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Transport pathways for macromolecules in the aortic endotheliumI. Transendothelial channels revealed by three-dimensional reconstruction using serial sections

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THE ANATOMICAL RECORD 236:653-663 (1993)
Transport Pathways for Macromolecules in the Aortic Endothelium:
1. Transendothelial Channels Revealed by Three-Dimensional
Reconstruction Using Serial Sections
Department of Veterinary Anatomy, Faculty of Agriculture, Iwate University, Ueda,
Morioka, Iwate (K.O., K.T.) and EM Application Laboratory, JEOL Ltd., Musashino,
Akishima, Tokyo (T.W.), Japan
Three-dimensional organization of structures labeled by
horseradish peroxidase as a tracer molecule in rat aortic endothelium was
examined to elucidate the transport pathways for macromolecules. Aortic
endothelium was divisible morphologically into four distinct parts, i.e., the
parajunctional region (JR), peripheral region (PR), organelle region (OR),
and nuclear region (NR). Almost all vesicles, intercellular clefts, and phagosomes were labeled by horseradish peroxidase. Peroxidase-positive vesicles tended to gather in the PR, occasional J R , and the upper part of the
NR. Ultrathin serial micrographs revealed the transcellular channels composed of vesicles in the PR and J R , and the paracellular channels composed of the intercellular cleft without constrictions of tight and gap junctions. Transcellular channels were subdivided into three morphologically
different types, where not only vesicles but invagination of abluminal membrane and intercellular cleft occasionally participated in their formation.
Three-dimensional reconstructions from three series of consecutive electron micrographs revealed that almost all peroxidase-positive vesicles
were connected directly or indirectly with the cell surface. These results
indicate that the transcellular and paracellular channels are the transport
pathways for macromolecules in the aortic endothelium and may suggest
that the “shuttle” hypothesis is unsuitable to explain transport system for
macromolecules because it postulates the existence of many free vesicles in
the cytoplasm. o 1993 Wiley-Liss, Inc.
Key words: Aortic endothelium, Permeability, Peroxidase perfusion, Rat,
Three-dimensional reconstruction, Transendothelial channel,
Transport pathway, Ultrathin serial section
The modulation of vascular permeability is recognized as one of the most important endothelial functions and has been studied particularly in the capillary
endothelium. Physiological measurements of the permeability properties revealed that the capillary wall
serves as a highly selective barrier and is regarded as
having the same function as an artificial membrane
with “small pores and a few large pores,” 4-8 nm and
20-30 nm in radius, respectively (Grotte, 1956; Pappenheimer, 1953; Rippe and Haraldsson, 1986; Rippe et
al., 1979; Taylor and Granger, 1984). The morphological equivalent of “small and large pores” has been
searched for intensively and intercellular clefts and
plasmalemmal vesicles have been considered as target
structures (Bundgaard, 1984; Simionescu, 1983; Taylor
and Granger, 1983; Wagner and Casley- Smith, 1981).
Recently, a number of possible transport pathways in
the endothelium have been illustrated in various kinds
of capillaries (Clough, 1991; Simionescu, 1983; Taylor
and Granger, 1983; Wagner and Casley-Smith, 1981).
In arterial endothelium, permeability properties are
not only important for transport of nutrients to the
intimal and medial smooth muscle cells, but also in the
close correlation between abnormal increases in endothelial permeability and atherogenesis (ROSS, 1986).
According to the physiological data by O’Donnell and
Vargas (19861, transport properties of arterial endothelium are similar to those of continuous capillary
endothelium, although morphological differences such
as endothelial thickness, intercellular junctional system, and vesicle density have been described between
them (Londoiio and Bendayan, 1990; Simionescu and
Simionescu, 1984). In addition, mechanical factors (i.e.,
blood flow, pressure oscillations, and sinusoidal
stretch) have a large influence on the rate of water-
Received October 20, 1992; accepted February 15, 1993.
Address reprint requests to Kazushige Ogawa, PhD, Department of
Veterinary Anatomy, Faculty of Agriculture, Iwate University,
3-18-8 Ueda, Morioka, Iwate 020,Japan.
soluble molecule transport in the arterial endothelium
(Chien, 1978) and may cause some differences in transport properties between arterial and capillary endothelium.
Morphological studies on the transport pathways for
macromolecules in the arterial endothelium have been
largely restricted to two-dimensional analyses based
on random thin sections. Bundgaard (1983) and
Frgkjaer-Jensen (1980) demonstrated that they provided less information compared to three-dimensional
analyses based on serial sections. This suggests that
insufficient morphological analyses have so far been
done in the arterial endothelium to understand its
transport properties for macromolecules. In the present
study, therefore, we wanted to elucidate transport
pathways in the aortic endothelium by three-dimensional reconstruction using ultrathin serial sections
and horseradish peroxidase (HRP) as a tracer molecule:
HRP labels both “small and large pores” owing to its
small radius of 2.98 nm. Since we have already demonstrated the existence of channels composed of fused
vesicles in the aortic endothelium (Ogawa et al., 1986),
our particular focus here is on the detailed nature of
channels of fused vesicles and intercellular clefts.
Tissue Preparation
Young adult Sprague-Dawley rats of both sexes
weighing about 130 gm were used in this study. Under
sodium pentobarbital anesthesia (Somnopentyl, Pitman-Moore, Inc., Washington Crossing; 0.1 ml per 100
gm body weight, by intraperitoneal injection), HRP
(type 11,Sigma Chemical Co., St. Louis) dissolved in 0.4
ml of physiological saline was injected into the saphenous vein with a concentration of 15-20 mg per 100 gm
body weight, and allowed to circulate for 2-5 min before sacrifice. The abdominal aorta was removed between celiac and renal arteries, and immersed for 120
min at 4°C in a mixture of 2% paraformaldehyde and
2.5% glutaraldehyde buffered with 0.1M cacodylate to
pH 7.4. During fixation, the aorta was cut into segments less than 2 mm in length. After fixation, the
tissues were rinsed for 3 hr a t 4°C in the same buffer
containing 8%sucrose. Non-frozen cross sections of 80100 pm in thickness were cut with a Microslicer
(Dosaka EM Co. Ltd., Kyoto) and incubated in the medium of Graham and Karnovsky (1966) for 30-60 min
at room temperature to visualize HRP. After rinsing,
sections were postfixed with 2% OsO, in the same
buffer for 120 min at 4”C,dehydrated through a graded
ethanol series followed by propylene oxide, and embedded in Spurr’s resin. Some sections were prestained en
bloc with 1%uranyl acetate for 60 min before dehydration. Control rats received physiological saline (0.4ml
per 100 gm body weight) instead of HRP to evaluate
the specificity of reaction products.
coated slightly with carbon and viewed with a JEM1200EX electron microscope at an acceleration voltage
of 60 KV especially focused on the plasmalemmal vesicles and intercellular clefts labeled with HRP. In some
grids, staining and/or carbon-coating were omitted. In
several portions photographed serially, plasma membrane and cytoplasmic organelles labeled with HRP
were traced on a transparent sheet from each print and
stored through a digitizing tablet in the computer.
Three-dimensional reconstruction was calculated using a JIM-5000 (JEOL Ltd., Akishima) high-speed image processing system with a three-dimensional reconstruction program. We reconstructed three endothelial
segments from three series of consecutive electron micrographs through the computer.
The magnification on the electron microscope was
calibrated with a carbon replica of an optical grating
(2,160 lines per mm). The thickness of serial sections
ranged between 20-25 nm according to the method of
Frgkjaer-Jensen (1980) which compares the number of
serial sections containing a single vesicle with the
maximum diameter of the same vesicle.
Localization of Horseradish Peroxidase and Its
Regional Difference
Aortic endothelium was divided into four distinct regions according to Simionescu et al. (1974) in continuous capillary endothelium, i.e., parajunctional region
(JR) located around the intercellular junction and characterized by low frequency or absence of vesicles; peripheral region (PR) located in the peripheral part of
the cell and characterized by large vesicle population;
organelle region (OR) located adjacent to the nucleus
and characterized by a high concentration of organelles; nuclear region (NR) located in the central
part and occupied chiefly by the nucleus itself. The last
three regions in the aortic endothelium were similar to
those in the capillary endothelium in their morphological features. Morphological characteristics of the aortic endothelium were recognized in the JR and summarized as follows. First, the JR was not always situated
between PRs as in the capillary endothelium. It held
one of six positions according to the combination of
adjoining regions and was frequently observed in three
of them as follows: JR lying between PRs; JR lying
between PR and OR; PR lying between ORs. Second,
JRs were subdivided into 2 groups (JR1 and JR2) characterized respectively by high- and low-vesicle population. There was every indication that the latter (JR2)
corresponded to the JR lying between PRs. Four distinct regions in the aortic endothelium are schematically summarized in Figure 1.
Reaction products of peroxidase were observed on the
external side of both luminal and abluminal membranes, in the intercellular clefts and the subendothe-
Serial Sectioning
Thin cross-sections of aortic endothelia were cut serially with a diamond knife mounted on an LKB U1trotome NOVA and collected on Formvar-covered grids
with a single-slot of 1 x 2 mm. The serial sections as
thin and uniform as possible were selected by judging
the interference color. The obtained serial sections
were stained with uranyl acetate and lead citrate,
Gap junction
Intercellular cleft
Internal elastic lamina
Subendothelial space
Fig. 1 . A diagram of four morphologically distinct regions in rat
aortic endothelium. JR1: parajunctional region lying between organelle regions characterizedby a high concentrationof vesicles. JR2:
parajunctional region lying between peripheral regions charac-
terized by a low concentrationof vesicles. NR: nuclear region occupied
by nucleus. O R organelle region characterized by a high concentration of organelles. PR parajunctional region characterizedby a high
concentration of vesicles.
lial space, and within almost all vesicles and most
phagosomes, whereas they were not found in the aortic
endothelium of the control animals. The reaction products revealed deep invaginations of the abluminal
membrane (Fig. 2). Peroxidase-positive vesicles tended
to gather in the PR, JR1, (Fig. 3) and the luminal part
of NR. Tight junctions and/or gap junctions were usually observed in the intercellular membranes and
formed a constriction less than 2 nm in the intercellular clefts unlikely to be permeable to HRP. Therefore,
the intercellular cleft was divided into luminal and abluminal parts, although the latter was often filled with
reaction products of peroxidase.
of intercellular cleft was recognized as a third type. The
transcellular channels connecting the luminal parts of
intercellular clefts with the abluminal space, however,
were not observed in the present study. In the highly
interdigitating portion, a chain of fused vesicles (or a
vesicle) was frequently (not rarely) found even in a
plane of a single section. This connected an abluminal
part of the intercellular cleft with an abluminal space
or another part of the abluminal intercellular cleft and
formed a shortcut pathway for macromolecules (Fig.
Intercellular clefts without the constriction were
sometimes found. They were about 20 nm in width
along most of its length (Figs. 6a, 7) and were regarded
as one of paracellular channels. Serial micrographs revealed that intercellular clefts varied conspicuously
from simple profiles to complicated profiles in intricacy
of interdigitation (Figs. 6, 7) and from permeable profiles to impermeable ones that are present at the intercellular junctions (Fig. 6).
Transendothelial Channels
Channels formed by vesicles through endothelial cell
cytoplasm (transcellular channels) inevitably contained the reaction products of peroxidase, but were
rarely found in a plane of a single section. They were
usually recognized only by three-dimensional organization of vesicles from serial electron micrographs. All
channels were recognized in the PR and JR of the endothelium. The present three-dimensional observation
revealed the existence of a t least three morphologically
different types of transcellular channels. The first type
is present in the PR. It consists of a chain of fused
vesicles and connects the luminal surface with the abluminal surface directly (Fig. 4). The second type is
also recognized in the PR and consists of a chain of
fused vesicles and invaginations of abluminal membrane forming a deep slit, not a tubule or a cone (Fig.
5). They fuse with each other and form the transcellular channel. The last type i F observed in the JR and
consists of a chain of fused vesicles (or a vesicle) and an
intercellular cleft. A chain of fused vesicles opens to the
luminal or abluminal surface and fuses with the intercellular membrane. This channel serves as a passage
route for macromolecules to bypass the intercellular
junctions (tight junction and/or gap junction). In the
serial micrographs of Figure 6, a vesicle opening simultaneously to the luminal space and the abluminal part
The three-dimensional reconstruction revealed that
almost all peroxidase-positive vesicles were connected
directly or indirectly (fused with other vesicles) with
the cell surface. The reconstruction also indicated that
vesicles had a tendency to gather in certain domains.
In Figure 8 of a stereopair including the JR, vesicles
were gathered in one endothelium between the luminal
membrane and the abluminal part of the intercellular
membrane. In this domain, vesicles opening to the lumen were close to the other vesicles opening to the
abluminal part of intercellular cleft, although a transcellular channel was not observed. Transendothelial
channels, i.e., transcellular and paracellular channels,
are schematically summarized in Figure 9.
Methodological Validity
According to the pore theory which describes the permeability properties of capillary walls, two systems of
Fig. 2. Deep invaginations with branches of abluminal membrane are filled with reaction products of
peroxidase in the organelle region of the endothelium. Bar: 0.5 +m.
Fig. 3. The first one of 11 serial electron micrographs used for three-dimensional reconstruction (Fig.
8).Peroxidase-positive vesicles are gathered in the parajunctional region lying between organelle regions. Bar: 0.5 km.
Fig. 4. Transcellular channel (arrow) composed of a chain of fused vesicles is recognized in the peripheral region of the endothelium. Bar: 0.2 pm.
pores, small pores and a few large pores, perforate capillary endothelium. The former with an effective radius
of 4-8 nm is assigned to a transport pathway for water
and small solutes up to the size of serum albumin (7 nm
in diameter). The latter with a radius of 20-30 nm
transports macromolecules (Grotte, 1956; Pappenheimer, 1953; Rippe and Haraldsson, 1986; Rippe et al.,
1979; Taylor and Granger, 1984). Besides cylindrical
channels about 12 nm in diameter, small pores were
also estimated to be slits about 8 nm in width. Previous
authors used many kinds of tracer molecules to determine the morphological counterparts in capillary endothelium (Bundgaard et al., 1979; Clough and Michel,
1981; Karnovsky, 1967; Simionescu et al., 1975). In the
present study, however, we adopted HRP as a tracer to
evaluate the permeability properties and t o identify
the transport pathways for macromolecules in the aortic endothelium because of the following reasons: (1)
HRP is an intermediate-sized tracer, about 6 nm in
diameter, and able to pass the endothelial layer
through both small and large pores; (2) HRP serves as
an enzymatic tracer and allows one to detect its precise
localization with the electron microscope by large
amounts of its reaction products. Actually, many authors have reported that HRP is observed in the intercellular clefts and vesicles of the arterial endothelium
(Florey and Sheppard, 1970; Huttner et al., 1973;
Okuda and Yamamoto, 1983; Schwartz and Benditt,
1972). Intercellular clefts and vesicles of the arterial
endothelium are regarded as counterparts of the small
and large pores of capillary endothelium (Bundgaard,
1984; Simionescu, 1983; Taylor and Granger, 1983;
Wagner and Casley-Smith, 1981). Much larger tracers
like ferritin (11 nm in diameter), however, can label
vesicles only (Baldwin and Chien, 1985; Huttner et al.,
1970,1973).In spite of these advantages, HRP shows a
significant disadvantage. Cotran and Karnovsky
(1967) reported that HRP induces histamine release
and leads to an increase in vascular permeability in
some species including Sprague-Dawley rats. This may
be caused by direct interaction of histamine with the
endothelium to open its intercellular junctions through
the endothelial contraction (Simionescu, 1983). However, this change in vascular permeability has occurred
only in venular endothelium (Cotran and Karnovsky,
1967) and has not been observed in arterial endothelium (Florey and Sheppard, 1970; Huttner et al., 1973).
These findings encouraged us to adopt HRP in the
present study. In addition, reaction products were not
observed in the aortic endothelium in our control experiments. This suggests that the reaction products
originate from the exogenous peroxidase and may exactly label all kinds of transport pathways for macromolecules in the aortic endothelium.
Almost all previous studies on the morphology of
transport pathways for macromolecules were based on
either tracer perfusion and random thin sectioning or
ultrathin serial sectioning in the method. The former
method is able to label vesicles taking part in the
transport of tracer molecules. However, it cannot draw
the organization of vesicles in the endothelium, because the diameter of vesicles is equal to the thickness
of conventional thin sections (Bundgaard, 1983). On
the other hand, the latter method tends to overestimate
three-dimensional organization of vesicles not always
participating in the transport for macromolecules.
Therefore, the combination of both methods as carried
out in the present study may allow one to elucidate in
Fig. 5. Serial electron micrographs in the peripheral region. A vesicle (arrow in Fig. 5A) connectedwith
invagination of abluminal membrane (arrow in Fig. 5B)fuses with another vesicle (*) opening to lumen
and forms a transendothelial channel. Bar: 0.2 pm.
Fig. 6. Five selected electron micrographs ( A 3rd; E D : 10th-12th;
E: 18th) from 18 serial sections. A vesicle opening to abluminal part
of the intercellular cleft (arrow in Figure 6b) fuses with luminal membrane (arrow in Figure 6d) and leads to a formation of a transcellular
channel. Another vesicle opening simultaneously to abluminal part of
the intercellular cleft and ablumen makes a shortcut pathway (arrow
in Figure 6a). Note that intricacy of interdigitation varies from a
complicated profile to a simple profile, and tight and gap junctions
recognized in Figures 6 b e are not observed in Figure 6a. Bar: 0.4 pm.
Fig. 7. Serial electron micrographs showing paracellular channel. Intercellular cleft without tight and
gap junctions is about 20 nm in width along most of its length and about 15 nm in some most narrowing
parts. Its intricacy of interdigitation varies from a mortise profile (Fig. 7A) to a simple profile. Bar: 0.2
Fig. 8. A stereopair of three-dimensional reconstruction in the parajunctional region. Note vesicles
gathered between the luminal membrane and abluminal part of the intercellular membrane in the
endothelium. Bar: 0.2 Fm.
detail the precise transport pathways for macromole- clefts, and vesicles opening to the lumen (Florey and
Sheppard, 1970; Huttner et al., 1973; Ogawa, unpubcules in the endothelium.
Immersion fixation was preferred in this experi- lished data). This makes it difficult to grasp the precise
ment because perfusion fixation tends to wash out transport pathways for macromolecules in these reHRP from the lumen, luminal region of intercellular gions.
66 1
Fig. 9. A diagram of three morphologically different types of transcellular channels and a paracellular channel in rat aortic endothelium. Type 1: transcellular channel formed by a chain of fused vesicles. Type 2: transcellular channel formed by a chain of fused vesicles
and invagination of abluminal membrane. Type 3: transcellular chan-
nel formed by intercellular cleft and a chain of fused vesicles (or a
vesicle), which connects lumen with abluminal part of the intercellular cleft or luminal part of the intercellular cleft with ablumen. PCC:
paracellular channel formed by intercellular cleft without tight and
gap junctions.
Transport Pathways for Macromolecules
neously to lumen and ablumen forms the transcellular
channel in the thin PR (Coomber and Stewart, 1986;
Cornillie and Lauweryns, 1984; Palade and Bruns,
1968; Simionescu et al., 1975). In the arterial endothelium, Ogawa et al. (1986) reported the channels composed of a chain of fused vesicles for the first time, but
there have been few confirmatory reports of this observation. Although Nag (1990) reported the channels in
the hypertensive rat arteriolar endothelium using the
same method as that of Ogawa et al. (1986), it is uncertain in our judgment whether the channel is precisely formed or not. As observed in the present study,
transcellular channels show much more complicated
profiles in naturally thick arterial endothelium. They
may behave more transiently and dynamically than
those in capillary endothelium because the arterial endothelium is influenced by greater mechanical factors
in blood vessels such as fast blood flow, high pressure
and flow oscillations, and intense sinusoidal stretch.
Consequently, they may be hardly observed in a single
conventional thin section unlike those in capillary endothelium, but they can be recognized by analyzing
ultrathin serial sections.
Most transcellular channels in capillary endothelium are usually present in the PR and composed only
of vesicles, although Frgkjaer-Jensen (1980) reported a
channel in the JR. In the aortic endothelium, transcellular channels are morphologically composed of invaginating abluminal membranes and intercellular clefts,
in addition to vesicles. This implies that these structures are valid and suitable for the connection of lumen
and ablumen across the long distance, the naturally
thick arterial endothelium.
We demonstrated that the discontinuity of intercellular junctions (tight junction and gap junction) occurs
in the aortic endothelium. This indicates the existence
of a paracellular channel, a transport pathway for macromolecules. In the arterial endothelium, it is widely
recognized that tight junctions show discontinuous
properties and disc-shaped gap junctions are intercalated or partly framed by tight junctional strands (Hutt-
Although Chien (1978) divided aortic endothelium
into four distinct parts according to Simionescu et al.
(19741, he did not show anything about the properties
of arterial endothelium. Therefore, we reported here
the morphological characteristics of the aortic endothelium with special reference to the JR as compared to
a continuous capillary endothelium.
In the present study, almost all vesicles, intercellular clefts, and phagosomes were labeled with HRP in
the aortic endothelium. This agrees well with previous
studies (Florey and Sheppard, 1970; Huttner et al.,
1973; Schwartz and Benditt, 1972). Since phagosomes
have a close relation to the intracellular digestive system, we excluded them from the discussion on transendothelial transport system for macromolecules.
Peroxidase-positive vesicles showed a tendency to assemble in the PR, in some JR where endothelium was
usually rather thick, and in the luminal portion of NR.
This may indicate that the transendothelial transport
system is located in the PR and JR. Vesicles in the OR
as well as NR cannot be expected to participate in this
transport system because physical hindrances such as
many organelles or nucleus and longer distances between lumen and ablumen exist in these regions. These
vesicles may serve to increase the cell surface area,
allowing changes in vascular diameter (Wolff, 1977) or
possessing elastic properties of endothelium (FrgkjaerJensen, 1984). They may also be involved in regulation
of cytosolic Ca concentration (Bundgaard, 1987). The
functional aspects of these vesicles need to be studied
Transendothelial channels can be regarded as one of
the transport pathways for macromolecules. We demonstrated the existence of three morphologically different types of transcellular channels in the aortic endothelium by ultrathin serial sectioning and HRP
labeling. In the capillary endothelium, many authors
have reported the transendothelial channels usually
found on a single thin section. In this case, a single
vesicle or a chain of fused vesicles opening simulta-
Chien, S. 1978 Transport across arterial endothelium. Prog. Hemost.
Thromb., 4:l-36.
Clough, G. 1991 Relationship between microvascular permeability
and ultrastructure. Prog. Biophys. Molec. Biol., 55:47-69.
Clough, G., and C.C. Michel 1981 The role of vesicles in the transport
of ferritin through frog endothelium. J. Physiol., 315:127-142.
Coomber, B.L., and P.A. Stewart 1986 Three-dimensional reconstruction of vesicles in endothelium of blood-brain barrier versus
highly permeable microvessels. Anat. Rec., 215:256-261.
Cornillie, F.J., and J.M. Lauweryns 1984 Transendothelial channels
in fenestrated endometrial blood capillaries of rats. Cell Tissue
Res., 237:371-373.
Cotran, R.S., and M.J. Karnovsky 1967 Vascular leakage induced by
Exp. Biol. Med., 126:
horseradish peroxidase in the rat. Proc. SOC.
Florey, Lord, and B.L. Sheppard 1970 The permeability of arterial
endothelium to horseradish peroxidase. Proc. Roy. Soc. Lond. B.,
FrZkjaerJensen, J. 1980 Three-dimensional organization of plasmalemmal vesicles in endothelial cells: An analysis by serial sectioning of frog mesenteric capillaries. J. Ultrastmet. Res., 73320.
Fr@kjaerJensen,J . 1984 The plasmalemmal vesicular system in striated muscle capillaries and in pericytes. Tissue Cell, 16:31-42.
Graham, R.C., and M.J. Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in the proximal tubules of
mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14:291-302.
Grotte, G. 1956 Passage of dextran molecules across the blood-lymph
barrier. Acta Physiol. Scand., Suppl., 21 1 :1-84.
Hiittner, I., M. Boutet, and R.H. More 1973 Studies on protein passage
through arterial endothelium: 11. Regional differences in permeability to fine structural protein tracers in arterial endothelium
of normotensive rat. Lab. Invest., 28:678-685.
Hiittner, I., R.H. More, and G. Rona 1970 Fine structural evidence of
specific mechanism for increased endothelial permeability in experimental hypertension. Am. J. Pathol., 61:395-412.
Hiittner, I., C. Walker, and G. Gabbiani 1985 Aortic endothelial cell
during regeneration. Remodeling of cell junctions, stress fibers,
and stress fiber-membrane attachment domains. Lab. Invest., 53:
Karnovsky, M.J. 1967 The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J . Cell Biol., 35:213236.
Londofio, I., and M. Bendayan 1990 Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and
mesothelial cells. Biol. Cell, 69~161-169.
Miihleisen, H., H. Wolburg, and E. Betz 1989 Freeze-fracture analysis
of endothelial cell membranes in rabbit carotid arteries subjected
to short-term atherogenic stimuIi. Virchows Archiv. B Cell
Pathol., 56:413-417.
Nag, S. 1990 Presence of transendothelial channels in cerebral endothelium in chronic hypertension. Acta Neurochirurgica, Suppl.,
51:335 -337.
ODonnell, M.P., and F.F. Vargas 1986 Electrical conductivity and its
use in estimating a n equivalent pore size for arterial endothelium. Am. J. Physiol., 250:H16-H21.
Ogawa, K.S., K. Fujimoto, and K. Ogawa 1986 Ultracytochemical
studies of adenosine nucleotidases in aortic endothelial and
smooth muscle cell. Ca+ +-ATPase and N a + , K+-ATPase. Acta
Histochem. Cytochem., 19:601-620.
Okuda, T., and T. Yamamoto 1983 The ultrastructural basis of the
permeability of arterial endothelium to horseradish peroxidase.
Freeze-fracture and tracer studies of rat thoracic aorta and basilar artery. Cell Tissue Res., 231:117-128.
Palade, G.E., and R.R. Bruns 1968 Structural modulations of plasmalemmal vesicles. J. Cell Biol., 37:633-649.
Baldwin, A.L., and S. Chien 1985 Effect of plasma proteins on endothelial binding and vesicle loading of anionized ferritin in rabbit Pappenheimer, J.R. 1953 Passage of molecules through capillary
walls. Physiol. Rev., 33:387-423.
aorta. Arteriosclerosis, 5:451-458.
Bundgaard, M. 1983 Vesicular transport in capillary endothelium: Rippe, B., and B. Haraldsson 1986 Capillary permeability in rat hindquarters as determined by estimations of capillary reflection coDoes it occur? Federation Proc., 42:2425-2430.
efficients. Acta Physiol. Scand., 127:289-303.
Bundgaard, M. 1984 The three-dimensional organization of tight
junction in a capillary endothelium revealed by serial-section Rippe, B., A. Kamiya, and B. Folkow 1979 Transcapillary passage of
albumin, effects of tissue cooling and of increases in filtration and
electron microscopy. J. Ultrastruct. Res., 88:l-17.
plasma colloid osmotic pressure. Acta Physiol. Scand., 105:171Bundgaard, M. 1987 Tubular invaginations in cerebral endothelium
and their relation to smooth-surfaced cisternae in hagfish (MyxRoss, R. 1986 The pathogenesis of atherosclerosis-an update. N.
ine glutinosa). Cell Tissue Res., 249:359-365.
Engl. J. Med., 314:488-500.
Bundgaard, M., J . Fr@kjaerJensen,and C. Crone 1979 Endothelial
plasmalemmal vesicles as elements in a system of branching in- Schwartz, S.M., and E.P. Benditt 1972 Studies on aortic intima: I.
vaginations from the cell surface. Proc. Natl. Acad. Sci. USA,
Structure and permeability of rat thoracic aortic intima. Am. J .
Pathol., 66:241-264.
ner et al., 1973, 1985; Miihleisen et al., 1989; Okuda
and Yamamoto, 1983). Our observation agrees well
with these reports. We also demonstrated that the intercellular clefts below the intercellular junctions are
usually filled with the reaction products of HRP. This
may be caused by the discontinuous tight and gap junctions and the transcellular channels connecting lumen
with intercellular cleft below the intercellular junctions. They may allow the intercellular cleft to be permeable to HRP due to the bypassing of the intercellular
junctions. Schwartz and Benditt (1972) grouped the
junctions between aortic endothelial cells into four categories, i.e., a single end-to-end or “butt” junction; a
simple “overlap” junction; a “mortise” junction; more
complicatedly folded junctions. Some investigators use
these categories as parameters of permeability for macromolecules through intercellular clefts. However, our
observation with serial electron micrographs such as in
Figures 6 and 7 suggest that the Schwartz and Benditt
groupings may not be valid because of the remarkable
variety in intricacy of these interdigitations.
It is widely accepted that “vesicular transport” is one
of the ways in which molecules cross the capillary wall.
Palade and Bruns (1968) and Simionescu (1983) proposed that plasmalemmal vesicles constitute a distinct
population of membranes and shuttle between luminal
and abluminal membranes to carry molecules in their
contents. This “shuttle” hypothesis presumes many
vesicles free in the cytoplasm. Recent studies by ultrathin serial sectioning have demonstrated that almost all plasmalemmal vesicles in the capillary endothelium open to the endothelial surface (Bundgaard et
al., 1983; Coomber and Stewart, 1986; FrokjaerJensen, 1980,1984). These workers have thrown doubt
upon the validity of this “shuttle” hypothesis. In the
aortic endothelium, our previous study (Ogawa et al.,
1986) demonstrated the different vesicle properties as
shown by enzyme activities between luminal and abluminal sides. Our present observation with three-dimensional reconstructions revealed that almost all peroxidase-positive vesicles were connected with the cell
surface (see Fig. 8). This suggests that the “shuttle”
hypothesis does not apply to the aortic endothelium. At
present, channels alone appear to be the means of
transendothelial transport system via plasmalemmal
vesicles in the aortic endothelium. However, since our
three-dimensional reconstructions of vesicles were carried out on a few limited arterial endothelial segments,
additional studies of other arterial endothelia should
be carried out to extend our understanding of the transport behavior of these morphological entities.
Simionescu, M., and N. Simionescu 1984 Ultrastructure of the microvascular wall: Functional correlations. In: The Cardiovascular
System, Handbook of Physiology, vol. IV, sect. 2, Microcirculation. E.M. Renkin and C.C. Michel, eds. Williams and Wilkins
Company, Baltimore, pp. 41-101.
Simionescu, M., N. Simionescu, and G.E. Palade 1974 Morphometric
data on the endothelium of blood capillaries. J. Cell Biol., 60:
Simionescu, N. 1983 Cellular aspects of transcapillary exchange.
Physiol. Rev., 63:1536-1579.
Simionescu, N., M. Simionescu, and G.E. Palade 1975 Permeability of
muscle capillaries to small heme-peptides: Evidence for the existence of patent transendothelial channels. J. Cell Biol., 64.586607.
Taylor, A.E., and D.N. Granger 1983 Equivalent pore modeling: Vesicles and channels. Federation Proc., 42.2440-2445.
Taylor, A.E., and D.N. Granger 1984 Exchange of macromolecules
across the microcirculation. In: The Cardiovascular System,
Handbook of Physiology, vol. IV,sect. 2, Microcirculation. E.M.
Renkin and C.C. Michel, eds. Williams and Wilkins Company,
Baltimore, pp. 467-520.
Wagner, R., and J.R. Casley-Smith 1981 Review: Endothelial vesicles.
Microvasc. Res., 21 :267-298.
Wolff, J.R. 1977 Ultrastructure of the terminal bed as related to function. In: Microcirculation. G. Kaley and B.M. Altura, eds. University Park Press, Baltimore, vol. 1, pp. 95-130.
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channel, using, dimensions, three, section, serial, transendothelial, macromolecules, transport, endotheliumi, revealed, pathways, aortic, reconstruction
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