Table Of Contentjf. Cell Sci. 76, 1-16 (1985)
Printed in Great Britain © The Company of Biologists Limited 1985
EXTRACELLULAR MATRIX INTERACTIONS WITH
THE APICAL SURFACE OF VASCULAR ENDOTHELIAL
CELLS
RANDALL H. KRAMER
Department of Anatomy and Division of Oral Biology, Schools of Dentistry and
Medicine, University of California, San Francisco, California 94143, U.SA.
SUMMARY
Cultured aortic endothelial cells, like their in vivo counterparts, form highly organized, confluent
monolayers of polarized epithelioid cells that secrete, exclusively at their basal surface, an
extracellular matrix to which they then attach. The influence of isolated subendothelial matrix
preparations on cell polarity and monolayer organization was studied by presenting fragments of the
matrix to confluent bovine aortic endothelial cell cultures. The matrix particles were immediately
bound to the apical aspect of the cell monolayer and induced rapid reorganization of the monolayer
into cells with a fibroblastoid morphology. To determine if fibronectin, the major glycoprotein of
the subendothelial matrix, could be involved in the observed apical cell surface - matrix interactions,
latex beads or small discs of Nucleopore filters were coated with the glycoprotein and presented to
confluent monolayers. In a fashion similar to that observed with matrix fragments, materials coated
with fibronectin caused focal reorganization of the cell layer. After contact with the coated beads,
the underlying endothelial cells flowed upward and spread over the entire bead, forming a canopy
of confluent cells that draped the particle. Contact of confluent monolayers with the coated filters
induced similar behaviour, except that monolayer reorganization into the fibroblastoid phenotype
was followed by emigration of the majority of underlying cells through the pores to the upper filter
surface, where they formed a new organized cell monolayer with the typical endothelial cell mor-
phology. Thus contact of the apical surface of endothelial cells with structures to which they adhere
initiates a rapid disruption of the organized cell monolayer, followed immediately by a concerted
effort of the local population to re-eatablish both cell polarity and monolayer contiguity. The
expression of this behaviour may be important during tissue remodelling that occurs in neo-
vascularization and during interactions with thromboemboli.
INTRODUCTION
The maintenance of cell polarity and contiguity by epithelia, endothelia and
mesothelia is behaviour crucial for the proper expression of differentiated functions.
It makes possible, for example, the unidirectional transport of solutes by epithelia and
the non-thrombogenic nature of vascular endothelium. While little is currently known
about the factors that regulate cell polarity, an obvious possibility is that environ-
mental signals help determine and maintain proper orientation of individual cells in
a given tissue. Such signals would include cell interactions with other cells and with
surrounding extracellular matrix and fluids.
Endothelia line all blood vessels and express unique cellular properties that endow
Key words: extracellular matrix, endothelial cell, cell polarity.
2 R. H. Kramer
the vasculature with a normally non-adhesive, non-thrombogenic and semi-
permeable surface. Most endothelia elaborate some form of extracellular matrix,
which is deposited exclusively at their basal surface and to which they strongly adhere.
Matrix secretion is thus a polarized event, and the pathogenic consequences of
intravascular matrix deposition are obvious, particularly in the light of the known
platelet-binding properties (thrombogenicity) of this material (Baumgartner,
Stemerman & Spaet, 1971). How does the apical (luminal) surface of the vascular
endothelial cell differ from the basal (abluminal) surface, and if differences exist, how
are they maintained?
This study investigated the adhesive properties of the apical cell surface of cultured
aortic endothelial cells. It was found that while the apical surface is normally quies-
cent, it can, under the appropriate conditions, behave as an actively adhesive surface.
Furthermore, contact of the apical surface with exogenously added extracellular
matrix or with artificial surfaces coated with a matrix protein induces a total disor-
ganization of the polarized cell layer, resulting in loss of the typical endothelial mor-
phology. The perturbed cells respond quickly to re-establish both their original
cell—cell interactions and an apical surface clear of adhesive structures.
MATERIALS AND METHODS
Cell culture
Calf bovine aortic endothelial (BAE) cells, obtained from Dr D. Gospodarowicz, University of
California, San Francisco, were passaged every 7 days at confluence, using 0-25 % trypsin in Ca2+-
and Mg2+-free phosphate-buffered saline (PBS) containing 2mM-EDTA. Dissociated cells were
plated on fibronectin-coated dishes at a split ratio of not more than 1 : 5 and cultured in the absence
of fibroblast growth factor in Dulbecco's Minimal Essential Medium (GIBCO, New York, NY)
supplemented with 10% calf serum (Sterile Systems, Logan, UT). The cloned cells have been fully
characterized and are positive for factor VHI-associated antigen (Gospodarowicz, Greenburg,
Foidart & Savion, 1981; Gospodarowicz, Moran & Braun, 1977; Gospodarowicz et al. 1980).
Fibronectin coating of culture dishes was performed by incubating a solution of purified fibronectin
in PBS at 100 /ig/ml for 1 h at 22°C. The dishes were then washed extensively with PBS. Fibronec-
tin was isolated from bovine or human plasma by elution from gelatin-Sepharose as previously
described by Ruoslahti, Hayman, Pierschbacher & Engvall (1982).
Preparation of isolated subendothelial matrix
BAE cells were seeded onto bovine fibronectin-coated 10 cm culture dishes at near-confluent
densities (5 X 10* cells/dish). After 24 h the medium was changed and ascorbic acid was added daily
at 50 ^g/ml. Culture medium was replaced every 3 days. After 7-10 days the subendothelial matrix
was isolated free of cells as described previously (Kramer, Gonzalez & Nicolson, 1980; Kramer &
Nicolson, 1979; Kramer, Vogel & Nicolson, 1982) but with slight modifications. Briefly, the cell
cultures were rinsed twice with PBS and twice with hypotonic buffer containing 10 mM-
TrisHCl (pH7-S), 0-05mM-CaCl 0-01% bovine serum albumin (BSA), and 0-lmM-
2>
phenylmethylsulphonyl fluoride at 37 °C. The dishes were incubated at 37 °C for 8—10 min with the
hypotonic buffer, after which the buffer was replaced with fresh hypotonic buffer containing 0-5 %
NP-40 non-ionic detergent. The dishes were rinsed once more with the detergent solution and then
washed five times with 10% calf serum in PBS. The resulting cell-free matrix was then removed
in large sheets by gently scraping the dish with a sterile rubber policeman. The matrices were
collected by centrifugation at 50^ for 10 min, then suspended in PBS containing 0-1 % BSA and
homogenized in a loose-fitting Dounce homogenizer (Bellco, Vineland, NJ), using five to ten
complete strokes. The resulting suspensions of matrix consisted of small fragments that averaged
Matrix-endothelial cell interactions 3
about 50—100/im wide. The matrix fragments were collected by low-speed centrifugation (150 £)
and rinsed three times with 0-1 %BSA in PBS.
Binding assays
For binding studies, subendothelial matrices were isolated as described above from BAE cultures
radiolabelled for 72 h in a leucine-free medium containing 5/xCi/ml of [3H]leucine and 5 % foetal
calf serum; ascorbic acid was added every 24h at 50/ig/ml. Bovine fibronectin, purified from
citrated plasma using gelatin-Sepharose (Ruoslahti, Hayman, Pierschbacher & Engvall, 1982), and
BSA (crystallized, Schwartz-Mann, Orangeburg, NY) were 125I-iodinated by the chloramine T
method (Greenwood, Hunter & Glover, 1963; Ruosiahti, Vuento & Engvall, 1978). The iodinated
fibronectin or BSA was adjusted with unlabelled fibronectin or BSA, respectively, to a specific
activity of 1 X 103 c.p.m./^g and diluted to 100^g/ml in 50/»i-Tris-HCl(pH7-5). Latex beads,
12 or 90 [im in diameter, were added to the protein solutions at a final concentration of 10 % (v/v)
and mixed by slow rotation for 1 h at 22 °C. The coated beads were then washed extensively with
lOmg/ml of unlabelled BSA in 50mM-Tris • HCl(pH7-5).
For binding assays, the coated latex beads were dispersed by brief sonication (Grinnell, 1980) and
diluted to a final concentration of 5 X 105 beads/ml in warm Iscove's-modified Dulbecco's Minimal
Essential Medium (GIBCO) containing 0-1 % BSA. Suspensions of radiolabelled matrix fragments
were prepared in the same medium and contained approximately 1-0 X 10s c.p.m./ml. Confluent
BAE cell monolayer cultures grown in 16-mm diameter cluster dishes (Costar, Cambridge, MA)
were rinsed twice with warm medium and received 0-5 ml of the suspension of coated latex beads
or homogenized matrix. In certain experiments the endothelial cell monolayers were pre-incubated
at 4°C for 30min before the binding assays that were performed at that temperature. At selected
times, triplicate dishes were washed gently twice with medium, and the radioactivity associated with
the cell layer was solubilized in 0-5 ml of 1 -5 M-NaOH and 0-1 % sodium dodecyl sulphate overnight
at 40 °C, neutralized with 1 -5 M-HCI, and counted by liquid scintillation spectrophotometry. Spon-
taneous solubilization of radiolabel from either the coated latex beads or the homogenized matrix
during the binding assay was less than 3-7 % of the total radioactivity added.
Microscopy
Cultures were fixed with 1 % glutaraldehyde in PBS at 37 °C for 15min and post-fixed with
1 % OsO 4 in PBS for 30 min at 22 °C. For scanning electron microscopy, the samples were processed
as previously described. Thick sections (0-5-2^m) of Araldite-embedded material were mounted
on glass slides and stained with 0-5 % toluidine blue. Immunofluorescent staining for bovine
fibronectin was performed on 7-day-old cultures of BAE cells seeded at confluence on Nucleopore
filters (5 /im diameter pores) precoated with human fibronectin, as described above for latex beads.
The antiserum specific for bovine fibronectin was a gift from Drs Eva Engvall and Erkki Ruoslahti
(La Jolla Cancer Foundation, La Jolla, CA) and was absorbed against human fibronectin. The
rabbit antiserum against fibronectin was used at 1 : 50 dilution, and fluorescein-conjugated goat anti-
rabbit immunoglobulin G (IgG) (Cappel, Cochranville, PA) was used as the second antibody at
1 : 200 dilution. Negative controls were performed in which normal, non-immune rabbit serum was
used in place of the anti-fibronectin antiserum. Specimens were mounted in PBS-buffered glycerol
and observed under epi-illumination.
RESULTS
The subendothelial matrix produced by the bovine aortic endothelial cells has been
extensively characterized in a number of studies. In addition to fibronectin (Birdwell,
Gospodarowicz & Nicolson, 1978; Kramer, Gonzalez & Nicolson, 1980; Sage,
Crouch & Bornstein, 1979), other glycoproteins such as laminin (Gospodarowicz et
al. 1981) and thrombospondin (Sageet al. 1979) are present. The major collagen type
found is type III, in addition to types I, IV and V (Sagee* al. 1979; Tseng, Savion,
Gospodarowicz & Stern, 1983). A high molecular weight heparan sulphate
R. H. Kramer
Fig. 1. Phase-contrast micrograph of fragments of bovine aortic endothelial (BAE) cell
extracellular matrix prepared by homogenization. Bar, 30 \un.
1001-
•a 60
o
m
40
20
12 18 24 30
Time (min)
Fig. 2. Time course of the binding of isolated BAE cell matrix fragments to confluent
BAE cell cultures. Matrices were prepared from [3H]leucine-labelled cultures and
homogenized to yield matrix fragments (see Fig. 1). Data points represent the average of
three dishes, in which the S.D. was less than 10% of raw data.
Matrix-endothelial cell interactions 5
proteoglycan is also associated with the isolated matrix (Kramer, Vogel & Nicolson,
1982). These components are arranged in a fibrillar structure that in confluent cul-
tures can be visualized by electron microscopy and immunocytochemical methods to
be only at the basal cell surface (Birdwell et al. 1978; Gospodarowicz, Greenburg,
Foidart & Savion, 1981).
Matrix fragments prepared from radiolabelled BAE cell cultures (Fig. 1) and
presented to confluent endothelial cell monolayers were quickly bound. The kinetics
of attachment was quite rapid and nearly complete after only 15 min incubation at
37 °C (Fig. 2); at 4 °C matrix binding did not occur, indicating that it is a temperature-
dependent process.
Immediately after the matrix fragments had attached to the cell monolayer, the cells
underwent a profound alteration in morphology. The normally organized arrange-
ment of the confluent epithelioid cell layer was transformed into fibroblastoid
cells having a mycelia-like appearance in which many of the original cell-
cell interactions were lost (Fig. 3). Similar results were obtained when extracellular
matrices were isolated from skin fibroblasts or aortic smooth muscle cells and seeded
onto BAE cell cultures. The disorganized cells exhibited increased motility, as obser-
ved by time-lapse microscopy (not shown). Adjacent areas of the BAE cell monolayer
that were not covered by the matrix sheets exhibited normal morphology. This
indicates that direct contact with the matrix is required for the altered cell shape and
that soluble factors released from the matrix are not inducing the morphological
change.
The disruption of the normally extensive cell—cell contacts was also evident when
the cultures were viewed in cross-section (Fig. 4). The exogenous extracellular matrix
was seen to be closely apposed at many sites to the apical surface of the endothelial
cells, and the normally extensive cell-cell junctions were lost (Fig. 4B). BAE cells
appeared to have established adhesions with the foreign matrix while still maintaining
adhesion with the original subendothelial matrix formed at their basal surface. It is
particularly noteworthy that at the edge of the matrix fragments endothelial cells
migrated from beneath the fragment and began to spread over its surface (Fig. 4c).
Eventually a confluent sheet of migrating endothelial cells continuous with the origi-
nal cell layer was observed to cover the new matrix (Fig. 4D). Most importantly, as
soon as they emerged from beneath the exogenous matrix, this advancing sheet of cells
exhibited normal morphology, with highly flattened and closely apposed cells.
Latex beads were used as a probe to study the adhesive properties of the BAE cell's
apical surface. Uncharged latex particles are normally non-adhesive (Grinnell, 1980),
but when coated with fibronectin, a major glycoprotein of the BAE cell's suben-
dothelial matrix (Birdwell et al. 1978; Gospodarowicz et al. 1980, 1981; Kramer et
al. 1980), the beads were rapidly bound to the BAE cell monolayers, and more than
70 % of the added beads were attached after 20 min incubation at 37 °C. In contrast,
latex beads coated with bovine serum albumin did not attach, even after several hours
of incubation. Furthermore, at 4°C the fibronectin-coated beads also failed to attach,
indicating that the attachment process was temperature-dependent. Latex beads
coated with the basement membrane-specific adhesive glycoprotein laminin also
R. H. Kramer
Fig. 3. Phase-contrast photomicrograph showing the response of BAE cells to contact at
their apical surface with isolated extracellular matrix. Fragments of isolated matrix
prepared from endothelial cell cultures were seeded onto the confluent endothelial cell
monolayer and incubated at 37 °C for 4h. Reorganization of the endothelial cells into
fibroblastoid cells has occurred beneath the matrix (arrowheads), while cells not covered
by the matrix fragment remain highly organized (top half of microscopic field). Bar,
75/tfn.
attached to BAE cell monolayers, but the rate of attachment was less than that obser-
ved with fibronectin-coated beads (unpublished results).
The normal ultrastructural features of the apical surface of cultured BAE cells, as
revealed by scanning electron microscopy, are shown in Fig. 5A. Typically, en-
dothelial cells are highly flattened, with no signs of microvilli or lamellopodia, and
form a cobblestone-like mosaic of non-overlapping cells with extensive cell-cell con-
tacts.
Examination by scanning electron microscopy of cultures incubated with
fibronectin-coated 12 /im diameter beads for various times is shown in Fig. 5B—D.
After incubation for 5 min, the attached latex particles appeared to be bound
predominately to the central portion of individual endothelial cells, and even at this
early time point there was an eruption of lamellopodia that began to spread up the
Matrix-endothelial cell interactions
B
Fig. 4. Light micrographs of thick sections of Araldite-embedded BAE cell cultures after
incubation with isolated extracellular matrices. BAE cell matrix fragments were seeded
onto confluent cultures of endothelium, as described for Fig. 3. The normal highly flat-
tened morphology of the endothelium (A) contrasts with the disorganized cell layer under-
lying matrix after 6h of incubation (B). In c endothelial cells (arrowhead) have begun to
migrate onto the top of the added matrix (12h). At 24h this migration is extensive (D).
Bar, 30 ^m.
surface of the sphere (Fig. 5B), becoming more extensive by 15 min (Fig. 5c). By 1 h
most of the beads were completely enveloped by endothelial cells (Fig. 5D) and
remained so even after 24 h of incubation (Fig. 6A). Occasionally, it was found that
the endothelial cells had spread completely over the latex bead and reformed a normal
monolayer. In this case the beads were found sandwiched between the cell layer and
the underlying extracellular matrix (Fig. 6B). This was particularly common when
beads of very large diameter (90 pan) were presented to endothelial cell cultures. Fig.
6c shows the arrangement of the endothelial cell layer after incubation with
fibronectin-coated particles 90 pan in diameter. After 24 h the endothelial cells had
migrated over the particle, creating a continuous canopy of highly flattened cells that
extended to the original cell layer. This process was usually complete after 12 h of
incubation.
R. H. Kramer
Fig. 5. Scanning electron micrographs of the binding of fibronectin-coated latex beads to
endothelial cell monolayers. Confluent BAE cell monolayers exhibit a smooth apical sur-
face (A). Fibronectin-coated beads (12/mi in diameter) were presented to BAE cell cul-
tures, and the monolayers were fixed and processed for examination after incubation for
5min (B), 15min (c), and 60min (D). Lamellopodia have gradually spread completely
over the bead surface. Bars: A, 10/mi; B,C,D, 5/an.
Matrix-endothelial cell interactions
Fig. 6. Light micrographs of semi-thin sections of plastic-embedded endothelial cell
monolayers incubated with fibronectin-coated latex beads. Confluent cell monolayers were
seeded with coated beads as described for Fig. 5. After 24 h of incubation with beads 12
/an in diameter (A, B) or 90 ^m in diameter (c), the cultures were processed. In A a single
endothelial cell has engulfed the 12 //m bead. Occasionally, the cells migrated over the
bead (B) separating the cell layer from its subendothelial matrix (arrowheads). A con-
tinuous cell monolayer has formed over the 90 jum diameter bead (c). Bars: A, B, 5 jUm; c,
20 fim.
When Nucleopore filters coated with plasma fibronectin were placed on top of
confluent monolayers of BAE cells, the underlying cells that came in contact with this
structure quickly transformed into a disorganized population of fibroblastoid cells
(Fig. 7A-D), similar to that observed when the cells were covered with fragments of
matrix (Fig. 3). However, with continued incubation the affected cells were observed
to migrate individually from beneath the filter through the 10 \im diameter pores and
arrive at the top surface of the filter (Fig. 7c,D). Usually, however, migrating cells
maintained some limited contact with adjacent cells in the original monolayer (Fig.
7c,D). After 5 h of incubation, more than 70 % of the cells originally beneath the filter
had migrated to the upper surface. By 24 h more than 90% had relocated and
established a new organized monolayer at the upper surface of the filter. Filters with
5 ,um diameter pores caused an identical response, but BAE cell migration through the
filter pores was somewhat slower than with the 10 fim diameter, filters. Filters coated
with bovine serum albumin neither attached to the monolayer nor induced monolayer
disorganization.
10 R. H. Kramer
Fig. 7
Description:the subendothelial matrix, could be involved in the observed apical cell surface - matrix interactions, latex beads or small discs of Nucleopore filters
