J. Embryol. exp. Morph., Vol. 15, 2, pp. 213-221, April 1966
With 2 plates
Printed in Great Britain
213
Regeneration from mesenteric arteries
in short-term culture
By J. D. FELDMAN & D. L. GARDNER 1
From the Department of Pathology, University of Edinburgh
The investigations which are described here were originally undertaken in
order to investigate the pathogenesis of early hypertension at a time when no
morphological changes were evident in adrenergic arteries on light microscopy.
Evidence of functional change was therefore sought by comparing the growth
patterns of cells from hypertensive and normal mesenteric arteries in culture.
The study of the cells which migrated from the explants form the basis for this
paper.
MATERIALS AND METHODS
Month-old Wistar laboratory-strain albino rats were used for the experiment.
Young rats were deliberately selected, as fat could be more cleanly dissected
away from vessel walls. Also: 'Tissues from old animals are often more difficult
to grow than tissues from young animals and exhibit a longer lag period before
growth commences' (Paul, 1965a).
Each week, over a period of 14 weeks, hypertension was induced in a group of
rats according to the method of Loomis (1946). One animal from this group was
matched for weight with an animal from a control group of rats in which a sham
operation had been performed. Nine explants from the branches of the superior
mesenteric artery of each animal were then set up in culture. Mesenteric vessels
were selected for this experiment as they are easily isolated, and at the same time
they are representative of visceral adrenergic arteries which are known to exhibit
pathological changes on light microscopy when exposed over a sufficient length
of time to a raised systemic blood pressure (Loomis, 1946; Masson, McCormack,
Dustan & Corcoran, 1958).
The removal of arteries was accomplished with the rats under ether anaesthesia, and all precautions to ensure sterility were observed. The abdominal
wall was opened in layers, and the coeliac gland exposed. The superior mesenteric
artery was then easily identified in close proximity to the gland, stripped of
surrounding mesentery, and clamped proximally. Blood was cleared from the
artery and its branches by injecting 1 ml of cold Hanks's solution into the
1
Authors' address: Hypertension Research Laboratory, Department of Pathology,
University New Buildings, Teviot Place, Edinburgh 8, Scotland.
214
J. D. FELDMAN & D. L. GARDNER
artery, distal to the clamp. Tissues were kept moist during dissection by dripping
Hanks's solution slowly from a reservoir bottle.
After teasing away surrounding mesentery, the artery and its branches were
quickly removed, and stripping of peri-arterial fat and fibrous tissue was completed under the dissecting microscope. The main branches were then cut into
segments of almost equal size with iris scissors, while the superior mesenteric
artery itself was steadied with forceps. Three segments were transferred by
pipette to each of three coverslips, and excess Hanks's solution removed. A
drop of cockerel plasma obtained by carotid artery bleeding (Paul, 19656) was
pipetted on to each segment of artery, and removed after a short period of time.
The fine film of plasma which remained was sufficient to anchor the vessels and
caused minimal interference when preparations were viewed under phasecontrast conditions at a later time (G. W. Pearce, personal communication).
Coverslips were finally inverted over culture slides with a flat central depression suitable for phase microscopy (supplied by Paul Rosenthal, N.Y.). The
medium used was composed of Eagle's (Burroughs and Welcome), calf serum
(Oxoid), and an extract of 10-day-old chick embryos, prepared according to the
method described by Paul (1965c). These were combined in a ratio of 8:1:1
(M. J. W. Faed, personal communication).
Cultures were usually observed over a period of 7 days, although some were
followed for up to 30 days. On the 4th day of culture the medium was removed
in part only, so that.some of the cell products of metabolism which had been
liberated into the medium during culture, and which were possibly essential for
the process of differentiation, remained. Fresh medium was added as a replacement.
PLATE 1
Fig. A. Phase-light cells, variously shaped, emerge from the end of an artery. The cells are
linked to one another by fine cytoplasmic processes. Three-day culture. Hypertensive rat.
Phase contrast, x200.
Fig. B. Detail of cells shown in fig. A. Nuclei and nucleoli are easily distinguished. Coarse
granules are scattered through the cytoplasm, apart from a complete or partial rim around
the nucleus. Phase contrast, x 400.
Fig. C. Phase-light cells from arterial end form a pavement-like pattern because of their
symmetrical shapes in close association. Three-day culture. Hypertensive rat. Phase contrast, x200.
Fig. D. Detail of cells shown in fig. C. The roughly pentagonal shape of the cells is more
obvious at this magnification. Phase contrast, x 400.
Fig. E. Profuse growth of cells from arterial end. A phase-dark tube is particularly well seen
in the middle of the field. Dark segments of the tube are separated from one another by
refractile globules. The nuclei cannot be easily distinguished in unstained preparations.
Seven-day culture. Hypertensive rat. Phase contrast, x 200.
Fig. F. Darkly stained tubes with prominent ellipsoid nuclei emerge from arterial end, and
contrast well with background of spindle and stellate cells containing many cytoplasmic
vacuoles. Eight-day culture. Control sham-operated rat. May-Griinewald/Giemsa stain,
x400.
J. Embryol. exp. Morph., Vol. 15, Part 2
J. D. FELDMAN & D. L. GARDNER
PLATE
facing p. 214
/. Embryo/, exp. Morph., Vol. 15, Part 2
J. D. FELDMAN & D. L. GARDNER
PLATE 2
facing p. 215
Regeneration from mesenteric arteries
215
RESULTS
Cells were observed daily under phase-contrast conditions at a magnification
of up to x 400. Higher powers could not be used as the thickness of the specimens then interfered with the phase system.
Two distinct groups of cells emerged from the ends of the arteries in culture:
(1) Phase-light cells
These cells migrated from the explant on about the 2nd day, singly or in
groups, and in different focal planes, presumably along the lines of the fibrin
clot. They were spindle, kite, triangular or stellate in shape (Plate 1, fig. A) and
contained a well-defined nucleus with one to four prominent nucleoli. The
nuclei occupied a central or paracentral position, and tended to be round or
oval in shape—conforming to the stellate or spindle form of the surrounding
cell. Mitotic figures were frequently observed. The nucleus was always surrounded by a number of coarse, refractile granules. These consistently left a
space, at first encircling, and later to one side of the nucleus (Plate 1, fig. B).
No Golgi apparatus or other cell organelle could be distinguished within the
space. Within 24-48 h the granules increased in number to such an extent that
they filled the previously pale-grey cytoplasm, and extended into the cell processes. The granules stained black with Sudan Black III, and indicated the
aggregation of fat droplets. Fine black granules situated peripheral to the coarse
granules described were thought to be mitochondria. No fibrils were seen
coursing through the cytoplasm, either under phase-contrast conditions (with
and without polarized light), or in stained preparations (haematoxylin and
eosin, May-Grunewald/Giemsa, Pollak's trichrome or Masson's trichrome).
PLATE 2
Fig. G. Phase-dark multicellular straps from arterial end connect with one another by means
of arched bands. The lowermost strap has a characteristically blunt end. Seven-day culture.
Hypertensive rat. Phase contrast, x 200.
Fig. H. Detail of interconnexion between strands of phase-dark cells. Seven-day culture.
Hypertensive rat. Phase contrast, x 400.
Fig. I. Central darkly stained strap is intermediate in development between the tube shown
in fig. F, where a single nucleus occupies the width of the tube at any given point, and the
thicker multicellular strap shown in fig. J. Six-day culture. Hypertensive rat. Pollak's trichrome stain, x 1000.
Fig. J. Multicellular strap from arterial end. Fibrils cannot be distinguished within cytoplasm. Six-day culture. Hypertensive rat. Pollak's trichrome stain, x 1000.
Fig. K. Twig-like arrangement of cells from side of artery. Three-day culture. Hypertensive
rat. Phase contrast, x 200.
Fig. L. Detail of cell from fig. K. The cell shown in this figure is not well focused in fig. K
as it is in a different focal plane from three other cells illustrated. At this magnification it can
be seen that the cell body is similar to the phase-light cell shown at the bottom offig.B. Phase
contrast, x400.
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J. D. FELDMAN & D. L. GARDNER
Cell processes were fine and branching, and often faded imperceptibly into the
medium, especially in older cells. Frequently the cell emerged from the explant
on a fine stalk which supported a cytoplasmic bulge containing the nucleus and
terminating in antler-like processes. The size of cell varied greatly depending on
the cell shape and the length of time in culture. Early cultures sometimes showed
thin needle-like processes emerging from the explant, in which no further
cellular detail was visible.
Most cells associated with one another in culture to form various patterns
such as a loose network interconnected by fine processes (Plate 1, figs. A, B)
or closer groups in which the cell bodies lay alongside one another. In the latter
case, if the cells were predominantly symmetrical in shape, a pavement-like
pattern resulted (Plate 1, figs. C, D). Sometimes cell boundaries became
indistinguishable, and a syncytium resulted. Radiating streamers of cells frequently developed after about 6 days in culture.
(2) Phase-dark cells
These cells emerged later than the phase-light cells, on approximately the
3rd to 4th day, and usually migrated out superiorly to the latter. The phasedark cells tended to arise singly more often than in groups, and appeared first
as dark tubes which terminated in blunt processes. The latter sometimes went
on to form branches which met with one another at an acute angle. Granules
appeared later and accumulated less rapidly than in phase-light cells. Although
cell boundaries were not obvious within a single tube, the poorly defined
nuclei were usually separated by a single refractile globule (Plate 1, fig. E). The
nuclei tended to occupy the width of the tube, and contained one to four
nucleoli. These features are better observed in stained preparations (Plate 1,
fig. F). Staining of tubes with silver nitrate did not produce any marking, which
supported our impression of a syncytium at this stage. After about 24 h in
culture, some of the tubes became linked by interconnecting arched bands; and
both the tubes and bands developed into thick, irregular, multicellular straps
terminating in blunt pseudopod-like processes (Plate 2, fig. G). After 2-3 days
in culture, it became increasingly difficult to follow the development of these
straps, as the proliferation of phase-light cells made observation of a strap
throughout its course impossible. As in the case of the phase-light cells, no
fibrils were seen in stained or unstained preparations (Plate 2, figs. H-J).
The cells which emerged from the sides of the arteries were for the most part
similar to the phase-light cells described previously. Only occasionally did a
phase-dark tube arise from the side of an artery, and when it did so it tended to
migrate out from near the cut end. This occurred particularly in a separate
group of experiments when glutamine was added to the medium, and in which
phase-dark tube and strap growth was also particularly abundant from the
ends of the arteries. Side cells did not very often arrange themselves in
pavement-like patterns. On the other hand, in earlier cultures, cells very often
Regeneration from mesenteric arteries
217
formed associations which resembled twigged branches (Plate 2, figs. K, L).
Later the cells tended to form loose networks or more closely arranged patterns
of spindle-shaped cells similar to those described from arterial ends.
Hypertensive arteries
There were no recognizable differences in the cell types that grew out from
normal and hypertensive arteries.
DISCUSSION
Since no differences were observed between the cells of arteries exposed to an
acute rise of pressure and those of controls, the discussion in this paper is
directed towards an interpretation of the character of cells emerging from the
arterial explants in culture.
Our phase-light cells are non-specific in their morphology, and also in the
patterns of association which they form together. Because many investigators
(Gaillard, 1935; Fell & Mellanby, 1953; Grobstein & Zwilling, 1953; Moscona,
1957; Rose, Pomerat, Shindler & Trunnell, 1958; Endo, 1960; Konigsberg,
1960, 1961) have shown that cells in culture are capable of differentiation when
a suitable culture environment, i.e. medium plus cellular population, is supplied,
we have concluded that the phase-light cells in our cultures represented ' covert
states of differentiation which, by current methods, are undetectable at the time
of analysis' (Grobstein, 1965).
The phase-dark cells differed from the phase-light cells in two important
aspects; viz. their cytoplasm was darker with fewer granules in older cells, and
also the cells came together in a definite, consistent and characteristic way, to
form first tubes and then multicellular, interconnecting cords, as described
above. These features are characteristic not only of our cells, but also of the
solid plexuses of cells which emerged from blood vessels in the embryonic chick
cultures of skin and subcutaneous tissues described by Lewis (1931). Lewis
considered it most likely that the outgrowing cords were of vascular endothelial
cells, which probably became highly differentiated at an early stage of development, and revealed their differentiated nature by their ability to form ' plexuses
of strands'. Because of the resemblance between the latter cells and our own in
both cytoplasmic appearance and cellular arrangement, we thought it quite
possible that the phase-dark cells produced in our culture environment similarly
represented vascular endothelial cells, a view supported by the work of Faed,
MacGregor & Gardner (1964) in which it is shown that the cells which grew
out from the cut ends of mesenteric arteries in culture ' emerged as strands or
tubes' and that 'fixed and sectioned material showed the strands to arise from
endothelium'. If this is so, it then becomes necessary to explain why it is that
cells from a fully developed animal reveal their differentiated nature in culture
by the formation of morphological patterns which we associate with function
at a much earlier stage of development.
218
J. D. FELDMAN & D. L. GARDNER
The answer may perhaps be found by examining the situations in which injury
to the body is followed by healing or regeneration, because in these instances
new vascular endothelium, and probably smooth muscle also, must be created
in the tissues of the fully formed animal. If we find that the formation of new
capillaries and possibly larger arteries under these conditions occurs in exactly
the same way as it does in the embryo, then it is possible to postulate that it is
the beginning of this regeneration that we are witnessing in the arteries of our
young adult rats in tissue culture, and that this is why the endothelial cells in
explants from adult tissues have differentiated in culture in exactly the same way
as they would do in an embryo.
Clark & Clark (1939) were able to study the manner in which new blood
vessels regenerate and become transformed into an adult pattern by observing
wound healing inside transparent chambers which had been inserted into the
ears of adult rabbits. They described the process of capillary development not
only in these adult animals, but also in amphibian larvae, and established that
the pattern of regeneration of blood vessels was similar in each; it is also very
similar to the picture we have obtained in culture. Their findings, then, tend to
support the view outlined above, while our studies, in turn, would support their
view that the actual initiation of endothelial sprouting is dependent on local
rather than circulatory factors, the latter being completely absent in our
experiments.
The subsequent stages of ontogeny of arteries in culture may require a more
complex arrangement of cellular architecture than that at present provided in
tissue culture, as the necessary induction of further differentiation may relate to
supracellular factors dependent on ' group relations amongst cells' (Grobstein,
1965). The provision of a three-dimensional framework by O'Neal et al. (1964),
for example, might have been the crucial factor which allowed for the differentiation of endothelial and smooth muscle within the dacron hubs which they
inserted into the centres of vascular prostheses in vivo. In order to isolate this
factor from the many others operating in vivo, such as, for example, the existence
of an established circulation, further studies using tissue culture techniques, with
environmental modifications and additions, may prove valuable.
Our failure to identify smooth muscle cells amongst those migrating from the
explant is probably due to the restriction of migration potential brought about
by the connective tissue binding these cells together in the explant (M. Matthews,
in preparation), although it could be explained, as Weiss (1949) has previously
suggested, by an absence in the culture medium of certain 'non-determinate
necessary factors' without which myofibrils cannot be formed in migrating
muscle cells. Because these factors may require time to accumulate in culture or,
alternatively, because migration of smooth muscle cells may only occur after a
latent period (Kasai & Pollak, 1964), some explants were observed for periods
of up to 30 days, but fibrils were not seen to develop.
It is possible, however, that some of the outgrowing phase-dark tubes repre-
Regeneration from mesenteric arteries
219
sent developing myoblasts, which' begin their programme of differentiation by
elongation' and 'at an early stage seem to interconnect in a syncytial manner'
(Arey, 1965).
With the time lapse cineLphotography studies of our cells which are at present
being carried out, it is hoped to investigate further not only the behaviour of the
phase-dark cells, but also to follow in more detail the development of the
various patterns of association of the phase-light cells, which have not been
discussed here.
SUMMARY
1. The pathogenesis of early hypertension was investigated by culturing
explants of mesenteric arteries from hypertensive and control sham-operated
rats.
2. The two types of cells which were seen to emerge from the explants when
viewed under phase-contrast conditions were called phase-light and phase-dark
cells respectively. No differences between cells from explants of hypertensive
and control animals were apparent.
3. The development of the phase-dark cells into a pattern of interconnecting,
arched cords was compared with the outgrowth obtained from embryonic
blood vessels in culture, and found to be similar. The resemblance between both
these outgrowths of cells, and the initial migration of cords of endothelial cells
from the developing blood vessels of embryos and the regenerating arteries in
healing wounds, led us to postulate that the phase-dark cells emerging from our
arteries may represent the first stage of arterial regeneration in culture.
4. We hypothesized further that the addition of certain factors to the culture
environment may result in the induction of the subsequent development of the
arteries.
RESUME
Regeneration des arteres mesenteriques dans les cultures
a court terme
1. La pathogenese de l'hypertension precoce a ete etudiee au moyen de
cultures d'explants d'arteres mesenteriques de rats hypertendus et de temoins
ay ant subi une operation simulee.
2. Les deux types de cellules qui ont ete vues emigrer des explants ont ete
appeles respectivement' cellules claires en phase' e t ' cellules sombres en phase',
d'apres leur aspect en contraste de phase. On n'a pas vu de differences
entre les cellules provenant d'explants d'animaux hypertendus et d'animaux
temoins.
3. Le developpement des cellules 'sombres en phase' en cordons anastomoses et arques, a ete compare avec celui des excroissances obtenues a partir
de vaisseaux sanguins embryonnaires en culture et s'est trouve semblable a lui.
220
J. D. FELDMAN & D. L. GARDNER
La ressemblance entre ces deux types d'excroissances cellulaires et la migration
initiale des cordons de cellules endotheliales a partir des vaisseaux sanguins
embryonnaires en cours de developpement, et les arteres en regeneration dans
les blessures en cours de cicatrisation, nous a amenes a supposer que les cellules 'sombres en phase' emigrant de nos arteres pourraient representer le
premier stade de la regeneration arterielle en culture.
4. Nous avons en outre exprime Phypothese que l'adjonction de certains
facteurs au milieu de culture peut aboutir a l'induction du developpement
consecutif des arteres.
We are grateful to Professor G. L. Montgomery for his criticism and to the technical staff
of the Pathology Department, particularly Miss L. D'Arcy. One of the authors (J. D. F.)
especially wishes to thank the scientists who have given so freely of their time in order to
debate the author's problems with her. The assistance given by Mr James Paul of the
University Department of Medical Photography is very much appreciated.
This work was directly supported by a grant from the British Heart Foundation. During
the period when this work was carried out the laboratory was in receipt of support from the
National Institute of Health, Washington (HE-05173).
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