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Ultrastructural studies on early elastogenesis.

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Ultrastructural Studies on Early Elastogenesis ’
W. H. FAHRENBACH. L. B. SANDBERG AND E. G. CLEARY’
Department of Electron Microscopy, Oregon Regional Primate Research
Center, Beaverton, Oregon; Departments of Biochemistry and
Experimental Biology, University of Oregon Medical School,
Portland, Oregon
ABSTRACT
Elastogenesis in the ligamentum nuchae of the fetal calf commences
with the extracellular deposition of hollow-appearing filaments, 130 A in diameter,
showing a banding pattern consisting of alternating segments, 50 A and 130 A long,
respectively. Similar filaments are found in t h e crude extract of t h e ligament. Mature
elastin forms within these masses of filaments a n d displays a n internal, branching
tangle of 30 A filaments. Elastogenesis is accompanied by a high level of cellular
activity involving acanthosomes. which have been interpreted a s a mechanism of
secretion of protein-polysaccharide complexes.
The fine structure of elastin has been
the subject of a number of studies (see reviews by Cox and Little, ’61; Partridge,
’62; Ayer, ’64), most of which have utilized various disruptive techniques followed by metal shadowing of the resulting
fragments i n preparation for electron microscopy. Xarrer (’60) studied the developing chick aorta in thin sections and
noted “small granules,” 130 A in diameter
” an apparent mis(his cited “130 m ~ is
print), located at the periphery of elastin.
He concluded that elastin was derived
from fibrils and from a homogeneous component. Kawase (’62), in a brief review,
reported that permanganate fixation of
ligamentum nuchae demonstrates the existence of beaded 100-200 A filament in
a reticular network in mature elastin.
Schwarz (’64), in a study of elastogenesis
in tissue culture, describcd in the first appearance of elastin as being in the shape
of droplets and observed 40-50 W filaments at the periphery of elastin and 80110 A filaments within it. He illustrated
tenuous connections between collagen fibrils and elastin and estimated purified
elastin to contain about 12% collagen,
although he was not able to demonstrate
any collagenous inclusions in elastin.
The present study was carried out on
the ligamentum nuchae of the fetal calf.
The amount of biochemical information
available on this tissue favors its selection
despite difficulty in obtaining prompt fixation. Our observations are concerned with
the filamentous structures associated with
ANAT. REC., 155: 563-576.
elastin as well as with secondary secretory
features of the fibroblasts which seem to
be concerned with the structural proteinpolysaccharide components of this connective tissue. For the sake of brevity these
protein-polysaccharide complexes will be
referred to as mucopolysaccharides.
MATERIALS AND METHODS
Samples of ligamentum nuchae of fetal
calf were obtained within 15 to 30 minutes
after death of the cow. Age of the fetus
was determined by the forehead to rump
length (Bogart, ’59). Ligaments of various ages were examined, but all micrographs in this paper have been obtained
from the ligament of a 180-day-old fetus,
since at that age a period of intensive elastogenesis begins (unpublished observations). Tissue was fixed in phosphatebuffered osmium tetroxide (Millonig, ’62)
and embedded in Araldite. Sections were
doubly stained with potassium permanganate (Lawn, ’60) and lead citrate (Reynolds, ’63). Other staining methods do not
reveal all the details shown here. Electron
microscopy was carried out with a Philips
EM 200 electron microscope.
*This study constitutes publication no. 171 from
the Oregon Regional Primate Research Center, supported in part by grant FR 00163 €rom the National
Institutes of Health, and In part by U.S.P.H.S. grant
AM-06318-02 to Dr. D. S. Jackson, in whose IaboratoTy
some of the work was performed. L. B. Sandberg 1s
a Postdoctoral Fellow of the U. S. Public Health
Service. E. G. Cleary is an Overseas Fellow of the
National Heart Foundation of Australia.
2 Present
addpess: Department of Experimental
Pathology, Australian National University, Canberra,
Australia.
563
564
W. 13. FAHRENBACH, L. B. SANDBERG AND E. G. CLEARY
A crude extract containing fibrillar components of the elastic ligament was prepared by freezing a ligament from a 180day-old fetus in liquid nitrogen and crushing it in a stainless steel mortar to a fine
granular form. The tissue then was homogenized in a Virtis homogenizer in five
volumes of tris-HC1 buffered normal saline.
The homogenate was allowed to swell
maximally (Jackson et al., '65) in the
buffer for 12 hours at 4°C with agitation
and then was centrifuged at 2,000 g for
20 minutes. The supernatant was suitably
diluted with the original buffer, applied
to carbon-coated grids, and negatively
stained with phosphotungstic acid.
barrel-shaped, 170-190 A long and 170220 A in diameter, joined to each other by
the shorter segments measuring about
100 A in length and 150 A in diameter
(fig. 11). Multiple branchings of the filaments are commonly encountered in negatively stained preparations. Crude extracts
do not contain recognizable fragments of
mature elastin. In the subsequent discussion the filaments will be referred to as
pre-elastin filaments in distinction to elastin fibers which may or may not have a
coating of the filaments.
Definitive elastin makes its appearance
within clumps of pre-elastin filaments in
the shape of small fibers also oriented in
the long axis of the ligament. These elastin
RESULTS
fibers, 700-1,000 A in diameter in the
The ligamentum nuchae of a 180-day- 180-day-old ligament, each surrounded by
old fetus is composed of about 28% col- pre-elastin filaments, form bundles and
lagen and 12% elastin by dry weight of anastomose with each other to fill gradthe tissue (unpublished observations). ually the interstices. A bundle like this,
Attenuated fibroblasts and their processes averaging 0.4 LI in diameter in the 180-daygive the tissue a relatively cellular aspect. old ligament, represents the early stage of
These cells conform to the description of the adult elastin fiber which measures befibroblasts by Movat and Fernando ('62)
tween 3.6 v and 9.9 LI in diameter both in
and Ross and Benditt ('64) with the ex- prepared elastin (Partridge, Davis and
ception that they form frequent deep em- Adair, ' 5 5 ) and in histological material
bayments (fig. 1, inset) containing devel- (unpublished observations).
oping elastin, a feature also observed in
The pre-elastin filaments are in intimate
tissue culture by Schwarz ('64).
association with the surface of the elastin
The first ultrastructural indication of but are not visible within it. Under the
elastogenesis consists of small extracellu- staining conditions employed, the elastin
lar accumulations of hollow-appearing fila- has a coarse granular texture (fig. 4).
ments, lying adjacent to fibroblasts and The granularity appears to be due to
more or less parallel to the long axis of the tightly tangled and branching filaments
ligament (fig. 3 ) , apparently identical to about 30 A in diameter. Short stretches
those reported by Maser ('65) in associa- of these filaments and multiple branching
tion with collagen and elastin. In the points are visible at high magnification
140-day-old fetus these filaments consti- (fig. 4, inset). Virtually no internal structute the predominant form of developing ture is visible in the mature elastin with
elastin. In cross-section the filaments have the usual uranyl acetate and lead-salt
a diameter of 130-140 A and a hollow- staining techniques.
Persistent and conspicuous assocation
appearing core of about 40 A. In longitudinal section they exhibit an indistinct of acanthosomes (also called spiny vesisegmentation. This substructure appears cles) with developing elastin (figs. 4, 7,
to consist of cylindrical or subspherical 9 ) led to a closer examination of the role
units, 130 A long and of equal diameter, of these structures. These vesicles have an
separated by more electron-opaque sec- internal lining of flocculent material and
tions about 50 A long. In a maximally are studded externally with blunt projecswollen, negatively stained preparation tions embedded in a homogeneous layer of
these filaments have a distinctly beaded lesser electron opacity. The length of
appearance and are the only recognizable these spines measures 100-150 A, the
structures aside from collagen fibrils. The lower figure predominating in the smallest
larger segments are now spherical or vesicles. The spines give frequently the
E. M . STUDIES O N ELASTOGENESIS
appearance of being looped or, alternately,
having a club-like shape, the end being
expanded and having a less opaque center.
The spines are probably the two-dimensional expression of a system of ridges on
the surface of the vesicle, as suggested by
Palay ('63). The membrane of the acanthosome is a unit membrane, about 65 A
thick, and stains intensely with permanganate.
The extensive Golgi cisternae of the
fibroblasts bud off large numbers of acanthosomes with a diameter of 600-900 A
(fig. 5). This activity appears to increase
as adult elastin is beginning to be formed
in significant quantities, that is, at a fetal
age of about 180 days. The content of
small acanthosomes is quite electron
opaque. Occasionally, the material responsible €or the opacity is contained within
Golgi cisternae before the membrane surface specialization peculiar to acanthosomes appears. Acanthosomes fuse with
each other, eventually forming bodies up
to about 2,300 A in diameter as they
approach the surface of the cell (figs. 4,
6, 7). Acanthosomes are liberated predominantly in the immediate vicinity of
developing elastin fibers. After fusing with
the cell membrane, the vesicle opens up
and flattens out, its membrane retaining
its distinctly greater electron opacity in
relation to the adjacent fibroblast membrane (figs. 8, 9). The filamentous content of the vesicles is frequently in contiguity with adjacent elastin. They also
are released near developing collagen,
though with much lower frequency (fig.
8). In this situation the contents of the
vesicles appear virtually identical to the
filamentous material associated with collagen fibrils, most probably a mucopolysaccharide coating. Acanthosomes also are
discharged quite frequently into the narrow gap between two adjacent fibroblasts.
A further, localized membrane differentiation has been observed and is interpreted as resulting from the release of
acanthosomes. Confined regions of the
cell membrane of the fibroblasts adjacent
to elastin exhibit a filamentous condensation subjacent to the membrane and a corresponding external coating of basement
membrane material, up to 1,000 A thick
(fig. 10). Similar patchy specialization
565
between adjacent fibroblasts consists of an
intercellular mass of faintly fibrillar material and submembranous condensations in
one or both of the contributing cells, often
resembling a zonula adhaerens (Farquhar
and Palade, '63). Both of these cases appear to be specializations for adhesion to
maintain a close relationship between
fibroblasts or elastin and adjacent fibroblasts.
DISCUSSION
The ultrastructure of the fibroblasts of
the ligamentum nuchae does not show significant differences from that of fibroblasts
not associated with elastogenesis. In view
of the presence of an apparently single cell
type it appears reasonable to suppose that
all components of this tissue, i.e. elastin,
collagen and mucopolysaccharides, are
secreted, perhaps in temporal or spatial
separation, by every cell. The presence of
acanthosomes is more conspicuous than
it is in fibroblasts in other locations and
will be discussed subsequently. The elastin-containing embayments in many fibroblasts might serve to contain a local concentration of a soluble elastin precursor
and, thereby, facilitate polymerization,
but this specialization does not appear to
be a necessity, as is amply demonstrated
by the illustrations.
Four types of fibrils have been observed
in the fetal ligamentum nuchae, namely
collagen, pre-elastin filaments, fine fibrils
of mature elastin, and diffuse mucopolysaccharide filaments. Collagen fibrils are
conspicuous by their total absence from
the interior of the developing elastin fibrils. As described herein, in the fetal
ligament at this age, the developing elastin
is spatially discrete from the developing
collagen. The elastin has not yet attained
a sufficient volume to entrap collagen by
fortuitous juxtaposition, as suggested by
Karrer ('60). The clear demonstration
that elastogenesis occurs in the absence of
the direct participation of collagen fibrils
is further and potent evidence against the
thesis that elastin is derived from collagen
and favors the separate identity of the two.
The supposition that up to 12% collagen
forms an integral part of elastin (Schwarz,
'64). is based on the 1.6% hydroxyproline
content of elastin (Partridge, '62). In view
566
W. H. FAHRENBACH, L. B . SANDBERG AND E. G . CLEARY
of the fact that purified elastin preparations show residual collagen contamination in the electron microscope (Dempsey
and Lansing, '52; Cox and Little, '61),
it is much more reasonable to question
whether elastin really contains hydroxyproline, or whether this amino acid is a
measure of the degree of collagen contamination in the elastin residue. Amino
acid analyses of elastins purified from the
fetal ligament at the age of 180 days, both
by repetitive autoclaving (Partridge and
David, '55) and by hot alkaline extraction
(Lansing, '51 ), give hydroxyproline values
of 1.7 and 2 . 0 % , respectively (Cleary et
al., '65). It is, thus, more reasonable to
assume, pending evidence to the contrary,
that the hydroxyproline is an integral part
of the elastin molecule, rather than to require the presence of a large "cryptic" collagen component.
Elastin is not a true elastomer in the
sense that rubber is. A true elastomer retains its elasticity even under conditions
of dehydration; its elasticity is, thus, inherent in its molecular structure, and it
forms a self-lubricating system under
shearing forces (Lloyd and Garrod, '46,
'48). Elastin, on the other hand, becomes
brittle when dehydrated and would, by
necessity, be a biphasic system. containing
the elastin polymer and a "lubricant," potentially water, but more probably a hydrated mucopolysaccharide as concluded
by Gotte, Serafini-Fracassini and Moret
('63) on the basis of extracts obtained
from purified elastin. The elastin polymer
is thought to consist of long, freely mobile
chains linked at relatively infrequent intervals to each other to prevent slippage
and to assure return to the same shape
after deformation (Lloyd and Garrod, '48).
On the basis of these considerations it is
tempting to speculate that the pre-elastin
filaments constitute a relatively highly organized, cylindrical array of elastin polymer chains, possibly deposited upon a preexisting backbone of mucopolysaccharide.
The constricted and globular segments of
the pre-elastin filaments may correspond
to regions of greater or lesser cross-linking
of the elastin molecules. A subsequent
physical or chemical change of the constituent chains may lead to the formation
of the branching tangle of thinner fila-
ments seen in the mature elastin. Increasing cross-linking of the elastin may cause
the eventual masking of all filamentous
aspects of elastin and its almost total lack
of stainability, as found in post-natal ligamentum nuchae (unpublished observations). The term tropo-elastin should be
reserved for a potential monomeric, soluble precursor which has, as yet, eluded
identification or isolation.
The fine filaments investing collagen
are probably an acid mucopolysaccharide,
since they are preserved and stained by
ruthenium red, a dye known to bind selectively to several acid mucopolysaccharides
(Luft, '64, '65; Luft, personal communication). Similar filaments are found in the
ligamentum nuchae adjacent to elastin
fibers and in the intercellular gap of zonulae adhaerentes. The seeming continuity
between elastin and collagen fibrils reported by Schwarz ('64) can probably be
accounted for by the superimposition of
mucopolysaccharide filaments.
The similarity between the investing
mucopolysaccharide of collagen and the
contents of the acanthosomes introduces
the somewhat perplexing problem of the
function of these vesicles. Acanthosomes
have been observed in a great variety of
tissues, such as mosquito oocytes, liver
parenchymal and Kupffer cells of the rat
and chicken (Roth and Porter, '62, '63,
'64), cockroach oocytes (Anderson, '64),
chick oocytes (Wyburn et al., '65), erythroblasts (Fawcett, '65), neuronal and glial
elements of the central nervous system
(Pday, '63; Rosenbluth and Wissig, '63;
Andres, '64; Takahashi and Ilama, '65;
Wolfe, '65), endothelium (Stehbens, '65),
fibroblasts (Goldberg and Green, '64) and
hypophyseal cells (Maillard, '63). Similarly, they have been named variously
alveolate, spiny, coated or acanthous vesicles, acanthosomes, dense micropinocytotic vesicles, v6sicules hQis6es de bgtonnets
or Stachelsaumblaschen. Convincing evidence has been advanced in several instances that the cell membrane differentiates locally into the membrane peculiar to
the acanthosome, infolds to form these
vesicles, which then fuse to form inclusion bodies containing a stored product
such as yolk (Rosenbluth and Wissig, '63;
Roth and Porter, '64; Wyburn et al., '65).
E. M. STUDIES ON ELASTOGENESIS
As a result, almost all occurrences of acanthosomes have been interpreted as being
involved in protein uptake. Palay (’63)
and others observed the conspicuous association of these vesicles with the Golgi system, which led to the tentative suggestion
that the acanthosomes originate in that
region (Andres, ’64) or are possibly the
expression of a circulating membrane system between the Golgi region and the surface (Palay, ’63). Convenient as a n interpretation of protein intake by a fibroblast
would be, circumstantial evidence speaks
against it i n the present case. The size of
the acanthosomes increases centrifugally,
the opposite of the condition existing in,
for example, yolk producing oocytes. Open
acanthosomes are not randomly distributed, as they are i n cells with acanthosomes of adsorptive function, but are preferentially situated in the locations mentioned previously, which are least likely to
have free access to the intercellular fluid.
Unless the unlikely possibility of subtractive polymerization of elastin is considered, the acanthosomes more probably contribute a substance to its formation. A
contribution of protein by the acanthosomes to the molecular structure of elastin
is highly improbable in view of the ubiquity of these vesicles.
The observed phenomena can be interpreted more readily as indicating secretion of a protein-polysaccharide complex
by way of acanthosomes. Goldberg and
Green (’64) have demonstrated considerable acanthosome activity in mouse fibroblasts and mention, in another context,
that these cells “are known to produce relatively large quantities of hexuronic acidcontaining mucopolysaccharides.” Synthesis of complex carbohydrates in the Golgi
region has been dcmonstrated by the use
of autoradiographic techniques (Peterson
and Leblond, ’64), Peterson-Neutra, ’65).
This secretory and sequestering role of the
Golgi saccules is more readily combined
with the evidence presented here, i n addition to corresponding to the traditional
concept of Golgi function. Consequently,
opened acanthosomes at the cell surface
are being interpreted in the present case
as signaling the release of mucopolysaccharides to bind adjacent cells together,
to form the ground substance associated
567
with collagen and to enter into the process
of elastogenesis i n a n unknown manner.
Mucopolysaccharides may play a role in
maintaining elasticity of mature elastin,
as suggested earlier in the hypothetical
model, or they may serve merely as a
means of maintaing close proximity between the cell surface and developing
elastin.
These considerations lead one to conclude that the membrane associated with
acanthosomes may be formed in various
tissues either in the Golgi region or at the
surface of the cell, and that the resultant
acanthosomes may migrate in either direction. It is probable that mucopolysaccharides are associated with each, though
they may be released i n one case and
ultilized as protein binding sites for pinocytosis in the other. It seems that a purely
pinocytotic function of the acanthosomes
would necessitate a forced interpretation
such as would follow in the present case,
namely adsorption of protein from the immediate vicinity of forming elastin, spontaneous splitting of large acanthosomes
and resorption of protein in the Golgi cisternae. The suggested membrane flow
concept of Palay (’63) becomes, thereby,
a more acceptable hypothesis and should
encourage further experimentation to test
the directionality of the process.
LITERATURE CITED
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vitellogenesis in the roach Periplaneta americann. J. Cell Biol., 20: 131-155.
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Ayer, J. P. 1964 Elastic tissue. Int. Rev. Conn.
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Bogart, R. 1959 Improvement of Livestock.
MacMillan Co., New Ynrk.
Cleary, E. G., D. S. Jackson and L. €3. Sandberg
1965 The changes in amino acid composition
of bovine ligament elastin with aging. Proc.
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Cox, R. C., and K. Little 1961 An electron microscope study of elastic tissue. Proc. Roy. SOC.
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Dempsey, E. W., and A. I. Lansing 1954 Elastic tissue. Int. Rev. Cytol., 3: 437-453.
Farquhar, M. G., and G. E. Palade 1965 Cell
junctions in amphibian skin. J. Cell Biol., 26:
263-292.
Fawcett, D. W. 1965 Surface specializations of
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75-91.
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W. H. FAHRENBACH, L. B. SANDBERG AND E. G. CLEARY
Goldberg, B., and H. Green 1964 An analysis
of collagen secretion by established mouse fibroblast lines. J. Cell Biol., 22: 227-258.
Gotte, L., A. Serafini-Fracassini and V. Moret
1963 The chemical composition of the NaC1.
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1965 The swelling of bovine ligamentum
nuchae as a function of pH. Biochem. J., 96:
813-8 17.
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staining. J. Cell Biol., 23: 54A.
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McCluskey, Academic Press, N. Y.,121-159.
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PLATES
PLATE 1
EXPLANATION OF FIGURES
1
Electron micrograph of part of a fibroblast i n the ligamentum nuchae
of the fetal calf. The fibroblast is surrounded by collagen fibrils in
various orientations and by small elastin fibers (arrows). Note fine
filamentous material associated with the elastin. The inset shows a n
embayment in a fibroblast with a small mass of developing elastin.
X 17,400; Inset X 15,500.
2
A developing elastin fiber, sectioned longitudinally, adjacent to the
surface of a fibroblast (bottom). Condensations of fibrillar material
are located subjacent to the membrane. Periodicity in the peripheral
pre-elastin filaments is barely perceptible (arrow). x 62,000.
3 High power micrograph of pre-elastin filaments showing beaded appearance (arrow). X 160,000.
5 70
E. M. STUDIES ON ELASTOGENESIS
W. H. Fahrenbach, L. B. Sandberg and E. G. Clcary
PLATE I
571
PLATE 2
EXPLANATION OF FIGURES
572
4
Cross-section of a developing elastin fiber. Smaller component fibers
are beginning to fusc and are surrounded by hollow-appearing prcelastin filaments. Note the large approaching acanthosome in thc
adjacent fibroblast. Collagen a t right and bottom. Shown i n the
inset are several branching filaments (arrows) of the mature elastin.
x 104,000; Inset x 330,000.
5
Micrograph of the peripheral area of Golgi zone. Numerous small
acanthosomes are being budded o f f or are lying freely i n the cytoplasm (arrows). x 58,000.
6
A number of acanthosomes presumably in the process of fusion. The
linear arrays of ribosomes are typically attached to membranes of
the endoplasmic reticulum. X 58,000.
E. M. STUDIES ON ELASTOGENESIS
W. H. Fahrenbach, L. B. Sandberg and E. G. Cleary
PLATE 2
573
PLATE 3
EXPLANATION
OF FIGURES
7 Acanthosome near liberation adjacent to a small mass of elastin.
x 83,000.
8
Open acanthosome i n close proximity to developing collagen. Note
the similarity of the extracellular coating of the acanthosome to the
“fuzz” associated with the collaaen. Also observe the darker membrane of the acanthosome turned out for a short distance (arrows).
x 79,000.
9
A number of acanthosomes (arrows) at or near liberation next to a
developing elastin fiber. No other acanthosomes were found in the
visible remainder of the cell. X 44,000.
10
Electron micrograph illustrating the typical differentiation of the
fibroblast adjacent to developing elastin. Note the superficial basement membrane material i n contiguity with the elastin and the
suhmembranous condensation. X 89,000.
11 A negatively stained preparation oE a centrifuged homogenate of
180-day-old fetal ligamentum nuchae, showing a collagen fibril and
numerous swollen pre-elastin filaments. The constricted regions uf the
beaded filament are indicated by short arrows, an1 the long arrow
points to a spherical segment. X 155,000.
574
E. M. STUDIES ON ELASTOGENESIS
W. H. Fahrenbach, L. B. Sandberg and E. G. Cleary
PLATE 3
575
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ultrastructure, elastogenesis, studies, early
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