THROMBOSIS RESEARCH Printed in the United
SECTION
THE
BLOOD
VESSEL
II, Vol. 8, Suppl. Pergamon Press,
States
WALL,
1.976 Inc.
VI
HEMORHEOLOGY,
AND
HEMODYNAMICS
CHARACTERISTIC ENDOTHELIAL JUNCTIONS IN DIFFERENT SEGMENTS OF THE VASCULAR SYSTEM* Maia Yale
Simionescu, Nicolae Simionescu and George E. Palade
University School of Medicine, New Haven, Connecticut
Section of Cell 06510, U.S.A.
Biology,
ABSTRACT Among the structural features potentially important in the control of the integrity and permeability of the endothelial lining are its intercellular junctions. The points of interest in relation to the endothelial junctions are:their permeability to molecules of different sizes, the extent of cell to cell adhesion, and the existence of intercellular communications. Recent data have shown that alterations in endothelial junctions permeability and integrity may play an important role in inflammation, atherosclerosis as well as in the production of both hemorrhage and throtiosis. The present study was carried out to obtain more information on the fine structural organization of the endothelial junctions in different segments of the vascular system. The identification of small vessels in sectioned tissues is sometimes uncertain and since it could be of considerable importance for structural-functional correlations, we have started to study well defined sequential segments of the microvasculature. Small units consisting of an arteriole, its capillaries and the emerging venule (ACV units) were identified in the rat omentum fixed _in situ. Such units were processed for thin sectioning and freeze-fracturing either as a whole or as isolated segments. On ACV units we have examined, so far, the organization of the endothelial junctions. The results showed that the intercellular junctions of the endothelium has continuous and elaborate tight junctions with enclosed large gap junctions (communicating junctions or maculae communicantes). The capillary endothelium is provided with tight junctions formed by staggered strands. The pericytic venules and the muscu* Parts of these results were presented at the 14th Annual Meeting of the American Society for Cell Biology, San Diego, Ca., 1974 (J. Cell Biol. 1974 63:316a (Abstr.)), and at the 15th Annual Meeting of the American Society for Cell Biology, San Juan, Puerto Rico, 1975.Full fledged papers have have been submitted for publication to the Journal of Cell Biology. 247
248
sup]‘l.
II
lar venules exhibit loosely organized endothelial junctions with low profile ridges and grooves, usually devoid of particles. No gap (communicating) junctions were found in capillaries and pericytic venules, whereas the muscular venules display small, isolated gap (communicating) junctions. By extending the investigation to the large vessels, we found out that the endothelium of arteries and aorta exhibitsa variable combination of well organized tight junctions and gap (communicating) junctions, whereas in the venous part of the circulation (veins, vena cava) there are less elaborate tight junctions and the gap (communicating) junctions are in low frequency. The loose venular junctions may explain low local resistance to stress and correlated increased permeability.
At the level of the microvasculature as well as in the large vessels, the endothelial lining plays two major roles:
1)
it insures a physical partition
between the blood components and the subendothelial structures; 2)
it medi-
ates the exchanges of substances between the blood and the interstitial fluid. Among the structural features potentially important in the control of these functions are the intercellular junctions of the endothelium. The points of interest in relation to the endothelial junctions are the following:
1)
the closure, or conversely the patency of the junctions to
molecules of different sizes; 2) 3)
the extent of cell to cell adhesion, and
the presence or absence of intercellular communications. Very little is known about the organization and the functional character-
istics of the endothelial junctions in different segments of the vasculature. Recent data have shown that alterations in the endothelial permeability may play an important role in microcirculatory disturbances as well as in atherosclerosis.
Evidence has been also accumulated indicating that'focal separa-
tion of the endothelial cells at the level of the junctions occurs preferentially in certain segments of the vasculature and is involved in inflammation and in the production of both hemorrage and thrombosis. The present study was carried out to obtain more information on the fine structural organization of the endothelial junctions in different segments of the vasculature.
Suppl.
II
ENDOTHELIAL
JUNCTIONS
Information about the organization of the intercellular junctions
249
can
be
obtained either from thin sectioned specimens or from freeze fracture preparations.
The latter was the method of choice in our study due to the extensive
membrane areas which are exposed by fracturing.
It has, however, the disad-
vantage that the true cell surface cannot be visualized. The inquiries were carried out on the following vessels in the rat:
the
aorta (elastic artery), the mesenteric artery (muscular artery), microvessels (arterioles, capillaries, venules), the mesenteric and renal veins and the vena cava inferior. For the microvasculature, small units, consisting of an arteriole, its capillaries, and the emerging venule (designated as A C V units) were fixed in situ in the omentum or the mesentery, identified under the dissecting mi-croscope, and removed as a whole.
Specimens of each vascular segment isolated
from these units were separately processed through thin sectioning or freezefracturing.
The total number of endothelial junctions examined in freeze-
cleaved preparations was 280. For the large vessels, samples of Q
3 lmm' were cut from a cylindrical
vascular segment and placed on a carrier with endothelial surfaces facing each other, one being painted dark; this way the fracture could be directed through the endothelial layer more precisely. The results showed that the vascular endothelium does not have junctional complexes of the type generally encountered in epithelia; in its case, the only junctional elements present are tight junctions and gap junctions (communicating junctions or maculae communicantes).
Their degree of development
and their interrelations vary characteristically from one vascular segment to another, as indicated synoptically in Table I. In the arterial part of the vasculature, the intercellular junctions of the endothelium consist of 2-4 ridges/grooves in the large arteries, or 2-6 ridges/grooves in the arterioles which are marked quasicontinuously by strands
2
yjo
M.
of particles.
SIMIONESCU
ET
A.L.
The ridges form a network in which most of the meshes are
occupied by gap junctions (communicating junctions).
fully
Variants in which tight
junctions as well as gap junctions occur separately are encountered in the aorta and the large arteries; they also have a smaller number of occluding junctional strands and less elaborately organized in depth than in the case of arterioles (Figs. 1 and 4). The intercellular junction of the endothelium of the capillaries consists of 2-5 staggered, continuous or quasi-continuous ridges/grooves, partly marked by particles, (the ridges appear on the A faces of the cleaved membranes and the grooves on the 3 faces; the particles appear associated with either the ridges or more often with the grooves).
Gap junctions (communicating junc-
tions) were absent at this level (Fig. 2). In the pericytic and muscular venules the junctions are reduced to discontinuous ridges/grooves either devoid of, or marked by few particles (Fig. 3).
Gap junctions (communicating junctions) are absent in the pericytic
venules and appear as rare, small plaques in the muscular venules.) In the large veins the tight junctions sometimes retain this discontinuous character, but the frequency of ridges and especially grooves with associated particles is higher.
They are less elaborate
than in arteries (Fig. 4),
but they have the tendency to be continuous on long distances (Fig. 5).
Gap
junctions (communicating junctions) are relatively large in veins. In addition, in the endothelium of mesenteric artery and of the vena cava we have found two different types of special junctions (Figs. 6 and 7):
11
the first type forms complex loops and consists of parallel, closely spaced strands of particles with intercalated narrow gap junctions (communicating junctions).
2)
The second type has the general configuration of multiple
loops formed by parallel rows of tightly packed particles on the A face or grooves on the B face. unknown.
The significance of these two types of junctions is
Suppl.
II
ENDOTHELIAL
JUNCTIONS
251
In general the endothelial junctions of the arterial part of the vascular system appear highly structured and are probably adapted to considerable mechanical stress.
As in other situations, the gap junctions (communicating junc-
tions) probably contribute to intercellular communication, and, in addition, to cell to cell adhesion. The endothelium of the venous part of the circulation has considerably less elaborate tight junctions which probably reflect adaptation to low blood pressure.
In the venules the occurrence of the least organized type of junc-
tion may explain low local resistance to stress and correlated increased permeability. It seems that gap junctions (communicating junctions) between endothelial cells occur only in the vascular segments provided with smooth muscle cells in their media. In capillaries the junctional elements appear to be structurally closer to those encountered in the arterial part of the vasculature except for the absence of gap junctions (communicating junctions). The salient findings in this inquiry are: (a)
the characteristics spe-
cialization of the intercellular junctions in different segments of the vasculature; (b)
the existence of simplified discontinuous junctions in post cap-
illary venules; and (c)
the frequent occurrence of gap junctions (communica-
ting junctions) especially in the arteriolar - arterial endothelium.
252
M.
SIMIONESCU
ET AL
+.I
I
I
I
8
I
!i a
I
I
Suppl.
II
ENDOTHELIAL
JUNCTIONS
Fig. 1 Rat omentum; isolated arteriole. The occluding junction appears on the B face (B) as a meshwork of continuous, interconnected grooves marked by rows of particles (arrows). Gap junctions (communicating junctions) (a) occupy some meshes of the occluding junction network, and are closely surrounded by strands of a gap junction exhibiting both A and B the latter. In &, faces, 1, plasmalemmal vesicles openings. X 92,000.
FIG. 2 Rat omentum: isolated blood capillary. The cleavage plane exhibits the A (A) and B (g) faces of the cell membranes of two adjoining endotheiial cells. In this replica the occluding junction is seen as a maze of branching and staggered grooves (arrows) on the B face. Most of them form a continuous network; a few appear disconnected (*>. The A face ridges (arrowheads) as well as the B face grooves (arrows) are marked by discontinuous rows of particles. x 59,000.
253
254
M.
SIMIONESCU
E'J?AI,
Suppl.
Fig. 3 Rat omentum: isolated pericytic venule. The cleavage plane exposes the A (A) and B (B> faces of the cell membrane of two adjoining endothelial cells. The endothelial junction appears on the A face as low profile ridges (arrowheads) marked by discontinuous rows of particles, and on the B face as shallow grooves devoid of particles (arrows); they are discontinuous in a few places only,in this case; in general the points of discontinuities are more frequent. X 62,000.
Fig. 4 Rat mesenteric artery. The cleavage plane exposes a composite junction with gap (communicating) junction plaques . The B face grooves appear either marked by particles (2) or are particle-free (2). In d1 and AZ, two small gap junctions. A, A face; B, B face. x 118,000.
I.1
Suppl.
ENDOTHELIAL
II
Fig.
4
255
JUNCTIONS
Fig.
5
FIGS.
6 and 7
Rat mesenteric artery (FIG. 6) and vena cava (FIG. 7). Special endothelial junction consisting on the A face of loops of parallel, closely packed rows of particles (pr) alternating with gap junctions (a). 4, A face. FIG. 6 X 76,000. FIG. 7 X 114,000.
SECTION
THE
BLOOD
VESSEL
II, Vol. 8, Suppl. Pergamon Press,
States
WALL,
1.976 Inc.
VI
HEMORHEOLOGY,
AND
HEMODYNAMICS
CHARACTERISTIC ENDOTHELIAL JUNCTIONS IN DIFFERENT SEGMENTS OF THE VASCULAR SYSTEM* Maia Yale
Simionescu, Nicolae Simionescu and George E. Palade
University School of Medicine, New Haven, Connecticut
Section of Cell 06510, U.S.A.
Biology,
ABSTRACT Among the structural features potentially important in the control of the integrity and permeability of the endothelial lining are its intercellular junctions. The points of interest in relation to the endothelial junctions are:their permeability to molecules of different sizes, the extent of cell to cell adhesion, and the existence of intercellular communications. Recent data have shown that alterations in endothelial junctions permeability and integrity may play an important role in inflammation, atherosclerosis as well as in the production of both hemorrhage and throtiosis. The present study was carried out to obtain more information on the fine structural organization of the endothelial junctions in different segments of the vascular system. The identification of small vessels in sectioned tissues is sometimes uncertain and since it could be of considerable importance for structural-functional correlations, we have started to study well defined sequential segments of the microvasculature. Small units consisting of an arteriole, its capillaries and the emerging venule (ACV units) were identified in the rat omentum fixed _in situ. Such units were processed for thin sectioning and freeze-fracturing either as a whole or as isolated segments. On ACV units we have examined, so far, the organization of the endothelial junctions. The results showed that the intercellular junctions of the endothelium has continuous and elaborate tight junctions with enclosed large gap junctions (communicating junctions or maculae communicantes). The capillary endothelium is provided with tight junctions formed by staggered strands. The pericytic venules and the muscu* Parts of these results were presented at the 14th Annual Meeting of the American Society for Cell Biology, San Diego, Ca., 1974 (J. Cell Biol. 1974 63:316a (Abstr.)), and at the 15th Annual Meeting of the American Society for Cell Biology, San Juan, Puerto Rico, 1975.Full fledged papers have have been submitted for publication to the Journal of Cell Biology. 247
248
sup]‘l.
II
lar venules exhibit loosely organized endothelial junctions with low profile ridges and grooves, usually devoid of particles. No gap (communicating) junctions were found in capillaries and pericytic venules, whereas the muscular venules display small, isolated gap (communicating) junctions. By extending the investigation to the large vessels, we found out that the endothelium of arteries and aorta exhibitsa variable combination of well organized tight junctions and gap (communicating) junctions, whereas in the venous part of the circulation (veins, vena cava) there are less elaborate tight junctions and the gap (communicating) junctions are in low frequency. The loose venular junctions may explain low local resistance to stress and correlated increased permeability.
At the level of the microvasculature as well as in the large vessels, the endothelial lining plays two major roles:
1)
it insures a physical partition
between the blood components and the subendothelial structures; 2)
it medi-
ates the exchanges of substances between the blood and the interstitial fluid. Among the structural features potentially important in the control of these functions are the intercellular junctions of the endothelium. The points of interest in relation to the endothelial junctions are the following:
1)
the closure, or conversely the patency of the junctions to
molecules of different sizes; 2) 3)
the extent of cell to cell adhesion, and
the presence or absence of intercellular communications. Very little is known about the organization and the functional character-
istics of the endothelial junctions in different segments of the vasculature. Recent data have shown that alterations in the endothelial permeability may play an important role in microcirculatory disturbances as well as in atherosclerosis.
Evidence has been also accumulated indicating that'focal separa-
tion of the endothelial cells at the level of the junctions occurs preferentially in certain segments of the vasculature and is involved in inflammation and in the production of both hemorrage and thrombosis. The present study was carried out to obtain more information on the fine structural organization of the endothelial junctions in different segments of the vasculature.
Suppl.
II
ENDOTHELIAL
JUNCTIONS
Information about the organization of the intercellular junctions
249
can
be
obtained either from thin sectioned specimens or from freeze fracture preparations.
The latter was the method of choice in our study due to the extensive
membrane areas which are exposed by fracturing.
It has, however, the disad-
vantage that the true cell surface cannot be visualized. The inquiries were carried out on the following vessels in the rat:
the
aorta (elastic artery), the mesenteric artery (muscular artery), microvessels (arterioles, capillaries, venules), the mesenteric and renal veins and the vena cava inferior. For the microvasculature, small units, consisting of an arteriole, its capillaries, and the emerging venule (designated as A C V units) were fixed in situ in the omentum or the mesentery, identified under the dissecting mi-croscope, and removed as a whole.
Specimens of each vascular segment isolated
from these units were separately processed through thin sectioning or freezefracturing.
The total number of endothelial junctions examined in freeze-
cleaved preparations was 280. For the large vessels, samples of Q
3 lmm' were cut from a cylindrical
vascular segment and placed on a carrier with endothelial surfaces facing each other, one being painted dark; this way the fracture could be directed through the endothelial layer more precisely. The results showed that the vascular endothelium does not have junctional complexes of the type generally encountered in epithelia; in its case, the only junctional elements present are tight junctions and gap junctions (communicating junctions or maculae communicantes).
Their degree of development
and their interrelations vary characteristically from one vascular segment to another, as indicated synoptically in Table I. In the arterial part of the vasculature, the intercellular junctions of the endothelium consist of 2-4 ridges/grooves in the large arteries, or 2-6 ridges/grooves in the arterioles which are marked quasicontinuously by strands
2
yjo
M.
of particles.
SIMIONESCU
ET
A.L.
The ridges form a network in which most of the meshes are
occupied by gap junctions (communicating junctions).
fully
Variants in which tight
junctions as well as gap junctions occur separately are encountered in the aorta and the large arteries; they also have a smaller number of occluding junctional strands and less elaborately organized in depth than in the case of arterioles (Figs. 1 and 4). The intercellular junction of the endothelium of the capillaries consists of 2-5 staggered, continuous or quasi-continuous ridges/grooves, partly marked by particles, (the ridges appear on the A faces of the cleaved membranes and the grooves on the 3 faces; the particles appear associated with either the ridges or more often with the grooves).
Gap junctions (communicating junc-
tions) were absent at this level (Fig. 2). In the pericytic and muscular venules the junctions are reduced to discontinuous ridges/grooves either devoid of, or marked by few particles (Fig. 3).
Gap junctions (communicating junctions) are absent in the pericytic
venules and appear as rare, small plaques in the muscular venules.) In the large veins the tight junctions sometimes retain this discontinuous character, but the frequency of ridges and especially grooves with associated particles is higher.
They are less elaborate
than in arteries (Fig. 4),
but they have the tendency to be continuous on long distances (Fig. 5).
Gap
junctions (communicating junctions) are relatively large in veins. In addition, in the endothelium of mesenteric artery and of the vena cava we have found two different types of special junctions (Figs. 6 and 7):
11
the first type forms complex loops and consists of parallel, closely spaced strands of particles with intercalated narrow gap junctions (communicating junctions).
2)
The second type has the general configuration of multiple
loops formed by parallel rows of tightly packed particles on the A face or grooves on the B face. unknown.
The significance of these two types of junctions is
Suppl.
II
ENDOTHELIAL
JUNCTIONS
251
In general the endothelial junctions of the arterial part of the vascular system appear highly structured and are probably adapted to considerable mechanical stress.
As in other situations, the gap junctions (communicating junc-
tions) probably contribute to intercellular communication, and, in addition, to cell to cell adhesion. The endothelium of the venous part of the circulation has considerably less elaborate tight junctions which probably reflect adaptation to low blood pressure.
In the venules the occurrence of the least organized type of junc-
tion may explain low local resistance to stress and correlated increased permeability. It seems that gap junctions (communicating junctions) between endothelial cells occur only in the vascular segments provided with smooth muscle cells in their media. In capillaries the junctional elements appear to be structurally closer to those encountered in the arterial part of the vasculature except for the absence of gap junctions (communicating junctions). The salient findings in this inquiry are: (a)
the characteristics spe-
cialization of the intercellular junctions in different segments of the vasculature; (b)
the existence of simplified discontinuous junctions in post cap-
illary venules; and (c)
the frequent occurrence of gap junctions (communica-
ting junctions) especially in the arteriolar - arterial endothelium.
252
M.
SIMIONESCU
ET AL
+.I
I
I
I
8
I
!i a
I
I
Suppl.
II
ENDOTHELIAL
JUNCTIONS
Fig. 1 Rat omentum; isolated arteriole. The occluding junction appears on the B face (B) as a meshwork of continuous, interconnected grooves marked by rows of particles (arrows). Gap junctions (communicating junctions) (a) occupy some meshes of the occluding junction network, and are closely surrounded by strands of a gap junction exhibiting both A and B the latter. In &, faces, 1, plasmalemmal vesicles openings. X 92,000.
FIG. 2 Rat omentum: isolated blood capillary. The cleavage plane exhibits the A (A) and B (g) faces of the cell membranes of two adjoining endotheiial cells. In this replica the occluding junction is seen as a maze of branching and staggered grooves (arrows) on the B face. Most of them form a continuous network; a few appear disconnected (*>. The A face ridges (arrowheads) as well as the B face grooves (arrows) are marked by discontinuous rows of particles. x 59,000.
253
254
M.
SIMIONESCU
E'J?AI,
Suppl.
Fig. 3 Rat omentum: isolated pericytic venule. The cleavage plane exposes the A (A) and B (B> faces of the cell membrane of two adjoining endothelial cells. The endothelial junction appears on the A face as low profile ridges (arrowheads) marked by discontinuous rows of particles, and on the B face as shallow grooves devoid of particles (arrows); they are discontinuous in a few places only,in this case; in general the points of discontinuities are more frequent. X 62,000.
Fig. 4 Rat mesenteric artery. The cleavage plane exposes a composite junction with gap (communicating) junction plaques . The B face grooves appear either marked by particles (2) or are particle-free (2). In d1 and AZ, two small gap junctions. A, A face; B, B face. x 118,000.
I.1
Suppl.
ENDOTHELIAL
II
Fig.
4
255
JUNCTIONS
Fig.
5
FIGS.
6 and 7
Rat mesenteric artery (FIG. 6) and vena cava (FIG. 7). Special endothelial junction consisting on the A face of loops of parallel, closely packed rows of particles (pr) alternating with gap junctions (a). 4, A face. FIG. 6 X 76,000. FIG. 7 X 114,000.
