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J Biol Chem, Vol. 273, Issue 46, 30719-30728, November 13, 1998
From the Cardeza Foundation for Hematologic Research and Division
of Hematology, Department of Medicine, Jefferson Medical College of
Thomas Jefferson University, Philadelphia, Pennsylvania 19107
The contact of fibrin with the apical
surface of human umbilical vein endothelial cells (HUVEC) can induce
capillary tube formation via the interaction of fibrin In the final steps of blood coagulation, the soluble plasma
protein fibrinogen is enzymatically converted by the action of thrombin
into insoluble fibrin monomers, which spontaneously polymerize to yield
a fibrin mesh containing entrapped platelets and red blood cells (1).
The fibrin blood clot plays a vital role in many physiologic and
pathophysiologic processes including hemostasis and thrombosis (1), and
in angiogenic processes such as wound healing and tumor growth (2, 3).
In these processes fibrin interacts with a variety of plasma proteins,
cells, and tissue matrix components. The main hemostatic function of
fibrin clot formation in vivo is to provide structural
support and scaffolding for the cellular and other blood elements
comprising the thrombus (1). Similarly, in the process of normal wound
healing, the main purpose of the fibrin clot that fills the wound is to
serve as a provisional matrix into which inflammatory cells migrate and
upon which new blood capillaries are formed (2). Fibrin formation is
also involved in pathological conditions, such as tumor growth, where
it is thought to provide a matrix for tumor cell proliferation and
possibly for maintenance of angiogenesis (2).
The interaction of the N-terminal region of fibrin with the endothelial
cell surface influences cell behavior in several respects. It can
disrupt the normal integrity of the endothelial cell monolayer (4), it
can stimulate secretion of stored von Willebrand factor (5) and it can
induce human umbilical vein endothelial cell (HUVEC)1 morphogenesis into
capillary tube-like structures (6, 7), thus initiating the process of
in vitro angiogenesis. These observations indicate that the
thrombin-mediated exposure of neo-epitopes at the N terminus of the
fibrin molecule confers fibrin, as opposed to fibrinogen, with the
ability to bind to endothelial cell surface receptors and thereby
induce the specific cellular responses referred to above.
We have previously shown that a specific form of fibrin designated
fibrin II, in which the neo-N terminus of the fibrin Because angiogenesis is a complex process, which likely involves
receptors from various cell adhesion molecule families (13, 14), we
also assessed the potential role of members of three families of
adhesion receptors including the integrins, the immunoglobulin gene
(Ig) superfamily, and the cadherins as putative endothelial cell
receptors for fibrin. Among the integrins, we focused on the
In the present investigation, we focused our attention on the
interaction of the N terminus of fibrin, namely the Reagents and Antibodies--
Medium 199 with Earle's salts and
Hepes buffer, Dulbecco modified Eagle's medium, and collagenase type I
were purchased from Life Technologies, Inc. RPMI 1640 medium was
purchased from Fisher Scientific. Carrier-free 125I (NaI in
NaOH) and the ECL detection system were purchased from Amersham
Pharmacia Biotech. Silicone oil (DC 550) was purchased from Dow Corning
Corp. through William K. Nye Inc. (New Bedford, MA). Lyophilized human
thrombin, Atroxin, a polyclonal pan-cadherin antibody against the
conserved cytoplasmic domain of the cadherin family, and a monoclonal
antibody (clone GC-4) against human N-cadherin were purchased from
Sigma. A second monoclonal antibody against N-cadherin (clone NCAD2)
and a monoclonal anti-ICAM-1 antibody (clone LB-2) were purchased from
Becton Dickinson (Bedford, MA). The monoclonal antibody (clone 75)
against residues 26-194 of human VE-cadherin was purchased from
Transduction Laboratories (Lexington, KY). A monoclonal antibody
against the human integrin Cell Culture--
Primary cultures of HUVEC were isolated from
human umbilical cords and maintained in culture as described (26).
Cells from passages 1-4 were used in this study and were periodically
checked for the expression of von Willebrand factor by
immunohistochemistry. Human aortic smooth muscle cells were cultured in
Dulbecco's modified Eagle's medium supplemented with glutamine,
penicillin/streptomycin, amphotericin, and 20% fetal calf serum. Cells
from passages 15-20 were used in this study. HADMEC were cultured in
RPMI 1640 medium supplemented with glutamine, penicillin/streptomycin,
and 5% fetal bovine serum on tissue culture dishes coated with 1%
gelatin and were confirmed to be endothelial cells by immunofluorescent
assessment of the expression of von Willebrand factor. Cells from
passages 10-15 were used in this study.
Preparation of the NDSK and Its Derivatives from
Fibrinogen--
Human fibrinogen was purified as described previously
(26). To prepare the NDSK, fibrinogen (10 mg/ml) was dialyzed into 70%
formic acid and was treated with cyanogen bromide (1.3 mg/100 mg of
fibrinogen) for 24 h at room temperature (27). The solution was
dialyzed into 50 mM Tris-buffer, pH 7.4, and contaminating hydrophobic fragments (28) were removed by centrifugation at 10,000 × g. The supernatant, containing two major
cleavage products, was chromatographed on a Sephadex G-100 column, and
fractions eluted after the void volume were pooled and analyzed by
SDS-PAGE. Purified NDSK was treated with thrombin (3 units/ml for
3 h at 37 °C) to generate NDSK II (NDSK lacking fibrinopeptides
A and B). NDSK I (NDSK minus fibrinopeptide A) was prepared by treating NDSK with Atroxin (2 µg/ml for 2 h at 37 °C), and NDSK 325, which lacks B Synthesis of the Binding Assays--
Binding of NDSK derivatives to HUVEC
suspensions was performed essentially as described previously for
fibrinogen binding to HUVEC (26). Briefly, HUVEC (~250,000 cells/ml)
suspended in buffer containing 1% bovine serum albumin and no added
cations, were incubated in 12-mm multiwells with the
125I-NDSKs at a final concentration of 20 nM.
The suspensions were incubated with horizontal shaking at 4 °C and
aliquots of suspension were removed in duplicate and added to
microcentrifuge tubes containing a mixture of 1 part light mineral oil
and 4 parts DC550 silicone oil (26). The cells were pelleted by
centrifugation at 12,000 × g for 10 min, the tips of
the tubes were amputated, and the cell-bound radioactivity was counted
by
Specific binding was calculated from the difference in total binding
and nonspecific binding. Reversibility of binding was assessed either
by dilution with excess binding buffer or by displacement with excess
unlabeled NDSK II. To assess the cation dependence of the system,
binding studies were carried out with binding buffer containing added
CaCl2 (2 mM), MgCl2 (2 mM), MnCl2 (0.5 mM), or EDTA (8 mM). To assess the integrity of the cell-bound
125I-NDSK, the radioactive pellet was extracted into
Laemmli sample buffer (32), electrophoresed, and subjected to
autoradiography as described below.
Potential inhibitory agents, such as antibodies (5-10 µg/ml) to
endothelial cell integrins, cadherins, or Ig superfamily molecules, or
specific fibrin-derived peptides such as
For the studies in monolayer, HUVEC were grown to confluence on 5-cm
wells (~500,000 cells/well), washed twice with 2 ml of serum-free
Medium 199 containing 1% bovine serum albumin, pH 7.4, and
preincubated with the same medium for 15 min at 37 °C prior to the
addition of the NDSK derivatives. All monolayer studies were carried
out at 37 °C. Nonspecific binding was determined as described above
for cell suspensions. Following incubation, duplicate wells were washed
twice with TBS and detached with TBS/EDTA, 900-µl aliquots of
detached cells were added to microcentrifuge tubes containing a mineral
oil/DC550 silicone oil mixture, and the cell-bound
125I-NDSK was quantitated as described above for the
suspension studies.
Estimation of Binding Parameters--
Scatchard plots of NDSK II
binding to HUVEC were analyzed by curve-fitting software developed by
Munson and Rodbard (34) for estimation of dissociation constants
(KD) and maximal binding
(Bmax). The data were analyzed initially using
both a two-site and a one-site model with nonspecific binding set at zero. Convergence of the data was obtained with only the one-site model. The data were subsequently analyzed by a one-site model with
nonspecific binding set as a parameter proportional to the ligand
concentration. The experimental estimates of nonspecific binding,
determined from the binding of labeled ligand in the presence of
100-fold molar excess of unlabeled ligand, coincided within 15% of
estimates by curve fitting using the one-site model.
Co-immunoprecipitation of VE-cadherin and NDSK
II--
Immunoprecipitation was performed as described previously (35)
with some modifications. HUVEC monolayers (10-15 T-75 flasks; ~30-45 × 10 6 cells) were washed three times with
TBS containing 2 mM CaCl2 and then scraped, on
ice, into TBS, pH 7.4, containing 2 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, 15 µg/ml aprotinin, and
15 µg/ml leupeptin (lysis buffer). The samples were centrifuged for 5-10 min at 1000 × g and the supernatants resuspended
in 0.5 ml of the same buffer containing 0.5% SDS and 1% Nonidet P-40,
put on ice for 15 min, and aspirated back and forth several times through a 27.5-gauge needle. After 15 min, lysis buffer containing only
1% Nonidet P-40 was added to bring the final concentration of SDS to
0.1%, the samples were allowed to stand on ice for 15 min and then
centrifuged at 12,000 × g for 15 min at 4 °C. To the supernatant, 125I-NDSK II was added to a final
concentration of 30 nM and the tubes were capped and
rotated end-over-end for 20-30 min at room temperature. The NDSK
II-bound lysate was then incubated for 2 h on ice with either an
anti-pan-cadherin IgG or a control rabbit IgG, both at a final
concentration of 50 µg/ml. Subsequently, 100 µl of the Protein
A-Sepharose suspension was added to each sample and incubated for 60 min on an end-over-end shaker. The incubation mixture was centrifuged
for 1-2 min at 12,000 × g and the pellet washed four
times with 1 ml of lysis buffer. After the final wash, the pellets were
boiled in Laemmli sample buffer (32), electrophoresed, and
immunoblotted as described above.
Co-capture of VE-cadherin and NDSK II--
Antibody capture
assays were performed as follows. To each 12-mm well of a Nunclon
multiwell dish, 500-µl aliquots of anti-pan-cadherin IgG or control
rabbit IgG (10 µg/ml TBS) were added and the dishes incubated for
2 h at 37 °C. Following aspiration of the antibody solutions,
each well was washed three times with 1 ml of TBS and residual protein
binding sites were blocked by incubation with 10% powdered milk in TBS
for 1-3 h at 37 °C. The wells were then aspirated and rinsed with
lysis buffer containing 0.1% SDS/1% Nonidet P-40, and 500-µl
portions of cell lysates (250,000-500,000 cells/ml), lysed as
described previously for the immunoprecipitation studies, were added
for 2 h at 37 °C. The lysates were then aspirated, and the
wells were washed and resupplemented with 500-µl aliquots of
detergent-containing lysis buffer. To measure total binding of the NDSK
fragments, 125I-labeled NDSK, NDSK II, or NDSK 325 were
added to the buffer at a final concentration of 20 nM for
30 min at room temperature. Nonspecific binding was determined by
preincubating certain wells for 20 min with a 100-fold molar excess of
the unlabeled derivatives prior to the addition of the respective
labeled derivatives. The wells were then washed three times with 1-ml
aliquots of detergent-containing lysis buffer to remove free
radioactivity, and the well contents were scraped into 1-ml portions of
1 M NaOH and counted by SDS-Polyacrylamide Gel Electrophoresis, Autoradiography, and
Western Blotting--
Integrity of the various NDSK derivatives was
examined by 12% SDS-polyacrylamide gels followed by either Coomassie
Brilliant Blue staining or immunoblotting with a monoclonal antibody
(T2G1) to the fibrin Statistical Analysis--
The n values for all
experiments range from three to six separate determinations of data
points measured in duplicate. Statistical analysis of the data was
performed using Student's t test (two-sample assuming
unequal variances) and single-factor analysis of variance with
Microsoft Excel Analysis Toolpak version 5.0 software (Gray Matter
International, Inc., Fremont, CA). Probability values of less than 0.05 (p < 0.05) were considered significant.
Analysis of NDSK Fragments--
Fibrinogen can be cleaved with
cyanogen bromide to generate a fragment termed the NDSK, which is
composed of the intact N termini of the A
The same NDSK fragments were transferred to nitrocellulose after
running on SDS-PAGE and Western blots were performed using a
monoclonal antibody (T2G1) to the Characterization of NDSK Binding to HUVEC--
Because fibrin II,
but not fibrin I or fibrin 325, can induce endothelial cell capillary
tube formation (7) which can be inhibited by antibodies to VE-cadherin
(9), we hypothesized that fibrin II may bind specifically to
endothelial cell receptors, possibly VE-cadherin. However, because our
preliminary binding studies were complicated by the problem of fibrin
polymerization,2 we chose to
study the binding of various NDSKs to HUVEC, eliminating the binding of
fibrin polymers since the NDSKs are soluble. Furthermore, since the
purified NDSKs do not contain the RGD sequences present in the native
molecule, potential RGD-dependent interactions are likely
eliminated. This approach also afforded us the opportunity to
characterize the binding determinants expressed at the N terminus of
fibrin and to aid in the identification of an endothelial cell fibrin receptor.
Time-course binding studies conducted over 3 h revealed that the
total binding of 125I-NDSK II (20 nM) to HUVEC
suspensions at 4 °C or monolayers at 37 °C reached a plateau
level in 10 min (Fig. 2). Therefore, in subsequent experiments, incubations were carried out for 20 min prior
to determination of particular binding parameters. Fig. 2 also
demonstrates that total binding in monolayer saturates at a lower
number of molecules per cell than in suspension (5,000 versus 40,000, respectively).
Specific binding of 125I-NDSK II by dilution was observed
to be reversible, reaching a maximum of 70% displacement after
2.5 h (Fig. 3). Reversibility by
competition, using excess unlabeled NDSK II, gave identical results
(data not shown). Moreover, the 125I-NDSK II recovered from
bound cells ran predominantly as a 58-kDa band, unchanged from the
appearance of the starting material (data not shown) indicating that
the radioactivity bound to HUVEC in these studies was NDSK II
which did not undergo significant degradation or
polymerization.
The relative affinities of the 125I-labeled and unlabeled
NDSK II were compared by conducting isotope dilution experiments in which the concentration of total NDSK II was kept constant at 25 nM while the proportion of unlabeled NDSK II to
125I-NDSK II was progressively increased. This type of
analysis revealed that the affinities of the two ligands were very
similar, as evidenced by a linear decrease in bound
125I-NDSK II as its proportion decreased (data not shown).
Saturability of NDSK II Binding to HUVEC--
Binding isotherms of
125I-NDSK II from 2.5 to 25 nM to HUVEC
suspensions were performed and analyzed by the LIGAND computer program (34). The data defined a single population of receptors that saturated
near 20 nM (Fig. 4), and
Scatchard data of five separate experiments revealed an apparent
dissociation constant (KD) of 7.7 ± 1.6 nM with approximately 21,000 ± 7000 binding
sites/cell (Fig. 4, inset).
The specific binding of 125I-NDSK II to HUVEC monolayers
reached a saturation between 10 and 20 nM (data not shown),
similar to that of the suspension studies (Fig. 4). However, the extent of binding was much less than in suspension and the accuracy of binding
at subsaturating concentrations was limited by the low number of counts
bound. Therefore, binding specificity was determined at saturating
concentrations as described below.
Specificity of NDSK II Binding to HUVEC--
To establish the
specificity of NDSK binding to HUVEC, the binding of each radiolabeled
NDSK fragment (20 nM) was measured in the absence or
presence of a 100-fold molar excess of its corresponding unlabeled
fragment. As demonstrated in Fig.
5A, only the binding of the
thrombin-cleaved 125I-NDSK II was significantly inhibited
by its cold fragment, revealing specific binding of 51 ± 6%.
Similar results were obtained with confluent HUVEC monolayers at
37 °C as shown in Fig. 5B, which demonstrates that the
specific binding of NDSK II was 69 ± 5%, but that the number of
molecules bound per cell was 10-fold less than in suspension, as
previously noted for the time-course experiments (Fig. 2). The
decreased number of NDSK II molecules in monolayer compared with
suspension is possibly attributable to a decrease in the number of
VE-cadherin molecules available for heterophilic binding as a result of
strong homophilic binding to other VE-cadherin molecules present at
intercellular contact sites.
Since neither NDSK nor NDSK I (which contain Divalent Cation Influence on NDSK II Binding to HUVEC--
Calcium
and manganese have been reported to influence fibrinogen binding to
NDSK II Binds to VE-cadherin on HUVEC--
In order to elucidate
the receptor to which NDSK II binds, several agents were tested for
their ability to inhibit the binding of 125I-NDSK II to
HUVEC. These included antibodies to the various cell adhesion molecules
known to be present on endothelial cells, specific peptides present
within fibrin(ogen), and heparin, which has recently been shown to
inhibit binding of fibrin fragments to HUVEC (33). The results of these
experiments revealed that binding of 125I-NDSK II to HUVEC
could not be inhibited by the antibodies 7E3 or LM 609 (data not
shown), both of which are directed against the integrin
As depicted in Fig. 7, of the antibodies
tested, only a monoclonal antibody directed against the first two
extracellular domains of VE-cadherin was capable of inhibiting
125I-NDSK II binding to HUVEC. In contrast, antibodies
against the extracellular domain of N-cadherin did not significantly
inhibit NDSK II binding (Fig. 7). Similarly, the binding of NDSK II to HUVEC was not influenced by monoclonal antibodies against PECAM-1 or
ICAM-1 (data not shown), indicating that these two members of the Ig
superfamily do not serve as receptors for the N terminus of fibrin
II.
Because the VE-cadherin antibody inhibited NDSK II binding, we further
analyzed the role of VE-cadherin in fibrin binding by studying the
association of NDSK II with cells that do not express VE-cadherin. For
these experiments, cultured vascular human smooth muscle cells were
lysed, run in SDS-PAGE, and immunoblotted using the VE-cadherin
antibody. These experiments showed that VE-cadherin was not expressed
by these cells (data not shown), as previously shown by other
investigators (9, 22). More importantly, these venous smooth muscle
cells did not show specific binding of 125I-NDSK II whether
in suspension at 4 °C or as monolayers at 37 °C (data not shown).
NDSK II Co-immunoprecipitates with HUVEC Cadherins--
To further
establish the role of VE-cadherin as the NDSK II receptor of
endothelial cells, we used a polyclonal pan-cadherin antibody to assess
whether 125I-NDSK II would co-immunoprecipitate from HUVEC
lysates along with VE-cadherin. The pan-cadherin antibody was used
since it is directed at the conserved cytoplasmic domain of the
cadherins and should therefore not interfere with the extracellular
NDSK II binding site, as would be expected for the monoclonal antibody which inhibited NDSK II binding (Fig. 7) and which is directed against
the extracellular domain of VE-cadherin. The Western blots of the
immunoprecipitated 130-kDa HUVEC cadherin are demonstrated in Fig.
8 (panels A and
B), using the polyclonal pan-cadherin and monoclonal
VE-cadherin antibodies, respectively. The large bands at the bottom of
lanes 2 and 3 in panel
B migrate at a molecular mass of 50 kDa and likely represent
the IgG of the immune complexes after being run under reducing
conditions. Panel C demonstrates that the pan-cadherin
antibody immunoprecipitated 59 ± 18% more radioactive NDSK II
than did a nonimmune IgG. Therefore, just as in the binding studies
(Fig. 5), the amount of specific binding of 125I-NDSK
II to isolated endothelial cell cadherins is approximately 50-60%.
Isolated HUVEC Cadherins Specifically Bind NDSK II--
To further
establish the identity of VE-cadherin as the NDSK II receptor of HUVEC,
we used a modified antigen capture assay in which HUVEC cadherins were
immobilized in wells coated with the polyclonal pan-cadherin antibody.
The Western blots of the immunocaptured 130-kDa HUVEC cadherin is
demonstrated in Fig. 9A using
the monoclonal VE-cadherin antibody. The results shown in Fig.
9B illustrate that the binding of 125I-NDSK II
to captured cadherins reached a plateau at 20 nM and that
specific binding, as determined by preincubation of wells with a
100-fold molar excess of unlabeled NDSK II, was 89 ± 5% (Fig.
9C). Fig. 9C also shows that the binding of
125I-NDSK and 125I-NDSK 325 to captured
cadherins was negligible, and that the binding of 125I-NDSK
II by material captured by nonimmune IgG was only 11 ± 8%,
similar to the level of nonspecific binding of NDSK II.
Thrombin-treated 125I-NDSK 325 also failed to bind to
captured cadherins (data not shown), indicating that exposure of the
neo-N terminus of the
The conclusion that the 125I-NDSK II was in fact bound to
captured endothelial cell VE-cadherin is supported by the observation that Western blot analysis of material scraped from the capture assay
plate coated with the pancadherin antibody revealed VE-cadherin that
was not detected from the plate coated with control rabbit IgG (Fig.
9A, lanes 2 and 3,
respectively). Although it is also likely that N-cadherin was captured
using the polyclonal pan-caderin antibody, captured N-cadherin could
not be demonstrated by Western blots possibly due to the low
sensitivity of the N-cadherin antibody as revealed by Western blot
analysis with HUVEC lysates (data not shown).
As a final demonstration of the role of VE-cadherin in binding of NDSK
II, an immortalized line of endothelial cells, known as HADMEC (41),
which are negative for VE-cadherin by immunoblot (Fig.
10A, lane
2) and immunofluorescence (data not shown), positive by
immunofluorescence for von Willebrand factor and N-cadherin (data not
shown), did not specifically bind 125I-NDSK II (Fig.
10B). These data firmly establish VE-cadherin as the NDSK II
receptor of endothelial cells.
The present paper focuses on the binding of soluble fibrin
derivatives to HUVEC. The analysis of binding of fibrin itself to
endothelial cells is difficult to quantitate since fibrin forms insoluble polymers after release of fibrinopeptides from fibrinogen (1). In order to circumvent the binding of polymerized fibrin, the
soluble NDSK of fibrin, which lacks the polymerization sites at the C
terminus of the fibrin molecule, was used to explore the structural
requirements of fibrin binding to endothelial cells in
vitro. NDSK is a fragment of fibrinogen, generated by cleavage with cyanogen bromide, which retains the N-terminal structural features
of each of the fibrin chains consisting of the dimeric fragment of A The results of our investigation clearly demonstrate that NDSK II,
which contains the neo-N-terminal Because several members of the endothelial cell integrin family are
known to bind fibrinogen (15, 16), and because the association of
matrix proteins with integrins plays an important role in the
development of new blood vessels (42-46), we initially evaluated the
potential of integrins to bind NDSK II. The NDSK II fragment used in
this study lacks the RGD sequences through which fibrinogen and many
other ligands bind to integrins (15, 16, 47, 48). Our results show that
none of the anti-integrin antibodies tested had any inhibitory effect
on NDSK II binding. Furthermore, the RGDS peptide, which inhibits
fibrinogen binding to integrins (15, 48), also failed to inhibit NDSK
II binding, suggesting that integrins did not directly participate in
NDSK II binding in our system.
Of the Ig superfamily molecules, PECAM-1 and ICAM-1 are known to play
important roles in endothelial cell function, with PECAM-1 mediating
cell-cell adhesion and leukocyte migration across interendothelial cell
junctions (17) and recently reported to play a role in angiogenesis
(10, 11), and ICAM -1 serving as a fibrinogen receptor that binds the
It has been recently demonstrated that CNBr-cleaved N-terminal
fragments of fibrin bind to HUVEC and that preincubation of the
N-terminal fragments with heparin inhibits the binding (33). These
findings suggest that the heparin binding domain, previously identified
as Cadherins are adhesive molecules present at adherens junctions, which
mediate cell-cell contacts via their extracellular domains while
anchoring to cytoskeletal structures via their cytoplasmic tails (24,
50-52). They play a major role in morphogenesis (50, 53, 54) and
development of blood vessels during embryogenesis (25). Vascular
endothelial cells express two different cadherins (20, 22), N-cadherin
and VE-cadherin. While N-cadherin appears to be diffusely distributed
over the plasma membrane (20), VE-cadherin is known to be located at
intercellular junctions of essentially all types of endothelium (23,
24) and has recently been shown to mediate endothelial cell capillary
tube formation in vitro either on its own (9) or in
conjunction with PECAM-1 (10).
We have recently demonstrated that a monoclonal antibody against
VE-cadherin, but not N-cadherin, prevents formation of endothelial cell
capillary tubes induced by sandwiching HUVEC between fibrin-fibrin or
collagen-collagen gels (9). Moreover, the VE-cadherin monoclonal antibody also produced disruption of preformed capillary tubes. These
data indicate that fibrin may function as a heterophilic ligand for
VE-cadherin on the endothelial cell surface. Indeed, the monoclonal
antibody that inhibited or disrupted tube formation (9) also inhibited
binding of NDSK II to endothelial cell suspensions or monolayers.
Although two monoclonal antibodies to N-cadherin failed to
significantly inhibit NDSK II binding to HUVEC, it is possible that
they recognize epitopes of the extracellular domain that do not
participate in this heterophilic interaction.
The VE-cadherin monoclonal antibody used in this investigation
recognizes amino acids 26-194, which bridge the first two
extracellular domains (EC1 and EC2) that are thought to contain the
region that may participate in homophilic interactions of VE-cadherin
at cell-cell junctions (24). To our knowledge, our finding of fibrin
binding to VE-cadherin describes the first heterophilic interaction for VE-cadherin. However, it should be noted that E-cadherin has recently been shown to participate in heterophilic interactions with internalin, a surface protein that mediates entry of Listeria
monocytogenes into epithelial cells (55), and also with the
The phosphorylation and intracellular protein associations of
VE-cadherin seem to influence the biology of endothelial cell monolayers (24). In our system, it is feasible that intracellular phosphorylation events influence the conformation of the extracellular domains of VE-cadherin to favor a heterophilic interaction with fibrin
as opposed to homophilic interactions with VE-cadherin molecules
present on adjacent cells. Such a switch in binding specificity has
been demonstrated with PECAM-1, where either the absence or
phosphorylation of a particular tyrosine residue within the cytoplasmic
domain of PECAM-1 changes its ligand specificity, possibly by promoting
dimerization, from heterophilic to homophilic binding (57). It is also
conceivable that dimerization of VE-cadherin may influence its ligand
specificity. Evidence to support this has been demonstrated with
C-cadherin, in which dimers of C-cadherin exhibit substantially greater
homophilic binding activity than do monomers, and the presence of
calcium in this system favors C-cadherin dimerization (58). We
speculate that a similar type of interaction may be occurring with
VE-cadherin, and thus postulate that the phosphorylation of VE-cadherin
or proteins associated with its cytoplasmic domain, allows a
conformational change that favors the formation of VE-cadherin
monomers, and thus promotes its heterophilic, calcium-independent
binding to the N terminus of fibrin.
The mechanisms involved in the interaction of fibrin with VE-cadherin,
which results in the formation of capillaries, have not been fully
elucidated. It is possible that the N-terminal region of fibrin
associates with apically expressed VE-cadherin molecules, which are not
present in intercellular junctions, and that this association induces
intracellular signals directly associated with endothelial cell
morphogenesis. It is also possible that the initial disruption of
endothelial cell monolayers, which occurs with overlying polymerized
fibrin (4), permits the interaction of fibrin with VE-cadherin
molecules present in the intercellular junctions, and that it is these
topographically restricted associations which are responsible for the
assembly of endothelial cells into capillary tubes.
In this investigation we have identified endothelial cell VE-cadherin
as a receptor for the We thank Andrew S. Likens for the artwork,
Rosemarie Silvano for preparation of the manuscript, and Bonny Lightner
and Heather Ryan for their experience in cell culture.
*
This work was supported in part by National Institutes of
Health Grant HL-20092 (to J. M.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Cardeza Foundation for
Hematologic Research, Thomas Jefferson University, 1015 Walnut St.,
Philadelphia, PA 19107. Tel.: 215-955-8458; Fax: 215-923-3836; E-mail:
jos.martinez{at}mail.tju.edu.
The abbreviations used are:
HUVEC, human
umbilical vein endothelial cells; VE-cadherin, vascular endothelial
cadherin; PECAM-1, platelet endothelial cell adhesion molecule-1; Ig, immunoglobulin family of cell adhesion molecules; ICAM-1, intercellular
cell adhesion molecule-1; HADMEC, human adipose-derived microvascular
endothelial cells; NDSK, N-terminal disulfide knot of fibrinogen; TBS, Tris-buffered saline.
2
T. L. Bach, C. Barsigian, C. H. Yaen,
and J. Martinez, unpublished observations.
Endothelial Cell VE-cadherin Functions as a Receptor for the
15-42 Sequence of Fibrin*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
15-42 with a
putative cell receptor (Chalupowicz, D. G., Chowdhury, Z. A.,
Bach, T. L., Barsigian, C., and Martinez, J. (1995) J. Cell Biol. 130, 207-215). To characterize this interaction, we
studied the binding of the thrombin-cleaved N-terminal disulfide knot
of fibrin (NDSK II), a dimeric fragment with exposed 15-42, to
HUVEC in three separate assay systems. Time-course binding of
125I-NDSK II to HUVEC monolayers or suspensions revealed
that binding was specific at 50-60%, as determined by the addition of
unlabeled NDSK II. Specific binding of 125I-NDSK II to
HUVEC was 70% reversible by dilution or by competition, and was found
to be divalent cation-independent. Binding plateaued after 10 min at a
saturation of 15-20 nM. Scatchard analysis using the
LIGAND computer program defined a single population of receptors with a
KD of 7.7 ± 1.6 nM and
approximately 21,000 ± 7000 binding sites/cell. N-terminal
disulfide knot derivatives in which 15-42 was absent (NDSK 325) or
unexposed (NDSK, NDSK I) did not show specific binding. Specific
binding of 125I-NDSK II could not be inhibited by RGDS or
by antibodies to the 
v3 or
1 integrins, PECAM-1, ICAM-1, or N-cadherin. In
contrast, a synthetic 15-42/ovalbumin conjugate inhibited total
125I-NDSK II binding by 47 ± 19% (corresponding to
95% of specific 125I-NDSK II bound) and a monoclonal
antibody to vascular endothelial cadherin (VE-cadherin) inhibited
binding by 35 ± 8% (corresponding to 70% of specific
125I-NDSK II bound). Another assay was based on the capture
of cadherins from HUVEC lysates by a polyclonal pan-cadherin antibody
immobilized on plastic dishes. Binding of NDSK II to the captured
cadherins was 89 ± 5% specific, while specific binding of NDSK
325 and NDSK was negligible. An immortalized line of human
adipose-derived microvascular endothelial cells, which express
N-cadherin but not VE-cadherin, demonstrated no specific binding of
NDSK II by the capture assay. These data define a novel interaction of
fibrin with VE-cadherin, which is mediated by the fibrin N-terminal
15-42 sequence, and may contribute to the mechanism through which
fibrin induces angiogenesis.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
chain
(15-42) is exposed, induces the formation of capillary tube-like
structures when in contact with the apical surface of HUVEC monolayers
(7). We have also more recently demonstrated that a monoclonal antibody
against the extracellular domain of VE-cadherin, or cadherin-5 (8),
inhibited fibrin II and collagen-induced capillary tube formation
whereas antibodies against N-cadherin, PECAM-1, or the
v3 integrin did not (9). In related
experiments, others have shown that antibodies against VE-cadherin or
PECAM-1 do not inhibit capillary tube formation when used individually, but do inhibit tube formation when incubated together with endothelial cells cultured on single-layer Matrigel (10). In contrast to the
aforementioned studies, which report the inability of PECAM-1 alone to
induce tube formation (9, 10), others have found PECAM-1 to play an
important role in both in vivo and in vitro angiogenesis (11). Taken together, these data suggest that VE-cadherin or PECAM-1 (both of which have a molecular mass of roughly 130 kDa) may
serve as endothelial cell receptors for matrix proteins, including but
not limited to fibrin, that play an important role in mediating
capillary tube formation. This idea is supported by the report that a
130-kDa protein can be purified from human umbilical vein endothelial
cell lysates by affinity chromatography over fibrin 15-42/Sepharose
(12). Based on these findings, the major hypothesis of the present work
was that the neo-N terminus of the fibrin II chain (15-42) can
bind to endothelial cell surface receptors, possibly VE-cadherin, and
that this binding may underlie the reported ability of VE-cadherin
antibodies to inhibit fibrin II-induced capillary tube formation
(9).
v3 (15) and
51 (16) integrins, which are known to bind fibrinogen. Within the Ig superfamily, in addition to PECAM-1, which participates in both heterophilic and homophilic interactions (17) as well as in capillary tube formation (10, 11), we also examined
ICAM-1, which acts as a fibrinogen receptor on endothelial cells and
mediates leukocyte adhesion to inflamed endothelium (18, 19). We also
examined the role of N-cadherin, which appears to be diffusely
distributed over the endothelial cell plasma membrane (20), and of
VE-cadherin, which, in addition to being specific to the vascular
endothelium (21, 22) and concentrated at cell-cell junctions (23, 24),
has also been shown to mediate vasculogenesis (25) and both in
vitro and in vivo angiogenesis (9, 10).
15-42 sequence, with an endothelial cell receptor by characterizing binding of the
N-terminal disulfide knot (NDSK) of fibrin to HUVEC. We have also
utilized a cadherin capture assay to analyze the direct interaction of
fibrin with isolated cadherins. Based on our results, we propose that
VE-cadherin serves as a fibrin-specific receptor on endothelial cells
and that the N terminus of the fibrin chain (15-42) functions as a heterophilic ligand for VE-cadherin.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
v3 (clone LM
609) was purchased from Chemicon International, Inc. (Temecula, CA),
and another against the integrin 1 subunit (clone P4C10)
was purchased from Life Technologies, Inc. Endothelial cell growth
factor was kindly provided by Dr. Kerri Pratt (Department of Surgery,
Thomas Jefferson University, Philadelphia, PA), and human aortic smooth
muscle cells were provided by both Dr. Kerri Pratt and Dr. A. Zalewski
(Division of Cardiology, Thomas Jefferson University, Philadelphia,
PA). An immortalized line of human, adipose-derived microvascular
endothelial cells (HADMEC) were provided by Dr. John T. Flynn
(Department of Physiology, Thomas Jefferson University, Philadelphia,
PA), and a polyclonal antibody to von Willebrand factor was kindly
donated by Dr. Dorothy Beacham (Cardeza Foundation, Thomas Jefferson
University, Philadelphia, PA). Monoclonal (clone 4G6) and polyclonal
(WACO) anti-PECAM-1 antibodies were gifts from Dr. Steven M. Albelda
(Department of Medicine, University of Pennsylvania, Philadelphia, PA).
A polyclonal antibody to the v3 integrin
and protease III from the venom of Crotalus atrox were
donated by Dr. P. Thiagarajan (Department of Medicine, University of
Texas Health Center, Houston, TX). Another monoclonal antibody against
v3 (clone 7E3) was a gift from Dr. Barry
Coller (Mount Sinai Medical Center, New York, NY), and a monoclonal
antibody to the fibrin sequence 15-42 was provided by Dr. B. J. Kudryk (New York Blood Center, New York, NY). All other reagents
were purchased from Fisher, Sigma, or Life Technologies, Inc.
1-42, was prepared by treatment of NDSK with protease III (8 µg/ml for 3 h at 37 °C) from C. atrox venom
(29). The identities of the NDSK derivatives were assessed by SDS-PAGE. For binding experiments, NDSK fragments were labeled with iodine 125, by the IODOGEN method (30), to a specific activity of 3000-5000 cpm/ng.
15-42 Peptide Sequence--
The 15-42
sequence, corresponding to the neo-N terminus of the fibrin chain
following cleavage of fibrinopeptide B by thrombin, was synthesized by
the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy of
solid-phase synthesis (31) using an Applied Biosystems 430A peptide
synthesizer and was purified by reversed-phase chromatography using an
Applied Biosystems semipreparative system equipped with a Rainin C18
column. The sequence of the purified 15-42 peptide was verified by
mass spectroscopy. The purified peptide was conjugated to ovalbumin by
glutaraldehyde cross-linking (12).
-scintillation. Nonspecific binding was determined by
preincubating cells for 20 min with a 100-fold molar excess of
unlabeled NDSK, NDSK I, NDSK II, or NDSK 325, prior to the addition of
the respective 125I-labeled derivatives.
15-42 (25 µg/ml) or RGDS
(0.5 mM), were preincubated with the cells for 20 min prior
to the addition of the 125I-NDSKs. For assessment of the
potential inhibitory role of heparin, heparin was preincubated at
increasing concentrations with the purified 125I-NDSK II
(33). Following incubation of HUVEC with the 125I-NDSK
fragments, binding was quantitated as described above.
scintillation.
15-21 sequence (36). Immunoreactive bands were visualized with a horseradish peroxidase-conjugated secondary antibody
and the ECL detection system. The 125I-labeled NDSK
derivatives were analyzed by autoradiography of dried gels. HUVEC and
HADMEC cadherins were separated on 7.5% polyacrylamide gels, under
reducing conditions, and Western blots performed using either the
monoclonal antibody against human VE-cadherin or the polyclonal
anti-pan-cadherin antibody, each at 10 µg/ml. Immunoreactive bands
were visualized using the ECL detection system.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, B, and chains held
together by disulfide bonds. The NDSK can be treated with thrombin,
Atroxin, or protease III to generate modified NDSK derivatives,
(i.e. NDSK II, NDSK I, or NDSK 325, respectively). Table
I summarizes the composition of these
derivatives. Each NDSK fragment, when run under nonreducing conditions
on 12% SDS-PAGE, appears as a heterogeneous group of bands as depicted
(Fig. 1A). The heavier,
lighter-staining bands have been observed in other studies involving
CNBr degradation of fibrinogen (28, 37), and are most likely due to the
presence of oxidized methionine residues that are resistant to CNBr
cleavage thus resulting in incomplete cleavage at the C termini of the fragments (37). The N-terminal residues, however, remain completely intact as demonstrated by N-terminal sequence analysis of fibrinogen NDSK, fibrin NDSK I, and fibrin NDSK II (37, 38). Thus, as the
N-terminal residues of fibrinogen-derived NDSK are cleaved with either
thrombin (Fig. 1, lane 2), Atroxin (Fig. 1,
lane 3), or protease III (Fig. 1, lane
4), the entire group of bands migrates at their new lower
molecular weights compared with the untreated fibrinogen NDSK (Fig. 1,
lane 1). The major bands of NDSK, NDSK II, NDSK
I, and thrombin-treated NDSK 325 migrate at 63.7, 58, 61, and 54 kDa,
respectively (Fig. 1A).
Generation and biochemical structure of NDSK derivatives
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Fig. 1.
. SDS-PAGE and immunoblot analysis of NDSK
fragments. The NDSK of human fibrinogen was generated by chemical
degradation with CNBr and was purified by Sephadex G-100
chromatography. The purified fibrinogen NDSK was treated for 3 h at
37 °C with thrombin, Atroxin, or protease III followed by thrombin
to generate NDSK II, NDSK I, or thrombin-treated NDSK 325, respectively. The NDSK fragments were run on 12% SDS-polyacrylamide
gels and visualized either by Coomassie staining (A) or by
Western blotting with the monoclonal antibody T2G1 against the human
fibrin 15-21 sequence (B). Positions of molecular markers
are indicated in the margin. Lane 1, NDSK at 63.7 kDa;
lane 2, NDSK II at 58 kDa; lane 3, NDSK I at 61 kDa; lane 4, thrombin-treated NDSK 325 at 54 kDa.
15-21 sequence in order to identify the antigenic exposure of this sequence (36). As shown in Fig.
1B, although present in NDSK (lane 1),
NDSK II (lane 2), and NDSK I (lane
3), the 15-21 sequence is available as an antigenic epitope only in NDSK II (lane 2), which was
generated by cleavage of fibrinopeptide B with thrombin.
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Fig. 2.
Time course of 125I-NDSK II total
binding to HUVEC. HUVEC suspensions (250,000 cells/ml) at 4 °C
or monolayers (500,000 cells/ml) at 37 °C were incubated with
125I-NDSK II (20 nM). At the indicated time
points, duplicate aliquots were collected and the bound ligand was
separated from free by centrifugation of cells through silicone oil,
followed by scintillation counting of cell pellets. The
ordinate to the left represents the scale of
total 125I-NDSK II bound to HUVEC suspensions (
); the
ordinate to the right represents the scale of
total 125I-NDSK II bound to HUVEC monolayers (
).
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Fig. 3.
Reversibility of
125I-NDSK II binding to HUVEC by
dilution. HUVEC suspensions (250,000 cells/ml) were
incubated at 4 °C with 125I-NDSK II (20 nM)
in two separate samples, one without and one with prior addition of
100-fold molar excess of unlabeled NDSK II. After 20 min, the cells
were pelleted by centrifugation, washed, and resuspended in the same
volume of buffer without added NDSK II. At sequential time points,
aliquots in duplicate were collected from each sample and bound ligand
was separated from free by centrifugation of cells through silicone
oil, followed by scintillation counting of cell pellets. Binding of
125I-NDSK II in the absence of unlabeled NDSK II
(Total Bound); binding of 125I-NDSK
II in the presence of 100-fold molar excess of unlabeled NDSK II
(Nonspecific). Binding was expressed as percentage of the
total 125I-NDSK II bound at time 0. The data shown is a
representative example of three separate experiments.
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Fig. 4.
Saturation isotherm and Scatchard analysis of
125I-NDSK II binding to HUVEC. Increasing
concentrations of 125I-NDSK II were incubated, without or
with prior addition of 100-fold excess unlabeled NDSK II, at 4 °C
with HUVEC (250,000 cells/ml) suspensions. Bound ligand was separated
from free by centrifugation of cells through silicone oil, followed by
scintillation counting of cell pellets. Specific binding () was
calculated by subtracting nonspecific binding (
), measured as the
residual binding in the presence of 100-fold molar excess of unlabeled
NDSK II at each concentration, from the total binding (
). The data
shown are a representative of five separate experiments.
Inset, Scatchard plot of NDSK II binding to HUVEC. Analysis
of the results using the nonlinear curve-fitting program, LIGAND (34),
defined a one-site binding isotherm for NDSK II with a
KD of 7.5 nM and
Bmax = 29,000 molecules/cell.
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Fig. 5.
Specific binding of various
125I-NDSK derivatives to HUVEC. Specific binding
of each 125I-NDSK derivative was determined by incubation
for 20 min at 4 °C with HUVEC suspensions (A) or at
37 °C with HUVEC monolayers (B), with
(Nonspecific) or without (Total Bound)
a prior 20-min incubation with a 100-fold molar excess of each
respective unlabeled NDSK fragment. 125I- NDSKs were added
to a final concentration of 20 nM. Binding of
125I-NDSKs was determined following centrifugation of cells
through silicone oil. NDSK II, thrombin-treated; NDSK, untreated; NDSK
I, Atroxin-treated; NDSK 325, protease III-treated. Values for total
and nonspecific binding represent the means ± S.D. of duplicate
measurements from three to six separate binding experiments; *,
p < 0.05.
15-42 masked either by
fibrinopeptides A and B or fibrinopeptide A alone, respectively), or
NDSK 325 (which lacks B15-42) manifest specific binding, these results suggest that the association of the N terminus of fibrin II
with the endothelial cell is most likely mediated by the 15-42 sequence, which becomes functionally unmasked only upon cleavage of
fibrinopeptide B (see Fig. 1B). To assess this possibility more directly, we conducted binding studies with excess 15-42 peptide as an inhibitor. When the 15-42 sequence was cross-linked to ovalbumin and preincubated with HUVEC suspensions, binding of
125I-NDSK II was inhibited to the level observed with
100-fold molar excess of unlabeled NDSK II, whereas inhibition with
ovalbumin alone was insignificant (Fig.
6). These data support the concept that
15-42 mediates specific binding of NDSK II to HUVEC.
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Fig. 6.
Inhibition of 125I-NDSK II
binding to HUVEC by a 15-42-ovalbumin conjugate. HUVEC
suspensions were preincubated with either unlabeled NDSK II (2 µM), the 15-42 peptide coupled to ovalbumin (25 µg/ml) or ovalbumin alone (25 µg/ml) for 20 min at 4 °C.
125I- NDSK II was added to a final concentration of 20 nM and allowed to proceed for 20 min at 4 °C. Binding of
125I-NDSK II was then determined following centrifugation
of cells through silicone oil. 125I-labeled NDSK II in the
absence of inhibitor (CONT), unlabeled NDSK II
(100X), 15-42 coupled to ovalbumin
(15-42-OVA), and ovalbumin (OVA). Binding was
expressed as counts per minute (CPM) of
125I-NDSK II bound to HUVEC. The data shown are means ± S.D. of duplicate measurements from four experiments. *,
p < 0.05.
1 and 3 integrins (16, 39); therefore, we considered whether these cations might also influence the binding of
NDSK II to HUVEC. In these experiments, 125I-NDSK II (20 nM) was incubated with HUVEC suspensions with added CaCl2, MgCl2, MnCl2, or EDTA, and
total binding was measured after 20 min of incubation at 4 °C. The
addition of divalent cations or EDTA failed to either stimulate or
inhibit 125I-NDSK II binding (data not shown), establishing
that NDSK II binding to HUVEC is a divalent cation-independent process.
v3 (15, 40). In agreement with these
findings, the RGDS peptide, which corresponds to the integrin-binding
domain of fibrinogen, also failed to inhibit NDSK II binding to HUVEC (data not shown). Taken together, these data support the conclusion that integrins did not mediate NDSK II binding to HUVEC under the
conditions tested. Unlike previous studies of fibrin N-terminal fragment binding to HUVEC (33), heparin (5-500 µg/ml) did not inhibit the binding of NDSK II to HUVEC in our system, suggesting that
the fibrin receptor is not a heparan proteoglycan and that binding of
heparin to the heparin-binding domain (15-42) did not interfere
with NDSK II binding to the fibrin receptor of endothelial cells.
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Fig. 7.
Effect of anti-cadherin monoclonal antibodies
on 125I-NDSK II binding to HUVEC. HUVEC
suspensions were preincubated with unlabeled NDSK II (2 µM), or monoclonal antibodies (5-10 µg/ml) against
N-cadherin (clone NCAD2) or VE-cadherin (clone 75) for 20 min prior to
addition of 125I-NDSK II (20 nM) and further
incubation for 20 min at 4 °C. Binding of 125I-NDSK II
was determined following centrifugation of cells through silicone oil.
125I-labeled NDSK II in the absence of antibody (CONT),
unlabeled NDSK II (100X), anti-VE-cadherin
(VE-CAD), anti-N-cadherin (N-CAD). Binding was
expressed as counts per minute (CPM) of
125I-NDSK II bound to HUVEC. The data shown are means ± S.D. of duplicate measurements from four experiments.
*p < 0.05.
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Fig. 8.
Co-immunoprecipitation of
125I-NDSK II with HUVEC cadherins. Confluent
HUVEC monolayers (~40 × 10 6 cells) were lysed in a
buffer containing 0.5% SDS and 1% Nonidet P-40 and brought to a final
detergent concentration of 0.1% SDS and 1% Nonidet P-40. The
particulate fraction was separated by centrifugation at 12,000 × g for 15 min, and the soluble fraction was incubated with
125I-NDSK II (30 nM) for 30 min at 25 °C.
Immunoprecipitations were carried out with 50 µg/ml of either an
anti-pan cadherin IgG (Pan-Cad IgG) or a control rabbit IgG
(Rabbit IgG). The immune complexes were collected
by precipitation with Protein A-Sepharose, subjected to SDS-PAGE under
reducing conditions on a 7.5% gel, transferred to nitrocellulose, and
immunoblotted with either the pan-cadherin polyclonal antibody
(A) or the VE-cadherin monoclonal antibody (B).
Lane numbers in both A and
B correspond to the following: lane 1,
control HUVEC lysate; lane 2, pan-cadherin IgG
immunoprecipitate; lane 3, rabbit IgG
immunoprecipitate. Co-immunoprecipitated 125I-NDSK II was
quantitated by scintillation counting of 10-µl aliquots of the
precipitated immune complexes (C). The data show the
means ± S.D. of four separate experiments expressed as percentage
of total 125I-NDSK II immunoprecipitated with a
pan-cadherin IgG.
chain was not involved in binding to
VE-cadherin. The higher level of specific binding of
125I-NDSK II in this system, as compared with the binding
suspension experiments and immunoprecipitations, is probably due to the
capacity of the immunocapture method to reduce nonspecific binding by
removal of other cell surface molecules that are washed away after
immunocapture.
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Fig. 9.
Saturation isotherm and specificity of
125I-NDSK II binding to captured HUVEC
cadherins. Multiwell dishes were coated with either a
pan-cadherin or a nonimmune IgG and incubated for 2 h at 37 °C
with HUVEC lysates solubilized in 0.5% SDS and 1% Nonidet P-40, as
described under "Experimental Procedures." The wells were washed,
incubated with 20 nM 125I-NDSK II for 20 min at
25 °C, washed again, and scraped into Laemmli sample buffer,
followed by SDS-PAGE (under reducing conditions on a 7.5% gel), and
Western blotted with the VE-cadherin monoclonal antibody
(A). Lane numbers correspond to the
following: lane 1, control HUVEC lysate;
lane 2, pan-cadherin IgG immunocaptured material;
lane 3, rabbit IgG immunocaptured material. The
pan-cadherin-coated wells were incubated with increasing concentrations
of 125I-NDSK II for 20 min at 25 °C, the wells were
washed, scraped into 1 M NaOH, and the amount of bound
125I-NDSK II analyzed by scintillation counting
(B). HUVEC were lysed and incubated in antibody-coated
wells, followed by the addition of 20 nM each of
125I-labeled NDSK II, NDSK, or NDSK 325 for 20 min at
25 °C (C). For determination of nonspecific binding,
preincubation with 100-fold excess of the unlabeled NDSK II
(Nonspecific) was carried out, for 20 min at 25 °C, prior
to the addition of 125I-NDSK II. The wells were washed,
scraped into 1 M NaOH, and analyzed by scintillation
counting. Values represent the means ± S.D. of duplicate
measurements from three to five experiments and are expressed as counts
per minute (CPM) of 125I-NDSK II bound to HUVEC
captured in wells coated with a polyclonal pan-cadherin IgG (, 
)
or rabbit IgG (
, 
). *, p < 0.05.
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Fig. 10.
Western blot and cadherin capture assays of
HUVEC and HADMEC. Confluent monolayers (~3 × 106 cells) of HUVEC and HADMEC were extracted into boiling
Laemmli sample buffer and run on 7.5% polyacrylamide gels under
reducing conditions. The proteins were transferred to nitrocellulose
paper, and Western blots were performed (A) using the
monoclonal antibody against human VE-cadherin at a concentration of 10 µg/ml to visualize VE-cadherin in HUVEC (lane
1) or HADMEC (lane 2). For the capture
assays (B), confluent HUVEC and HADMEC monolayers were lysed
in a buffer containing 0.5% SDS and 1% Nonidet P-40 (250,000-500,000
cells/ml), as described under "Experimental Procedures," and
incubated in wells coated with pan-cadherin IgG for 2 h at
37 °C. The wells were washed and incubated with 20 nM
125I-NDSK II for 20 min 25 °C (Total
Bound). Preincubation with 100-fold molar excess of the
unlabeled NDSK II (Nonspecific) was carried out in certain
wells prior to the addition of 125I-NDSK II for 20 min at
25 °C. The wells were washed, scraped into 1 M NaOH, and
analyzed by scintillation counting. Values represent the means ± S.D. of duplicate measurements from four separate experiments and
are expressed as counts per minute (CPM) of
125I-NDSK II bound to HUVEC or HADMEC captured in wells
coated with pan-cadherin IgG. *, p < 0.05.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1-51, B 1-118, and 1-78 (27, 38). Various forms of the NDSK
lacking either fibrinopeptides A and B (NDSK II), fibrinopeptide A only
(NDSK I), or the sequence B1-42 (NDSK 325) can be prepared by
treatment of the NDSK of fibrinogen with either thrombin, Atroxin, or
protease III, respectively.
15-42 sequence, specifically binds with high affinity to HUVEC suspensions in a time- and
concentration-dependent manner, which is saturable,
reversible, and cation-independent. In contrast, the derivatives in
which 15-42 is not exposed (NDSK and NDSK I) or is absent (NDSK
325) did not specifically bind to HUVEC. Furthermore, binding of NDSK
II was inhibited by a 15-42/ovalbumin conjugate, indicating that
this sequence is responsible for mediating the fibrin-endothelial cell
interaction. The free peptide itself possesses low functional activity
(5, 12), perhaps because it differs in conformation from its native
dimeric form in the fibrin molecule. NDSK II also specifically bound to
HUVEC monolayers in a time-dependent matter, which
demonstrated less nonspecific binding than in suspension, although the
total number of molecules bound per cell was lower. This may be due to
the participation of a portion of the available VE-cadherin molecules
in the formation of intercellular contacts. It should be emphasized
that the binding of NDSK II to HUVEC is clearly distinct from our
previous report (26) of fibrinogen binding to HUVEC, which requires
2 h to plateau, is only partially reversible, and requires
divalent cations. Binding is also distinct from that of NDSK fragment
binding to macrophages (28), which occurs predominantly through the
neo-N terminus of the chain.
117-133 sequence at the C terminus of fibrinogen (18, 19). In our
present study, however, we found that antibodies against PECAM-1 or
ICAM-1 did not inhibit the binding of NDSK II to endothelial cells, in
agreement with our previous report of the failure of anti-PECAM-1
antibodies to inhibit fibrin II-induced capillary tube formation (9).
Of note, the 117-133 sequence that binds to ICAM-1 is lost upon
cleavage of fibrinogen with cyanogen bromide, thus eliminating the
possibility of its interference in the binding of NDSK II to
endothelial cells.
15-42 (49), is likely involved in mediating the observed binding
of fibrin fragments, potentially to a heparan sulfate-containing
proteoglycan (33). In contrast, in our system, preincubation of
purified NDSK II with heparin did not have any appreciable effect on
the binding of NDSK II. These differences may be due to differences in
the experimental systems. Alternatively, it may be that the binding of
NDSK II to HUVEC VE-cadherin has a higher affinity than does the
putative proteoglycan-like receptor, and so is preferentially active in
our studies.
E7 integrin of mucosal T lymphocytes
(56). Based on our present evidence, the EC1 and/or EC2 domains of
VE-cadherin participate in a heterophilic interaction with the
15-42 sequence of fibrin. This conclusion is based on the findings
that (a) binding of NDSK II to HUVEC was inhibited by a
monoclonal antibody against VE-cadherin, (b) immunoprecipitation experiments with a pan-cadherin antibody
co-immunoprecipitated NDSK II with HUVEC cadherins, and (c)
NDSK II bound with high specificity to isolated HUVEC cadherins that
had been captured by a pan-cadherin antibody. It is important to note
that the degree of specific NDSK II binding to captured cadherins was
89% compared with 51% for NDSK II binding to whole cells. Perhaps, by
isolating the cadherins, potential interference from cellular
components that participate in nonspecific interactions with NDSK II
was removed. Finally, NDSK II did not bind to cadherins captured from HADMEC, which contain N-cadherin but not VE-cadherin as assessed by
Western blot and immunofluorescence experiments, thus demonstrating indirectly that NDSK II functions specifically as a heterophilic ligand
for VE-cadherin. The results of our investigation are consistent with
the report of the isolation of an unidentified 130-kDa protein from
HUVEC lysates, which is clearly not PECAM-1, by affinity chromatography
over fibrin 15-42/Sepharose (12).
15-42 sequence at the neo-N terminus of the
fibrin chain exposed upon thrombin-mediated cleavage of
fibrinopeptide B. Thus, we have identified both a new fibrin-specific receptor as well as the first heterophilic ligand for VE-cadherin that
demonstrates binding independent of divalent cations. Because of the
central role of fibrin in blood coagulation, wound healing, and tumor
growth and of VE-cadherin in endothelial cell function, these findings
have broad implications in the understanding of the fibrin association
with vascular endothelium in processes such as hemostasis and
thrombosis, as well as in signaling cascades which may play a role in a
variety of endothelial cell functions including the induction of
capillary-tube formation during the angiogenic process.
ACKNOWLEDGEMENTS
FOOTNOTES
Dunglison M.D./Ph.D. candidate; recipient of a predoctoral
fellowship from the American Heart Association, Southeastern
Pennsylvania Affiliate.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y.-H. Guo, I. Hernandez, B. Isermann, T.-b. Kang, L. Medved, R. Sood, E. J. Kerschen, T. Holyst, M. W. Mosesson, and H. Weiler Caveolin-1-dependent apoptosis induced by fibrin degradation products Blood, April 30, 2009; 113(18): 4431 - 4439. [Abstract] [Full Text] [PDF]
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 M. Guo, D. Daines, J. Tang, Q. Shen, R. M. Perrin, Y. Takada, S. Y. Yuan, and M. H. Wu Fibrinogen-{gamma} C-Terminal Fragments Induce Endothelial Barrier Dysfunction and Microvascular Leak via Integrin-Mediated and RhoA-Dependent Mechanism Arterioscler. Thromb. Vasc. Biol., March 1, 2009; 29(3): 394 - 400. [Abstract] [Full Text] [PDF]
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R. A. Campbell, K. A. Overmyer, C. R. Bagnell, and A. S. Wolberg Cellular Procoagulant Activity Dictates Clot Structure and Stability as a Function of Distance From the Cell Surface Arterioscler. Thromb. Vasc. Biol., December 1, 2008; 28(12): 2247 - 2254. [Abstract] [Full Text] [PDF]
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D. Vestweber VE-Cadherin: The Major Endothelial Adhesion Molecule Controlling Cellular Junctions and Blood Vessel Formation Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): 223 - 232. [Abstract] [Full Text] [PDF]
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 J. Cho, J. L. Degen, B. S. Coller, and D. F. Mosher Fibrin but Not Adsorbed Fibrinogen Supports Fibronectin Assembly by Spread Platelets: EFFECTS OF THE INTERACTION OF {alpha}IIb{beta}3 WITH THE C TERMINUS OF THE FIBRINOGEN {gamma}-CHAIN J. Biol. Chem., October 21, 2005; 280(42): 35490 - 35498. [Abstract] [Full Text] [PDF]
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A. M. Belkin, G. Tsurupa, E. Zemskov, Y. Veklich, J. W. Weisel, and L. Medved Transglutaminase-mediated oligomerization of the fibrin(ogen) {alpha}C domains promotes integrin-dependent cell adhesion and signaling Blood, May 1, 2005; 105(9): 3561 - 3568. [Abstract] [Full Text] [PDF]
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I. P. Michael, G. Sotiropoulou, G. Pampalakis, A. Magklara, M. Ghosh, G. Wasney, and E. P. Diamandis Biochemical and Enzymatic Characterization of Human Kallikrein 5 (hK5), a Novel Serine Protease Potentially Involved in Cancer Progression J. Biol. Chem., April 15, 2005; 280(15): 14628 - 14635. [Abstract] [Full Text] [PDF]
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 G. Bazzoni and E. Dejana Endothelial Cell-to-Cell Junctions: Molecular Organization and Role in Vascular Homeostasis Physiol Rev, July 1, 2004; 84(3): 869 - 901. [Abstract] [Full Text] [PDF]
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 R. A. Adams, M. Passino, B. D. Sachs, T. Nuriel, and K. Akassoglou Fibrin Mechanisms and Functions in Nervous System Pathology Mol. Interv., June 1, 2004; 4(3): 163 - 176. [Abstract] [Full Text] [PDF]
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 V. Fontaine, Z. Touat, E. M. Mtairag, R. Vranckx, L. Louedec, X. Houard, B. Andreassian, U. Sebbag, T. Palombi, M.-P. Jacob, et al. Role of Leukocyte Elastase in Preventing Cellular Re-Colonization of the Mural Thrombus Am. J. Pathol., June 1, 2004; 164(6): 2077 - 2087. [Abstract] [Full Text] [PDF]
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F. Zhang, J. Cheng, N. R. Hackett, G. Lam, K. Shido, R. Pergolizzi, D. K. Jin, R. G. Crystal, and S. Rafii Adenovirus E4 Gene Promotes Selective Endothelial Cell Survival and Angiogenesis via Activation of the Vascular Endothelial-Cadherin/Akt Signaling Pathway J. Biol. Chem., March 19, 2004; 279(12): 11760 - 11766. [Abstract] [Full Text] [PDF]
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M. Cieslak, J. Niewiarowska, M. Nawrot, M. Koziolkiewicz, W. J. Stec, and C. S. Cierniewski DNAzymes to beta 1 and beta 3 mRNA Down-regulate Expression of the Targeted Integrins and Inhibit Endothelial Cell Capillary Tube Formation in Fibrin and Matrigel J. Biol. Chem., February 22, 2002; 277(9): 6779 - 6787. [Abstract] [Full Text] [PDF]
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M. Corada, F. Liao, M. Lindgren, M. G. Lampugnani, F. Breviario, R. Frank, W. A. Muller, D. J. Hicklin, P. Bohlen, and E. Dejana Monoclonal antibodies directed to different regions of vascular endothelial cadherin extracellular domain affect adhesion and clustering of the protein and modulate endothelial permeability Blood, March 15, 2001; 97(6): 1679 - 1684. [Abstract] [Full Text] [PDF]
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 B. Angst, C Marcozzi, and A. Magee The cadherin superfamily: diversity in form and function J. Cell Sci., January 2, 2001; 114(4): 629 - 641. [Abstract] [PDF]
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S. L. Harley, J. Sturge, and J. T. Powell Regulation by Fibrinogen and Its Products of Intercellular Adhesion Molecule-1 Expression in Human Saphenous Vein Endothelial Cells Arterioscler. Thromb. Vasc. Biol., March 1, 2000; 20(3): 652 - 658. [Abstract] [Full Text] [PDF]
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S. M. Dallabrida, M. A. De Sousa, and D. H. Farrell Expression of Antisense to Integrin Subunit beta 3 Inhibits Microvascular Endothelial Cell Capillary Tube Formation in Fibrin J. Biol. Chem., October 6, 2000; 275(41): 32281 - 32288. [Abstract] [Full Text] [PDF]
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