Saccharide expression on wounded endothelial cell monolayers in vitro
R. Y. BALL 1 , R. W. STODDART 2 , C. J. P. JONES 2 and M. J. MITCHINSON 1
' Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQP, UK
Department of Pathology, University of Manchester Medical School, Stopford Building, Oxford Road, Manchester MI3 9PT, UK
z
Summary
Monolayer cultures of porcine aortic endothelial
cells were used as a model of the endothelium of
large arteries. Linear wounds were produced in
such cultures by scraping and the subsequent sequence of events in nearby cells was analysed. The
earliest detectable event was cellular spreading at
the margins of the wounds (2h) followed by cell
migration (starting at 6-8 h) and cell proliferation
in regions adjacent to the wound (16 h and later).
Cell spreading was associated with the appearance
of saccharides selectively at the spreading margins
of the cells, which bound the lectins, ConA, LCA
and PSA, and were sensitive to a-mannosidase.
Terminal a-mannosyl residues were therefore present.
The appearance of these saccharides suggests a
mechanism by which monocytes might adhere to
and/or migrate through the endothelium of vessels
at sites of cellular response to injury.
Introduction
to be modified whenever cellular surfaces alter. Moreover, since their biosynthesis depends upon the integrity
of endomembrane systems, the chemical structures that
they exhibit may be particularly sensitive to alterations of
membrane turnover and flow (Stoddart, 1984). In a
study of endothelial damage in pigeons fed cholesterol,
Lewis and colleagues (1982) have obtained direct evidence for changes in the 'glycocalyx' of the endothelial
cells, especially over early lesions.
For these reasons, a study was made of the saccharides
of membrane glycoproteins of wounded porcine aortic
endothelial cell cultures. When the cultures were confluent, linear wounds were made and differences between
cells growing into the denuded area and those distant
from the injury were studied using a panel of biotinylated
lectins.
There is now abundant evidence that the lipid-laden
foam cells of human fatty streaks and advanced atherosclerotic plaques are macrophages (Schaefer, 1981; Assmann, 1982; Vedeler et al. 1984; Aqel et al. 1984, 1985;
Klurfeld, 1985; Jonasson e* a/. 1986; Gown etal. 1986).
Ultrastructural studies of diet-induced atherosclerosis in
laboratory animals have suggested that the intimal macrophage foam cells are derived from circulating monocytes
that adhere focally to the endothelium (Gerrity, 1981;
Joris et al. 1983; Faggiotto et al. 1984; Schwartz et al.
1985). This adhesion appears to occur particularly at foci
of increased endothelial cell turnover (Lewis et al. 1985;
Walker et al. 1986), perhaps as a result of desquamating,
but non-denuding, injury (Walker et al. 1986).
In vivo, monocytes adhere preferentially to nearby
viable endothelial cells after superficial denuding endothelial injury in the aorta of hypercholesterolaemic, but
not normocholesterolaemic, rabbits (Walker & Bowyer,
1984). When confluent cultures of endothelial cells are
wounded in vitro, monocytes adhere better to the cells at
the wound edge than to the confluent monolayer away
from the site of injury (de Bono & Green, 1984; Di
Corleto & de la Motte, 1985). These observations suggest
that differences in the surfaces of endothelial cells migrating into areas of injury are important in the binding of
monocytes. Clearly this might have important implications for both atherogenesis and inflammation.
Carbohydrates are major components of the outer faces
of plasmalemmata (Cook & Stoddart, 1973) and are likely
Journal of Cell Science 93, 163-172 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Key words: endothelium, regeneration, lectins, ConA,
macrophages.
Materials and methods
Preparation of cultures of porcine aortic endothelial
cells
Segments of descending thoracic aorta about 15-20 cm long
were obtained from pigs aged up to six months at slaughter (W.
J. Adkins Ltd, Cambridge). Both ends of the vessel were
clamped with artery forceps before its removal from the carcase.
The aorta was then conveyed to the laboratory, its exterior
moistened by medium (Medium 1) composed of Hanks'
balanced salts solution (HBSS: Flow Laboratories, Irvine,
Scotland) containing: penicillin (100i.ii.ml~ 1 ), streptomycin
sulphate (100/igmP 1 ), chloramphenicol (5^gml - 1 ) and
amphotericin B (lO^gmP 1 ). The branches of the aorta were
163
then ligated, plastic cannulae were tied into both its ends and
the vessel was perfused with sufficient Medium 1 to remove
blood and clots. Approximately 15-20 ml of crude collagenase
(type IA: Sigma Chemical Co., Poole, Dorset) in Medium 1
( 0 2 % , w/v) were then instilled into the lumen, the cannulae
were plugged and the aorta was incubated at 37°C for 30min.
The contents of the aorta were then drained into a 50 ml
centrifuge tube containing 10 ml of culture medium (Medium
2): 10% (v/v) foetal calf serum in Dulbecco's modification of
Eagle's medium containing sodium bicarbonate (3-7mgml~ ),
2mM-L-glutamine, and antibacterial and antifungal agents as
already described. The aortic lumen was then perfused with a
further 20-25 ml of Medium 1 in small samples to recover as
many endothelial cells as possible. During perfusion, the aortic
wall was gently massaged and the vessel rotated slowly. The
endothelial cell suspension was centrifuged at up to 115g-for
lOmin, the pellet was resuspended at room temperature in 5 ml
of Medium 2, transferred to a tissue culture flask (25 cm2 Falcon
Plastics) and incubated at 37°C in an atmosphere of 5 %
CO2/95% air (v/v) for 5-6 h. The culture was then washed
with three 5-ml samples of HBSS. Five ml of fresh Medium 2
were then added and the culture was returned to the incubator.
The medium was changed the next morning and thereafter at
intervals of 2-3 days.
Confluent cultures were subcultured. They were flooded
with trypsin-EDTA (Flow laboratories) and the excess was
immediately removed. The cultures were then maintained at
room temperature until the cells detached (usually within
5-10 min). The cells were resuspended in an appropriate
volume of culture medium and subcultured at a split ratio of
1:3. The medium was changed next day. For studies of cellular
morphology, lectin binding and rate of wound closure, the cells
from the second to sixth subcultures were maintained on
22 mm X 22 mm glass coverslips in tissue culture dishes
(35 mmX 10 mm: Falcon Plastics) containing 1 ml of Medium 2.
Linear wounds were made in confluent cultures by lightly
drawing the tip of a plastic cell scraper, 15 mm wide, across the
centre of the coverslip. The medium was changed immediately
after wounding.
Measurements of luound closure
Measurements of the width of the wound were made on living
cultures using an inverted phase-contrast microscope and stage
micrometer. Several measurements were made at arbitrarily
selected locations from each of several coverslips in each
experiment. Since the observations from one experiment were
not significantly different from those of another, all the results
were pooled. Measurements were made using the same cultures
at selected times up to 36 h after injury.
Preparation of cultures for lectin-binding studies
At selected times (2-48 h) after wounding, cultures were
washed three times with 1-ml samples of 0-01 M-phosphatebuffered saline (PBS), pH7-6, air-dried, freeze-dried overnight
(Freezemobile 6 freeze-drier: Virtis, Gardiner, NY, USA) and
then fixed for 30 min in 10% phosphate-buffered formol-saline
(PBFS; 40% aqueous formaldehyde: PBS (1:9, v/v)). The
preparations were then air-dried and wrapped in foil until the
time for lectin-binding studies.
Staining with biotinylated lectins
All biotinylated lectins and the avidin-peroxidase conjugate
were obtained from the Sigma Chemical Co. Coverslips bearing
wounded cultures were affixed to glass microscope slides with
XAM mountant (Gurr, BDH, Poole, Dorset) and allowed to
dry at 37°C overnight. Endogenous peroxidase activity was then
blocked with a reagent consisting of 0-4% (v/v, from concentrated) HC1 and 0-5% (v/v, from 100 vol.) H 2 O 2 in absolute
methanol, for 30 min. After washing in distilled water, followed
by 0-05M-Tns-buffered saline, pH7-6 (TBS), the cultures
were treated for 30 min with biotinylated lectin at lO^gml" 1 in
0-05M-TBS supplemented with 1 mM-CaCI2. The Tris buffer
had the following composition: Tris base (60-57gl~'), NaCl
(81gl~') in glass-distilled water, with the pH adjusted to 7-6
with concentrated HC1; this yielded a stock solution of 0 5 M
with respect to Tris; dilution of 1vol. with 9vols of glassdistilled water gave 0-05 M-TBS; the pH remained at 7 6 . The
low concentration of the biotinylated lectin was used to avoid
problems of precipitation found at higher concentrations.
The panel of lectins used and their specificities are shown in
Table 1. After washing in 0-05 M-TBS + 1 mM-CaCl2, the cultures were incubated with avidin-peroxidase at 2/Jgm\~l in
0-125 M-TBS containing 0-347 M-NaCl for 45 min. They were
then washed in 005 M-TBS and the sites of lectin binding were
revealed by exposure of the cultures for 5 min to a reagent
consisting of p-dimethyl aminobenzidene (0-05%, v/v) and
H 2 O 2 (0-015%, v/v, from 100 vols) in 0-05 M-TBS. The gold
sulphide-silver intensification procedure of Newman and colleagues (1983) was then applied and, after washing in 1 % (v/v)
Table 1. Lectins used, their specificities and competing sugars
Label
B
F B
F
F B
B
B
F
ConA
PSA
LCA
ePHA
1PHA
WGA
PWM
SB A
DBA
HPA
AHA
VVA
MPA
BSA
F B
LTA
F
F
F
F
F
F
B
B
B
B
B
B
Acronym
Source
Canavalia ensifonnis
Pisuin satwum j
Lens culinaris J
Phaseolus vulgaris
Phaseolus vulgaris
Triticum vulgaris
Phytolacca americana
Glycine max
Dolichos biflorus
Helix pomatia
Arachis hypogaea
Vicia villosa (B4 isolectin)
Madura pomifera
Criffoma simplicifolia
(B4 form of isolectin 1)
Tetragonolobus purpureus
Major saccharide specificity
R. Y. Ball et al.
Final molarity of
competing sugar
Q'-D-mannopyranose/a'-D-glucopyranose
Similar, but not identical, to ConA
See text for details
Bisected 2/3 antennary, N-linked
Non-bisected 3/4 antennary, N-linked
Di-A'-acetyl chitobiose/some sialyl groups
Di-A'-acetyl chitobiose
ff-D-A'-acetyl galactosamine
cr-D-A'-acetyl galactosamine
£V-D-Ar-acetyl galactosamine
ff-D-galactosyl l,3-A'-acetyl galactosamine
a'-D-A'-acetyl galactosaminyl 1,3-galactose
Q'-D-galactose
Q'-D-galactose
a'-Methyl-D-mannopyranoside
a'-Methyl-D-mannopyranoside
a'-Methyl-D-mannopyranoside
N/A
N/A
Tri. A'-acetyl chitotriose
N/A
A'-acetyl-D-galactosamine
A'-acetyl-D-galactosamine
A'-acetyl-D-galactosamine
D-galactose
A'-acetyl-D-galactosamine
D-galactose
D-galactose
100 mM
100 mM
100 mM
N/A
N/A
0-5 mM
N/A
10 mM
10 mM
10 mM
100 mM
10 mM
10 mM
10 mM
a--L-fucose
L-fucose
100 mM
B, biotinylated lectin; F, fluorochrome-labelled lectin; N / A , not applicable.
164
Co:'mpeting sugar
acetic acid, the preparations were dehydrated, cleared and
mounted in XAM.
Staining with fluorochrome-labelled lectins
Fluorochrome-labelled lectins (Table 1) were obtained from the
Sigma Chemical Co. HPA was used in a rhodamine isothiocyanate conjugate; all the others conjugated with fluorescein isothiocyanate. In order to test for the competitive binding of lectins,
coverslip cultures were attached to slides and subjected to the
blockade of endogenous peroxidase, as above. After washing in
distilled water, they were stained with standing drops of
fluorochrome-labelled lectins at 500/igml~ for 5 min. The
high concentration was to allow maximum opportunity to detect
competition. The excess of lectin was washed away in a stream
of running tap water, followed by two washes in distilled water,
and each culture was wet-mounted with a drop of distilled water
and a coverslip. The cultures were then examined on a
fluorescence microscope to check both for the binding of each
lectin and its pattern. Control cultures, untreated with lectins,
showed no autofluorescence.
The coverslips were then floated off under distilled water and
the cultures were treated with biotinylated ConA at 10/igml""1,
revealed, intensified and mounted, a9 above.
Controls of lectin binding
Where possible, controls of lectin binding were made using the
appropriate competing sugars (Table 1), with the sugar added
simultaneously with the lectin. Further controls of the staining
procedures were made, variously, by omitting lectin, avidin—
peroxidase, />-dimethyl aminobenzidene, H2O2 or the intensification procedure.
The use of a'-mannosidase formed a further, rigorous control
(below).
a-Mannosidase treatment
Digestion with a'-mannosidase from Jack bean (Sigma Chemical Co.) was performed on some cultures prior to staining. The
enzyme was applied, as a large standing drop, at 0'18 unit ml"
in 0-1 M-sodium citrate buffer (pH4-5) for l h at 25CC, in a
humidity chamber. After digestion, the enzyme was recovered
from each specimen separately and tested for activity in the
standard assay (below). The enzyme-treated cultures were
washed with water and 0-05M-TBS (pH7-6) and then used for
the binding of biotinylated lectins, as above.
Before use, the preparation of a'-mannosidase was checked
for the presence of o--glucosidase, /3-galactosidase, /5-Ar-acetyl
hexosaminidase and O'-L-fucosidase, as well as a'-mannosidase,
using appropriate nitrophenyl glycosides (Colowick & Kaplan,
1972) and standard spectrophotometric assays at 410 nm.
Briefly, o-nitrophenyl /3-galactoside was used for assaying for
/8-galactosidase, and the appropriate ^-nitrophenyl glycosides
for all other assays. Substrates were used at final concentrations
of 2-27 niM for /J-galactosidase, 0-5 mM for a"-glucosidase,
0'35mM for a'-L-fucosidase, and 0-33 mM for a'-mannosidase
and /S-N-acetyl hexosaminidase activities; all were in excess.
The a'-mannosidase preparation was assayed at 2-S6X10" units
of mannosidase per ml of incubation solution. Each incubation
was of 3 ml final volume. The conditions used were as follows:
for a--glucosidase, 0-lM-sodium phosphate buffer (pH6-8) at
37°C; for a'-mannosidase, 0-1 M-sodium citrate buffer (pH4-5)
at 25 °C; for a-L-fucosidase, 0-2 M-sodium citrate buffer
(pH 6-5, containing a trace of bovine serum albumin) at 25°C;
for /3-galactosidase and /3-iV-acetyl hexosaminidase, 0-1Msodium phosphate buffer (pH7-3, containing 1-lmM-magnesium chloride and lOmM-mercaptoethanol) at 37°C.
The a'-mannosidase was also tested for other glycosidase
activities expressed in citrate buffer (pH4-5) at 25°C as above.
Likewise, the a'-mannosidase activity remaining on the slides
after the digestions was assayed. The preparation of a'-mannosidase was free of detectable proteolytic activity and of concanavalin A (ConA).
Microscopy
Specimens stained by the peroxidase-based procedure were
examined on a Leitz Laborlux 12 microscope with a light blue
filter (Agfa correction filter, daylight to artificial) at magnifications of up to X400. Photography was carried out on a Nikon
Optiphot microscope, using an F3-35A camera and UFX-II
exposure system together with blue and green filters and Kodak
Technical Pan (2415) film.
Specimens stained with fluorochrome-labelled lectins were
examined on a Vickers Photoplan microscope, using epiillumination and the primary and barrier filters appropriate for
use with fluorescein and rhodamine, respectively, at a magnification of X200. Photography was not undertaken.
Autoradiography
Autoradiography was undertaken to determine the uptake of
tritiated thymidine. Four sets of cultures were incubated in each
4-h period after wounding, 0-4 h, 4-8 h, 8-12 h, 16-20 h,
24-28 h and 36-40 h, in Medium 2 containing [6-3H]thymidine
(1 or2/iCiml~ : Amersham International, Amersham, Bucks).
The medium was changed at the start of each 4-h period. After
the incubation with tritiated thymidine, the cultures were
extensively washed with PBS and then fixed with 5 % (v/v)
PBFS. The preparations were then washed with several
changes of distilled water and prepared for autoradiography.
The emulsion (K5; Ilford, Mobberley, Cheshire) was exposed
for 48 h before developing. Preparations were counterstained
with either haematoxylin and eosin or with the
May-Grunwald-Geimsa stain. Autoradiographs were scored in
fields 0-23 mm wide X 0-46mm long using an E lla eyepiece
graticule (Graticules Ltd, Tonbridge, Kent), fitted to a microscope with X10 eyepiece and X25 objective lenses. The fields
were measured from the leading edges of cells growing into the
wound. The field was then moved sideways (90° to the wound)
so that the adjacent area was counted and so on until six
contiguous fields, extending to 1-38 mm from the wound edge,
were scored. Random areas, many millimeters from the wound,
were also scored to provide an indication of the labelling index
in the confluent part of the culture distant from the injury. For
each culture, 12 measured strips and numerous distant fields
were counted. Results from the four determinations for each
time period were not significantly different one from another
and were therefore pooled. The results are expressed as a
labelling index ratio, which relates the labelling index in the
defined field to the labelling index in the confluent culture
distant from the injury.
Results
Rate of mound closure
Careful use of the cell scraper produced a denuding
wound to the endothelial cell culture the length of the
coverslip and having parallel sides about 1-5 mm apart
(Fig. 1). There was usually a clean separation of cells at
the edges, but occasionally focal compression artefacts
were produced. Within 2h of wounding, cells at the
wound edge had produced short pseudopodia, some
narrow, others broad and fan-shaped. By 4h after
Endothelial cell sacchatides
165
12
7
1-6-
fm
24
36
6
81 46
5
2 0-8-
60
425
3-
0-4
0
4
8
12
16
20
24
28
Time after wounding (h)
32
36
Fig. 1. Width of wound in endothelial cell monolayer (in
mm) plotted against time after wounding (in h). The results
are expressed as mean ± S.D. and are pooled from several
experiments. The number of observations at each time is
indicated.
wounding these changes had become more obvious and
the cells appeared to be aligning themselves with their
long axes at 90° to the wound. After 6-8 h there was
definite migration of cells into the denuded area with a
slight reduction in its width (Fig. 1). Migration of cells
into the wound continued steadily over the next day or so
and by 36 h after injury, the wound was only about 40 %
of its original width (Fig. 1). While the cells were
migrating into the wound, they maintained their transverse orientation. The cells were relatively thin and
elongated and separated by clear gaps; this contrasted
with nearby quiescent cell9, which were usually large,
polygonal and touching one another.
Au toradiography
Labelling cells in the S phase of the cell cycle with
tritiated thymidine (l-2juCiml~') for 4h produced a
heavy nuclear staining after autoradiography. There was
no difficulty in distinguishing labelled from unlabelled
cells. The background labelling index in parts of the
confluent culture distant from the wound was fairly
similar at all times (4-38 ±1-99%, mean ± S.D., 24
observations).
For at least the first 12 h after injury, cells in the
vicinity of the wound showed no increase in the absolute
level of tritiated thymidine labelling and the labelling
index did not rise above that of distant confluent areas
(Fig. 2). By 16-20h, however, cells in the regenerating
epithelium at the wound edge had a labelling index more
than twice that of confluent areas and by 24-28 h the
labelling index ratio in the regenerating epithelium was
5-61 ±0-92 (Fig. 2). During the latter time interval,
about one quarter of the cells within the first 0-23 mm
strip of cells growing into the wound was in DNA
synthesis. Labelled cells were scattered among the cells
growing into the wound; there was no obvious pattern to
their distribution within this regenerating endothelium.
Increased labelling was confined to the regenerating
epithelium. Cells in fields that did not include regenerating cells (e.g. more than 0-46 mm from the wound edge at
24-48 h) failed to show a significant increase in the
tritiated thymidine labelling index ratio (Fig. 2) and the
166
17
R.Y. Ball et al.
Uiil
B
D
Fig. 2. The [ 3 H]thymidine labelling index ratio of cultured
endothelial cells after injury. Labelling was carried out at
0-4h (A), 4-8h (B), 8-12h (C), 16-20h (D), 24-28h (E)
and 36-40 h (F) after wounding. At each time six adjacent
fields of cells, each 0-23 mm wide, were assessed. The field at
the wound edge is indicated (e). The results are expressed as
mean ± S.D. with four observations for each. The
approximate extent of the regenerated endothelium is
indicated at the top of the figure at 8, 12, 17, 24 and 36 h
after injury. Each bar is in proportion to the width of
regenerated endothelium and the values derived are 0-08,
0-20, 0'30, 0-33 and 0-45 mm, respectively, at the specified
time intervals. At 16-20 h (D) the regenerated endothelium
was slightly wider than the field counted and at 36-40 h (F) it
was about the same width as the first two fields assessed.
absolute level of label did not rise above that at the time of
wounding.
Lectin binding
All of the lectins used showed some degree of staining of
the cell bodies, though to markedly differing extents
(Table 2) and with varying patterns. No inconsistencies
were found where the same lectin was used both as
biotinylated and fluorochrome-labelled conjugates and
the controls with competing sugars all showed substantial
or total suppression of staining.
Staining at the cellular margins towards the wound was
much more restricted and was seen only with ConA,
LCA, PWM and, slightly, with WGA. The staining was
evident by 2h from injury (Fig. 3) and was much more
marked by 4h (Fig. 4). Thereafter, a slight separation of
cells further away from the wound became evident and
this was accompanied by an increasing binding of ConA
to their edges. No other lectins, save LCA, PWM and
WGA, showed any such effect (Fig. 5). Twelve hours
after wounding, some cells had moved clear of their
neighbours and were advancing into the wound; several
of these showed distinct polarity. In such cases ConA
showed a stronger staining towards the advancing edge of
the cell (Fig. 6), while a few lectins (Table 2) gave a more
intense staining of the trailing edge.
The staining with ConA at the cellular margins was
very labile to a'-mannosidase, which abolished it completely. The enzyme contained no other detectable glycosidases or proteolytic activity, no evidence of ConA
Table 2. Binding of lectins to various areas of
regenerating endothelial cells
Table 3. Interference of binding of ConA to
regenerating endothelial cells by various other lectins
Competition with ConA
Staining of cells
Lectin
Wound edge
Cell body
Trailing edge
ConA
PSA
LCA
ePHA
1PHA
WGA
PWM
SBA
DBA
HPA
AHA
VVA
MPA
BSA
LTA
2
—
1
-
3
1
2
2
2
+/-
1
2*
1
2.
1
1
I
1
—
—
1
1
1
2
+/-
+/1
-
+/-
i
1
+/+/1
1
—
—
—, no stain; + / —, possible faint stain; 1, weak definite stain;
2, moderate stain; 3, strong stain.
•This staining was resistant to, and intensified after, treatment
with a'-mannosidase.
contamination, after exhaustive studies in other tissues,
and remained active after treatment of the sections. The
staining of the cell body with ConA was much less
susceptible to a'-mannosidase and was only slightly
reduced.
In the competition studies, several lectins showed
some degree of interference with the binding of ConA to
the cell bodies, though none wholly suppressed it,
whereas at the cellular margins only PWM showed
competition; LCA and WGA, notably, did not (Table 3).
Discussion
The response of endothelial cells at the edges of the
Competing lectin
PSA
LCA
ePHA
1PHA
WGA
PWM
DBA
HPA
AHA
BSA
LTA
Cell body
+
+
+
+/+
+
+
Wound edge
+
+
+ , strong competition, i.e. staining with ConA is lessened; +/—,
weak competition, i.e. staining with ConA is slightly lessened; —, no
observable change in ConA staining.
injuries was rapid. Within 2—4 h the cells were beginning
to spread and by 6-8 h there was migration. DNA
synthesis occurred by 16-20 h, and reached a maximum
after 1 day. Similar observations have been made on
wounded human and bovine endothelial cultures (Sholley et al. 1977; Schwartz et al. 1980). Substantial
repopulation of narrow wounds in cultures of human
endothelium can occur even in the absence of cellular
DNA synthesis (Sholley et al. 1977). Although the
wounds in the present study were comparatively wide,
the time course of the cellular changes resembled quite
closely that of endothelial cells adjoining a narrow
superficial aortic injury in the rabbit in vivo (Ramsay et
al. 1982).
The changes in lectin binding occurred early and
paralleled the cytological manifestations of cellular migration. Binding of ConA to the leading edges of polar
cells and margins of spreading cells was seen 2-4 h after
Fig. 3. Endothelial wound edge
2h after injury. The endothelial
cells are beginning to spread
into the wound and their
margins stain moderately with
biotinylated ConA. Nearby cells
show intense staining of their
bodies with biotinylated ConA.
X400.
Endothelial cell saccharides
167
Fig. 4. Endothelial wound edge
4h after injury. There is further
spreading and some separation
of the endothelial cells, which
show conspicuous binding of
biotinylated ConA to their
leading edges. X400.
injury, occurring as the cells were beginning to migrate
and spread, but hours before increased DNA synthesis.
The binding of ConA to the cell margins suggests the
presence of cr-D-mannopyranosyl or (r-D-glucopyranosyl
residues (Goldstein & Hayes, 1978) either at non-reducing terminals of oligosaccharides or in a 1,2 linkage at
accessible internal sites (Goldstein et al. 1967, 1974;
Goldstein & Hayes, 1978; Kornfeld et al. 1971; Kornfeld
& Ferris, 1975). It is also likely that if mannosyl residues
were involved, they constituted parts of N-linked oligosaccharides of glycoproteins (Kornfeld & Kornfeld,
1980; Stoddart, 1984). This is also consistent with the
observed binding of LCA, PWM and WGA at the cellular
margins, though ePHA and 1PHA, both of which bind to
subsets of complex N-linked sequences (Cummings &
Kornfeld, 1982; Hammarstrom et al. 1982; Yamashitaef
al. 1983), showed no significant staining at the edges of
the wound. The absence of staining with PSA is a little
surprising, since it is considered to have properties very
like those of LCA (Trowbridge, 1974; Debray et al.
1981; Debray & Montreuil, 1983), but biochemical
differences in their properties are known (Kornfeld et al.
1981).
The staining of cellular margins with WGA was not
affected by pretreatment with neuraminidase, implying
that it was binding not to sialyl residues, but possibly
with the di-jV-acetyl chitobiosyl sequences of N-linked
oligosaccharides. PWM has a requirement for a closely
Fig. 5. Endothelial wound edge
12 h after injury. The
preparation has been stained
with biotinylated ePHA. There
is moderately intense staining of
the cell body but none of the
edge of the cell migrating into
the wound. X648.
168
R.Y. Ball et al.
6
related, but not identical structure (Yokoyama et al.
1978; Katagiri et al. 1983). Hence the weak competition
seen between PWM and ConA is not inconsistent with
the absence of detectable competition between WGA and
ConA.
The rather weak binding of LCA to the cellular
margins and its failure to compete with ConA suggest
interaction with a saccharide for which it had a much
lower affinity constant than did ConA. In view of the
structural requirements for the binding of each lectin
(Goldstein & Hayes, 1978; Kornfeld et al. 1981), this
would be consistent with the interacting saccharide being
of the high mannose type.
The binding of ConA to cellular margins was extremely susceptible to cr-mannosidase, an exo-glycosidase
(Li & Li, 1972). This showed that tf-D-mannopyranosyl
residues were present at non-reducing terminals and that
the sites of interaction with ConA were either all at such
terminals, or were both terminal and at internal sites that
became labile as the terminal residues were removed.
Only structures of the high mannose, complex and
intermediate types have Q'-D-mannopyranosyl residues at
their non-reducing terminals and the latter two categories
have some internal residues of this sugar, which are
substituted by ^-D-2-deoxy, 2-acetamido glucopyranosyl
groups, or oligosaccharides on the basis of this (Kornfeld
& Kornfeld, 1980; Stoddart, 1984). These would be
refractory to cv-mannosidase. It is unlikely, therefore,
that their binding of ConA would be so reduced by pretreatment with cr-mannosidase as was seen here. Likewise, partially glucosylated high mannose structures, or
phosphorylated forms of the 'lysosomal' type (Kornfeld &
Kornfeld, 1980; Stoddart, 1984) would be incompletely
susceptible to cr-mannosidase. It is therefore likely that
the oligosaccharides expressed at the margins of the
spreading endothelial cells were of the simple high
mannose type and it is certain that they possessed a-D-
Fig. 6. Endothelial wound edge
12 h after injury. There is
strong staining by biotinylated
ConA of the advancing edge of
the endothelial cell migrating
into the wound. X648.
mannopyranosyl residues at some or all of their nonreducing terminals.
At all times, the patterns of lectin binding to the cell
bodies differed from those found at the cell margins,
suggesting the presence of different categories of
N-linked oligosaccharides and of mucin types in lesser
abundance. Competition studies gave no evidence that
any of these saccharides were in close association with the
ConA-binding saccharides of the cellular margins,
though competition was found with ConA-binding sites
on the cell bodies. These sites differed from those at the
margins, being only partially susceptible to a'-mannosidase. They probably represented a heterogeneous population of high mannose, complex and possibly, phosphorylated structures. Some of them were associated
with granules and vesicles inside the cells.
Electron microscopic studies (unpublished) have
shown that the binding of ConA at the cellular margins is
at both upper and lower cell surfaces, with some staining
of elements of endomembranes near the spreading edge.
This may mean that the expression of a'-mannosidasesensitive ConA-binding saccharides at the cell surface is a
consequence of the recruitment of components of internal
membranes into the plasmalemma, which occurs during
cellular spreading (Singer & Kupfer, 1986). It could also
reflect a change in the relative rates of membrane flow,
processing reactions and glycosyl transfer. Whatever the
mechanisms involved, the Con-A recognition sites of
these saccharides obviously persist for only short periods,
because the cell membrane moves backwards during
locomotion (Singer & Kupfer, 1986), and yet the saccharides concerned were only demonstrable near the leading
edge.
The proportion of cells synthesizing DNA in the
confluent part of the endothelial cell culture in vitro
distant from the wound was high compared with the
overall proportion in aortic endothelium in situ in various
Endothelial cell sacchaiides
169
species (Florentin et al. 1969a; Payling Wright, 1972;
Caplan & Schwartz, 1973; Schwartz & Benditt, 1976,
1977; Reidy & Schwartz, 1983). However, very high
labelling indices may occur focally in the normal aortic
endothelium in vivo (Schwartz & Benditt, 1976) and
enhanced endothelial cell turnover occurs in various
pathological states (Florentin et al. 19696; Payling
Wright, 1972; Schwartz & Benditt, 1977; Reidy &
Schwartz, 1981, 1983; Walker et al. 1986). The background level of DNA synthesis in these experiments was
therefore no greater than that sometimes found in vivo.
Wounding the cultures increased the thymidine labelling
of cells migrating into the wound, but not of those more
distant, in keeping with the suggestion that cellular
migration is a trigger to DNA synthesis (Schwartz et al.
1980).
The alteration of saccharides on migrating or spreading
endothelial cells might have important implications for
the adhesion of monocytes to vascular endothelium in
vivo. Macrophages express receptors for cr-D-mannosyl
and tt-L-fucosyl residues (Sharon, 1984; Largent et al.
1984; Mokoena & Gordon, 1985), but blood-borne
monocytes appear to lack these (Mokoena & Gordon,
1985). Kataoka & Tavassoli (1985) found that monocytes
cultured in wells coated with a microexudate only developed mannosyl receptors after 4-7 days. This may not,
however, necessarily mean that circulating monocytes
never acquire such receptors, but that when they do they
attach to endothelial surfaces and emigrate. cv-Mannosyl
receptors might develop as a result of physiological
exposure to mediators of monocyte activation, or could
be age-dependent.
Monocyte emigration occurs in the microvasculature at
sites of remodelling or injury and in large vessels at sites
of endothelial injury. Macrophages appear to get trapped
in the intima of large arteries and therefore accumulate in
early atheromatous plaques (Faggiotto et al. 1984;
Schwartz et al. 1985; Aqel et al. 1985). Several mechanisms have been postulated for the emigration of monocytes in atherosclerosis. One view is that it results from
release of chemoattractants from the vessel wall (e.g. see
Gerrity et al. 1985), but it is at least equally likely that
initial adhesion is mediated by molecules on the surface of
endothelial cells. If such molecules were initially represented equally on the luminal and abluminal aspects of
the cells, but some of those on the luminal aspect were
masked or removed by blood flow, a gradient might be
created that would promote emigration. Among candidate molecules for such a mechanism are the saccharides
studied here. The location of a"-mannosyl residues at the
margins of endothelial cells makes them particularly
attractive as components of this putative emigratory
mechanism, because monocytes seem to adhere preferentially near the edges of endothelial cells in culture
(Pawlowski et al. 1988). Terminal a'-L-fucosyl residues,
widely expressed on endothelial cells (Holthofer et al.
1982; Moller & Lennert, 1984; Walker, 1985; Little ef al.
1986; Hultberg et al. 1988) do not show this characteristic marginal distribution.
Cell-cell interactions involving saccharides and lectinlike molecules are phylogenetically widespread (Brandley
170
R. Y. Ball et al.
& Schnaar, 1986) and the attachment of monocytes to
endothelium is likely to involve some such conserved
mechanisms. The selective expression of terminal amannosyl residues at spreading endothelial margins
suggests such a possibility. A recent study has shown that
neutrophil adhesion to venules is inhibited by a mannose
6-phosphate-rich yeast polysaccharide (Lewinsohn et al.
1987). It has not been established whether adhesion of
monocytes to endothelial cells near sites of injury is
inhibited by blockade of this putative mechanism. Such
experiments may be relevant to the development of
atherosclerosis and may help to clarify the basic mechanisms of leucocyte emigration from blood vessels.
R.Y.B. was supported by the Wellcome Trust. The work was
also supported in part by the British Heart Foundation and East
Anglian Regional Health Authority. Dr B. M. Herbertson
provided valuable advice on autoradiography. We gratefully
acknowledge the assistance of Jacqui H. Enright.
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{Received 15 November 1988 - Accepted 25 Januar\< 1989)