system has potential
usefulness for further biochemical and morphological
studies of the phenomenon of induction of structural
change in blood vessels by increased circumferential
wall tension, particularly for those studies requiring
acute and accurately measurable changes in wall tension and/or change in the humoral environment.
10.
11.
12.
13.
14.
15.
16.
17.
.
18.
19.
20.
21.
22.
23.
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W R Hume
Hypertension. 1980;2:738-743
doi: 10.1161/01.HYP.2.6.738
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