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Association of DNA synthesis and apparent dedifferentiation of aortic smooth muscle cells in vitro
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
12,
354-362
(1970)
Association of DNA Synthesis and Apparent Dedifferentiation of Aortic Smooth Muscle Cells in Vitro’ K. E. FRITZ, J. Departments
JARMOLYCH,
AND
A. S.
of Pathology, Veterans Administration Albany Medical College, Albany, N.
Received
January
DAOUD
Hospital,
and
Y.
20, 1970
Smooth muscle cells (SMC) of medial explants of swine thoracic aortas appear to undergo in the first few days of culture rapid dedifferentiation toward more primitive forms. The number of cells synthesizing DNA in the explants increases as the number of well differentiated SMC decreases. Electron microscopic autoradiography revealed that more of the cells incorporating JH-thymidine were poorly differentiated SMC. Thus it appears that in this in vitro situation dedifferentiation of SMC precedes rapid cell proliferation. In early cholesterol-induced atherosclerosis in swine many partially or completely undifferentiated cells are present. The results of the current in vitro study suggest that the undifferentiated cells in the atherosclerotic lesions could arise from mature SMC through dedifferentiation.
In a previous study (Jarmolych et al., 1968) we have reported that medial explants of the thoracic aorta of the swine, cultured in semisynthetic medium, developed a new peripheral growth by about the 4th day, which increased in amount at least up to 21 days when the experiment was terminated. Electron microscopic studies showed that in the early cultures most cells of the peripheral growth had no filaments or other specialized features characteristic of smooth muscle cells (SMC) and could not be classified as to origin (fibroblastlike and undifferentiated or primitive cells). Light microscopy autoradiographic studies after “H-thymidine pulse labeling revealed numerous labeled nuclei in the peripheral growth. In older cultures filaments were demonstrable in most cells, and by the Zlst day almost all cells had substantial numbers of fusiform densities and of filaments consistent with myofilaments. Autoradiographic studies of these older cultures showed a decrease in number of labeled cells concomitant with the increase in specialized features. From
the above observations
we formulated
the following
two
hypotheses
which are illustrated diagrammatically in Fig. 1. First, SMC in the original explant can undergo dedifferentiation in the first few days toward more primitive forms giving rise to the undifferentiated cells in the early peripheral growth which subsequently redifferentiate toward SMC. Second, rapid cell proliferation does not occur until extensive dedifferentiation has taken place. In Part I of the current study the first hypothesis was tested in three ways. (1) Swine aortic explants were examined by electron microscopy after O-4 days I This study was supported Brown-Hazen Fund.
by USPHS
Grant
H-7155,
354
VA Research
Funds
and a grant
from
the
DNA
SYNTHESIS
AND
CELL
DEDIFFERENTIATION
355
MBuusLA5T-LIuE CELLS
FIG. 1. This schematic diagram illustrates our conception of the relationship between DNA synthesis in smooth muscle cells (SMC) and their degree of differentiation. Mature SMC in the explant proper undergo gradual dedifferentiation toward more primitive cells, as indicated by the heavy outside arrows. First there is a decrease in the filamentous component of SMC with concomitant increase in organelles (modified SMC). Second, all filaments disappear, and the cells assume the forms of fibroblast-like or completely undifferentiated (“primitive”) cells. By electron microscopic autoradiography, ‘H-thymidine incorporation was demonstrated only in fibroblast-like or primitive cells (dotted nuclei). Conversely primitive and fibroblast-like cells appear to differentiate toward mature SMC, as demonstrated in the peripheral growth and indicated in this diagram by the light, inner arrows.
in culture for degree of differentiation of SMC. Results were confirmatory of observations made in the previous study suggesting progressive loss of filaments and increasing numbers of “primitive” undifferentiated cells. (2) Swine aortic explants that had been pulse labeled with 3H-thymidine were serially sectioned and examined by light microscopy in order to evaluate spatial relations of labeled cells to vasa vasorum. Results of such study should aid in evaluating
356
FRITZ,
JARMOLYCH,
AND
DAOUD
the contribution of the cells of the vasa vasorum to the undifferentiated cell population. (3) To determine whether medial SMC could be the sole source of outgrowth of cells, similar explants from the rabbit aortic media, which has no vasa vasorum, were studied. In Part II of the current study the second hypothesis was tested in two ways. (1) Cells pulse labeled with “H-thymidine were examined by electron microscopic autoradiography on the 2nd and 4th days to ascertain their degree of differentiation as compared to unlabeled cells. (2) Labeled cells were counted in swine aortic explants that had been pulse labeled with “H-thymidine after O-4 days in culture. The purpose of the latter procedure was to see whether the proportion of labeled cells increased as the number of well differentiated cells in the population decreased. MATERIALS
AND
METHODS
From the thoracic aorta of swine and rabbits, tubular segments 5.0 cm long were excised under sterile conditions 5-15 minutes after sacrifice. The swine were approximately 6-month-old slaughterhouse animals, weighing 60-75 kg. The rabbits, New Zealand white, weighed 2-3 kg. The segments were cleared of blood and extraneous material and transferred to ice-cold M-199 medium containing penicillin-streptomycin (100 U/ml) (Jarmolych et al., 1968). They were cut into rings, 1.0 cm long. The adventitia and outer third of the media were stripped and discarded. The ring was then everted and the intima with subjacent media stripped and discarded, leaving only the midportion of the media. Light microscopy confirmed that there was no residual intima or adventitia. A few intramedial vasa vasorum were present in the specimens from swine while those from rabbits contained none. The remaining medial tissue was rinsed in fresh medium and transferred to a Petri dish containing medium, where it was cut into 1 X 5 or 1 X l-mm segments. Segments were transferred to small (30-ml) plastic culture bottles containing 5.0 ml of a growth medium composed of M-199 supplemented with 20% swine serum and penicillin-streptomycin (100 U/ml) for swine explants and with 20% fetal calf serum for rabbit explants. The atmosphere in the bottle was replaced by 5% CO2 in air and the bottle sealed. Under these conditions the initial pH was 7.4. The bottles were incubated at 37%. The growth medium was changed and the atmosphere adjusted three times per week. Part I Explants from pig aorta. Over 100 segments from 19 swine were grown for 1, 2, 4, or 9 days and were then processed for light microscopy including autoradiography. In addition, noncultured segments of some of these aortas were processed in the same manner (0 day). Two segments each from 0, 1, 2, 4, and 9 days were serially sectioned. Segments from two of these 19 aortas cultured for 0, 1, 2, or 4 days were processed for routine electron microscopy. They were initially fixed in glutaraldehyde, postfixed in Os04, and embedded in Maraglas or Epon. Segments destined for autoradiography were processed as described below in Part II.
DNA
SYNTHESIS
AND
CELL
DEDIFFERENTIATION
357
Explants from rabbit aorta. Explants from five rabbits were grown up to 21 days and studied by routine light microscopy only. Part II
Segments taken from one of the two aortas used for routine electron microscopy were also exposed to “H-thymidine, 1.5 mCi/ml, for 2 hours at 37OC and processed for light microscopic autoradiography according to the method of Kopriwa and Leblond (1962). For electron microscopic autoradiography the procedure was as follows: segments were rinsed in fresh, nonradioactive medium, fixed in glutaraldehyde, utilizing 20 changes of fixative followed by 20 changes of buffer, postfixed in OsO*, and embedded in Maraglas or Epon. Thin sections were transferred to collodion-covered slides and stained with uranyl acetate and lead citrate. A layer of carbon, approximately 60 8, thick, was deposited on the slide in a high vacuum evaporator, and the slides were then coated with Kodak NTE emulsion concentrated according to the method of Salpeter and Bachman (1964). After exposure at 4°C in a nitrogen atmosphere for 12-14 weeks and development in Kodak Dektol following gold latensification, the specimens were transferred to grids for examination in an RCA EMU 3F microscope at initial magnification of X 5600. Light microscopic evaluation of cells synthesizing DNA was done on representative sections. The total number of cells and the number of labeled cells were obtained in three areas: middle and two ends of the section. The result was expressed as the ratio of labeled to total cells counted (in percent). RESULTS Part 1 Electron Microscopy. Ultrastructural study of representative samples of uncultured segments (0 days) showed that the cells were mainly mature smooth muscle cells (Fig. 2). The nucleus was generally elongated with condensation of chromatin at the periphery. The bulk of the cytoplasm was occupied by filaments, with numerous fusiform densities especially marked in the vicinity of the plasmalemma. A few vasa vasorum with typical endothelial cells having a thick basement membrane and tight junctions were also noted in some sections. After culture for 24 hours the SMC, in most instances, showed very few changes from the mature SMC described above. However, some showed a slight increase in the number of organelles, mainly free ribosomes, and a few revealed a fairly extensive increase in granular endoplasmic reticulum. The filaments, which were generally still the major cytoplasmic component, were somewhat decreased in amount. Only a few necrotic cells showing loss of integrity of organelles and disruption of cell or nuclear membranes were present. The endothelial cells of the vasa vasorum were still recognizable as such, with few or no morphological changes apparent. After 2 days in culture most SMC showed a decrease in filaments and a concomitant increase in organelles. In many cases these changes were extensive, and the number of organelles had increased so much that only a few remaining
358
FRITZ,
JARMOLYCH,
AND
DAOUD
FIG. 2. Mature smooth muscle cells (SMC) from an uncultured segment (0 day) of middle media of swine thoracic aorta. The bulk of the cytoplasm appeared fdamentous (Fill with numerous fusiform densities (FD). Organelles are sparse and localized mainly around the nucleus (N). The cell has a basement membrane (BM) and contains micropinocytotic vesicles (MPV). Virtually all of the SMC in the media of the normal swine aorta have this appearance. M, mitochondria; GER, granular endoplasmic reticulum; R, ribosomes. x 10,800.
DNA
SYNTHESIS
AND
CELL
DEDIFFERENTIATION
359
filaments could be seen at the periphery of the cells. Several cells appeared to have no filaments. Some of these undifferentiated cells had a partial basement membrane similar to that seen in the mature SMC. In the area of the vasa vasorum there appeared to be an increase in the number of cells, most of which retained some of the morphological characteristics of endothelial cells, such as the tight junction. In addition, fibroblast-like cells, probably perithelial, were present. The degree of cellular degeneration was more severe than at 1 day. After culture for 4 days in most areas the majority of cells showed no filaments. Many cells were elongated and resembled fibroblasta including an extensive granular endoplasmic reticulum, while the others showed few organelles but large numbers of free ribosomes, thus resembling primitive cells. Occasional cells had a partial basement membrane. In other areas the changes were less pronounced and the cells had some cytoplasmic SMC characteristics such as filaments, while in rare areas the SMC were only minimally altered. Degenerative changes were variable but tended to be more severe than at 2 days. Light microscopic studies of serial sections of swine explants. A few vasa vasorum could be identified in each explant cultured O-4 days if serial sections were studied. The numbers varied from one section to another and from one segment to another. In almost all samples, sections containing no vasa vasorum were observed. In some of the segments up to 10 and sometimes 20 consecutive sections were free of these structures. Autoradiography showed that 3H-thymidine labeled cells were distributed unevenly throughout the sections, but the number of labeled cells was not related to the presence or absence of vasa vasorum. Among the explants examined at 9 days, two had no vasa vasorum at all. These two had a peripheral outgrowth of the same magnitude as those with vasa vasorum. The numbers of labeled cells in the explant proper and in the peripheral growth did not appear to be related to the presence or absence of vasa vasorum. Rabbit explant study. As was previously mentioned, the aortic medial explant from rabbits had no vasa vasorum at all. Nevertheless, by about the 14th day these explants developed an outgrowth similar (by light microscopy) to that of pig explants. Part II Electron microscopic autoradiography. Because of the scarcity of labeled nuclei observed by light microscopy, no electron microscopic autoradiography of the zero and l-day explants was done. In 2-day explants no labeled nuclei were found, probably due to sampling. In 4-day explants approximately 11% of the nuclei examined were “H-thymidine labeled. All cells with a labeled nucleus were of the fibroblast-like (Fig. 3) or primitive type. Filaments consistent with myofilaments were not unequivocally identified in cells with a labeled nucleus, though a few highly suggestive structures were noted in occasional cells. Count of labeled cells by light microscopy. The results of the count of labeled cells in light microscopic autoradiography are presented in Table I. The number of labeled cells increased from an occasional cell at 24 hours to approximately 11% at 4 days. The distribution of labeled cells was, in general, random
360
FRITZ,
JARMOLYCH,
AND
DAOUD
FIG. 3. Electron microscopic autoradiograph of a fibroblast-like cell. The reduced silver grains (Gr) typical of NTE emulsion after development in Dektol and gold latensification are confined to the nucleus (N). The cytoplasm contains an extensive network of granular endoplasmic reticulum (GRR). This cell is from a 4-day explant‘and is typical of the type most frequently observed at this period. X 18,500.
DNA SYNTHESIS
TABLE RATIO
OF NUMBER COUNTED
No. of days Control 1 2 4
361
AND CELL DEDIFFERENTIATION I
OF 3H-T~~~~~~ LABELED CELLS TO TOTAL NUMBER CELLS (IN PERCENT) IN SWINE MEDIAL EXPLANTS
Total cells counted
Labeled cells
1412 1430 1297 1078
0 5 40 121
throughout the sections. However, variation from area to area in the same section.
OF
Percentage 0.0 0.4 3.0 11.2
existed from section to section and
DISCUSSION We found in this study, as previously, that essentially all cells in the original explant were packed with filaments but that by 4 days most of the .cells had few or none. Several explanations can be suggested to account for this observation. One is that the mature SMC present at the outset had died and been replaced by an overgrowth of endothelial cells from the vasa vasorum, but studies of serial sections of swine aortic explants indicate no significant role for cells from the vasa vasorum. In the rabbit explants no vasa vasorum were seen so that this source was eliminated, and yet there was an outgrowth similar to that seen with swine aortic explants, which do contain vasa vasorum. Another possible explanation for the increase in the number of cells without filaments is that filaments were lost through a process of degeneration. Some of the cells in the explants appeared to be degenerating and others were disintegrated. However, the fact that 11% were synthesizing DNA at the time of pulse labeling on the 4th day and that these 11% appeared by electron microscopy to be similar to most of the unlabeled cells makes it seem unlikely that degeneration per se accounts for the less differentiated appearance of these cells. A more likely explanation, consistent with our ultrastructural findings, is that loss of filaments progresses from day to day in viable SMC of the explants. Thus, data obtained in the current study are compatible with the hypothesis that the medial SMC in the explants undergo rapid dedifferentiation. The second hypothesis stated in the introduction also is supported by the observations. The proportion of labeled cells increased as the number of well differentiated SMC decreased. Furthermore, none of the labeled cells examined by electron microscopy was a well differentiated SMC. In fact, no filaments consistent in appearance with myofilaments were identified in any labeled cell. Thus it appears that at least in this in vitro situation dedifferentiation of SMC occurs before rapid cell proliferation takes place. In early gross atherosclerotic lesions induced in swine by high cholesterol diets (Daoud et al., 1969) and in the healing process of arterial injury (Murray et al., 1966), many of the cells have only sparse filaments and some appear to
362
FRITZ,
JARMOLYCH,
AND DAOUD
have none. The results of the current in vitro study suggest that these cells in the lesions could arise from mature SMC by dedifferentiation. The observations made in this study combined with those of the previous study of aortic explants (Jarmolych et al., 1968) provide evidence to support the scheme illustrated in Fig. 1. Such a cycle is also consistent with observations made on other types of specialized cells (Doljanski, 1930; Grobstein, 1959). REFERENCES A. S., JONES, R., and SCOTT, R. F. (1968). Dietary-induced atherosclerosis in miniature swine. II. Electron microscopy observations: characteristics of endothelial and smooth muscle cells in the proliferative lesions and elsewhere in the aorta. Exp. Mol. Pathol. 8, 263-301. DOLIANSKI, L. (1930). Sur le rapport entre la proliferation et l’activite pigmentogene dans les cultures d’epithelium de l’iris. C. R. Sot. Biol. 105, 343-345. GROBSTEIN, C. (1959). Differentiation of vertebrate cells. In “The Cell” (J. Bracket and A. E. Mirsky, eds.), Vol. I, pp. 437-496. Academic Press, New York. JARMOLYCH, J., DAO~D, A. S., LANDAU, J., FRITZ, K. E., and MCELVENE, E. (1968). Aortic media explants. Cell proliferation and production of mucopolysaccharides, collagen and elastic tissue. Exp. Mol. Pathol. 9, 171-188. KOPRIWA, B. M., and LEBLOND, C. P. (1962). Improvements in the coating techniques of radioautography. J. Histochem. Cytochem. 10, 269-284. MURRAY, M., SCHRODT, G. R., and BERG, H. F. (1966). Role of smooth muscle cells in healing of injured arteries. Arch. Pathol. 82, 138. SALPETER, M. M., and BACHMANN, L. (1964). Autoradiography with electron microscope. A procedure for improving resolution, sensitivity and contrast. J. Cell Biol. 22,469-477. Scm, R. F., JONES, R., DAOUD, A. S., ZLJMBO, O., COULSTON, F., and THOMAS, W. A. (1967). Experimental atherosclerosis in rhesus monkeys. II. Cellular elements of proliferative lesions and possible role of cytoplasmic degeneration in pathogenesis as studied by electron microscopy. Exp. DAOUD,
Mol.
Pathol.
7, 34-57.
AND
MOLECULAR
PATHOLOGY
12,
354-362
(1970)
Association of DNA Synthesis and Apparent Dedifferentiation of Aortic Smooth Muscle Cells in Vitro’ K. E. FRITZ, J. Departments
JARMOLYCH,
AND
A. S.
of Pathology, Veterans Administration Albany Medical College, Albany, N.
Received
January
DAOUD
Hospital,
and
Y.
20, 1970
Smooth muscle cells (SMC) of medial explants of swine thoracic aortas appear to undergo in the first few days of culture rapid dedifferentiation toward more primitive forms. The number of cells synthesizing DNA in the explants increases as the number of well differentiated SMC decreases. Electron microscopic autoradiography revealed that more of the cells incorporating JH-thymidine were poorly differentiated SMC. Thus it appears that in this in vitro situation dedifferentiation of SMC precedes rapid cell proliferation. In early cholesterol-induced atherosclerosis in swine many partially or completely undifferentiated cells are present. The results of the current in vitro study suggest that the undifferentiated cells in the atherosclerotic lesions could arise from mature SMC through dedifferentiation.
In a previous study (Jarmolych et al., 1968) we have reported that medial explants of the thoracic aorta of the swine, cultured in semisynthetic medium, developed a new peripheral growth by about the 4th day, which increased in amount at least up to 21 days when the experiment was terminated. Electron microscopic studies showed that in the early cultures most cells of the peripheral growth had no filaments or other specialized features characteristic of smooth muscle cells (SMC) and could not be classified as to origin (fibroblastlike and undifferentiated or primitive cells). Light microscopy autoradiographic studies after “H-thymidine pulse labeling revealed numerous labeled nuclei in the peripheral growth. In older cultures filaments were demonstrable in most cells, and by the Zlst day almost all cells had substantial numbers of fusiform densities and of filaments consistent with myofilaments. Autoradiographic studies of these older cultures showed a decrease in number of labeled cells concomitant with the increase in specialized features. From
the above observations
we formulated
the following
two
hypotheses
which are illustrated diagrammatically in Fig. 1. First, SMC in the original explant can undergo dedifferentiation in the first few days toward more primitive forms giving rise to the undifferentiated cells in the early peripheral growth which subsequently redifferentiate toward SMC. Second, rapid cell proliferation does not occur until extensive dedifferentiation has taken place. In Part I of the current study the first hypothesis was tested in three ways. (1) Swine aortic explants were examined by electron microscopy after O-4 days I This study was supported Brown-Hazen Fund.
by USPHS
Grant
H-7155,
354
VA Research
Funds
and a grant
from
the
DNA
SYNTHESIS
AND
CELL
DEDIFFERENTIATION
355
MBuusLA5T-LIuE CELLS
FIG. 1. This schematic diagram illustrates our conception of the relationship between DNA synthesis in smooth muscle cells (SMC) and their degree of differentiation. Mature SMC in the explant proper undergo gradual dedifferentiation toward more primitive cells, as indicated by the heavy outside arrows. First there is a decrease in the filamentous component of SMC with concomitant increase in organelles (modified SMC). Second, all filaments disappear, and the cells assume the forms of fibroblast-like or completely undifferentiated (“primitive”) cells. By electron microscopic autoradiography, ‘H-thymidine incorporation was demonstrated only in fibroblast-like or primitive cells (dotted nuclei). Conversely primitive and fibroblast-like cells appear to differentiate toward mature SMC, as demonstrated in the peripheral growth and indicated in this diagram by the light, inner arrows.
in culture for degree of differentiation of SMC. Results were confirmatory of observations made in the previous study suggesting progressive loss of filaments and increasing numbers of “primitive” undifferentiated cells. (2) Swine aortic explants that had been pulse labeled with 3H-thymidine were serially sectioned and examined by light microscopy in order to evaluate spatial relations of labeled cells to vasa vasorum. Results of such study should aid in evaluating
356
FRITZ,
JARMOLYCH,
AND
DAOUD
the contribution of the cells of the vasa vasorum to the undifferentiated cell population. (3) To determine whether medial SMC could be the sole source of outgrowth of cells, similar explants from the rabbit aortic media, which has no vasa vasorum, were studied. In Part II of the current study the second hypothesis was tested in two ways. (1) Cells pulse labeled with “H-thymidine were examined by electron microscopic autoradiography on the 2nd and 4th days to ascertain their degree of differentiation as compared to unlabeled cells. (2) Labeled cells were counted in swine aortic explants that had been pulse labeled with “H-thymidine after O-4 days in culture. The purpose of the latter procedure was to see whether the proportion of labeled cells increased as the number of well differentiated cells in the population decreased. MATERIALS
AND
METHODS
From the thoracic aorta of swine and rabbits, tubular segments 5.0 cm long were excised under sterile conditions 5-15 minutes after sacrifice. The swine were approximately 6-month-old slaughterhouse animals, weighing 60-75 kg. The rabbits, New Zealand white, weighed 2-3 kg. The segments were cleared of blood and extraneous material and transferred to ice-cold M-199 medium containing penicillin-streptomycin (100 U/ml) (Jarmolych et al., 1968). They were cut into rings, 1.0 cm long. The adventitia and outer third of the media were stripped and discarded. The ring was then everted and the intima with subjacent media stripped and discarded, leaving only the midportion of the media. Light microscopy confirmed that there was no residual intima or adventitia. A few intramedial vasa vasorum were present in the specimens from swine while those from rabbits contained none. The remaining medial tissue was rinsed in fresh medium and transferred to a Petri dish containing medium, where it was cut into 1 X 5 or 1 X l-mm segments. Segments were transferred to small (30-ml) plastic culture bottles containing 5.0 ml of a growth medium composed of M-199 supplemented with 20% swine serum and penicillin-streptomycin (100 U/ml) for swine explants and with 20% fetal calf serum for rabbit explants. The atmosphere in the bottle was replaced by 5% CO2 in air and the bottle sealed. Under these conditions the initial pH was 7.4. The bottles were incubated at 37%. The growth medium was changed and the atmosphere adjusted three times per week. Part I Explants from pig aorta. Over 100 segments from 19 swine were grown for 1, 2, 4, or 9 days and were then processed for light microscopy including autoradiography. In addition, noncultured segments of some of these aortas were processed in the same manner (0 day). Two segments each from 0, 1, 2, 4, and 9 days were serially sectioned. Segments from two of these 19 aortas cultured for 0, 1, 2, or 4 days were processed for routine electron microscopy. They were initially fixed in glutaraldehyde, postfixed in Os04, and embedded in Maraglas or Epon. Segments destined for autoradiography were processed as described below in Part II.
DNA
SYNTHESIS
AND
CELL
DEDIFFERENTIATION
357
Explants from rabbit aorta. Explants from five rabbits were grown up to 21 days and studied by routine light microscopy only. Part II
Segments taken from one of the two aortas used for routine electron microscopy were also exposed to “H-thymidine, 1.5 mCi/ml, for 2 hours at 37OC and processed for light microscopic autoradiography according to the method of Kopriwa and Leblond (1962). For electron microscopic autoradiography the procedure was as follows: segments were rinsed in fresh, nonradioactive medium, fixed in glutaraldehyde, utilizing 20 changes of fixative followed by 20 changes of buffer, postfixed in OsO*, and embedded in Maraglas or Epon. Thin sections were transferred to collodion-covered slides and stained with uranyl acetate and lead citrate. A layer of carbon, approximately 60 8, thick, was deposited on the slide in a high vacuum evaporator, and the slides were then coated with Kodak NTE emulsion concentrated according to the method of Salpeter and Bachman (1964). After exposure at 4°C in a nitrogen atmosphere for 12-14 weeks and development in Kodak Dektol following gold latensification, the specimens were transferred to grids for examination in an RCA EMU 3F microscope at initial magnification of X 5600. Light microscopic evaluation of cells synthesizing DNA was done on representative sections. The total number of cells and the number of labeled cells were obtained in three areas: middle and two ends of the section. The result was expressed as the ratio of labeled to total cells counted (in percent). RESULTS Part 1 Electron Microscopy. Ultrastructural study of representative samples of uncultured segments (0 days) showed that the cells were mainly mature smooth muscle cells (Fig. 2). The nucleus was generally elongated with condensation of chromatin at the periphery. The bulk of the cytoplasm was occupied by filaments, with numerous fusiform densities especially marked in the vicinity of the plasmalemma. A few vasa vasorum with typical endothelial cells having a thick basement membrane and tight junctions were also noted in some sections. After culture for 24 hours the SMC, in most instances, showed very few changes from the mature SMC described above. However, some showed a slight increase in the number of organelles, mainly free ribosomes, and a few revealed a fairly extensive increase in granular endoplasmic reticulum. The filaments, which were generally still the major cytoplasmic component, were somewhat decreased in amount. Only a few necrotic cells showing loss of integrity of organelles and disruption of cell or nuclear membranes were present. The endothelial cells of the vasa vasorum were still recognizable as such, with few or no morphological changes apparent. After 2 days in culture most SMC showed a decrease in filaments and a concomitant increase in organelles. In many cases these changes were extensive, and the number of organelles had increased so much that only a few remaining
358
FRITZ,
JARMOLYCH,
AND
DAOUD
FIG. 2. Mature smooth muscle cells (SMC) from an uncultured segment (0 day) of middle media of swine thoracic aorta. The bulk of the cytoplasm appeared fdamentous (Fill with numerous fusiform densities (FD). Organelles are sparse and localized mainly around the nucleus (N). The cell has a basement membrane (BM) and contains micropinocytotic vesicles (MPV). Virtually all of the SMC in the media of the normal swine aorta have this appearance. M, mitochondria; GER, granular endoplasmic reticulum; R, ribosomes. x 10,800.
DNA
SYNTHESIS
AND
CELL
DEDIFFERENTIATION
359
filaments could be seen at the periphery of the cells. Several cells appeared to have no filaments. Some of these undifferentiated cells had a partial basement membrane similar to that seen in the mature SMC. In the area of the vasa vasorum there appeared to be an increase in the number of cells, most of which retained some of the morphological characteristics of endothelial cells, such as the tight junction. In addition, fibroblast-like cells, probably perithelial, were present. The degree of cellular degeneration was more severe than at 1 day. After culture for 4 days in most areas the majority of cells showed no filaments. Many cells were elongated and resembled fibroblasta including an extensive granular endoplasmic reticulum, while the others showed few organelles but large numbers of free ribosomes, thus resembling primitive cells. Occasional cells had a partial basement membrane. In other areas the changes were less pronounced and the cells had some cytoplasmic SMC characteristics such as filaments, while in rare areas the SMC were only minimally altered. Degenerative changes were variable but tended to be more severe than at 2 days. Light microscopic studies of serial sections of swine explants. A few vasa vasorum could be identified in each explant cultured O-4 days if serial sections were studied. The numbers varied from one section to another and from one segment to another. In almost all samples, sections containing no vasa vasorum were observed. In some of the segments up to 10 and sometimes 20 consecutive sections were free of these structures. Autoradiography showed that 3H-thymidine labeled cells were distributed unevenly throughout the sections, but the number of labeled cells was not related to the presence or absence of vasa vasorum. Among the explants examined at 9 days, two had no vasa vasorum at all. These two had a peripheral outgrowth of the same magnitude as those with vasa vasorum. The numbers of labeled cells in the explant proper and in the peripheral growth did not appear to be related to the presence or absence of vasa vasorum. Rabbit explant study. As was previously mentioned, the aortic medial explant from rabbits had no vasa vasorum at all. Nevertheless, by about the 14th day these explants developed an outgrowth similar (by light microscopy) to that of pig explants. Part II Electron microscopic autoradiography. Because of the scarcity of labeled nuclei observed by light microscopy, no electron microscopic autoradiography of the zero and l-day explants was done. In 2-day explants no labeled nuclei were found, probably due to sampling. In 4-day explants approximately 11% of the nuclei examined were “H-thymidine labeled. All cells with a labeled nucleus were of the fibroblast-like (Fig. 3) or primitive type. Filaments consistent with myofilaments were not unequivocally identified in cells with a labeled nucleus, though a few highly suggestive structures were noted in occasional cells. Count of labeled cells by light microscopy. The results of the count of labeled cells in light microscopic autoradiography are presented in Table I. The number of labeled cells increased from an occasional cell at 24 hours to approximately 11% at 4 days. The distribution of labeled cells was, in general, random
360
FRITZ,
JARMOLYCH,
AND
DAOUD
FIG. 3. Electron microscopic autoradiograph of a fibroblast-like cell. The reduced silver grains (Gr) typical of NTE emulsion after development in Dektol and gold latensification are confined to the nucleus (N). The cytoplasm contains an extensive network of granular endoplasmic reticulum (GRR). This cell is from a 4-day explant‘and is typical of the type most frequently observed at this period. X 18,500.
DNA SYNTHESIS
TABLE RATIO
OF NUMBER COUNTED
No. of days Control 1 2 4
361
AND CELL DEDIFFERENTIATION I
OF 3H-T~~~~~~ LABELED CELLS TO TOTAL NUMBER CELLS (IN PERCENT) IN SWINE MEDIAL EXPLANTS
Total cells counted
Labeled cells
1412 1430 1297 1078
0 5 40 121
throughout the sections. However, variation from area to area in the same section.
OF
Percentage 0.0 0.4 3.0 11.2
existed from section to section and
DISCUSSION We found in this study, as previously, that essentially all cells in the original explant were packed with filaments but that by 4 days most of the .cells had few or none. Several explanations can be suggested to account for this observation. One is that the mature SMC present at the outset had died and been replaced by an overgrowth of endothelial cells from the vasa vasorum, but studies of serial sections of swine aortic explants indicate no significant role for cells from the vasa vasorum. In the rabbit explants no vasa vasorum were seen so that this source was eliminated, and yet there was an outgrowth similar to that seen with swine aortic explants, which do contain vasa vasorum. Another possible explanation for the increase in the number of cells without filaments is that filaments were lost through a process of degeneration. Some of the cells in the explants appeared to be degenerating and others were disintegrated. However, the fact that 11% were synthesizing DNA at the time of pulse labeling on the 4th day and that these 11% appeared by electron microscopy to be similar to most of the unlabeled cells makes it seem unlikely that degeneration per se accounts for the less differentiated appearance of these cells. A more likely explanation, consistent with our ultrastructural findings, is that loss of filaments progresses from day to day in viable SMC of the explants. Thus, data obtained in the current study are compatible with the hypothesis that the medial SMC in the explants undergo rapid dedifferentiation. The second hypothesis stated in the introduction also is supported by the observations. The proportion of labeled cells increased as the number of well differentiated SMC decreased. Furthermore, none of the labeled cells examined by electron microscopy was a well differentiated SMC. In fact, no filaments consistent in appearance with myofilaments were identified in any labeled cell. Thus it appears that at least in this in vitro situation dedifferentiation of SMC occurs before rapid cell proliferation takes place. In early gross atherosclerotic lesions induced in swine by high cholesterol diets (Daoud et al., 1969) and in the healing process of arterial injury (Murray et al., 1966), many of the cells have only sparse filaments and some appear to
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have none. The results of the current in vitro study suggest that these cells in the lesions could arise from mature SMC by dedifferentiation. The observations made in this study combined with those of the previous study of aortic explants (Jarmolych et al., 1968) provide evidence to support the scheme illustrated in Fig. 1. Such a cycle is also consistent with observations made on other types of specialized cells (Doljanski, 1930; Grobstein, 1959). REFERENCES A. S., JONES, R., and SCOTT, R. F. (1968). Dietary-induced atherosclerosis in miniature swine. II. Electron microscopy observations: characteristics of endothelial and smooth muscle cells in the proliferative lesions and elsewhere in the aorta. Exp. Mol. Pathol. 8, 263-301. DOLIANSKI, L. (1930). Sur le rapport entre la proliferation et l’activite pigmentogene dans les cultures d’epithelium de l’iris. C. R. Sot. Biol. 105, 343-345. GROBSTEIN, C. (1959). Differentiation of vertebrate cells. In “The Cell” (J. Bracket and A. E. Mirsky, eds.), Vol. I, pp. 437-496. Academic Press, New York. JARMOLYCH, J., DAO~D, A. S., LANDAU, J., FRITZ, K. E., and MCELVENE, E. (1968). Aortic media explants. Cell proliferation and production of mucopolysaccharides, collagen and elastic tissue. Exp. Mol. Pathol. 9, 171-188. KOPRIWA, B. M., and LEBLOND, C. P. (1962). Improvements in the coating techniques of radioautography. J. Histochem. Cytochem. 10, 269-284. MURRAY, M., SCHRODT, G. R., and BERG, H. F. (1966). Role of smooth muscle cells in healing of injured arteries. Arch. Pathol. 82, 138. SALPETER, M. M., and BACHMANN, L. (1964). Autoradiography with electron microscope. A procedure for improving resolution, sensitivity and contrast. J. Cell Biol. 22,469-477. Scm, R. F., JONES, R., DAOUD, A. S., ZLJMBO, O., COULSTON, F., and THOMAS, W. A. (1967). Experimental atherosclerosis in rhesus monkeys. II. Cellular elements of proliferative lesions and possible role of cytoplasmic degeneration in pathogenesis as studied by electron microscopy. Exp. DAOUD,
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