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Morphometry of tubular bodies in endothelial cells in normal stable isolated perfused and edematous dog lungs.

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THE ANATOMICAL HECORD 196295 -300 (1980)
Morphometry of Tubular Bodies in Endothelial
Cells in Normal, Stable Isolated Perfused,
and Edematous Dog Lungs
PETER B. BERENDSEN AND DAVID 0. DEFOUW
Department of Anatomy, College of Medicine and Dentistry of New Jersey,
Newark, New Jersey 07103
ABSTRACT
The volume densities and dimensions of tubular bodies (WeibelPalade bodies) in dog pulmonary capillary endothelial cells were examined by
means of stratified random sampling and electron microscopic morphometry. The
0.2%.
mean volume density of tubular bodies in normal (control) lungs was 0.43 I
The mean volume densities of tubular bodies in stable, isolated lungs perfused for
?hhour, 1hour, and 2 hours and in lungs made edematous by increasing hydrostatic
pressure or by decreasing oncotic pressure did not vary significantly from that of
normal lungs. The mean thickness of the endothelium measured a t the middle of
the tubular bodies of normal dog lungs was nearly twice the mean thickness of the
overall capillary endothelial cell sample. The mean endothelial thickness across
tubular bodies from stable and from edematous isolated perfused lungs did not
differ significantly from that of the control group. The mean width of tubular bodies
from normal dog lungs was 0.25 I0.06pm and the mean length was 0.81 t
0.61 pm. The mean widths and lengths of tubular bodies from stable and from
edematous isolated perfused dog lung endothelial cells did not differ statistically
from those of normal dog lungs. Thirty percent of the tubular bodies in the sample
were found to be adjacent to a mitochondrion in the same plane of section. Tubular
bodies contained both tightly packed and loosely grouped tubules. It is concluded
that the tubular bodies in canine pulmonary endothelial cells remain stable during
the perfusion of isolated lungs and in oncotic and hydrostatic edema of isolated
perfused lungs.
Membrane bounded groups of tubules were
first reported to be present in endothelial cells
by Weibel('64), Weibel and Palade ('641, and by
Stehbens ('65). Although Rhodin ('68) has proposed that these structures be termed "specific
endothelial granules," they have often been
called "Weibel-Palade bodies." Kawamura et
al. ('74)have termed them "tubular bodies," the
term by which they will be identified in this
paper. They have been identified within the
endothelial cell cytoplasm of normal blood vessels, ranging in size from capillaries to the
aorta in many species (Weibel, '64; Stehbens,
'65; Fuchs and Weibel, '66; Cauna and Hinderer, '69; Sengel and Stoebner, '70; Steinsiepe
and Weibel, '70; Kojimahara, '77). Tubular
bodies have been noted to be retained in cultured umbilical endothelial cells (Takeshige
and Fujimoto, '77; Haudenschild et al., '75).
Similar structures have been reported in endothelia of humans with various diseases (Ki-
000-3276X/80/1963-0295$01.40
0 1980 ALAN
R. LISS, INC.
mura et al., '75; Garancis et al., '71; Nieland et
al., '72; Haustein, '73; Jerusalem et al., '74;
Macadam et al., '75; De Martino et al., '69; Jao
et al., '77; Feiner and Gallo, '77; Daniels et al.,
'74). They are also reported to be present in the
endothelia of intracranial blood vessels
(Herrlinger et al., '74; Hirano et al., '73; Hirano
et al., '74; Kawamura et al., '74; Hirano and
Matsui, '75; Gessega and Anzil, '75; Hassoun et
al., '78).
Morphometric study of rat aortas demonstrated reduction in the number of tubular
bodies following incubation in epinephrine.
This finding lead Burri and Weibel('68) to suggest that the tubular bodies are related to the
production of a procoagulative substance.
The isolated, perfused, dog lung is frequently
used as a subject €or experimental studies of
endothelial transport (e.g., Per1 et al., '75, '76).
Received May 3, 1 9 7 9 accepted August 29, 1979.
295
296
PETER B. BERENDSEN AND DAVID 0. DEFOUW
The reaction of the tubular body component of
pulmonary endothelial cells may affect the results of these experimental procedures. Therefore, this study reports the volume densities of
tubular bodies in the isolated perfused lungs as
determined by established stereological procedures.
MATERIALS AND METHODS
The materials for this study were obtained
from the preparations previously reported by
DeFouw and Berendsen ('78a,b; '79). In brief,
four groups of preparations were used. The first
group consisted of normal lungs obtained from
three adult mongrel dogs (approximately 20 kg
body weight) of either sex, killed with a n overdose of pentobarbital (2 g). The trachea and
lungs were exposed, removed from the thorax,
and immediately fixed by the endotracheal instillation of cold 2% glutaraldehyde in 0.15 M
NaHCO,, buffer, pH 7.4, at a pressure of 20 cm
H,O, followed by immersion in fixative.
The second group consisted of stable isolated
perfused lungs prepared from six mongrel dogs.
Following stable perfusion for %, 1, or 2 hours,
two lung preparations a t each time period were
fixed as above with glutaraldehyde.
In the third group, edema was produced by
perfusion of the isolated lungs with fluid containing decreasing concentrations of macromolecules. In the fourth group, edema was produced by sustained elevation of the outflow
pressure. In these last two groups, the lungs
were fixed at the end of two hours. For full
details, see DeFouw and Berendsen ('78a,b,
'79).
Stratified random sampling (Weibel, '73) was
used as follows. Each pair offixed lungs was cut
into twelve longitudinal slices and two tissue
blocks were selected from each slice by the use
of a numbered overlay and random number
table. Each of the 24 blocks was divided in half,
with one half prepared for light microscopy and
the other half used for electron microscopy. The
specimens used for electron microscopy were
post fixed in 1% osmium tetroxide in 0.15 M
NaHCO, buffer, pH 7.3, for one hour, were then
dehydrated in solutions of ethanol of graded
concentrationsand then in propylene oxide and
embedded in Epon (Luft, '61). Each of the 24
original tissue specimens per lung gave 10-15
Epon blocks and thus provided approximately
240-360 blocks per test lung. From the primary
sample of 24 original tissue specimens per lung,
10 were selected randomly and one Epon block
from each of these ten was prepared for electron
microscopy. This produced ten grids per dog
lung. Thin sections (60-9Onm) were cut on
LKB or Sorvall MT-2 ultramicrotomes, stained
with uranyl acetate and lead citrate (Reynolds,
'631, and examined with a Philips EM-300. Ten
electron micrographs of each block were taken
at x 21,600 by photographing the upper left
corners of sequential grid squares. This provided a stratified random sample of 1,500 electron micrographs. Each micrograph was
analyzed with a test grid having 168 equidistant points and t h e point counts were
recorded for estimating the volume densities of
the endothelial cytoplasmic organelles. This
stratified procedure produced a sample size sufficient to yield a n expected error of less than 5%
(Weibel, '73) in the estimation of stereologic
parameters.
The significance of differences between
means of the morphometric parameters for the
normal (control) and stable and edematous isolated perfused lungs were evaluated by a twotailed Student's t-test (Freund, '67).
RESULTS
The volume densities, i.e., proportion of
endothelial cell cytoplasm occupied by microtubules and tubular (Weibel-Palade) bodies in
capillary endothelial cells of normal, stable isolated perfused, and edematous isolated perfused dog lungs are given in Table 1.The mean
volume densities of tubular bodies and microtubules within normal lungs did not differ significantly from those observed in stable isolated-perfused or in the hydrostatic and oncotic, edematous isolated perfused lungs in this
study.
The thickness of t h e endothelial cells
through the middle of seventy tubular bodies
was measured from the luminal to abluminal
surfaces (Table 2).
TABLE 1. Volume densities of tubular
bodies and microtubules'
Lung type
Normal
Stable isolated perfused
?hhour
1 hour
2 hours
mean of ?hhour, 1 hour,
2 hours
Hydrostatic edema
Oncotic edema
'Expressed as %) of cytoplasm
Mean
&
0.43
2
1 SEM
0.29%
0.63 .t 0.28%
0.46t 0.5%
0.25 i 0.05%
0.45 f 0.19%
0.56 k 0.09%
0.46 .t 0.21%
297
TUBULAR BODIES IN NORMAL AND PERFUSED DOG LUNGS
TABLE 2. Measurements of endothelium and tubular bodies’
Normal
Thickness of endothelium
at tubular bodies (pm)
Range (pmi
Width of tubular bodies (pm)
Range (pm)
Length of tubular bodies (pm)
Range (pm)
Perfus&
0.52 ? 0.16
0.93 0.33
0.25 t 0.06
0.37 0.15
0.81 ? 0.61
2.8
0.33
~
~
~
0.63
f 0.23
1.16 - 0.33
0.29 f 0.14
0.70 - 0.14
0.80 ? 0.60
2.56 - 0.23
Hydrostatic
edema
0.47
?
0.16
0.70 - 0.42
2 7 ? .04
.33 - .23
.79 t .13
.88 - .65
Oncotic
edema
0.56
0.93
?
0.19
-
0.33
0.29
f
0.08
0.042 - 0.19
1.09 5 0.60
2.19 - 0.47
‘Mean values t 1 SEM
2Mean of %, 1,2 hr
No statistically significant differences were
identified among the mean endothelial thicknesses through tubular bodies in lungs of the
groups studied.
In many micrographs, the sectioned air-blood
barrier had a n interstitial compartment which
was thicker on one side of the capillary than on
the other due to greater amount of interstital
matrix, fibers, and cells. The opposite, “thin”
side of the air-blood barrier consisted of opposed
type 1 epithelial and endothelial cells with a
basal lamina between. Tubular bodies in the
endothelium adjacent to the “thick’portion did
not vary in number or form compared to those
within the endothelium of the thin portion.
Clusters or groups of tubular bodies were not
common.
The lengths and widths of the tubular bodies
in the normal and experimental groups are
given in Table 2. Widths were measured from
luminal to abluminal sides of each tubular
body. Approximately two-thirds of the tubular
bodies had lengths that were more than twice
the width (Fig. 2). The remainder were irregularly rounded (Fig. 3). Approximately 30% of
the tubular bodies had a mitochondrion in close
association (Figs. 2,3,6) in the plane of section.
Ten percent of the tubular bodies were near
endothelial cell nuclei (Fig. 4).
Each tubular body was bounded by an irregular membrane which was often indistinct. This
enclosed tubules of 14-18 nm diameter which
were usually seen in cross section. Each tubule
had a core that was denser than the surrounding matrix. The tubules within some tubular
bodies were irregularly spaced (Fig. 5). Most
tubular bodies contained tightly packed hexagonally arranged tubular units (Fig. 11, with a
center-to-center spacing of approximately
25 run.
Microtubules were infrequent in the cytoplasm but were readily identifiable in longitudinal orientation (Fig. 3). Cross sectioned cyto-
plasmic microtubules were distinguished from
the circular profiles of pinocytotic vesicles by
the smaller diameter of the former.
DISCUSSION
The mean thickness of pulmonary capillary
endothelium through the midpoint of the tubular bodies observed in this sample ofpulmonary
alveolar capillaries was 0.52 +- 0.16 pm. This is
slightly less than twice the mean overall thickness of the endothelium, which we have reported to be 0.30 & 0.02 pm in normal dog lungs
and 0.35 0.02 pm during the two hour stable
perfusion period (DeFouw and Berendsen,
’78a). Although the two hour perfusion period
has been reported to produce a statistically significant increase in overall endothelial thickness (DeFouw and Berendsen, ’78a), the mean
increase in thickness through the tubular
bodies was not statistically significant. This
indicates that the alveolar capillary endothelial cells at the tubular bodies are thicker
than elsewhere, and these sites tend to remain
stable during perfusion and during experimental edema of isolated lungs.
The volume densities of tubular bodies in
alveolar capillary endothelial cells reported in
this study are similar to those reported in rat
capillaries (Fuchs and Weibel, ’66)and slightly
higher than those in frog capillaries (Steinsiepe
and Weibel, ’70). A much higher volume density of tubular bodies (Burri and Weibel, ’68)
has been reported in normal rat aortic endothelial cells. Burri and Weibel (’68) reported a
relative decrease in volume density of these
structures during incubation of aorta in Ringer’s solution, possibly owing t o endothelial
swelling. In this study, the failure of either
perfusion for two hours or the production of
edema to have statistically significant effects
on the volume density of tubular bodies suggests that the tubular bodies are relatively sta-
*
298
PETER B. BERENDSEN AND DAVID 0. DEFOUW
Fig. 1. Tubular body (arrow) in the endothelium of an isolated dog lung perfused for ?4 hour.
X
173,000.
Fig. 2. Elongated tubular body (arrow) adjacent to a mitochondrion (m). Oncotic edema. x 173,000.
Fig. 3. Rounded tubular body (arrow)with a mitochondrion (m) and a microtubule (mt) adjacent. Stable perfusion for %
hour. x 173,000.
Fig. 4. Rounded tubular body (arrow) near the nucleus of an endothelial cell. Stable perfusion for ?4 hour. x 173,0130,
Fig. 5. Tubular body (arrow) with loose grouping of tubules. Stable perfusion for ?4 hour. x 173,000.
Fig. 6 . Tubular body (arrow) with tight packing of tubules. Oncotic edema. x 173,000.
TUBULAR BODIES IN NORMAL AND PERFUSED DOG LUNGS
ble during the perfusion process under the experimental conditions used here.
The mean width of tubular bodies in normal
dog pulmonary capillary endothelial cells in
this study is in agreement with the width of
tubular bodies reported in human skin capillaries (Zelickson, '661, frog vessels (Steinsiepe
and Weibel, '70), and in rabbit and human eye
capillaries (Matsuda and Sugiura, '70). The
mean widths reported here are smaller than
those observed in dog aortic endothelial cells
(Sun and Ghidoni, '69). Most investigators give
lengths of tubular bodies ranging from maxima
of 0.7 pm to 3.2 pm (Weibel and Palade, '64;
Zelickson, '66; Steinsiepe and Weibel, '70; Matsuda and Sugiura, '70; Macadam et al., '75) in
blood vessels of various species. These lengths
are comparable to the maxima in the present
report. Although the tubular bodies are assumed to be rod-like, a flattened plaque or other
shape is possible. Determination of shape must
await serial reconstruction. Mitochondria occupy only 2 5 3 . 5 % of the endothelial cytoplasmic volume in capillaries of normal and
isolated perfused dog lungs (DeFouw and
Berendsen, '78a). Thirty percent of the tubular
bodies in this sample were adjacent to mitochondria in the same plane of section. With the
0.4-0.5% volume density of tubular bodies, the
probability that mitochondria and tubular
bodies would be contiguous on a random basis
should be very small. There may be a functional
relationship between the two. The 70% of tubular bodies that did not have mitochondria in the
plane of section may have had contiguous mitochondria above or below the plane of section.
The diameters of tubules within the tubular
bodies in this study were similar to those reported in endothelia of various organs by others
(Weibel and Palade, '64; Zelickson, '66; Herrlinger et al., '74; Macadam et al., '75). Although
some authors have given center-to-center dimensions between tubules (Weibel and Palade,
'64), the tubules within the tubular bodies observed in this study varied in compactness. The
more loosely grouped tubules were similar to
those termed type 1by Sun and Ghidoni ('691,
whereas the more closely compacted clusters of
tubules were called type 2 by those authors.
The former appearance was similar to that of
tubular structures seen in human tumor cells
reported by Uzman et al. ('711, and endothelia
of patients with systemic lupus erythematosus
(Garancis et al., '71; Nieland et al., '72; Naustein et al., '73; Jerusalem et al., '74; Macadam
et al., '75).
Loosely grouped tubules within tubular
bodies have been noted within the endoplasmic
reticulum of tumor cells (Uzman et al., '741, and
299
an origin of tubular bodies from the Golgi apparatus has been suggested (Sengel and Stoebner, '70; Matsuda and Sugiura, '70; Kojimahara, '77). Tubular bodies have been reported
not to react when cytochemicallytested for the
presence of acid phosphatase (Lemeunier, et
al., '69) and are therefore probably not related
to lysosomes. Burri and Weibel('68) have suggested that these structures may be related to
the production of a procoagulative substance.
This study demonstrates that tubular bodies
have stable volume densities and dimensions in
stable and edematous isolated perfused lung
preparations and suggests that isolated perfused lung preparations may be useful in future
studies of their function.
ACKNOWLEDGMENTS
The authors are grateful to Doctor F.P.
Chinard and the late Dr. W. Per1 for their collaboration and to Doctors P. Chowdhury and A.
Ritter for their assistance.
This work was supported in part by Public
Health Service grants HL-19571 and HL12879.
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