DNA Synthesis and Mitoses
in Coronary Collateral Vessels of the Dog
By Wolfgang Schaper, Marc De Brabander, and Paul Lewi
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ABSTRACT
DNA synthesis and mitotic activity in coronary collateral arterioles was
assessed in dogs at different time intervals after gradual ameroid constriction of
the left circumflex coronary artery. Labeling of nuclei and the mitotic index
were highest at 3 weeks after implantation of the constrictor and. gradually
declined thereafter. Labeling persisted for at least 8 weeks but radioactive
DNA was not found 12 months after coronary artery constriction nor in controls
or animals with sham operations. Proliferative activity was, at any time
interval, highest at the level of the smallest diameters of the collateral vessels.
Labeled nuclei and mitoses were found in endothelial, medial, and adventitial
cells. Myocardial mesenchymal cells also incorporated tritiated thymidine. The
data provide evidence that after constriction of a major coronary artery, the
coronary collateral vessels enlarge by an active growth process that follows the
basic laws of cell kinetics.
KEY WORDS
autoradiography
DNA synthesis
ischemia
cell division
coronary collateral circulation
ameroid constriction
vessel wall growth
• Myocardial ischemia leads to an enlargement of collateral vessels in man (1-5) as well
as in animals (6-10). However, uncertainties
exist as to the mechanism of this increase in
vascular caliber. Early but scanty observations
(11) showed that the collateral vessels are
overstretched arterioles 'lacking an arterial
coat" and state that these vessels are preexistent and open up under the influence of
pressure gradients and metabolic and neurogenic factors (12). However, several reports
from our laboratory (7,13-21) have suggested
that collaterals grow actively rather than
expand passively when the surrounding cardiac muscle is made ischemia These observations were based on the chance finding of
smooth muscle cells in mitosis and on the fact
that the volume of the vascular wall increases
(14,17).
The final and formal evidence ~of arterial
growth in the presence of myocardial ischemia
was, however, never established. The material
From the Cardiovascular Department of Janssen
Pharmaceutica, Research Laboratoria, B-2340, Beerse,
Belgium.
Received November 26, 1970. Accepted for
publication March 17, 1971.
Circulation Research, Vol. XXVlll,
Ju
1971
presented in this study provides evidence that
DNA synthesis and mitoses are responsible for
the growth and expansion of collateral vessels.
This was achieved by the detection of
radioactive DNA in enlarged collaterals of
dogs with chronic coronary artery occlusion
after injection of a labeled precursor of DNA,
tritiated thymidine. Furthermore, the determination of the labeling index as a function of
time after experimental coronary artery occlusion made possible an estimate of the speed
with which arterial smooth muscle cells are
able to reproduce.
Method
Details of the surgical procedure have been
described elsewhere (17). In brief, ameroid
constrictors (22) were implanted around the
circumflex branch of the left coronary artery of
randomly selected mongrel dogs of either sex with
an average weight of about 18 kg. The ameroid
plastic is a hygroscopic material which swells
with uptake of tissue fluid. Since the material is
encased by a stainless steel ring, it constricts the
coronary artery over a period of 2% weeks (7).
After the implantation, which was carried out
under anesthesia and artificial respiration using
clean surgical techniques, the animals were
allowed to recover. Three, 4, 8, and 52 weeks
after the operation, groups of dogs were again
671
SCHAPER, DE BRABANDER, LEWI
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•
•
<
Arteriographic demonstration of a large coronary
collateral bypassing a chronic coronary artery occlusion.
The collateral is divided into stem (S), midzone (M),
and reentry (R) by the heavy bars. Fine and heavy
bars indicate the borders of vascular tissue blocks.
The plane of sectioning is always at the reentry end
of the blocks. LAD =; left anterior descending coronary
artery; LCCA = left circumflex coronary artery; AC
= constrictor.
anesthetized, the chest was opened under
artificial respiration and a heart-lung preparation
was set up. Three millicuries of tritiated
thymidine1 were injected into the venous reservoir
and the heart-lung preparation was continued for
approximately 3 hours. Then the venous reservoir
was emptied and the heart was perfused with
oxygenated Tyrode's solution to remove as much
blood and free radioactivity as possible. The heart
was then fixed by perfusion with 4% phosphatebuffered formaldehyde, rinsed again with Tyrode's solution to remove the non-tissue-bound
formaldehyde, and a suspension of micronized
barium sulfate in a 9% gelatin solution was
infused into the coronary arteries via the aorta
under a pressure of 100 mm Hg. The white
barium mass made enlarged collaterals clearly
visible to the naked eye, but arteriograms were
also taken in order not to miss endomural
collaterals (Fig. 1). All identifiable collaterals
lObtained from the Radiochemical Centre, Amersham, U. K.
were excised over their entire length from stem to
reentry into the recipient artery.
According to Longland's definition (23) the
"stem" is a subbranch of a coronary artery from
which the collateral artery originates. The
"midzone" is the part of the collateral located
between stem and reentry and is expected to
show the most obvious and dramatic growth
transformation because of its very small initial
diameter (40 fim on the average). The reentry is
a subbranch of the occluded coronary artery.
These collaterals were cut perpendicular to their
longitudinal axis and divided into small blocks of
about 5 mm. Paraffin sections of about 6 ttm were
cut and stained with PAS and hematoxylin-eosin.
The sections prestained with PAS were coated in
the darkroom
with liquid nuclear emulsion
(Ilford L 4 ) 2 and stored in light-tight containers
according to Schaper and Van Even (24) for 4
weeks at 4°C. After exposure, the slides were
developed for 3 minutes in Kodak D19 developer,
fixed, washed and counterstained with Harrishematoxylin.
Radioactive DNA was made demonstrable by
reflected light microscopy according to Rogers
(25) with the Leitz Ultropak System.
In six animals, demecolcine (Colcemid)3 in a
dose of 0.5 mg/kg was injected intravenously
approximately 3 hours before the heart-lung
preparation was started.
In addition to the 11 animals with chronic
coronary artery occlusion, 3 normal dogs and 3
dogs with sham operations were used as controls.
In dogs with sham operations, the same surgical
procedure was followed with the exception of the
constrictor implantation. Three weeks after the
sham operation, a heart-lung preparation was set
up as described above, and the same amount of
tritiated thymidine was added to the venous
reservoir.
Identical heart-lung preparations were made in
the three normal dogs. In the normal dogs and in
the dogs with sham operations, vessels comparable in size with enlarged collaterals (between 400
and 1600 /jjn) and vessels from the nonenlarged
arteriolar collateral network were excised and
processed as described above. In addition to, the
two types of control experiments, normal vessels
(i.e., unrelated to collaterals) from nonoccluded
coronary arteries were taken from the hearts with
chronic coronary artery occlusions. These vessels
were also processed for autoradiography.
Determination of the Labeling Index.—Labeled nuclei are easily identifiable in emulsioncovered histological sections with reflected light
microscopy (Fig. 2). All labeled nuclei in each
2
Ilford,
3
Essex, England.
Ciba AG, Switzerland.
Circulation Research, Vol. XXVIII,
June
1971
673
DNA SYNTHESIS IN CORONARY VESSELS
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FIGURE 2
Autoradiographic demonstration of radioactive DNA in nuclei of coronary vascular smooth
muscle cells in part of a collateral vessel studied 3 weeks after constrictor implantation.
Clusters of developed silver grains (bright white) indicate the presence of radioactive DNA
in 3 nuclei of smooth muscle cells. The tissue was stained with periodic acid-Schiff and
counterstained with Harris-hematoxulin. The section was photographed with transmitted
light (tissue) and with reflected light (silver grains) illumination. The lumen (L) is filled with
micronized barium sulfate-gelatin.
vascular tissue section (thickness, 6 am) were
counted, listed, and classified as endotnelial cells,
smooth muscle cells, adventitial fibroblasts, and
myocardial mesenchymal cells. All nuclei of the
tunica intima and of the tunica media, labeled
and not labeled, were also counted and classified
into these cellular categories. The labeling index
is defined as the ratio of labeled to nonlabeled
nuclei times 100. Mitoses were counted in a
similar way (Fig. 3), and the mitotic index was
calculated as the ratio of mitoses to total
number of nuclei times 100.
The relation between the labeling and the
mitotic index was determined from a midzone
tissue block from which 40 serial sections were
cut. Section 1 was used for autoradiography and
the labeling index was determined. Section 2 was
stained with hematoxylin-eosin and the mitoses
were counted. Section 3 was used for autoradiography, etc. This alternating procedure was found
necessary because mitoses are less readily detectable in emulsion-covered sections.
Results
Labeling Index as a Function of Time —
Circulation Research, Vol. XXVIH, June 1971
The largest number of labeled nuclei was
found in the group of animals 3 weeks after
constrictor implantation (Figs. 4 and 5). In
animals studied at later sampling intervals (4
and 8 weeks after the operation) labeling had
declined.
Parallel with the decrease in the number of
DNA-synthesizing cells, the total cell population of the collaterals rose from an initial value
of about 30 cells/6 /xm tissue section to about
300 cells 8 weeks after constrictor implantation (Fig. 6). The decline in the number of
DNA-synthesizing nuclei is statistically significant (P = 0.0040) when the dogs studied 3
weeks after constriction are compared with
those studied 8 weeks after constriction.
Labeled nuclei could not be detected in dogs
studied 1 year after the operation, nor were
they found in normal dogs or in dogs with
sham operations. Labeled nuclei were also
lacking in the normal coronary vessels of the
SCHAPER, DE BRABANDER, LEWI
674
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FIGURE 3
Mitosis of a smooth muscle cell in metaphase (arrow). Tissue was stained with hematoxylineosin. Section was taken from a collateral vessel of a dog heart 3 weeks after implantation of
the constrictor.
animals with chronic coronary artery occlusion.
The percent of labeled nuclei over the
entire length of the excised collaterals at the 3week sampling interval was -3.5% of all
endothelial and medial cells. The percent of
labeled midzone nuclei was 5.5S5, indicating
that the frequency of DNA-synthesizing nuclei
falls off toward both ends of the midzone. The
highest frequency of labeling ever observed in
a 3-week vascular segment was 8%, an average
of 20 serial sections taken in sequence from a
midzone block. The highest absolute number
of labeled nuclei in a single 6 /tin section of a
collateral midzone was 43 labeled nuclei in a
vessel wall with an internal diameter of
310 fim.
Labeled nuclei were also found in the
adventitia and in the myocardium surrounding the growing vessels. These cells could
easily be identified as fibroblasts when found
in the advential space or as mesenchymal
myocardial cells. However, labeled cardiac
muscle cells could not be identified with
certainty. Occasionally it was possible to
identify radioactive DNA in the nuclei of
myocardial capillaries. In these cases, identification was aided by the "buckshot" appearance of the perfusion-fixed tissue: all capillaries were fixed in the widely patent state.
Frequency of Mitoses as a Function of
Time.—Six animals received demecolcine,
which stops mitoses in the metaphase. Only
nuclei in the metaphase were counted in
hematoxylin-eosin stained sections or in sections with Weigert's hematoxylin-elastica van
Gieson. Although the time during which
demecolcine could halt mitoses was about 6
hours, the number of counted mitoses was
surprisingly low. At the 3-week sampling, 23
mitoses were found in 22 sections (average
number of counted sections per heart), i.e.,
roughly 1 mitosis/section of 6 /an.
At the 8-week sampling, the number of
mitoses decreased to 8/22 sections. There
were no mitoses in normal dogs or in dogs
with sham operations. Mitotic activity had
Circulation Research, Vol. XXVIU, /«»* 1971
675
DNA SYNTHESIS IN CORONARY VESSELS
500 -.
400 -
300 -
-
200 H
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100-
1 2
3
4
5
6
7
8
weeks post op.
after coronary artery occlusion, one must be
certain that a comparable number of vessels is
studied, that a comparable number of blocks is
taken from each heart, and that a comparable
number of sections is prepared from these
blocks. Table 1 shows that sampling errors
have not been made.
The reliability of the tritiated thymidine
technique as a measure of cell proliferation
has been sufficiently established elsewhere
(27). Furthermore, the observed increase in
total cell number, together with the occurrence of mitosis, indicates that the incorporation of radioactive thymidine is due to
reduplication of DNA in preparation for
nuclear division and not to metabolic DNA
turnover (28).
Several studies have failed to show uptake
of tritiated thymidine or mitotic activity by
cells of the normal vascular wall (29, 30).
FIGURE 4
Number of labeled nuclei and labeling index as a
function of time after implantation of the constrictor.
Total amount of vascular label (endothelial plus
medial plus adventitial labeled nuclei) per heart, which
is the average per 24.5 vascular sections (Table 1).
Each point represents one heart. Note the decrease in
labeling as a function of time after operation. The
difference of the means between 3 and 8 weeks has
a random probability of 0.004.
also ceased completely in dogs with chronic 1year coronary artery occlusion.
Most of the mitoses were found in the
tunica intima and in the tunica media of the
collaterals. Often it was not possible to classify
these mitoses into endothelial or smooth
muscle cells because of the dedifferentiated
state that is a normal phenomenon during this
stage of cellular reproduction.
Discussion
It is difficult to draw quantitative conclusions from histological material and even more
difficult to test these results statistically. A
tissue section is only a sample that the
investigator hopes is representative of at least
the block of tissue from which it was taken.
When comparing coronary vessels from different animals and at different time intervals
Circulation Research, Vol. XXVlll,
June 1971
o
2 10
• Midzone
o Total collateral
V
•54-
2-
1 2
3
4
5
6
7
w e e k s post op.
8
FIGURE S
The number of labeled smooth muscle and endothelial
nuclei per section was divided by the total number
of nuclei (labeling index in percent). Solid line: labeling index as a function of time in midzone pieces only.
Broken line: labeling index over the entire length of
the collateral vessel (stem plus midzone plus reentry).
SCHAPER, DE BRABANDER, LEWI
676
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Increase in the total population of endothelial and
smooth muscle nuclei per midzone section as a function of time. The total cell population rises from a
normal value of 30 (=midzones of normal hearts,
obtained from ±40p. vessels) to a final value of 300.
The difference of the means between 3 and 8 weeks
has a random probability of 0.0000012.
Labeling of endothelial cells, which have to
withstand the shear forces of the pulsatile
pressure and flow, was detected in the guinea
pig by Wright (31) after infusion of tritiated
thymidine. The only study that succeeded in
demonstrating labeled cells throughout the
vascular tree of normal adult animals was that
of Spaet and Lejnicks (32). However, the
authors stated that the animals, laboratory
rabbits, continue to gain weight as they age,
even after complete maturity. Hence, the
vascular bed is also growing.
In our control experiments, labeled cells
were never found in normal coronary arteries
and arterioles after 3 to 4 hours of exposure to
tritiated thymidine in a heart-lung prepara-
TABLE 1
Analysis of Possible Sampling Errors
3 wk
Collaterals/heart
Blocks/heart
Vascular sections/heart
6
20
24.5
8 wk
6
22
*
5
18.3
21.7
*Number of pieces too small for comparison.
The number of collaterals, blocks, and vascular sections per heart were not significantly different from
each other in the 3, 4, and 8 week periods.
tion. Hence the cells of the vascular wall in
normal adult animals should be classified as a
stable or normally nongrowing cell population
(33).
On the basis of labeling studies with
tritiated thymidine in rats at 3 days and 6
months after birth, Leblond (33) arrived at
an experimental classification of cell population types. Cell populations which had retained for 6 months most of the label received
at 3 days but showed no new labeling when
given an injection of tritiated thymidine 6
months after birth were called the stable or
nongrowing cell populations. Striated, smooth,
and cardiac muscle and the intermediate-sized
neurons of the brain belong to these populations. Cell populations labeled at 3 days as
well as at 6 months were called expanding cell
populations. Cell populations which took up
tritiated thymidine at both intervals but
showed no label 6 months after injection
received at 3 days were called renewing cell
populations.
When we apply this classification to our
model of vascular growth, we reach the
following conclusion. Under the influence of
chemical (hypoxia) and physical (13) factors,
a stable or nongrowing cell population (vascular smooth muscle) suddenly adopts the
behavior of an expanding cell population—the
vessel grows larger and the number of cells
increases. In the absence of stimulation
(diminished tissue hypoxia due to collateral
growth) the expanding cell population reverts
to a stable nongrowing cell population. It is
quite obvious that the system shows the
features of a self-controlling growth process
(34). As with partial hepatectomy and other
experimental models of this type, there is an
initial fast mitotic response to a certain
stimulus that quickly reaches a maximum and
is followed by a gradual decline toward
normal activity as the size of the population
reaches critical values.
In our model, however, neither the exact
onset of cell division and the moment of peak
activity nor the exact time when cell turnover
has reached its normal value again is known
because these points were not included in the
Circulation Research, Vol. XXVIU, June
1971
677
DNA SYNTHESIS IN CORONARY VESSELS
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observation period. Most probably, cell division started before 3 weeks after implantation
of the constrictor. Indeed, occlusion of the
circumflex artery has been shown statistically
(7) to occur at 2/2 weeks after the operation.
Because this occlusion is most probably the
trigger that induces collateral growth, maximal stimulation should be expected at that
moment. It seems likely, moreover, that even
before total occlusion, when the stenosis has
reached a critical value, cell growth has
already started.
Vascular DNA synthesis and subsequent
mitosis lead to an expansion of the initial cell
population by a factor of approximately 10
(Fig. 6). This increase is due solely to mitosis
(details of the calculations leading to this
conclusion are presented in the Appendix).
The agreement between the percent of
labeled nuclei and the increase in the cell
population is sufficient reason to accept the
hypothesis that enlargement of collaterals is
the result of active growth rather than passive
stretch. The relatively long duration of the S
phase (the time necessary to duplicate DNA)
of the proliferative cycle of the arterial smooth
muscle cells (22 hours, see Appendix) explains
why the collateral circulation cannot prevent
myocardial infarction when a large coronary
artery is suddenly occluded. However, when
such an occlusion develops more slowly, a
sizeable amount of cardiac muscle can be
saved through growth transformation of the
collateral circulation. Autopsy findings have
shown that this can also occur in the human
heart (6).
Only speculations can be offered at present
as to which biochemical agents and which
physical forces initiated vascular growth. We
believe that the mechanisms which are responsible for coronary autoregulation (35)
may play a role whenever the dilative
stimulus reaches a certain intensity and lasts
for a sufficiently long time.
Appendix
Because the cells of the vascular tree are
generally believed to form a stable nongrowing population and hence normally do not
Circulation Research, Vol. XXVlll,
June 1971
synthesize DNA, it seemed of interest to
estimate the length of the S phase of the cell
division cycle in a case in which these cells do
form new DNA.
Let N (t) be the total number of cells at
time *, / (*) the percent of cells with labeled
nuclei at time t, tH the duration of phase S in
hours, and s(t) the rate of the transition of
cells into phase S at time t (hr1). Then the
percent of cells in phase S at any time t is
given approximately by
s(t) - ts • 100,
provided s(t) does not change significantly
over the period ts.
When 3H-thymidine is administered for 3
hours, the percent of labeled cells will be
defined by
f(t) = s(t)-(te +3)-100.
The rate of increase of cells can be
computed from
dN = s(t)-N(t)-dt
and
dN
0.01
f(t)-dt
By integration we obtain
If S (to,t) denotes the area under the curve
of percent labeled nuclei versus weeks after
implantation, we may substitute
[ f(t). dt= (24 x 7 ) .
S(to,t)
•> o
= 168 -S(to,t).
Hence
from which we can derive
In
1.68
S(to,t) - 3.
N(t)
N(t0)
(1)
SCHAPER, DE BRABANDER, LEWI
678
Using decimal logarithms, this expression is
equivalent to
*'
0.730 -S(to,t)
logN(t)-\ogN(t0)
A
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Using total cell number and labeling index
of endothelium and media and interpolation
between 4 and 8 weeks, we obtained an
approximate value of 22 hours for the duration
of the synthetic phase of the cell division
cycle.
Although this is a large value, it falls within
the range of previous observations concerning
mammalian cells (36) and it is not in
contradiction with the observed increase in
cell number. Of course it is a crude estimate
and it is impossible to take into account the
number of cells leaving the population by
destruction or migration. Observations of
cellular degeneration (37) suggest that this
actually does occur. This would mean that,
according to the above equation, the duration
of the S phase is in fact shorter than the value
we calculated.
Acknowledgment
The skillful help of Mr. R. Xhonneux, who
performed all the operations, and Mr. P. Van Even,
who made all histological sections and autoradiographs, is gratefully acknowledged.
References
1. ZOUL, P.M., WESSLER, S., AND SCHLESINGEH,
M.J.: Interarterial coronary anastomoses in the
human heart, with particular reference to
anemia and relative cardiac anoxia. Circulation
4:797-815, 1951.
2. PAULIN, S.: Interarterial coronary anastomoses in
relation to arterial obstruction demonstrated in
coronary arteriography. Invest Radiol 2:147159, 1967.
3. FULTON, W.F.M.: The Coronary Arteries. Springfield, 111., Charles C Thomas, 1965, pp 72128.
4. BLUMCART, H.L., SCHLESINGER, M.J., AND ZOLL,
P.M.: Angina pectoris, coronary failure and
acute myocardial infarction: Role of coronary
occlusions and collateral circulation. JAMA
116:91-97, 1941.
5. GENSINI, G.G., AND BRUTO D A COSTA, B.C.:
Coronary collateral circulation in living man.
Amer J Cardiol 24:393-400, 1969.
6. BLUMGART, H.L., Zoix, P.M., FREEDBERG, A.S.,
AND GILLIGAN, D.R.: Experimental production
of intercoronary arterial anastomoses and their
functional significance. Circulation 1:10-27,
1950.
7. SCHAPER W: Collateral circulation in the canine
coronary system. Thesis, Faculty of Medicine,
University of Louvain, 1967.
8. ECKSTEIN, R.W., GREGG, D.E., AND PRITCHARD,
W.H.: Magnitude and time of development of
the collateral circulation in occluded femoral,
carotid and coronary arteries. Amer J Physiol
132:351-361, 1941.
9. ECKSTEIN, R.W.: Effect of exercise and coronary
artery narrowing on coronary collateral circulation. Circ Res 5:230-235, 1957.
10. GREGC, D.E.: Coronary Circulation in Health and
Disease. Philadelphia, Lea and Febiger, 1950,
pp 187-202.
11. MAUTZ, F.R., AND GREGG, D.E.: Dynamics of
collateral circulation following chronic occlusion of coronary arteries. Proc Soc Exp Biol
Med 36:797-801, 1937.
12. GREGG, D.E., AND FISHER, L.C.: Blood supply to
the heart. In Handbook of Physiology, sec 2,
vol 2, Circulation, edited by W.F. Hamilton
and P. Dow. Washington, D. C , American
Physiological Society, 1963, pp 1517-1584.
13. SCHAPER, W.: Tangential wall stress as a molding
force in the development of collateral vessels in
the canine heart. Experientia 23:595, 1967.
14. SCHAPER, W., AND VANDESTEENE, R.: Rate of
growth of interarterial anastomoses in chronic
coronary artery occlusion. Life Sci 6:16731680, 1967.
15. SCHAPER, W.: Collateral circulation after experimental coronary artery occlusion. Acta Cardiol,
suppl 13, pp 74-79, 1969.
16. SCHAPER, W., JAGENEAU, A., AND XHONNEUX, R.:
Development of a collateral circulation in the
pig and dog heart. Cardiologia 51:321-335,
1967.
17. SCHAPER, W., SCHAPER, J., XHONNEUX, R., AND
VANDESTEENE, R.: Morphology of intercoronary anastomoses in chronic coronary artery
occlusion. Cardiovasc Res 3:315-323, 1969.
18. SCHAPER, J., BORCERS, M., AND SCHAPER, W.:
Ultrastructure and histochemistry of developing intercoronary anastomoses. In Proc 5th
Europ Congr Cardiol, Athens, Hellenic Society
of Cardiology, 1968, pp 321-326.
19. SCHAPER, W.: Ischemia-induced DNA-synthesis
in coronary collateral vessels (abstr). Circulation 41-42, (suppl III) 111:206, 1970.
20. SCHAPER J., BORCERS, M., AND SCHAPER, W.:
Coronary collateral vessels: An electron-microscopic study. Amer J Cardiol, in press.
21. SCHAPER, W.: Physiology of the collateral
circulation in the normal and hypoxic myocardium. Ergeb Physiol, in press.
Circulation Reieercb, Vol. XXVIII, June 1971
DNA SYNTHESIS I N CORONARY VESSELS
22.
LTTVAK,
J.,
SIDERIDES,
L.E.,
AND VINEBERG,
A.M.: Experimental production of coronary
artery insufficiency and occlusion. Amer Heart
J 53:505-518, 1957.
23. LONGLAND, C.J.: Collateral circulation of the
limb. Ann Roy Coll Surg Engl 13:161-183,
1953.
24.
25.
26.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
27.
28.
29.
SCHAPER, W., AND VAN EVEN, P.: Zur exposition
und Entwicklung autoradiographischer Praparate. Pflueger Arch 312:126-128, 1969.
ROGERS, A.W.: Techniques of Autoradiography.
Amsterdam, Elsevier Publishing Co., 1967.
SIEGEL, S.: Nonparametric Statistics. New York,
McGraw Hill, 1956.
CLEAVER, J.E.: Thymidine metabolism and cell
kinetics. North-Holland Research Monographs.
Frontiers of Biology, vol. 6. Amsterdam, NorthHolland Publishing Co., 1967.
PELC, S.R.: Incorporation of labeled precursors of
DNA in non-dividing cells, in Cell Proliferation, edited by L.F. Lamerton. Oxford,
Blackwell Scientific Publishers, 1963, pp 94109.
SPRARAGEN, S.C., BOND, U.P., AND DAHL, L.K.:
Role of hyperplasia in vascular lesions of
cholesterol-fed rabbits studied with thymidineH 3 autoradiography. Circ Res 11:329-336,
1962.
Circulation Research, Vol. XXV1I1, June 1971
679
30. ALTSCHUL, R.: Endothelium: Its Development,
Morphology, Function and Pathology. New
York, Macmillan, 1954.
31. WRIGHT, H.P.: Endothelial mitosis around aortic
branches in normal guinea pigs. Nature
(London) 220:78-79, 1968.
32.
SPAET, T.H., AND LEJNICKS, I.: Mitotic activity
of rabbit blood vessels. Proc Soc Exp Biol Med
125:1197-1201, 1967.
33. LEBLOND, C.P.: Classification of cell populations
on the basis of their proliferative behavior. Nat
Cancer Inst Monogr 14:119-150, 1964.
34. RILEY, P.A.: Heuristic model of mitotic autoregulation in cell populations. Nature (London)
223:1382-1383, 1969.
35. BERNE, R.M.: Cardiac nucleotides in hypoxia:
Possible role in regulation of coronary blood
flow. Amer J Physiol 204:317-322, 1963.
36.
MAURER, W., AND SCHULTZE, B.: Uberblick iiber
autoradiographische Methoden und Ergebnissen zur Bestimmung von Generationszeiten
und Teilphasen von tierischen Zellen mit
markiertem Thymidin. Acta Histochem 8:7387, 1969.
37. BORGERS, M., SCHAPER, J., AND SCHAPER, W.:
Acute vascular lesions in developing coronary
collaterals. Virchow Arch [Path Anat] 351:111, 1970.
DNA Synthesis and Mitoses in Coronary Collateral Vessels of the Dog
WOLFGANG SCHAPER, MARC DE BRABANDER and PAUL LEWI
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Circ Res. 1971;28:671-679
doi: 10.1161/01.RES.28.6.671
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