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Pericyte of a teleost fishUltrastructure position and role in neoplasia as revealed by a fish model.

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Pericyte of a Teleost Fish: Ultrastructure,
Position, and Role in Neoplasia as Revealed
by a Fish Model
U.S. Environmental Protection Agency, Environmental Research Laboratory,
Gulf Breeze, Florida 32561
The morphology and position of the pericyte, a periendothelial
cell, is described for a teleost fish, Cyprinodon variegatus. This cell was found
attached to the abluminal surfaces of capillaries, venules, and arterioles of the
submucosa of the midgut of the fish. The cell was encompassed by a thin basal
lamina, possessed numerous plasmalemmal vesicles, a “sole region” which contained thinner actin-like filaments and possibly thicker myosin-like filaments, and
ranged in form from ovoid to stellate, with long cytoplasmic extensions t h a t partially covered the endothelium of the associated microvessel. The pericyte of C.
variegatus has been shown to give rise to hemangiopericytomas (experimentally
induced with diethylnitrosamine) and possibly to pericytomas. The range of phenotypic expression of these pericyte-derived neoplasms is broad, and dependent
upon the degree of differentiation of their constituent cells which range from clear
cell pericytes to myofilamentous laden cells that resemble smooth muscle cells. In
this regard and in regard to its normal ultrastructural morphology, and anatomical position, in relationship to microvasculature in this fish, the cell is very similar to other vertebrate pericytes. Limited evidence suggests that small fish species
may be excellent study models for further elucidation of pericyte form, function,
and role in disease.
Much of what is known about the pericyte, a periendothelial cell of mesenchymal origin, has been learned
from studies of mammalian microvasculature. Sims
(1986) reviewed information available on pericytes in
the literature, and pointed out the apparent occurrence
of pericytes in most vertebrate species from fishes to
mammals. The absence, however, of a detailed description of the pericyte of teleost fishes became apparent
following a reading of Sims’ paper and a review of more
recently published papers. Several authors describe
vasculature studies of certain fishes and briefly refer to
pericytes, but none of them offer a detailed, ultrastructural description of the pericyte itself (Morris and Allbright, 1979; Casley-Smith, 1987; Garcia and AlvarezUria, 1986).
Because of the recent increased interest in the role of
pericytes in certain neoplastic processes in both mammals (Sims, 1986) and fishes (Schultz and Schultz,
1985; Couch and Courtney, 1987), a study of the ultrastructure of the normal pericyte of a teleost fish, the
sheepshead minnow (Cyprinodon variegatus) was undertaken. The specific purposes of this study were to 1)
describe the ultrastructure of the fish pericyte and to
compare it to that of other vertebrate pericytes, and 2)
provide a characterization of the normal fish pericyte
for comparison to pathological forms of the cell occurring in certain fish neoplasms that may serve as study
models for homologous mammalian neoplasms.
Juvenile and adult sheepshead minnows from laboratory stocks were used. These fish were reared from
eggs in the laboratory, were fed on a commercial flake
food as juveniles and adults, and kept in flowing sea
water (salinity ranged from 15 to 25%0).Portions (crosssectional segments of 2-5 mm) of the anterior midgut,
approximately 2-3 cm posterior to the esophagus, were
taken by dissection. A well-defined stomach is lacking
in Cyprinodon as it is in other members of the teleost
family Cyprinodontidae. Light microscopic (LM) study
of histological cross-sections (Harris hematoxylin and
eosin stained sections) of these portions revealed that
the lamina propria and submucosa of the intestine
were, normally, moderately vascularized, containing
arterioles, venules, capillaries, and larger veins and
arteries. Pericyte-like cells were observed, with LM,
attached to the outer layers of the arterioles, venules
and capillaries. Therefore, the portions (thick cross sections) of the middle intestine were minced in and fixed
in 3%glutaraldehyde buffered with sodium cacodylate.
Pieces less than 1 mm3 were postfixed in cacodylate
buffered 1%osmium tetroxide, washed, dehydrated in
acetone series, and embedded in Spurr’s medium. Thin
sections were stained in uranyl acetate and lead citrate
Received J u n e 23, 1989; accepted December 15, 1989.
and examined and photographed with a Zeiss EM10
electron microscope.
Hemangiopericytoma (neoplasm) material was obtained from fish specimens exposed to N-nitrosodiethylamine from another study (Couch and Courtney,
1987), and processed for LM and EM a s stated above for
normal tissues.
Structure and Characteristicsof Normal Pericytes
Pericytes were found abutting the outer layer of capillary walls in hematoxylin stained sections of the submucosa of the midintestinal wall of the sheepshead
minnow (Fig. 1).With LM, the perikarya and nuclei of
these cells were most obvious, and their most characteristic LM feature was their intimate association with
the outer surface of the vessel (Fig. 2). In searching for
and locating pericytes with LM, i t was at times easy to
confuse unattached but closely abutted fibroblasts with
pericytes. Further, from the luminal direction of the
very thin-walled vessels, endothelial cell nuclei could
be confused with those of actual pericytes. Usually, f i broblasts are further removed from the basal lamina of
vessels than are pericytes. Endothelial cell nuclei usually protrude toward the lumen of the vessel.
Electron microscopy (EM) permitted precise identification of the teleost pericyte. With EM, the C. uariegatus pericyte appeared a s a variable shaped cell in
profile, ranging in form from squamous to plump ovoid,
to elongated and stellate (Figs. 3-5). Cytoplasmic extensions of certain pericytes in cross sections were
found to reach considerable distances around the embraced capillary (as revealed in cross sections of capillaries) (Fig. 3).A basal lamina (-25-75 nm thick) was
found to cover the pericyte and to extend beneath and
between it and underlying endothelial cells (Figs. 4,5),
except where the pericyte closely contacted the endothelial cell. This thin basal lamina, which was continuous with the thicker basal lamina of the vessel (up to
-100 nm thick), was a chief identifying characteristic
of the pericyte (as it is of the pericytes in other vertebrates).
The general shapes of the perikarya of the cells depended on the variable shapes of the large, heterochromatic nuclei (Figs. 43) that they possessed. Nuclei
ranged in profile forms from discoid to ellipsoid to almost round, with each form having clefts or indentations at their periphery. The nuclear heterochromatin
was distributed strongly peripheral in most mature
pericytes, with occasional nuclei demonstrating nucleoli, as well as diffuse central and peripheral heterochromatin (Fig. 4).
The most striking cytoplasmic feature of the C. uariegatus pericyte was the presence of many plasmalemma1 vesicles. They were distributed inside of and, free
of, or connected to the plasmalemma around the circumference of the cell, but were rarely on that side
opposed to the endothelium of the vessel (Figs. 4,5).
Opposing endothelial cells had identical plasmalemma1 vesicles (size and form) inside their entire circumferences (Fig. 4). The connection of some plasmalemma1 vesicles with the plasmalemma was obvious in
some cells (Fig. 5 ) , whereas other vesicles were obviously detached and found deeper in the cytoplasm (Fig.
4). Mitochondria were obvious in some pericytes; in
others, they were not seen commonly in presented profiles. Thin and thick filaments, probably actin and/or
myosin, were in the peripheral cytoplasm, usually adjacent to endothelial surfaces (Fig. 5). A region similar
to the “sole region” in other pericytes, as described by
Bruns and Palade (19681, also was observed in C. uariegatus pericytes. As in their findings, i t was near the
surface, overlying the endothelium, and contained
some filaments, but not plasmalemmal vesicles (Figs.
43). Some cells attached to larger vessels, probably
postcapillary venules, were pericyte-like and contained
much more of the thin and thick filaments which suggested a possible contractile function for the cells.
Location and Relationship of Cyprinodon variegatus
Pericytes to Vascular and Nonvascular Tissues
Pericytes appeared to be distributed modestly in
numbers on blood vessels in the submucosa of C. uariegatus, with, usually, no more than two visible in a
single section of vessel with LM (Figs. 1,2). EM, however, went much further than LM toward demonstrating distribution and abundance of pericytes and their
cytoplasmic extensions (Fig. 3) on the microvessels.
Usually, the position of the pericyte perikaryon, in
relation to the underlying endothelial cells, was directly above the junction of two endothelial cells (Figs.
3,4).In some cases, a brief cytoplasmic extension of the
pericyte protruded a short distance into the junctional
gap between endothelial cells. At that point there was
no evidence of basal lamina material between the pericyte and endothelium, and the relationship between
pericyte and endothelial cell was that of a loose “peg
and socket” contact. Rarely, a small cell junction between pericyte and underlying endothelial cell was observed in the “sole region” a s seen in Figure 4.
As mentioned earlier, many pericytes gave rise to
long cytoplasmic extensions that embraced or encircled
the capillary to which they were attached. Many of
these extensions (from the perikaryon) became very
thin (Fig. 3) at their extremities, but continued to be
enclosed by the basal lamina shared with the vessel.
In the submucosa of the gut of C. uariegatus, there is
considerable amount of loose connective tissue (both
cellular, i.e., fibroblasts, and acellular, i.e., collagen).
Collagen fibrils were intimately associated with the
basal lamina covering the free surface of many pericytes (Figs. 3 3 ) . Long, sinuous strands of fibroblast
cytoplasm, and perikarya of fibroblasts were found
nearby in the loose collagenous matrix (Figs. 3,5).
Relationships of Pericytes to Known Disease in
Cyprinodon variegatus
As one result of studies on developing small fish species as models for vertebrate cancer research, Couch
and Courtney (1987) described pericytomatous neoplasms induced with n-nitrosodiethyl-amine in C. uariegatus. One of these neoplasm types was diagnosed a s
hemangiopericytoma. This neoplasm occurred in the
abdominal cavity, liver, and on the body wall, retroperitoneally. Another neoplasm type, from the same
study, was described a s a pericytoma, originating from
cells on the outer layer of veins in the liver. Both of
these chemically induced neoplasms could have arisen
from pericytes or related cells, and contained transitional cells or smooth muscle cells of mesenchymal-
Fig. 1. Submucosal capillary in Cyprznodon uariegatus; note the
nucleated red blood cells (RBCs) in lumen that is approximately
equivalent to one RBC in thickness; perikaryon of probable pericyte
(P)abutting the abluminal surface of the vessel. Harris hematoxylin
and eosin (H&E), x 250.
Fig. 2. Higher magnification of capillary, in cross section, in submucosa of C . uariegatus; the nuclei of two probable pericytes (P) are
visible; again, note the RBCs (two) in lumen of capillary, relatively
thin endotbelium (E), and surrounding connective tissue elements
including fibroblasts (F). x 1,200.
Fig. 3. Low-magnification electron micrograph demonstrates the
distribution of pericytes around a microvessel in the submucosa; note
profiles of two pericytes (P) associated with what is probably a capillary (judged by diameter, and RBC capacity). Cytoplasmic extensions
of the pericytes appear on the outer border of the vessel and can be
seen extending from the pericyte cell body (arrows). Note that the
perikarya of pericytes may lie directly over the junctions of endothelial cells. x 5,000.
pericyte origin as described by Sims (1986) in his discussion of the transition of pericytes to smooth muscle
cells, as demonstrated by Meyrick and Reid (1978).
The broader histologic pattern of the hemangiopericytoma consisted of tracts of spindle-shaped cells forming whirling or storiform pattern or a variable tract
pattern (Fig. 6). In some of the tracts, the most numerous cells had very filamentous, eosinophilic cytoplasm
indicating t ha t considerable amounts of myofilamen-
tous material were present. These tracts appeared to
consist mainly of smooth muscle cells and had the appearance of leiomyosarcomas (Fig. 7). The specific,
smaller pattern diagnostically characteristic of the hemangiopericytoma in the same neoplasms consisted of
spindle-shaped cells with clear to mildly eosinophilic
cytoplasms arranged concentrically around patent or
collapsed venules, arterioles or capillaries (Fig. 7).
Only neoplastic tissues originally fixed for LM and em-
Fig. 4. EM of an elongate pericyte on basal lamina above endothelium of capillary; note the strong pattern of heterochromatin in the
nucleus; note the thin basal lamina (bl) covering the pericyte; and
that the numbers ofplasmalemmal vesicles (PV) are abundant in both
the pericyte and endothelial cells. The “sole region” (SR) may be iden-
tified by the absence of plasmalemmal vesicles. Occasionally this region will have brief cell contacts between pericyte and endothelial cell
as illustrated just below lower SR arrow. Nucleated RBC is present in
the lumen of the capillary. x 25,000.
bedded in paraffin was available for EM; some of this
was reprocessed for EM. In resulting electron micrographs, presumed “ghosts” of elongated pericytes constituted the whorls of cells surrounding collapsed and
patent blood vessels.
The pattern in the hepatic pericytoma was that of
spindle-shaped cells arranged in layers with greater
density of cells near the adventitia of larger hepatic
veins (Couch and Courtney, 1987). These layers ex-
tended to and compressed hepatic parenchyma. Some
amorphous, acellular material was found between the
layers of these cells, and the cells which originated on
the walls of longer veins had filamentous, eosinophilic
cytoplasms. These cells were probably pericytes, andlor
cells in transition to smooth muscle (Sims, 1986; Meyrick et al., 1981; Meyrick and Reid, 1978), or smooth
muscle cells that were neoplastic (i.e., forming a leiomyosarcoma or leiomyoma vs a pericytoma).
Fig. 5. Portion of cytoplasm of pericyte; some vesicles in cytoplasm
are attached to plasmalemma (PV); note presence of some mitochondria and thick and thin filaments (arrows) in cytoplasm near
endothelium (E); collagen is found above pericyte in loose connective
tissue of the submucosa; basal lamine (bl) material is found on outer
surface of pericyte and between i t and endothelial cell. x 32,000.
morphological differences were observed. Cyprinodon
variegatus pericytes possessed the key identifying features of other vertebrate pericytes: they 1)were encompassed within a basal lamina that was continuous with
the basal lamina of the adjacent vessel; 2) contained
many plasmalemmal vesicles, 3) possessed a n apparent
“sole region” with filaments but no vesicles in that region, and 4)were capable of giving rise to neoplasms
that possess the characteristics of hemangiopericytomas or pericytomas found in other vertebrate species.
Further, their relationship with underlying endothelial cells was similar to that for other vertebrate pericytes, and some evidence was observed that suggests
that the fish pericyte may be a source for developing
Sims (1986), in his review, listed all published, detailed, ultrastructural studies of pericytes for vertebrates (his Table 1).The only cold-blooded vertebrates
studied in detail and listed in regard to pericytes were
frogs (Brundgaard and Frokjaen-Jensen, 1982; Michel
et al., 1984). As mentioned previously, three fish studies done since 1979 mentioned and figured pericytes,
but presented no detailed description of them. The
present study permitted a n ultrastructural comparison
of a teleost pericyte with other vertebrate pericytes.
The C. variegatus pericytes were very similar to other
vertebrate pericytes, and no remarkable qualitative,
Fig. 6. Low-magnification LM of hemangiopericytoma from C. uariegatus (attached to liver) exposed to N-nitrosodiethylamine; note interlacing pattern formed by tracts of neoplastic pericytes (TI. The
pattern and tinctorial quality resembles that of leiomyosarcoma in
some areas of the neoplasm. x 560.
Fig. 7. Higher-magnification LM of hemangiopericytoma from C.
uariegatus exposed to N-nitrosodiethylamine. Note the concentric or
whirling pattern of pericytes around patent or collapsed blood vessels
(arrows). However, a t this higher magnification, the tracts of cells
still reveal the storiform pattern (TI. Many of the cells have pink
staining, filamentous cytoplasm (H&E), and thus, portions of the neoplasm resemble leiomyosarcoma. x 1,400.
stages of intermediate cells and smooth muscle cells, as
they are in mammals; i.e., they may develop into cells
that contain more myofilamentous material than most
pericytes (these cells could be either normal cells or
transformed neoplastic cells).
Dardick et al. (1989)pointed out the need to be aware
of the range of differentiation assumed by neoplastic
cells in human hemangiopericytoma, and t h a t these
cells were capable of a possessing or lacking many
features of pericytes. They further noted t h a t in many
hemangiopericytomas there were admixtures of
smooth muscle-like cells and pericyte-like cells.
Perhaps vertebrate leiomyosarcomas and leiomyomas are merely differentiation-dependent variants of
pericytic neoplasms (cells involved may possess none,
some, or much of the actin and myosin-like filaments,
in their cytoplasms, and may represent pericytes or
pericyte-derived cells differentiating into intermediate
or smooth muscle cells).
The close similarity in cellular form, histological position, and histopathology of the fish and mammalian
pericyte, as shown in this study, provides some basis
for future comparisons of normal and pathologic expression of pericytes among and between closely related and more distantly related vertebrate species. If
terms from mammalian oncology are to be applied to
identification of lower vertebrate neoplasms (e.g., fish
hemangiopericytoma), then basic, accurate descriptions of the normal cell types (cell of origin) thought to
give rise to those neoplasms must be provided for the
lower vertebrate species in order to provide confidence
that the same or homologous entities are being discussed.
Selected species of small teleost fishes may be excellent models in which to study the differentiation of
pericytes into smooth muscle cells, and in which to
study the pathological changes in, and caused by, pericytes in the vertebrate animals.
Bruns, R.R., and G.E. Palade 1968 Studies on blood capillaries. I.
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Brundgaard, M., and J. Frokjaen-Jensen 1982 Functional aspects of
the ultrastructure of terminal blood vessels: a quantitative study
on consecutive segments of the frog mesenteric microvasculature.
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Casley-Smith, J.R. 1987 The phylogeny of the fine structure of blood
vessels and lymphatics: similarities and differences. Lymphology,
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hepatocarcinogenesis in estuarine sheepshead minnow (Cyprinodon uariegutus): neoplasms and related lesions compared with
mammalian lesions. J. Natl. Cancer Inst., 79t297-321.
Dardick, I., S.P. Hammar, and B.W. Scheithauer 1989 Ultrastructural
spectrum of hemangiopericytoma: a comparative study of fetal,
adult, and neoplastic pericytes. Ultrastruct. Pathol. 13t111-154.
Garcia, J.M., and M. Alvarez-Uria 1986 Intraventricular blood vessels of the hypothalamus of Salmo gairdneri and Gambusiu uffinis. Anat. Rec., 214t76-81.
Meyrick, B., K. Fujiwara, and L. Reid 1981 Smooth muscle myosin in
precursor and mature smooth muscle cells in normal pulmonary
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pulmonary arterial circulation. Lab. Invest., 38t188-200.
Michel, M.E., N.L. Shinowara, S. Odman, and S.I. Rappaport 1984
Morphology of endoneurial blood vessels of frog sciatic nerve during vascular perfusion. Microvasc. Res., 28t220-232.
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and Molec. Pathology 42t320-330.
S h s , David E. 1986 The pericyte-a review. Tissue Cell, 218:175199
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fishultrastructure, pericytes, mode, neoplasia, teleost, role, revealed, fish, positional
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