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On the proliferative activity of arterial smooth muscle cells in rat postnatal ontogenesis Auto-historadiographic studies
297
Atherosclerosis,
23 (1976)
0 Elsevier Scientific
297-304 Publishing Company,
Amsterdam
-Printed
in The Netherlands
ON THE PROLIFERATIVE ACTIVITY OF ARTERIAL SMOOTH MUSCLE CELLS IN RAT POSTNATAL ONTOGENESIS AUTO-HISTORADIOGRAPHIC Z. YURUKOVA*,
P. HADJIISKY,
Centre de Recherches Cardiologiques H6pital Boucicaut, Paris (France) (Received (Accepted
STUDIES
J. RENAIS
and L. SCEBAT
Claude Bernard, Laboratoire
associk 208 du C.N.R.S.,
27th May, 1975) 17th July, 1975)
[3H] Thymidine incorporation in myocyte nuclei and mitoses in the media were studied in rats aged 10 and 45 days and 6 and 12 months. DNA synthesis and mitoses were more evident in muscular arteries, the abdominal aorta and the external third of the media than in elastic arteries, the thoracic aorta and the internal two-thirds of the media. The incorporation of [j H] thymidine in mediacytes was significant up to the age of puberty; it greatly decreased at 6 months and had completely disappeared by 12 months. Thus, the growth in the adult aortais due more to a hypertrophy of the myocytes and an augmentation of the extra-cellular conjunctive constituants than to a hyperplasia. Key words:
Ageing - Aorta - DNA synthesis - Gradients of growth -Media Muscular arteries -Post-natal development - [3H] thymidine
- Mitoses -
Introduction The proliferative capacity of the smooth muscle cells (SMC) of the arterial media is a problem of major theoretical interest, because of the recently demonstrated role of SMC and its still poorly understood origin in the genesis of the reparative intimal thickening [1,9,16,18] and cellular atherosclerotic lesions [ 12,13,26,32] . Smooth muscle cells, as highly differentiated cells with specific function, are regarded as a stable cell population of the mature organism [ 10,231, with a restricted division-capacity. However, in recent years numerous * Supported by a scholarship from the DGRST of the French Government: Cardiovascular Diseases, Medical Academy. Sofia 31, Bulgaria. Address for reprints: Dr. P. Hadjiisky, HBpital Boucicaut, Paris 75015.
present
adress:
Department
Of
298
reports have been published on medial smooth muscle cell proliferation in a variety of arterial lesions, for instance in experimental cholesterol atherosclerosis in rabbits [3,25,30,31], swine [7,39] and monkeys [28], in experimental hypertension [ 6,201, and following nonspecific experimental injury [9,11,19]. Very few studies have been devoted to the growth and the proliferative capacity of intact mammalian arteries on the medial SMC, [4,34,2,24], which encouraged us to undertake this study. As an index of proliferation, mitotic figures are rare in a normally stable cell population such as the arterial SMC. Hence, the autoradiographic visualization, using [ 3H] thymidine, of DNA-synthesis in cell nuclei preparing themselves for division affords a more dynamic yet simpler approach to the study of the proliferative activity of these cells. Since the period for DNA synthesis is considerably longer than the duration of the mitotic phase, it is evident that the possibility of finding [ 3H] thymidine-labelled cell nuclei is much greater than that of observing mitotic figures. For that reason, the number of 3H-labelled nuclei is a more sensitive indicator of cell renewal than the number of mitoses alone. Material and methods Twelve Wistar albino rats of varying ages were used: newborn (10 days), prepubertal (45 days), adult (6 months) and old (10 months). All animals received a single 3 /&i/g body weight intraperitoneal injection of tritiated thymidine (CEA-France, spec. activity 27.0 Ci/mM). In order to exclude any alteration in uptake associated with diurnal variations in mitotic rhythm and DNA synthesis [27], the labelled nucleoside was injected at 9.30 a.m. in every instance. An hour and a half later all animals (except the newborn) were perfused with 1.5% glutaraldehyde solution in Sorensen buffer (pH = 7,3) for 12 min under Nembutal narcosis. The aorta and the iliac, renal and carotid arteries were removed and fixed for 30 min in 1.5% glutaraldehyde solution in Sorensen buffer and then for 2 hours in Bouin’s fixator (aqueous solution). Fragments of all arteries together with specimens from the duodenum and spleen, which served as controls, were embedded in the same celloidin-paraffin block from which 4 pm thick histologic sections were cut. Autoradiograms were prepared with Kodak stripping film lK-10 and exposed at 4°C in a dry atmosphere for 4 weeks. After development in Kodak D-19 developer they were stained through the photoemulsion with 1% methylgreen-pyronine solution. Under oil immersion all [“HI thymidine-labelled nuclei and mitoses in the media of the examined arteries were counted. Only cells with 5 or more silver grains per nucleus were scored as radioisotope-labelled. Since the mean values of labelled and dividing nuclei are very small, it was not possible to determine the [3H] thymidine index by the conventional method, Therefore, the number of labelled nuclei of the medial smooth muscle cells was determined per standard area of arterial media. In the aortic wall this area was measured with an ocular grid. In the other, smaller arteries, consecutive transverse sections were used and the medial surface was estimated by means of an ocular micrometer and by the formula C = n/4 [02 {D + (tl + t2)}” J , D being the total diameter of the transsected artery, and
299
t1 and t2 the diameter of the media in two opposite artery segments. The results obtained were expressed as number of labelled nuclei and mitoses per 100,000 pm* of arterial medial surface. In haematoxylin-eosin stained paraffin sections of the thoracic aorta wall of all experimental animals, the total number of nuclei of smooth muscle cells per 100,000 pm2 of medial surface was also determined. Results and discussion No spontaneous vascular lesions were observed in any of the examined arterial segments of the experimental animals. Evidence of proliferation was therefore interpreted to be due solely to normal processes of cell renewal. In evaluating the autoradiograms of the arterial walls of experimental animals it appeared that the medial SMC showed a high proliferative activity in the early phases of postnatal development. The number of DNA-synthesizing nuclei of albino rat arterial SMC was very large in all examined arteries as early as the 10th day of life. In the various elastic arteries (a. iliaca, a. renalis, a. carotis) it was approximately equal - about 180-200 DNA-synthesizing nuclei per 100,000 pm2 of medial surface. What was impressive however, was the marked difference in the number of [3 H] thymidine-labelled nuclei in the aortic segments. In the abdominal aorta the number of DNA-synthesizing nuclei (132/ 100,000 pm2) was about three times greater than in the thoracic aorta (47/ 100,000 pm2 ). The number of labelled nuclei in our experimental series showed an abrupt fall up till the 45th day of life to as low as 14-9/100,000 pm2 in the elastic arteries and 2-1/100,000 pm2 in the aorta. After this age the increasing thickness of the arterial media was primarily due to SMC enlargement and increase in the amount of the intercellular substance. In adult 6-month-old animals, the number of DNA-synthesizing nuclei in the media was reduced to as low as 4-7/100,000 pm2 in the elastic arteries and l-5/100,000 pm2 in the abdominal aorta. In lo-month-old animals [3 H] thymidine-labelled nuclei were very rarely encountered (less than l/100,000 pm”). The role of cell enlargement for the increasing thickness of the arterial media after puberty was also apparent from the decreasing mean number of SMC nuclei per standard area of the thoracic aortic media which occurred with advancing age (Fig. 1). The number of mitoses in the medial SMC of the arteries was only appreciably higher in newborn animals (see Table 1 and Fig. 2). In the later stages of postnatal development of the albino rat it was very rare. This did not allow a definite conclusion to be drawn on the relation between the number of mitoses and the number of DNA-synthesizing nuclei, or the determination of whether DNA synthesis is regularly followed by cell division. However, the number of mitoses in our experimental series was approximately 10 times smaller than the number of DNA-synthesizing nuclei. These data match the results of studies on myocardial muscle cell proliferation in the albino rat which showed that both in newborn and in adult animals the number of mitoses in the myocardium is approximately 10 times smaller than that of t3H] thymidine-labelled nuclei [ 21,291. What is striking in assessing the autoradiograms of the arterial walls is the difference of labelling intensity of DNA-synthesizing smooth muscle nuclei
aorta thoracica
0
?? aorta
orterio art&o arteria
x
0 .
abdominalis iliaca carotis renalis
10 20304045days Fig.
1. Age-dependent
6
bebaviour
7
8
9
10 Age
of DNA-synthesizing
(rWnth.5) nuclei
in rat arterial
media.
with silver grains to the emulsion in the different arteries and in individual animals. The silver grains above the cell nuclei whose number reflects the degree of [3H] thymidine incorporation varied strongly from 5 to 8 supranuclear grains to large accumulations; it was thus impossible to make an exact count (Figs. 3 and 4). In general, the mean number of silver grains per nucleus tended to be reduced in the course of the postnatal development, i.e. with advancing age (Fig. 4C). This phenomenon is in keeping with the reported decrease in the number of DNA-synthesizing nuclei and in nucleic acid content [24] as a function of rat age. It has also been observed in the myocardial myofibres during the postnatal growth of albino rats [22]. A study of the localization of DNA-synthesizing and metaphasic nuclei showed that they are unevenly distributed in the media of the arteries of newborn animals, with perhaps some predilection for its external layers (Fig. 3). In
& k5300
-
: E g- 200\ ._ !+! 0 1 z
100-
L.
I.
Fig.
2.
Change
I,,
I
I
“6
7
8
I*
102OJO4045days with
age
in
the
mean
number
Y of
10 Age (months) cell
nuclei
per
100,000
in
rat
thoracic
amtic
media.
301
Fig. 3. Numerous tritium-labelled nuclei in arterial smooth muscle cell of 10 days of albino rate. A: renal iliac artery; C: carotid artery. Autoradiogram; methyl-green pyronin stain, X 1000.
artery; B:
prepubertal and adult experimental animals (45 days and 6 months old) the preferential localization of labelled nuclei in the external media layers was also observed. Such an arrangement of active nuclei in the arterial wall can be understood if we take into account the fact that the diverse histoenzymatic activities are more marked in the external third of the aortic media in young animals [14,15,17]. From the above data it appears that during the postnatal ontogenesis, mitotic division of medial SMC is essential for the growth of the arterial wall and the increase in its medial layer thickness. Medial SMC division activity is particularly high during the period of rapid growth (the first few days after birth and up till puberty). Thanks to this, arterial dimensions keep up with increasing body mass. During this period, the activity of respiratory enzymes and ATPase is more elevated in the abdominal aorta than in the thoracic aorta [14,17]. This phenomenon, along with the greater [3 H] thymidine incorporation ascertained by the present study, indicates that DNA synthesis and aortic growth are more rapid before puberty in the abdominal aorta. It is due to this greater activity that the aorta can increase in length. After puberty the increase in arterial wall thickness almost exclusively depends on medial SMC enlargement and deposition of connective tissue matrix in the intercellular spaces of the media. Evidence for this can be seen in the decreasing cellularity of the media with
302
Fig. 4. Tritium-labelkd nuclei in medial smooth muscle cells of iliac artery in (A) 45-day-old and (B, C) 6-month-old albino rats. Note the scarcity of silver grains over the nucleus in 4C (arrow). Autoradiogram, methyl-green pyronin stain, X 1000.
advancing age, demonstrated in the present study and in the studies of other workers [ 51, as well as by the very small number of DNA-synthesizing cells in adult experimental animals. In 6-month-old albino rats whose age approximately corresponds to 20 years in man [8,27], at a time when the total thickness of the arterial media has reached adult levels and its further increase is negligible, labelled nuclei in the media are rarely encountered and obviously have no bearing on the growth processes. The results obtained in the present study are similar to those established in an analogous experimental study of DNA-synthesizing nuclei in the cellular elements of intact albino rat myocardium, where a sharp decline in the curve of DNA-synthesizing and dividing nuclei was also observed with advancing age [ 21,221. The present results indicate that the arteries of albino rats comprise a rather stable population of cells. But even under normal conditions, in all periods of rat adult life, the intact arterial wall is the site of some (though limited) DNAsynthesis and associated mitotic cellular activity. Acknowledgements The authors are grateful to Professor W. Bernhardt and his staff at the E.M. Villejuif’s Laboratory of the Cancer Research Institute for their help and encouragement, to Professor J.P. Dadoune (Laboratory of Histology, Faculty
303
of Medicine, Paris) for the generous advice given during the study, J. Dutartre for her excellent technical assistance.
and to Mrs
References 1 Buck, R.C., lntimal thickening after ligature of arteries, Circulat. Res., 9 (1961) 418426. 2 Berry, C.L., Looker, T. and Germain. J., The growth and development of rat aorta, Part 1 (Morphological aspects), J. Anat. (London), 113 (1972) l-16. 3 Cavallero, C., Turolla. E. and Ricevuti, G., Cell proliferation in the atherosclerotic plaques of cholesterol-fed rabbits, Part 1 (Colchicine and c3Hl thymidine studies, Atherosclerosis, 13 (1971) 9-20. 4 Cliff, W.J.. The aortic tunica media in growing rats studied with the electron microscope, Lab. Invest., 17 (1967) 599-615. 5 Cliff. W.J., The aortic tunica media in ageing rats, EXP. Molec. Path., 13 (1970) 172-189. 6 Crane, W.A.J. and lngie. D.J.. Tritiated thymidine uptake in rat hypertension, Arch. Path., 78 (1964) 209-221. 7 Florentin, R.A., Nam, S.Ch.. Lee. K.T.. Lee. K.J. and Thomas, W.A., Increased mitotic activity in aortas of swine (after three days of cholesterol feeding), Arch. Path., 88 (1969) 463469. 8 Freis. E.D., In: K. Okamoto (Ed.), Spontaneous Hypertension, Springer Verlag, Berlin. 1972, p. 231. 9 Geer, J.C. and Haust, M.D.. Smooth Muscle Cells in Atherosclerosis, Karger, Basel, New-York. 1972. 10 Grundman, E. and Seidel, H.J., Die reparative Parenchymregeneration. In: F. Biichner et al. (Eds.), Handbuch der allgemeinen Pathologie, Vol. 6 (Entwicklung, Wachstum, Geschwiilste), Part 2 (Regeneration, Hyperplasie, Cancerisierung), Springer-Verlag, Berlin, Heidelberg, 1969. PP. 108-112. 11 Hassler, O., The origin of the cells constituting arterial i&ma thickening - An experimental autoradiographic study with the use of 3H-thymidine, Lab. Invest.. 22 (1970) 286-293. 12 Haust, M.D. and More, R.H., Significance of smooth muscle cells in atherogenesis. In: R.J. Jones (Ed.), Evolution of the Atherosclerotic Plaque, Chicago University Press, Chicago, Ill., 1963. p. 51. 13 Haust, M.D., More, R.H. and Movat, H.Z.. The mechanism of fibrosis in atherosclerosis, Amer. J. Path., 35 (1959) 265-273. 14 Hadjiisky, P., Renais, J. and Scebat, L., Activite’s hydrolasiques de l’aorte de lapin jeune et adulte, Bull. Ass. Anat. (Nancy), 148 (1970) 372-383. 15 Hadjiisky. P., Renais, J. and Scebat, L., Histochimie et histoenzymologie du m6tabolisme glucidique de l’aorte de Callus gallus, Arch. Mal. Coeur (AthBroscl.), 63 (1970) 40-52. 16 Hadjiisky, P.. Renais, J. and Scebat, L., Histochimie et histoenzymologie de l’e’paississement diffus de l’intima aortique chez le lapin immunisk contre l’aorte de rat, Paroi Art&ielle (Paris), 1 (1) (1973) 39-52. 17 Hadjiisky, P., Renais, J. and Scebat, L.. Ddveloppement et s&escence de l’aorte de rat - Histochimie et histoenzymologie comparative, Atherosclerosis, 22 (1975) 19-38. 18 Jurukova, Z. and Rohr, H.P.. Beitrag zur Bildung bindegewebiger Matrix in glatten Muskelzellen, Path. Europ., 3 (1968) 551-570. 19 Jurukova. Z., Reparative regeneration of smooth muscle tissue. Compt. Rend. Bulg. Acad. Sci., 27 (1974) 275-278. 20 Jurukova, Z. and Kiprov, D., Role of hyperplasia in hypertensive arterial lesions -An autoradiographic study with jH-thymidine, Compt. Rend. Bulg. Acad. Sci., In press. 21 Klinge, O., Proliferation und Regeneration am Myocard, Z. Zellforsch.. 80 (1967) 488-517. 22 Kunz, J., Keim, U. and Fuhrmann, I., Die Proliferation van Muskelzellen und Bindesgewebszellen des Rattenherzens w&rend des postnatalen Wachstums. EXP. Path.. 6 (1972) 270-277. 23 Leblond. C.P.. Messier. B. and Kopriwa, B., Thymidine-“H as a tool for the investigation of the renewal of cell populations, Lab. Invest., 8 (1959) 296-308. 24 Looker, T. and Berry, C.L.. The growth and development of rat aorta, Part 2 (Changes in nucleic acid and scleroprotein content), J. Anat. (London), 113 (1972) 30-34. 25 McMillan. G.C. and Stary. H.C.. Preliminary experience with mitotic activity of cellular elements in the atherosclerotic plaques of cholesterol-fed rabbits studied by labelling with tritiated thymidine. Ann. N.Y. Acad. Sci., 149 (1968) 699-709. 26 Parker, F. and Odland, G.F., A correlative histochemical, biochemical and electron-microscopic study of experimental atherosclerosis in the rabbit aorta with special reference to the myointimal cells, Amer. J. Path.. 48 (1966) 197-216. 27 Pilgrim, C., Erh, W. and Maurer, W., Diurnal fluctuations in the numbers of DNA synthesizing nuclei in various mouse tissues, Nature (London). 199 (1963) 863-870. 28 Ritchie, R.E., Ybunger, R.. Scott, H.W. and Stephenson, S., Radioautographic studies of the proliferative phase of primate atherogenesis, Surg. Forum, 14 (1963) 306-318. 29 Rumyantzev, P.P., Autoradiographic study of DNAsynthesis and nucleus division in cardiac muscle during embryonic and postnatal histogenesis, Arkh. Anat. Gistol. Embriol., 47 (1964) 59-65.
304 30
Spraragen, S.C., Bond, V.P. and Dahl. L.K., Role of hyperplasia in vascular lesions of cholesterol-fed rabbits studied with thymidine-3H autoradiography. Circulat. Res., 11 (1962) 329-337. 31 Stary, H.C., Cell proliferation in the experimental atheroma as revealed by radioautography after injection of thymidine3H, Circulation, 36. Suppl. 2 (1967) 11. 32 Still, W.H.J. and Dennison, S.M., Reaction of the arterial intima of the rabbit to trauma and hyperlipemia. EXP. Molec. Path., 6 (1967) 143-155. 33 Thomas, W.A.. Florentin, R.A., Nam, S.C., Kim Dong, N., Jones Rose. N.. and Lee, K.T.. Proliferative phase of atherosclerosis in swine fed cholesterol, Arch. Path., 86 (1968) 621-643. 34 Wolinsky, H., Comparison of medial growth Of human thoracic and abdominal aortas, Circulat. Res.. 27 (1970) 531-538.
Atherosclerosis,
23 (1976)
0 Elsevier Scientific
297-304 Publishing Company,
Amsterdam
-Printed
in The Netherlands
ON THE PROLIFERATIVE ACTIVITY OF ARTERIAL SMOOTH MUSCLE CELLS IN RAT POSTNATAL ONTOGENESIS AUTO-HISTORADIOGRAPHIC Z. YURUKOVA*,
P. HADJIISKY,
Centre de Recherches Cardiologiques H6pital Boucicaut, Paris (France) (Received (Accepted
STUDIES
J. RENAIS
and L. SCEBAT
Claude Bernard, Laboratoire
associk 208 du C.N.R.S.,
27th May, 1975) 17th July, 1975)
[3H] Thymidine incorporation in myocyte nuclei and mitoses in the media were studied in rats aged 10 and 45 days and 6 and 12 months. DNA synthesis and mitoses were more evident in muscular arteries, the abdominal aorta and the external third of the media than in elastic arteries, the thoracic aorta and the internal two-thirds of the media. The incorporation of [j H] thymidine in mediacytes was significant up to the age of puberty; it greatly decreased at 6 months and had completely disappeared by 12 months. Thus, the growth in the adult aortais due more to a hypertrophy of the myocytes and an augmentation of the extra-cellular conjunctive constituants than to a hyperplasia. Key words:
Ageing - Aorta - DNA synthesis - Gradients of growth -Media Muscular arteries -Post-natal development - [3H] thymidine
- Mitoses -
Introduction The proliferative capacity of the smooth muscle cells (SMC) of the arterial media is a problem of major theoretical interest, because of the recently demonstrated role of SMC and its still poorly understood origin in the genesis of the reparative intimal thickening [1,9,16,18] and cellular atherosclerotic lesions [ 12,13,26,32] . Smooth muscle cells, as highly differentiated cells with specific function, are regarded as a stable cell population of the mature organism [ 10,231, with a restricted division-capacity. However, in recent years numerous * Supported by a scholarship from the DGRST of the French Government: Cardiovascular Diseases, Medical Academy. Sofia 31, Bulgaria. Address for reprints: Dr. P. Hadjiisky, HBpital Boucicaut, Paris 75015.
present
adress:
Department
Of
298
reports have been published on medial smooth muscle cell proliferation in a variety of arterial lesions, for instance in experimental cholesterol atherosclerosis in rabbits [3,25,30,31], swine [7,39] and monkeys [28], in experimental hypertension [ 6,201, and following nonspecific experimental injury [9,11,19]. Very few studies have been devoted to the growth and the proliferative capacity of intact mammalian arteries on the medial SMC, [4,34,2,24], which encouraged us to undertake this study. As an index of proliferation, mitotic figures are rare in a normally stable cell population such as the arterial SMC. Hence, the autoradiographic visualization, using [ 3H] thymidine, of DNA-synthesis in cell nuclei preparing themselves for division affords a more dynamic yet simpler approach to the study of the proliferative activity of these cells. Since the period for DNA synthesis is considerably longer than the duration of the mitotic phase, it is evident that the possibility of finding [ 3H] thymidine-labelled cell nuclei is much greater than that of observing mitotic figures. For that reason, the number of 3H-labelled nuclei is a more sensitive indicator of cell renewal than the number of mitoses alone. Material and methods Twelve Wistar albino rats of varying ages were used: newborn (10 days), prepubertal (45 days), adult (6 months) and old (10 months). All animals received a single 3 /&i/g body weight intraperitoneal injection of tritiated thymidine (CEA-France, spec. activity 27.0 Ci/mM). In order to exclude any alteration in uptake associated with diurnal variations in mitotic rhythm and DNA synthesis [27], the labelled nucleoside was injected at 9.30 a.m. in every instance. An hour and a half later all animals (except the newborn) were perfused with 1.5% glutaraldehyde solution in Sorensen buffer (pH = 7,3) for 12 min under Nembutal narcosis. The aorta and the iliac, renal and carotid arteries were removed and fixed for 30 min in 1.5% glutaraldehyde solution in Sorensen buffer and then for 2 hours in Bouin’s fixator (aqueous solution). Fragments of all arteries together with specimens from the duodenum and spleen, which served as controls, were embedded in the same celloidin-paraffin block from which 4 pm thick histologic sections were cut. Autoradiograms were prepared with Kodak stripping film lK-10 and exposed at 4°C in a dry atmosphere for 4 weeks. After development in Kodak D-19 developer they were stained through the photoemulsion with 1% methylgreen-pyronine solution. Under oil immersion all [“HI thymidine-labelled nuclei and mitoses in the media of the examined arteries were counted. Only cells with 5 or more silver grains per nucleus were scored as radioisotope-labelled. Since the mean values of labelled and dividing nuclei are very small, it was not possible to determine the [3H] thymidine index by the conventional method, Therefore, the number of labelled nuclei of the medial smooth muscle cells was determined per standard area of arterial media. In the aortic wall this area was measured with an ocular grid. In the other, smaller arteries, consecutive transverse sections were used and the medial surface was estimated by means of an ocular micrometer and by the formula C = n/4 [02 {D + (tl + t2)}” J , D being the total diameter of the transsected artery, and
299
t1 and t2 the diameter of the media in two opposite artery segments. The results obtained were expressed as number of labelled nuclei and mitoses per 100,000 pm* of arterial medial surface. In haematoxylin-eosin stained paraffin sections of the thoracic aorta wall of all experimental animals, the total number of nuclei of smooth muscle cells per 100,000 pm2 of medial surface was also determined. Results and discussion No spontaneous vascular lesions were observed in any of the examined arterial segments of the experimental animals. Evidence of proliferation was therefore interpreted to be due solely to normal processes of cell renewal. In evaluating the autoradiograms of the arterial walls of experimental animals it appeared that the medial SMC showed a high proliferative activity in the early phases of postnatal development. The number of DNA-synthesizing nuclei of albino rat arterial SMC was very large in all examined arteries as early as the 10th day of life. In the various elastic arteries (a. iliaca, a. renalis, a. carotis) it was approximately equal - about 180-200 DNA-synthesizing nuclei per 100,000 pm2 of medial surface. What was impressive however, was the marked difference in the number of [3 H] thymidine-labelled nuclei in the aortic segments. In the abdominal aorta the number of DNA-synthesizing nuclei (132/ 100,000 pm2) was about three times greater than in the thoracic aorta (47/ 100,000 pm2 ). The number of labelled nuclei in our experimental series showed an abrupt fall up till the 45th day of life to as low as 14-9/100,000 pm2 in the elastic arteries and 2-1/100,000 pm2 in the aorta. After this age the increasing thickness of the arterial media was primarily due to SMC enlargement and increase in the amount of the intercellular substance. In adult 6-month-old animals, the number of DNA-synthesizing nuclei in the media was reduced to as low as 4-7/100,000 pm2 in the elastic arteries and l-5/100,000 pm2 in the abdominal aorta. In lo-month-old animals [3 H] thymidine-labelled nuclei were very rarely encountered (less than l/100,000 pm”). The role of cell enlargement for the increasing thickness of the arterial media after puberty was also apparent from the decreasing mean number of SMC nuclei per standard area of the thoracic aortic media which occurred with advancing age (Fig. 1). The number of mitoses in the medial SMC of the arteries was only appreciably higher in newborn animals (see Table 1 and Fig. 2). In the later stages of postnatal development of the albino rat it was very rare. This did not allow a definite conclusion to be drawn on the relation between the number of mitoses and the number of DNA-synthesizing nuclei, or the determination of whether DNA synthesis is regularly followed by cell division. However, the number of mitoses in our experimental series was approximately 10 times smaller than the number of DNA-synthesizing nuclei. These data match the results of studies on myocardial muscle cell proliferation in the albino rat which showed that both in newborn and in adult animals the number of mitoses in the myocardium is approximately 10 times smaller than that of t3H] thymidine-labelled nuclei [ 21,291. What is striking in assessing the autoradiograms of the arterial walls is the difference of labelling intensity of DNA-synthesizing smooth muscle nuclei
aorta thoracica
0
?? aorta
orterio art&o arteria
x
0 .
abdominalis iliaca carotis renalis
10 20304045days Fig.
1. Age-dependent
6
bebaviour
7
8
9
10 Age
of DNA-synthesizing
(rWnth.5) nuclei
in rat arterial
media.
with silver grains to the emulsion in the different arteries and in individual animals. The silver grains above the cell nuclei whose number reflects the degree of [3H] thymidine incorporation varied strongly from 5 to 8 supranuclear grains to large accumulations; it was thus impossible to make an exact count (Figs. 3 and 4). In general, the mean number of silver grains per nucleus tended to be reduced in the course of the postnatal development, i.e. with advancing age (Fig. 4C). This phenomenon is in keeping with the reported decrease in the number of DNA-synthesizing nuclei and in nucleic acid content [24] as a function of rat age. It has also been observed in the myocardial myofibres during the postnatal growth of albino rats [22]. A study of the localization of DNA-synthesizing and metaphasic nuclei showed that they are unevenly distributed in the media of the arteries of newborn animals, with perhaps some predilection for its external layers (Fig. 3). In
& k5300
-
: E g- 200\ ._ !+! 0 1 z
100-
L.
I.
Fig.
2.
Change
I,,
I
I
“6
7
8
I*
102OJO4045days with
age
in
the
mean
number
Y of
10 Age (months) cell
nuclei
per
100,000
in
rat
thoracic
amtic
media.
301
Fig. 3. Numerous tritium-labelled nuclei in arterial smooth muscle cell of 10 days of albino rate. A: renal iliac artery; C: carotid artery. Autoradiogram; methyl-green pyronin stain, X 1000.
artery; B:
prepubertal and adult experimental animals (45 days and 6 months old) the preferential localization of labelled nuclei in the external media layers was also observed. Such an arrangement of active nuclei in the arterial wall can be understood if we take into account the fact that the diverse histoenzymatic activities are more marked in the external third of the aortic media in young animals [14,15,17]. From the above data it appears that during the postnatal ontogenesis, mitotic division of medial SMC is essential for the growth of the arterial wall and the increase in its medial layer thickness. Medial SMC division activity is particularly high during the period of rapid growth (the first few days after birth and up till puberty). Thanks to this, arterial dimensions keep up with increasing body mass. During this period, the activity of respiratory enzymes and ATPase is more elevated in the abdominal aorta than in the thoracic aorta [14,17]. This phenomenon, along with the greater [3 H] thymidine incorporation ascertained by the present study, indicates that DNA synthesis and aortic growth are more rapid before puberty in the abdominal aorta. It is due to this greater activity that the aorta can increase in length. After puberty the increase in arterial wall thickness almost exclusively depends on medial SMC enlargement and deposition of connective tissue matrix in the intercellular spaces of the media. Evidence for this can be seen in the decreasing cellularity of the media with
302
Fig. 4. Tritium-labelkd nuclei in medial smooth muscle cells of iliac artery in (A) 45-day-old and (B, C) 6-month-old albino rats. Note the scarcity of silver grains over the nucleus in 4C (arrow). Autoradiogram, methyl-green pyronin stain, X 1000.
advancing age, demonstrated in the present study and in the studies of other workers [ 51, as well as by the very small number of DNA-synthesizing cells in adult experimental animals. In 6-month-old albino rats whose age approximately corresponds to 20 years in man [8,27], at a time when the total thickness of the arterial media has reached adult levels and its further increase is negligible, labelled nuclei in the media are rarely encountered and obviously have no bearing on the growth processes. The results obtained in the present study are similar to those established in an analogous experimental study of DNA-synthesizing nuclei in the cellular elements of intact albino rat myocardium, where a sharp decline in the curve of DNA-synthesizing and dividing nuclei was also observed with advancing age [ 21,221. The present results indicate that the arteries of albino rats comprise a rather stable population of cells. But even under normal conditions, in all periods of rat adult life, the intact arterial wall is the site of some (though limited) DNAsynthesis and associated mitotic cellular activity. Acknowledgements The authors are grateful to Professor W. Bernhardt and his staff at the E.M. Villejuif’s Laboratory of the Cancer Research Institute for their help and encouragement, to Professor J.P. Dadoune (Laboratory of Histology, Faculty
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of Medicine, Paris) for the generous advice given during the study, J. Dutartre for her excellent technical assistance.
and to Mrs
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