Journal of Cell Science 104, 1073-1081 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
1073
Immunoelectron characterisation of the inter-endothelial junctions of
human term placenta
Lopa Leach1,*, Peter Clark1, Maria-Grazia Lampugnani2, Alicia G. Arroyo3, Elisabetta Dejana2
and J. Anthony Firth1
1Department
of Anatomy and Cell Biology, St. Mary’s Hospital Medical School, Imperial College of Science, Technology and
Medicine, Norfolk Place, London W2 1PG, UK
2Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy
3Servicio de Inmunologia, Hospital de la Princesa, Universidad Autonoma de Madrid, Madrid, Spain
*Author for correspondence
SUMMARY
The molecular constituents of the paracellular clefts in
human placental microvessels were investigated using
antibodies against PECAM-1, pan-cadherin, A-CAM
(N-cadherin), cadherin-5 and two types of integrins
(those recognised by antibodies to the 1 chain and
v 3). Ultrastructural localisation of these molecules in
ultrathin frozen sections of human term placentae was
attempted using colloidal gold immunocytochemistry,
after establishing their presence by indirect immunofluorescence.
At the light microscopical level, the endothelial paracellular clefts were found to be immunoreactive to the
antibodies against PECAM-1, cadherin-5 and pan-cadherin, but not the integrins. The latter showed diffuse
distribution in the endothelium and in the abluminal
interstitial space. PECAM-1 and pan-cadherin were also
seen in the cytoplasm and luminal surface of the
endothelium. Immunoelectron studies revealed that the
cadherins and PECAM-1 were present in the wide
regions of the paracellular clefts, but not in tight junc-
tional regions. Using immunocytochemistry, these wide
junctional areas were found to be associated with the
cytoskeletal linking molecules vinculin and α-actinin.
These regions may therefore contain adherens-type
junctions. Cadherin-5, localised by two different monoclonal antibodies, 7B4 and TEA, was the only antigen
which was cleft-specific, the others also being seen in the
cytoplasm of the microvascular endothelium. Cadherin5 and pan-cadherin were co-localised in the same wide
junction, but were usually seen to occupy different
microdomains of, and different wide zones of, the same
cleft. The cell adhesion molecules localised in the paracellular wide junctions of the human placental microvessels may play a role in maintaining the intercellular
spacing between endothelial cells, and may be part of a
paracellular “fibre matrix” with permeability-restricting properties.
INTRODUCTION
available as to which extracellular components are present
within the cleft and how they are connected with the membranes. Three-dimensional modelling of the cleft geometry
has led workers to postulate the existence of bridging molecules within the cleft in order to maintain uniform width
in the face of changing pressure (Hsiung and Skalak, 1984;
Silberberg, 1988). The existence of such “linkers” spanning
the clefts in the wide zones has been reported (Firth et al.,
1983; Leach and Firth, 1992; Schulze and Firth, 1992), but
the nature of their component molecules remains unknown.
Knowledge of the molecular composition of endothelial
junctions is required to understand the organisation of paracellular junctions and how the paracellular cleft provides a
permeo-selective barrier between the blood and the underlying tissue.
So far, a few integral membrane proteins have been
One of the main pathways for transfer of water and
hydrophilic solutes across continuous non-brain capillaries
is the paracellular cleft. A typical endothelial cleft shows
one or more zonulae occludentes or tight junctions interspersed with wide zones of uniform width. The tight junctional regions have been shown to be discontinuous along
the axial length of the capillary and contain a separation of
4 nm at the point of apposition of the plasma membranes
of adjacent cells (Karnovsky, 1967; Firth et al., 1983;
Bundgaard, 1984; Ward et al., 1988; Leach and Firth,
1992). The wide zones are thought to contain a glycocalyx-like extracellular matrix which, in series with the tight
junctional regions, determines the selectivity of the cleft
(Curry and Michel, 1980). Little information is currently
Key words: endothelium, intercellular junctions, cell adhesion
molecules, cadherin, immunogold cytochemistry, placenta
1074 L. Leach and others
described in the paracellular cleft regions of endothelial
cells. PECAM-1 (Newman et al., 1990), also called CD31
(Simmons et al., 1990; Muller et al., 1989) or endo-CAM
(Albelda et al., 1990); the integrin heterodimers α5β1 and
α2β1 (Lampugnani et al., 1991); V-cadherin (Heimark et
al., 1990), N-cadherin/A-CAM (Volk and Geiger, 1984)
and pan-cadherin (Geiger, 1990) and most recently an
endothelial-specific cadherin called cadherin-5 (Lampugnani et al., 1992) have been identified. Endothelial cadherins, which display Ca2+-dependent homophilic binding
specificity, are thought to be components of the endothelial plaque or adherens-type junction situated in the wide
zones of the paracellular cleft (Heimark et al., 1990).
Peripheral constituents of this junction are plakoglobin, vinculin, α-actinin and actin microfilaments (Franke et al.,
1988; Magee and Buxton, 1991). Cadherins are therefore
thought to be essential for the formation of cell-cell associations (Takeichi, 1991) and regulators of the permeability properties of the vasculature (Lampugnani et al., 1992).
Integrins, which usually mediate cell-matrix contacts, have
also been shown to mediate intercellular adhesion between
endothelial cells; the integrins α5β1 and α2β1, but not other
members of the β1 subfamily, have been located at cell-cell
contact sites (Lampugnani et al., 1991). These authors have
also shown that integrins possessing the αv chain, with the
exception of the heterodimer αvβ3, are present at cell-cell
borders of umbilical vein endothelial cell monolayers and
in explanted islets of umbilical vein endothelium. PECAM1 is an integral membrane glycoprotein found on the surface of platelets, at endothelial intercellular junctions in culture, and on cells of myeloid lineage. It too has been
implicated in formation of cell-cell junctions, since cells
transfected by PECAM-1 demonstrate calcium-dependent
aggregation and possess junctions highly enriched in
PECAM-1 (Albelda et al., 1991).
To ascertain whether these molecules are present in the
paracellular clefts of continuous non-brain capillaries, we
have used an immunoelectron histochemical method
applied to ultrathin frozen sections of the term human placenta. Antibodies against vinculin and α-actinin were also
used to determine the nature of the intercellular junctions.
Double immunolabelling with anti-pan-cadherin and cadherin-5 antibodies was carried out. The human placenta was
chosen because of its extensive microvascular bed which is
readily accessible to perfusion studies in vitro. The paracellular clefts of the human placental microvessels (Fig. 1)
resemble continuous non-brain capillaries, both in structure
(Leach and Firth, 1992) and permeability characteristics
(Eaton et al., 1993).
MATERIALS AND METHODS
Tissue preparation
Lobules of human term placentae obtained from placentae (n = 4)
delivered by Caesarian section were (1) washed thoroughly in cold
PBS and immersion fixed in 4% p-formaldehyde for 60 min and
(2) extracorporeally perfused for a 20 min equilibration period
(Leach and Firth, 1992) and perfusion-fixed for 30 min with 4%
p-formaldehyde, excised and immersion-fixed for a further 60 min.
After washing in PBS, some tissue was frozen in iso-pentane and
cryosectioned (10 µm thickness) for indirect immunofluorescent
labelling. For ultracryomicrotomy, tissue pieces were sandwiched
in 8% gelatin and cryoprotected by agitating in 2.3 M sucrose for
2 h. They were then frozen on metal stubs by plunging in liquid
nitrogen. Ultrathin frozen sections (45-60 nm thickness) were cut
using a Reichert-Jung FC4E cryo-ultramicrotome.
Fig. 1. (A) Light micrograph of a 1 µm thick resin section, stained with Toluidine Blue, showing organisation of placental terminal (T),
intermediate (I) and stromal (S) villi. Fetal capillaries (fc) can be seen underlying the syncytiotrophoblast (sn). Occasional
cytotrophoblasts (c) can be seen in the terminal and intermediate villi. The interstitial space (i) contains a variety of mesenchymal cells.
Bar, 100 µm. (B) Electron micrograph of a ~70 nm thick, ferrocyanide-mordanted resin section showing a placental terminal villus. The
fetal microvessel (FC), can be seen to be lined with thin unfenestrated endothelial cells (e) with numerous paracellular clefts (arrow)
(Leach and Firth, 1992). The syncytiotrophoblast (sn) contains numerous microvilli which project into the maternal blood space and the
interstitial space (i) is rich in collagen. Bar, 0.1 µm. (C) High power electron micrograph of a human placental microvascular paracellular
cleft. The cleft consists of wide zonular regions of uniform width interspersed with tight junctional regions (arrows) where the adjacent
endothelial membrane leaflets are seen in close apposition. Bar, 0.1 µm. (Fig. 1A was provided by E. D. A. Wescott.)
Cell adhesion molecules in placental microvessels 1075
Antibodies
Immunofluorescence
Antibodies against PECAM-1 (mouse clone 9G11, undiluted culture supernatant, British Biotechnology, Oxford, UK), A-CAM
(mouse ascitic fluid, dilution 1:100; Sigma, UK), pan-cadherin
(rabbit polyclonal serum, dilution 1:100, gift from Dr T. Volberg,
Weizman Institute, Israel; Geiger et al., 1990), β1 integrin chain
(mouse clone Lia 1.2, undiluted culture supernatant; Arroyo et al.,
1992), αvβ3 integrin complex (mouse clone LM609 against both
subunits, ascitic fluid, dilution 1:100; Cheresh, 1987), vinculin
(mouse ascitic fluid, dilution 1:100; Sigma, UK), α-actinin (rabbit
polyclonal serum, dilution 1:100; Sigma, UK), human cadherin-5
(both mouse clone 7B4, undiluted culture supernatant; Lampugnani et al. (1992) and clone TEA 1/31, purified IgG, 43 µg/ml,
characterised as shown below) were used.
7B4 and TEA recognised the same antigen in human cultured
endothelial cells from umbilical cord vein. Indeed (1) both 7B4
and TEA immunoprecipitated a protein of identical molecular
mass and (2) TEA could not immunoprecipitate any protein from
a cell extract previously immunodepleted with 7B4 (Fig. 2). TEA
could still immunoprecipitate the expected band from a cell extract
immunodepleted with a negative antibody (Fig. 2). Human umbilical cord vein endothelial cells were cultured, labelled and
immunoprecipitated as already described in detail (Lampugnani et
al., 1992).
The frozen cryosections were labelled by a modified indirect
immunofluorescent method (Coons et al., 1955), where the sections were immersed in 0.01 M glycine for 10 min to eliminate
free aldehyde groups, washed in PBS and incubated in normal
human serum (1:50 dilution) for 30 min to prevent non-specific
activity and block human Fc receptors which may cross-react with
the rabbit secondary antibody. The sections were then incubated
in the primary antibody for 1 h at 37°C, washed thoroughly in
PBS containing 0.1% BSA, and placed for 30 min at 37°C in
FITC-labelled rabbit anti-mouse IgG (1:100 dilution, Sigma, UK)
for the monoclonals, and FITC-labelled goat anti-rabbit IgG
(Sigma) for the rabbit anti-pan-cadherin. The frozen sections were
mounted in Mowiol 4-88 (Hoechst, Frankfurt, Germany) viewed
under a Zeiss epifluorescence microscope and photographed on
Kodak T-Max 400 films.
kDa
Immunoelectron cytochemistry
The ultrathin frozen sections of the human term placentae were
labelled by an indirect colloidal gold immunolabelling technique
(Geuze et al., 1981; Leach et al., 1989). Briefly, the sections were
transferred through drops of 0.1 M glycine (10 min) and 0.1%
BSA in PBS before incubation with normal human serum (1:50
dilution, 30 min). They were then incubated in drops of the chosen
primary antibody overnight in a humid chamber at 4°C, washed
through several drops of PBS containing 0.1 M BSA, and placed
on either 10 nm colloidal gold-conjugated goat anti-mouse IgG or
goat anti-rabbit IgG (1:25 absorbance) for 30 min. For double
immunolabelling experiments the sections were incubated with a
mixture of the primary antibodies, TEA and anti-pan-cadherin, in
similar conditions as above. The secondary antibodies were a mixture of 5 nm colloidal gold-labelled goat anti-rabbit IgG and 10
nm colloidal gold-labelled goat anti-mouse IgG. The sections were
washed throughly, post-fixed in 2% glutaraldehyde, stained by
placing on drops of basic uranyl acetate (pH 8, 10 min), followed
by 2% aqueous uranyl acetate (10 min), and taken through drops
of 1.5% methyl cellulose (Fluka, Switzerland) on ice. The sections were air dried and viewed under a Jeol 100X electron microscope. Photographs were taken at ×30 000, ×50 000, ×100 000
and ×160 000 magnifications.
Controls
For controls, sections for both methods were incubated in (1) PBS,
(2) rabbit non-immune serum (dilution 1:100, Sigma, UK) and (3)
human serum albumin (mouse Ig, dilution 1:100, Sigma, UK)
instead of the primary antibody.
RESULTS
Fig. 2. Immunoprecipitation analysis of 35S-methionine-labelled
human umbilical cord vein endothelial cells. TEA mAb
immunoprecipitated a band of 140 kDa apparent molecular mass,
as did 7B4 mAb. TEA could not recognise any specific band in a
cell extract previously immunodepleted with 7B4. NI, a mAb
against CD2 lymphocyte antigen, not expressed by EC, was used
as a negative control. The positions of marker proteins are
indicated.
Immunofluorescence studies
The paracellular clefts of fetal microvessels were found to
be immunoreactive to mAbs 7B4 and TEA, and antiPECAM-1 (Fig. 3A,B,C,D). Immunoreactivity to anti-pancadherin was localised both in cleft regions and in the
endothelium (Fig. 3E). No immunoreactivity to the antibodies against the β1 and αvβ3 integrins was seen in the
paracellular clefts, immunofluorescence being localised diffusely in the endothelium and in the abluminal interstitial
space (Fig. 3F,G).
Immunoelectron studies
Immunogold studies revealed that cadherin-5 (7B4 and
TEA antigen), pan-cadherin and PECAM-1 (Fig. 4A-F)
1076 L. Leach and others
Fig. 3. Fluorescent micrographs of frozen sections (10 µm thickness) of term human placenta showing immunolocalisation of cadherin5, PECAM-1, pan-cadherin, β1 and αvβ3 using primary antibodies at a concentration of 25-50 µg/ml and a FITC-labelled secondary
antibody. (A) Cadherin-5, localised by the mAb 7B4, can be seen as discrete lines corresponding to the paracellular clefts of a stromal
villous vessel and in clefts of smaller vessels of intermediate and terminal placental villi. (B) Capillaries within terminal placental villi
can be seen to contain cadherin-5, localised by the mAb TEA, in the paracellular cleft regions. (C,D) Vessels in stromal villi showing
localisation of PECAM-1 in the paracellular clefts and in the cytoplasm of smaller microvessels in intermediate villi. (E) Microvessels
of intermediate and terminal villi showing localisation of pan-cadherin on the luminal surface and in the cytoplasm. (F) β1 integrin can
be seen to have a diffuse localisation in the microvessels and in the surrounding interstitial layer. (G) A large stromal vessel showing
αvβ3 having a similar diffuse localisation in the endothelium and surrounding interstitial layer. (H) Control micrograph showing the
absence of staining in a stromal villous vessel that had been incubated with PBS instead of the relevant primary antibody. Bar, 100 µm.
Cell adhesion molecules in placental microvessels 1077
Fig. 4. Electron micrographs showing ultrathin frozen sections of human term placentae which had been immunoreacted with primary
antibodies against cell adhesion molecules. The antigens were subsequently localised with colloidal gold (10 nm)-conjugated secondary
antibodies. (A) Intercellular cleft showing cadherin-5 (using mAb 7B4) at wide junctional regions (arrows) of the cleft. Bar, 0.2 µm.
(B,C) Intercellular clefts at a higher magnification showing localisation of cadherin-5 within the intercellular space of wide zones and
associated on or close to the membrane leaflets; (B) using mAb TEA; bar, 0.1 µm; arrow, tight junctional region; (C) using mAb 7B4;
bar, 0.2 µm. (D) Montage of an intercellular cleft showing extensive pan-cadherin labelling of the cytoplasmic surface of wide regions.
Bar, 0.1 µm. (E) Micrograph showing PECAM-1 in the cleft region (arrow), within the cytoplasm and on the luminal surface. Bar, 0.1
µm. (F) PECAM-1 can be seen along the luminal surface of the endothelium. Bar, 0.1 µm. e, endothelium; lu, lumen of vessel.
1078 L. Leach and others
Fig. 5
Cell adhesion molecules in placental microvessels 1079
were present in or adjoining the wide junctional zones of
the paracellular clefts but not in the tight junctional regions.
Pan-cadherin and PECAM-1 were also localised in the
luminal membrane regions of the endothelium and within
the cytoplasm of the endothelium (Fig. 4E,F). A-CAM was
found only very occasionally in the wide junctions. It was
also seen in the abluminal surface and cytoplasm of the
endothelium (Fig. 5A,B).
Only the antigen localised by the mAbs TEA and 7B4
was found to be inter-endothelial cleft specific and was
localised in the extracellular space within the clefts, or on
or close to the plasma membranes of the wide zones (Fig.
4B,C). Gold labelling was found at the cytoplasmic surface
of wide zones with the antibody against pan-cadherin (Fig.
4D). Double immunolabelling with TEA and anti-pan-cadherin showed an extensive labelling with anti-pan-cadherin,
whilst the distribution of the TEA antigen was more discrete in the paracellular cleft (Fig. 5C,D). TEA antigen and
pan-cadherin were occasionally co-localised in the same
wide zones, but were mostly found in different wide zones
of the same cleft.
The cytoplasmic surface of the wide zones appeared
more electron dense. Both vinculin and a-actinin were
found associated with these regions (Fig. 5E,F); the latter
was also present near abluminal membrane regions and at
sites more distant than vinculin from the wide junctional
regions. Placentae which were immersion-fixed immediately after delivery, or which were perfused for equilibriation prior to fixation, both showed similar immunoreactivity; the latter showed less signs of ultrastructural damage.
Controls
No staining was seen when PBS or non-immune serum was
used instead of a primary antibody (Figs 3H, 5H). When
anti-human serum albumin was used, gold particles were
Fig. 5. Electron micrographs of ultrathin frozen sections of human
term placentae which had been immunoreacted with primary
antibodies against cell adhesion molecules, cytoskeletal linking
molecules and human serum albumin. The antigens were
subsequently localised with 10 nm colloidal gold-conjugated
secondary antibodies for single-labelling experiments and
localised with 5 nm and 10 nm colloidal gold-labelled secondary
antibodies for double-labelling experiments. Bars, 0.1 µm. (A) ACAM can be seen in the abluminal surface of the endothelium and
near the intercellular cleft (arrow). (B) A-CAM can be seen in the
cytoplasm of the endothelium. (C) Double-immunolabelling with
mAb TEA and anti-pan-cadherin; TEA antigen can be seen to
occupy a different wide zone (arrow) than those seen to contain
pan-cadherin. (D) Double-immunolabelling showing both TEA
antigen and pan-cadherin co-localised in the same wide zone
(arrow). Pan-cadherin can also be seen to occupy a different wide
zone. (E) Paracellular cleft showing vinculin in the cytoplasmic
surface of the wide zone. (F) α-actinin can be seen near the wide
junctional region of the cleft (arrow), close to the abluminal
membrane and associated with the luminal membrane.
(G) Albumin can be seen on the luminal surface of the
endothelium and in cytoplasmic vesicles (arrows).
(H) Micrograph of endothelium taken from placenta incubated in
non-immune serum as a control - the cleft, cytoplasm, luminal and
abluminal surfaces show no gold-labelling. A tight junctional
region (arrow) is clearly visible. e, endothelium; i, interstitial
space; lu, lumen.
seen within cytoplasmic vesicles and at the luminal surface
of the endothelium (Fig. 5G). No staining was seen associated with the intercellular clefts.
DISCUSSION
Our results are the first to show, at the ultrastructural level,
that the paracellular clefts of intact microvessels contain
cadherins, recognised by anti-pan-cadherin (Geiger et al.,
1990), anti-A-CAM/N-cadherin (Volk and Geiger, 1984)
and anti-cadherin-5 (Lampugnani et al., 1992), and also the
cell adhesion molecule PECAM-1 (Newman et al., 1990).
Immunolocalisation at the ultrastructural level established
that these molecules, seen throughout the paracellular
regions by immunofluorescence, were present in discrete
membrane microdomains in the wide junctional regions of
the clefts. These wide zones appear similar to zonulae
adherentes, both in morphological appearance and by their
association with peripheral cytoplasmic molecules such as
vinculin and α-actinin.
The integrins, immunolocalised by antibodies against the
β1 chain and the vitronectin receptor αvβ3, were not present in the endothelial clefts of human placental capillaries. The absence of αvβ3 in the clefts was to be expected,
since the αvβ3 complex has been reported to be absent in
cell-cell contacts of both umbilical vein explants and umbilical vein endothelial cell monolayers (Lampugnani et al.,
1991), thus providing a negative control. The absence of
staining seen when the antibody against the β1 chain was
used is puzzling, since two members of this family, α5β1
and α2β1, have been located by us at cell-cell contact sites
of umbilical vein cells (Lampugnani et al., 1991). The
observed negative immunoreactivity could be due to differences between endothelial cells in vitro and in intact capillaries, or to differences between endothelia in the umbilical vein and placental microvessels.
Of all the antigens localised, cadherin-5 (localised by
both mAb 7B4 and mAb TEA) appears to be cleft-specific,
whilst pan-cadherin, A-CAM and PECAM-1 were also present at the luminal surface and in the cytoplasm of the
endothelium. A-CAM showed only occasional labelling at
the intercellular junctions compared to the junctional
labelling obtained with the pan-cadherin. Salomon et al.
(1992) have reported a similar extrajunctional distribution
of A-CAM and pan-cadherin and a similar disparity in junctional labelling of the two in human umbilical cord endothelial cells. They suggest that different cadherins, coexpressed in the same endothelial cells, may undergo
differential surface distribution. A similar differentiation
may be at play in endothelia of intact capillaries. The presence of PECAM-1 in these microvascular junctions suggests that PECAM-1 may also be involved in endothelial
cell-cell adhesion in vivo, since PECAM-1 has been shown
to influence aggregation of transfected cells in vitro
(Albelda et al., 1991) and is present in intercellular junctions of human umbilical vein endothelial cells. PECAM1 is also present on surfaces of peripheral blood monocytes,
neutrophils, platelets and certain T cell subsets, suggesting
a role in adhesive events taking place during thrombosis
and wound healing. The presence of PECAM-1 on the lumi-
1080 L. Leach and others
nal membrane microdomains of the placental microvessels
was therefore to be expected. Since PECAM-1 is expressed
on the surface of endothelia and blood cells which do not
spontaneously aggregate, cell-cell adhesion mediated by
this molecule must require activation. The calcium ion
dependence of PECAM-1 cell-cell adhesion (Albelda et al.,
1991) also suggests that the interaction is a heterophilic one,
though the counter receptor is unknown.
In this study, TEA antigen was co-localised with pancadherin in the cleft regions, further enhancing the view
that this molecule does belong to the cadherin family (Lampugnani et al., 1992) but it was also seen to occupy different microdomains of the same cleft when compared to
pan-cadherin in double immunolabelling procedures. This
may be due to the greater avidity of the anti-pan-cadherin
antiserum combined with the effect of steric hindrance
during competition for the same sites, or because the placental endothelial clefts also contain other cadherins distinct from TEA antigens which are being localised by the
pan-cadherin antibody. The antigen localised with the pancadherin antibody was typically at the cytoplasmic side of
the clefts; the antibody was one directed against the conserved C-terminal cytoplasmic domain of cadherins (Geiger
et al., 1990). TEA antigen was localised in the extracellular space or associated with the plasmalemmal leaflets of
the wide zones. The monoclonal antibody TEA recognises
the extracellular sequence of the isolated transmembrane
cadherin-5 (proven by flow cytometry analysis and
immunofluorescence, which show that TEA binds and
stains all contacts of non-permeabilised endothelial cells;
M. G. Lampugnani, unpublished observations). The ultrastructural localisation in our study supports these observations.
Using cultured human umbilical vein endothelial cells,
Lampugnani et al. (1992) have shown that addition of a
monoclonal antibody to cadherin-5 increases the permeability of confluent layers. The antigen is localised at cell
boundaries only where cells are in contact, their distribution being modified by agents which enhance permeability
such as tumour necrosis factor, thrombin and elastase. The
authors also showed that cadherin-5 was restricted to the
vascular endothelial layer of the vessels in a wide range of
different tissues examined, and the antigen was not detected
in any of the epithelial type tissues tested. Other cadherins,
localised by anti-pan-cadherin, have been implicated in the
induction of adherens junctions (Salomon et al., 1992;
Takeichi, 1988). They have been shown to bind with cytoplasmic anchoring molecules, the catenins (Kemler and
Ozawa, 1989), and are associated with vinculin and αactinin to the F-actin cytoskeleton which influences cell
shape and alters permeability (Yuruker and Niggli, 1992).
The role of cadherins in regulating permeability of intact
capillaries, especially the endothelial cleft-specific cadherin-5, warrants investigation. The exclusive localisation
of cadherin-5 in the endothelial adherens-type junctions
may reflect specific functional and structural properties of
these junctions distinct from those of epithelia.
In this study we have begun to examine the molecular
architecture of the wide junctions of the placental interendothelial clefts, which appear to be similar to epithelial
adherens-type junctions in that they both possess cadherins
and associate with cytoskeletal linking molecules. These
zones differ from typical epithelial adherens junctions in
possessing the Ig superfamily molecule, PECAM-1.
PECAM-1 may not be involved directly in cell adhesion at
junctional regions, its possible function being to provide,
along with other endothelial cell adhesion molecules, an
adhesive surface for extravasation of leukocytes via paracellular pathways. The cadherins investigated in this study
may, by virtue of their known adhesive properties, their
localisation in the wide junctional regions of the placental
microvessels and their functional properties ascertained
from experiments with cell cultures (Lampugnani et al.,
1992; Salomon et al., 1992), play a role in maintaining junctional organisation and capillary permeability. These molecules may, along with other cell adhesion molecules, such
as PECAM-1 and other cadherins, be the bridging molecules postulated to be necessary to maintain the uniform
width observed for endothelial wide junctions (Silberberg,
1988). They may also be the anchoring proteins which
together with extracellular glycoproteins form the fibre
matrix postulated to be necessary for regulating capillary
permeability and integrity (Curry and Michel, 1980).
This work was funded by the Wellcome Trust. We wish to
thank Professor Richard Beard, Department of Obstetrics and
Gynaecology, St. Mary’s Hospital Medical School, London, and
the midwives of the Aleck Bourne Labour Ward, St. Mary’s Hospital, for their helpfulness and efficiency in supplying us with
freshly delivered human placentae. Fig. 1A was provided by E.
D. A. Westcott.
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(Received 23 October 1992 - Accepted 17 December 1992)