[CANCER RESEARCH 32,77-82,January1972J
The Proliferation of Capillary Endothelial Cells'
Ian F. Tannock2andShigejiroHayashi3
Section of Experimental Radiotherapy, University of Texas, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025
SUMMARY
Recognition of capillary endothelial cells was facilitated by
a special
staining
procedure
developed
previously.
Thymidine-labeling indices for endothelial cells were estimated
in a number of unstimulated mouse tissues and were found to
be in the range 0 to 2 .4%. There was no increase in mean
labeling index up to 3 weeks after 2000 rads or up to 2 weeks
after 4000 rads irradiation of muscle, skin, or bone. After
fracture of the femur, the labeling index of capillary
endothelial cells in regenerating callus increased to about 10%
at 3 to S days after fracture and decreased to zero by Day 16.
The duration of DNA synthesis in the fractured femur was
estimated in a double-labeling experiment to be 7 ±2 hr. The
results of a repeated labeling experiment suggested a turnover
time of 80 ±25 hr on the 6th day after fracture, with a wide
distribution of intermitotic times about the mean value. At 3
days after injury, when the labeling index is higher, the
corresponding estimate of turnover time is about 50 hr: this
value is close to a previous estimate of turnover time for
capillary endothelial cells of a mammary tumor growing in the
same strain of mouse.
have investigated the proliferation of endothelial cells in mice
by the techniques of thymidine autoradiography. Data are
presented for endothelial cells in a variety of normal tissues, in
tissues damaged by irradiation, and in the callus formed after
fracture of the femur.
PROUFERATION
OF
ENDOTHELIAL
UNSTIMULATED TISSUE
CELLS
IN
Materials and Methods. Specific-pathogen-free C3H/He mice
were used in this and all other experiments. Mice of both sexes
were used ; they were 8 to 12 weeks old and weighed 25 to 30
g. The thymidine-labeling index was estimated for capillary
endothelial cells in a number of normal tissues. Five mice were
each given i.p. injections of 50 jiCi of TdR-3H4 and were
killed 1 hr later. The following tissues were excised and placed
in neutral formol-0.9% NaCl solution: liver, kidney, stomach,
small intestine (jejunum), skin, muscle (both cut from the
right leg), and right femur. Bones were decalcified, and 4-u
paraffin sections were cut on precleaned glass slides. Sections
were dewaxed, stained overnight in Luxol fast blue solution,
and then stained with the periodic acid-Schiff reaction (31).
Autoradiographs were prepared by dipping in Ilford K5
INTRODUCTION
emulsion, and exposure times of about 1 month were used.
Slides were developed in Kodak D 19 developer and fixed in
Kodak fixer. After being washed for I hr, the sections were
The proliferation of capillary endothelial cells plays a
stained with Harris' hematoxylin; they were left unmounted
central role in such diverse processes as wound healing, organ
because we have sometimes observed grain fading in mounted
transplantation, tumor growth, and the response of tissue to
radiotherapy. Indeed, the rate of extension of the capillary
sections.
bed may be the limiting factor for each of these processes,
Endothelial cells usually were heavily labeled (>20
inasmuch as the survival of any tissue is critically dependent
grains/cell), and background was low ( < 2 grains/cell).
Endothelial cells were recognized and included in cell counts if
on a supply of nutrients through the vascular system.
they were within the walls of blood vessels which were only 1
However, despite the extensive studies of cell proliferation in
many types of normal and malignant tissue, there have been
cell thick (i.e., endothelium of arterioles and venules was
excluded) and if the nuclei were long, thin, and completely
few systematic
investigations
of the normal rate of
surrounded
by red-stained
periodic acid-Schiff-positive
proliferation
of capifiary endothelial cells or of their
proliferative response to stimuli. In this study, we have used a cytoplasm. The presence of blue-stained erythrocytes within
special staining technique (29, 31) to obtain maximum
the lumen assisted with recognition of capifiaries; however, we
contrast between capillaries and other tissue elements, and we
included empty capillaries in the counts if we were certain of
their identity. Color photomicrographs of sections stained by
‘The
work was supported
inpartby USPHS ResearchGrantsCA the above method have been published elsewhere (29).
5047 and CA 6294.
Results. Estimates of labeling index for each tissue are
2 Recipient
of
a Research
Training
Fellowship
of
the
International
Agency for Research on Cancer, during the tenure of which this work presented in Table 1; the low values imply a slow rate of
was done. To whom correspondence should be addressed, at proliferation. Endothelial mitoses were rarely recognized in
Department of Radiology, Hospital of the University of Pennsylvania, the sections.
Philadelphia, Pa. 19104.
In many murine tissues, the duration of the S phase (Ta)
3Present address: Department of Radiology, Kyoto Prefectural
University
of Medicine,
Hirokouji,
Kawaramachi,
Kamikyo-ku,
Kyoto, has a relatively constant value in the range 7 to 9 hr (5, 24). If
Japan.
Received June 3, 1971; accepted August 27, 1971.
JANUARY
4The abbreviation used is: TdR-3H, tritiated thymidine.
1972
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77
Ian F. Tannock
Ts 15within
and Shigejiro Hayashi
this range for endothelial
cells (see also below),
approximate estimate of their turnover
obtained with the formula (28):
an
time (7') may be
L.I. = X(Ts)/(7)
(A)
Here A is a factor that for slowly proliferating populations has
a value close to log@2and L.I. is the labeling index.
For a labeling index of 0.4%, Equation A gives a turnover
time of about 8 weeks; for the higher labeling index of
endothelial cells lining the Haversian canals of the femur
(2.4%), the corresponding value of T is about 10 days. These
values must, however, be regarded as approximate as the
duration of the S phase was not measured. Also, precise
determination of labeling indices with values as low as those in
Table 1 requires the counting of very many cells, and
difficulties of recognition render this impractical.
PROLIFERATION OF ENDOTHELIAL CELLS AT SHORT
INTERVALS AFTER IRRADIATION
were anesthetized with sodium pentobarbitol, and radiation
was administered with a cesium irradiator, with the use of 2
opposed
sources at a dose rate of 1050 rads/min.
Thymidine-labeling indices for capillary endothelial cells in the
skin, muscle, and femur were evaluated from groups of 3 or 4
mice that were killed at intervals up to 3 weeks after
irradiation.
The techniques of histology and autoradiography were as
above. In this and subsequent experiments, slides were
randomized
to prevent subjective bias when scoring
autoradiographs.
Results.
Estimates
of labeling index for capillary
endothelial cells at intervals after irradiation are shown in
Table 2. No labeled cells were found on the 1st day after
irradiation, and DNA synthesis might be inhibited for a short
period. However, despite the use of high doses of irradiation,
there was no apparent damage to endothelial cells and no
increase in the rate of proliferation within the interval studied.
PROUFERATION OF ENDOTHELIAL
MECHANICAL INJURY
Materials and Methods. The right legs of 40 mice were
irradiated with a single dose of either 2000 or 4000 rads. Mice
Table 1
Labeling indices of endothelial cells in unstimulated tissues
of
of
cells
labeled
index
TissueNo.
countedNo. cellsLabeling
(%)Liver50020.4Kidney50020.4Stomach
mucosa)29400.0Small
(serosa and
mucosa)50020.4Skin
intestine (serosa and
leg)56220.4Muscle
(from the
leg)30000.0Bone
(from the
(Haversian canals and other5501
32.4capillaries
in the femur)
CELLS AFTER
Materials and Methods. Proliferation of endothelial cells in
regenerating callus after fracture of the femur was used as a
model system for studying the response to mechanical injury.
Mice were anesthetized with sodium pentobarbitol, and their
right femurs were fractured by gentle digital pressure at the
middiaphysis (17, 32). No attempt was made to splint or fix
the fracture . Callus formed rapidly around the sites of
fracture. Serial radiographs were taken, and the early callus
developed into bony callus after about 3 weeks. Sufficient
endothelial cells to determine labeling index could be found in
regenerating callus from the 5th day after fracture, and the
labeling index was estimated on Days 5, 7, 10, 13, and 16 after
fracture. Counts of 100 to 500 cells. were used to determine
labeling index, except on Day 16 when fewer endothelial cells
could be recognized. The probable labeling index on Day 3
Table2
Labeling indices ofendothelial cells after irradiation
Days after
2000 (%)1Muscle
radsTissueLabeling―
after
4000 radsTissueLabeling―
index
Skin
02Muscle Bone0
0
01Muscle
Skin
Bone0
0
Skin
25Muscle Bone0
1
32Muscle
Skin
Bone1
0
Skin
Bone0
0
35Muscle
Skin
Bone1
1
410Muscle
Skin
Bone0
0
512Muscle
Skin
Bone0
1
022Muscle
Skin
Bone0
0
4
a Each estimate
78
index (%)Days
is based
on counts
of 100 endothelial
cells.
CANCER RESEARCH VOL.32
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Proliferation
of Capillary Endothelial
Cells
Table3
Proportions of labeled cells in animals given injections of TdR-3H on Days 3 and 5
Forfurther
details,
seethetext.
Total
cells positive labeled
countedTotal cells%
cellsFive
2.5―Five
mice given injections on Day 3 and killed on Day 52766222.5
micegiveninjections
on Day 5 andkilled
on Day 73466418.5
±
±2.1
“Mean
±S.E.
cells, a repeated thymidine-labeling experiment was started on
the 5th day after fracture. Thirty mice were given repeated
injections of 25 jzCi TdR-3H at 6-hr intervals, and 3 mice were
killed 1 hr after each injection. Autoradiographs
were
prepared, and the proportions of labeled cells were determined
from randomized
sections.
Results. The relation between the labeling index of capillary
endothelial cells in the regenerating callus and time after
fracture
0
4
8
days o(ter
12
fracture
Chart 1. The relation between thymidine-labeing index of capillary
endothelial cells and time after fracture of the femur. ., values derived
from an experiment where there was a 48-hr interval between labeling
and death of the animal.
@
was estimated by giving 5 animals injections of TdR-3 H and
killing them 48 hr later on Day 5 ; the proportion of labeled
cells was compared with that for 5 animals given injections on
Day 5 and killed on Day 7.
The duration of DNA synthesis (Ta) of endothelial cells was
estimated on the 5th day after fracture by a double-labeling
method (1 , 3). Five mice were given injections of 10
pCi' 4C-labeled thymidine; this was followed 1 hr later by an
injection of 250 j.zCi TdR-3 H and the mice were killed 1 hr
after the 2nd injection. Sections were cut and stained and were
first autoradiographed by the usual procedure of dipping in
Ilford KS emulsion diluted 1: 1 in distilled water. After
exposure for 1 week, slides were developed, fixed, and stained
with hematoxylin. They were then immersed in an absorbing
solution [37.5% collodian (flexible) U.S.P., dissolved in a
solvent of ether:ethyl alcohol (3: 1)1 and autoradiographed for
a 2nd time in undiluted emulsion. After exposure for 4 weeks,
slides were finally developed and fixed. Grains from tritium
j3-particles appeared only in the lower emulsion, and the high
dose used ensured dense labeling of positive cells. Only
j3-particles from ‘
4C penetrated the absorbing layer to give a
spray of grains in the upper emulsion centered on the labeled
cell. Autoradiographic resolution was poorer for
C grains in
the upper emulsion than for tritium grains in the lower
emulsion; however, by adjustment of the focus of the
microscope, it was usually possible to recognize cells labeled
with 3H alone, with ‘4Calone, or with both isotopes.
For estimation of the turnover time of capillary endothelial
is shown
in Chart
1. The
mean
labeling
index
decreased from a mean value of about 9% on Day 5 to zero on
Day 16. When animals were given injections of TdR-3H on
Days 3 and 5 , and the proportions of labeled cells estimated
after 48 hr, the results were as shown in Table 3.
The proportion of labeled cells for animals killed on Day 7
at 48 hr after labeling is about twice the labeling index for
cells injected at the same time (Day 5), with animals killed
after 1 hr. An increase by a little less than 2 is to be expected
if all labeled cells divide shortly after labeling and then become
distributed around the cycle. The greater proportion of labeled
endothelial cells in animals given injections of thymidine at 3
days after fracture suggests a higher rate of proliferation at
that time; the 1-hr labeling index should then be about 11%
(Chart 1).
The results of the double-labeling experiment were as
follows: number of cells counted, 542; number of cells labeled
with both
isotopes
(N), 46; number
of cells labeled with 3H
alone (n1 ), 7; number of cells labeled with ‘
4C alone (n2), 8.
The probable duration of DNA synthesis may then be
calculated from the formulae:
(B)
Ts =(N+n1)/(n1)(N+n2)/(n2)hr
The best estimate is obtained by combining the 2 expressions
to give:
Ts(2N+ni
The standard
approximately
S From
equation
+n2)/(n1
+n2)=7.l
hr
(C)
error of the mean estimate of T@ is
2 hr.5 The estimated duration of DNA
C
Ts _ 1 =(2N)/(n1 +n2)
Thus
log@(T@_ 1) = log@2—log@[(n,/N) + (n2/N)J
Differentiating
@Ts
@(n,
IN)+ @(n,/N)
Tsl
(n,/PT)+(n2/N)
Binomial estimates for the standard errors of , IN and n2IN, ie.:
@(n,
IN ) = (@J;@@;
(N —,)/(N3'2)
give i@Ts 2.0 hr.
JANUARY 1972
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79
Jan F. Tannock
and Shige/iro Hayashi
synthesis, 7 ±2 hr, is within the range found for most mouse
tissues (5 , 24).
The results of the repeated labeling experiments are shown
in Chart 2 . By making some simplifying assumptions about the
distribution of phase times, it is possible to estimate turnover
time from these data. Since the method involves fitting a curve
to a range of points, the estimated value is more reliable than
that based only on the labeling index and duration of DNA
synthesis. The simplest model is to assume that the duration of
G1 , S, and G2 phases is constant; however, where distributions
of phase times have been estimated for slowly proliferating
tissues by the method of labeled mitoses, the distribution of
1 phase
time
in
particular
has
usually
been
found
to
be
rather broad . A more reasonable model is to consider that the
@
80
60
40
duration of S and G2 phases is constant but that cells are
triggered randomly into DNA synthesis from a pool of cells in
a resting (or G0) phase (4); this model is equivalent
to a very
wide distribution of G1 phase duration. A curve derived for
this model with parameter values T@ ‘@
hr, T@ 80 hr is shown
in Chart 2.
The theoretical curve of Chart 2 tends slightly to
underestimate the percentage of labeled cells at short intervals
after the first thymidine injection and to overestimate the
percentage of labeled cells at later intervals; this effect is
greater for a curve derived by assuming constant phase
durations. Small differences of this type between experimental
points and derived curves are to be expected because no
allowance was made for the decreasing rate of cell
proliferation over the 48-hr period of the experiment (Chart
1). The estimated value of turnover time is an average value,
probably representative for the 6th day after fracture. The
shape of the derived curve (Chart 2) is quite sensitive to the
value of T, once the value of T@ known; however, it is quite
insensitive to proportional variations in T and T@. Thus, since
the mean value of T8 and its standard error were estimated by
double labeling to be 7 ±2 hr, the corresponding mean value
of T and its standard error is about 80 ±25 hr. At 3 days after
fracture, when the labeling index is about 1 1%, the turnover
time is probably about 50 hr.
20
DISCUSSION
0
T
t
t
t
6
12
18
24
30
36
42
48
The
hours after first thymidrte injecton
present
capillary
Chart 2. The proportions of labeled capifiary endothelial cells in
regenerating callus following repeated injections of TdR-3H. The
estimates
endothelial
of
cells
in
thymidine-labeling
unstimulated
index
mouse
tissue
for
were
mostly in the range 0 to 0.4%, and values of turnover time are
pfl)bably about 2 months or more. Our mice, weighing 25 to
experiment was begun 5 days after fracture of the femur. Each point
30 g, grew slowly, but
represents a separate animal and was derived from counts of 50 to 200
cells. The curve was derived for a model derived in the text.
However, estimates of the distance between tattoo marks on
the legs have demonstrated
slight growth of bones in mice of
mainly
by accumulation
of fat.
Table4
Published estimates of thymidine-labeling index for endothelial cells in unstimulated tissues
LabelingTissueSpeciesindex
(%)ReferenceCapillaries
(9)Capillaries
of retinaMouse0.01Engerman
myocardiumMouse0.14Capillaries
of
(8)myocardium,
of endocardium,Mouse0.7—
epicardiumMouseAlveolar
and
(25)distinguished)Kidney
cells (types notMouse0.7Shimkin
et aL
1.9Edwards
and Klein
et at
rat
4-mo.rat0.9
0.25Phillips
(7)Capillaries
rat3.4Crane
arteriesAdult
(26)stated)Arteries
(anatomical site notRabbit0.6―Spaet
(23)Mesenteric
stromal cells4-wk
and Leong
and Dutta
and Lejnieks
veinsRabbit0.1—1.1―AortaRabbit0.003—0.08―Coronory
and
vesselsRabbit0bSpraragen
(27)AortaRabbit0.02Gaynor
(13)Large
kidney0.14—0.17AortaGuinea
vessels in lung and
(20)Abdominal
[email protected]@Florentin
et aL
pig0.4—2.7Payling
Wright
et aL (11)
a Labeling index estimated
after infusion of TdR-3 H for 7 hr.
b One labeled cell per 200 high-power fields.
c Labeling index determined
after in vitro incubation
with TdR-3 H
80
CANCER RESEARCH VOL.32
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Proliferation
@
@
this size (S. Hayashi, unpublished information); this growth
may be reflected by the higher labeling index (and presumably,
therefore, shorter turnover time) observed for endothelial cells
in the femur. In Table 4 we have listed published estimates of
thymidine-labeling index for endothelial cells in a variety of
species; most of the values obtained in mature animals are less
than 1%, confirming that unstimulated endothelium generally
has a slow turnover.
It has been proposed by many authors that capillary damage
might be responsible for some of the dose-limiting late effects
observed in normal tissues after radiotherapy. Changes in
capifiary function, such as penneability or blood flow, are
often observed shortly after irradiation (6, 15 , 18), but the
lysis of capillary endothelial cells is seldom seen. However,
Phillips (2 1, 22) has reported that rats irradiated with 1600
rads or more, and protected from acute death by hypoxia,
died at 3 to 6 months from acute radiation pneumonitis, with
complete loss of endothelium from the alveolar capillaries.
Strong correlations between visible capifiary damage and late
radiation necrosis of brain (19) or late myocardial fibrosis (10)
have also been observed.
From the present data and from values of labeling index
listed in Table 4, it appears that the turnover time of
unstimulated capifiary endothelium is typically a few months.
In some tissues an increased rate of proliferation has been
observed at short intervals after irradiation (2), but we found
no significant changes in labeling index up to 2 to 3 weeks.
Other authors (10, 16) have also found no tendency for
increased proliferation of endothelial cells at least up to 40
days after irradiation. The interval between irradiation and
observation of acute capillary damage might therefore reflect
the time for a critical number of damaged endothelial cells to
enter mitosis and then become pycnotic. This effect is
probably cumulative, with other endothelial cells being
stimulated to divide in an attempt to replace those that have
died at mitosis. Thus, Fajardo and Stewart (10) have observed
some increase in thymidine-labeling index at 40 to 70 days
after 2000 rads irradiation to rabbit hearts, and this increased
proliferative activity is followed by the appearance of
myocardial
fibrosis. Cumulative
pycnosis of capillary
endothelial cells could lead to complete breakdown of the
capillary circulation, and nutritional failure might then lead to
the delayed radiation necrosis of parenchymal cells that has
been observed in many tissues.
The maximum labeling index of capillary endothelial cells in
response to the mechanical injury of fracture of the femur
(about 11%) was the same as the labeling index observed
previously in a transplanted mouse mammary tumor growing
in the same C3H strain of mouse (30); the corresponding value
of turnover time was about 50 hr. In the tumor,
well-nourished carcinoma cells had a much greater rate of
proliferation and slowing of tumor growth resulted from
nutritional failure as the separation between neighboring
capillaries increased (30). In the regenerating callus, the
periostiocytes have likewise been reported to have a higher
rate of proliferation than endothelial cells (32). Thus the
proliferation of capifiary endothelial cells may be rate limiting
for both tumor growth and healing of fracture.
The relation between labeling index and time after fracture
(Chart 1) suggests that the proliferation of endothelial cells
of Capillary Endothelial
Cells
might be dependent on the release of a stimulatory factor (or
repression of an inhibitor) which then decays as healing takes
place. Continuous proliferation of endothelium in the tumor
at a rate equal to the maximal
response
to injury would
be
consistent with the continuous release of an angiogenesis
factor by tumor cells. There is now convincing evidence for a
stimulatory angiogenesis factor released by tumor cells. In
experiments where tumor pieces were implanted in Millipore
filters, vascularization in surrounding normal tissue was
observed (12, 14); implantation of normal tissue within such
filters gave negative results (12). The stimulatory factor was
recently isolated from several tumors and was found to be a
soluble molecule the important component of which was an
RNA-protein complex (12). However, if the same factor is
responsible for angiogenesis in response to injury, it must also
be present in normal tissue, or its production must be
stimulated by the injury. The lack of an acute proliferative
response
to damage by radiation
suggests that the release of an
angiogenesis factor might follow the expression rather than the
induction
expression
irradiation
of vascular damage.
With fracture
of damage is immediate
of the femur the
and acute, but after
it appears to be delayed and chronic.
ACKNOWLEDGMENTS
We thank Dr. Herman Suit for his advice and encouragement.
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CANCER RESEARCH VOL.32
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The Proliferation of Capillary Endothelial Cells
Ian F. Tannock and Shigejiro Hayashi
Cancer Res 1972;32:77-82.
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