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INTRODUCTION |
The study of vascular development is important because of the
fundamental embryological principles that underlie this complex and
essential process, and the potential for therapeutic manipulation of
vascular formation in human disease processes. The assembly of
endothelial cells into vascular structures requires specific signals
from luminal surface receptors that are activated by soluble ligands,
and signals from the extracellular matrix mediated through receptors on
the abluminal surface of the endothelial cell (1, 2).
Virtually all molecules of the matrix are able to communicate to cells
by binding to integrins (3, 4). The integrin receptors are
noncovalently associated heterodimeric glycoproteins that reside in the
cell membrane where their cytoplasmic domains connect to elements of
the cytoskeleton. These molecules support a variety of functions,
including cell attachment and migration, and activation of cytoplasmic
signaling molecules (4, 5). A number of different integrin receptor
complexes have been identified on the surface of endothelial cells, and
linked to specific functions (2). Data from recent experiments indicate
that interaction of the
v
3 receptor with its ligands is critical
for tumor and cytokine-induced angiogenesis, through a cell survival
function mediated by this integrin (6). Also,
v
3-mediated
signaling through the Ras-mitogen activated protein kinase
(MAPK)1 pathway, specifically
through sustained activation of MAPK, appears to be critical for the
angiogenic program (7).
To search for new molecular pathways of vascular development, we have
recently characterized a locus identified by an enhancer trap event in
a line of transgenic mice (8). The gene located in this locus, Del1,
was shown to be expressed in an endothelial-restricted pattern in early
embryonic development. The proteins encoded in the locus were
characterized by genomic and cDNA cloning. The isoform encoded by
the common transcript, Del1 major (Del1 MJR), was found to contain a
signal sequence, three EGF-like repeats, and two discoidin I-like
domains. A less frequent transcript, Del1 minor (Del1 MNR), was noted
to be truncated in the amino-terminal discoidin I-like domain due to
alternative splicing. Both transcripts had an RGD motif in the B loop
of the second EGF-like repeat. Preliminary data suggested that Del1 MJR
is a matrix protein, and that the RGD motif could mediate endothelial
cell attachment through integrin ligation. In vitro and
in vivo functional studies suggested that Del1 may have a
complex role in the regulation of vascular formation, serving to
inhibit endothelial cell differentiation or contribute to the complex
process of vascular remodeling. Such a functional role was not easily
reconciled with the molecular interaction of Del1 with the
v
3
integrin receptor.
To further characterize the ability of Del1 to initiate
integrin-mediated events, and to investigate whether Del1 ligation of
integrin receptors triggers signaling pathways similar to other RGD
ligands, a series of experiments were conducted with recombinant baculovirus proteins. Cell attachment assays revealed that Del1 could
mediate endothelial cell attachment at concentrations similar to that
observed with known matrix proteins such as vitronectin (VN) and
fibronectin (FN), and that the majority of this activity was mediated
through
v
3. Also, Del1 was shown to mediate endothelial cell
migration through binding to this integrin. Endothelial cells on Del1
were found to form focal complexes that contained phosphorylated proteins characteristic of integrin signaling. In addition, studies employing an in ovo chick CAM assay have documented a novel
angiogenic activity of Del1 that is dependent on
v
3 signaling.
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EXPERIMENTAL PROCEDURES |
Matrix Components and Chemicals--
Human VN (Life
Technologies, Inc., Grand Island, NY) was resuspended in Dulbecco's
phosphate-buffered saline (DPBS) to a final concentration of 14 µM and stored frozen until further use. Cycloheximide, FN, protease inhibitors such as leupeptin, aprotinin, and
phenylmethylsulfonyl fluoride, the tyrosine phosphatase inhibitor
sodium orthovanadate, and protein A-Sepharose CL-4B were all purchased
from Sigma.
RGD Peptides and Antibodies--
Synthetic peptides were
purchased from Life Technologies, Inc. and correspond to sequences
GRGDSP, GRGESP, GPenGRGDSPCA (Pen RGD), where Pen stands for
penicillamine. The Del1 peptide represents amino acids 89-106,
CEISEAYRGDTFIGYBCK, was synthesized as a linear molecule, and dissolved
in dimethyl sulfoxide. Other peptides were dissolved in DPBS at 2 mg/0.1 ml, and stored until further use. Monoclonal antibodies to
integrins
v
3 (clone LM609),
v
5 (clone PIF6),
1 (clone
JB1a),
2
1 (VLA 2),
v (clone CLB-706),
2 (clone JBS2), and
6 (clone CLB-701) were purchased from Chemicon International,
Temecula, CA (9-12). A monoclonal antibody to human
3 integrin
(GPIIIa) residues 348-426 (clone AP3) was a generous gift from Dr.
Peter J. Newman, The Blood Center of Southeastern Wisconsin. Monoclonal
antibodies to vinculin (clone VIN-11-5), talin (clone 8d4), and
fluorescein isothiocyanate-conjugated phallicidin were purchased from
Sigma (13, 14). Monoclonal anti-phosphotyrosine antibody (clone 4G10),
anti-Shc polyclonal antibody, and anti-MAPK polyclonal antibody were
purchased from Upstate Biotechnology, Lake Placid, NY (15).
Phospho-specific MAPK antibody was purchased from New England Biolabs,
Beverly, MA. Polyclonal anti-p125FAK (C-20) was purchased
from Santa Cruz Biotechnology (16). Cy3-conjugated goat
anti-rabbit and goat anti-mouse immunoglobulins were
purchased from Jackson Immunoresearch Laboratories, West
Grove, PA.
cDNA Constructs and Recombinant Protein
Expression--
Recombinant bacterial Del1 protein was produced in
Escherichia coli as described previously (8). Recombinant
Del1 proteins were also expressed in baculovirus. cDNA fragments
encoding the Del1 MJR, Del1 MNR, and Del1 MJR protein with RAD
substituted for the RGD motif (Del1 RAD) were cloned into the shuttle
vector pACGP67B and transfected into Sf9 cells with the
Baculogold reagents (Pharmingen, San Diego, CA). Del1 RAD was derived
by polymerase chain reaction mutagenesis as per established protocols
(17). Recombinant baculovirus expressing Del1 proteins were generated and amplified as per the suppliers instructions (Pharmingen, San Diego,
CA). Protein expression was confirmed by Western blotting of lysates of
infected Sf9 cells with polyclonal antisera to Del1. Recombinant
protein was purified with His-Bind resin (Novagen, Madison, WI), and
subsequently evaluated by SDS-PAGE according to well established
methodology (18).
Human 143B osteosarcoma cells were engineered to express high levels of
the murine Del1 MJR, through stable transfection and selection of
clones by Northern and Western blot (8). A 7-step column based
purification strategy was developed to purify the expressed Del1 MJR
protein from conditioned media derived from these cells, employing
heparin-Sepharose, gel filtration, and ion exchange resins. Mammalian
cell protein purified in this fashion was estimated to be at least 95%
pure by SDS-PAGE, and documented to be endotoxin free by limulus assay.
Cell Culture--
Bovine aortic endothelial cells (BAEC) and
human umbilical vein endothelial cells (HUVEC) were a kind gift from
Dr. D. Vaughan (Vanderbilt University, Nashville, TN). BAEC were
maintained in complete DMEM supplemented with 10% fetal bovine serum,
100 units/ml penicillin, and 100 µg/ml streptomycin. HUVEC were
maintained in EGM medium (Clonetics, San Diego, CA) supplemented with
20% fetal bovine serum and growth factors. Cells were cultured on 1%
gelatin-coated tissue culture dishes and employed in experiments between passages 4-7.
Adhesion Assays--
Test adhesive substrates were diluted in
DPBS to stated concentrations, and 50 µl/well were added to 96-well
Maxisorp plates (Nunc, Naperville, IL) and placed overnight at 4 °C.
After coating, wells were rinsed with DPBS, and nonspecific sites were
blocked with 10 mg/ml BSA at 37 °C for 1 h. In some
experiments, cells were pretreated with 25 µg/ml cycloheximide for
2 h and maintained in the drug during the entire assay period.
Cells were detached with minimum trypsinization (1 to 2 min), followed
by addition of soybean trypsin inhibitor at a concentration of 0.5 mg/ml. Pelleted cells were washed with trypsin inhibitor in DPBS, spun down again, and resuspended in Dulbecco's modified Eagle's medium (DMEM) with 1 mg/ml BSA. Approximately 30,000 cells/100 µl were applied to each well and allowed to adhere for 60-90 min at 37 °C.
In experiments where RGD peptides or soluble adhesive ligands were
used, cells were preincubated with the respective reagents for 30 min
at 37 °C prior to addition to the wells. For experiments with
integrin receptor blocking antibodies, cells were mixed with the
appropriate antibody with no preincubation, and allowed to attach to
the matrix for 15 min at 37 °C. For all experiments, nonadherent
cells were removed with warm DPBS, and the remaining cells were fixed
with 4% paraformaldehyde for 10 min. Cells were stained with 0.5%
toluidine blue for 5 min and rinsed with water. Stained cells were
solubilized with 1.0% SDS and quantified on a microtiter plate reader
at 595 nm. Experiments described were performed in quadruplicate and
repeated a minimum of three times. Results from a representative
experiment are presented as mean and standard deviation. The adhesion
assay protocol was validated in our experimental conditions as
described previously (19).
Migration Assays--
Test substances were diluted to
appropriate concentrations in DMEM containing 200 µg/ml BSA and
placed in the bottom wells of a modified Boyden chemotaxis chamber
(Neuro Probe, Cabin John, MD), unless otherwise indicated. Standard
assays were performed with BAEC exactly as reported by Liaw et
al. (20). Blocking actions of monoclonal antibodies were evaluated
in migration experiments with HUVEC using transwell dishes as described
previously (21). Del1 (100 nM) was employed in assays with
50 µg/ml anti-
v
3 (clone LM609), 25-50 µg/ml anti-
v
5
(clone P156), 1:20 dilution anti-
1 (clone JB1a), and 20-40 µg/ml
anti-
2
1 (JBS2). Integrin receptor blocking antibodies and cells
were applied to the wells simultaneously. Each experiment was performed
in quadruplicate and repeated at least three times.
Immunofluorescence Analysis--
Coverslips were coated
overnight at 4 °C with the adhesive substrates diluted in DPBS at a
final concentration of 100 nM, washed 3 times with DPBS,
and blocked with 10 mg/ml BSA in DPBS for 3 h at 37 °C. After
growing cells on the coverslips for 3 h, they were fixed with 4%
paraformaldehyde at room temperature for 15 min, permeabilized with
acetone for 3 min, and stained with fluorescein
isothiocyanate-conjugated phallicidin. For vinculin, phosphotyrosine,
and anti-
3 clone (AP3) staining, cells were fixed with 4%
paraformaldehyde at room temperature for 15 min and permeabilized for 2 min with 0.2% Triton X-100 in 50 mM Tris, pH 7.5. The
monoclonal anti-vinculin antibody (VIN-11-5, Sigma) was used at a 1:60
dilution, the anti-phosphotyrosine monoclonal antibody at a dilution of
1:100, and anti-
3 at 1:50 dilution. For talin staining, cells were
fixed with methanol for 10 min on ice and the anti-talin monoclonal
used at a 1:100 dilution. Application of all monoclonal antibodies was
followed by a secondary Cy3-conjugated goat anti-mouse antibody
(Jackson Laboratories, Inc.). All antibodies were applied in DPBS
containing both 3% BSA and 2% goat serum. All primary and secondary
antibody incubations were carried out for 1 h each at
37 °C.
Immunoprecipitations and Western Blotting of Phosphorylated
Proteins--
Thirty-five-mm tissue culture dishes (Costar, Cambridge,
MA) were coated overnight at 4 °C with VN, FN, or Del1 in DPBS at 100 nM final coating concentration. Subconfluent BAEC were
serum starved with 0.5% serum for 24 h. Before addition of cells,
coated dishes were washed 3 times with DPBS and nonspecific sites were blocked with 10 mg/ml BSA in DMEM for 1 h at 37 °C. BAEC were harvested as described for the adhesion assay, suspended in DMEM containing 1 mg/ml BSA and allowed to stand for 20 min at 37 °C. Approximately 1.5-2 × 106 cells were applied to each
dish and allowed to adhere. After 30, 60, or 120 min at 37 °C, cells
were lysed with ice-cold RIPA lysis buffer or MAPK buffer (22). One
group of serum-starved BAEC were washed with DPBS and lysed in the
dish, and referred to as attached cells.
Cell lysates were clarified by centrifugation at 15,000 × g at 4 °C. Supernatants precleared with protein
A-Sepharose CL-4B (Sigma) for 1 h at 4 °C were subjected to
immunoprecipitation with anti-phosphotyrosine (clone 4G10) or
anti-p125FAK (C-20) antibodies overnight at 4 °C. The
following day, immune complexes were harvested after a 1-h incubation
with protein A-Sepharose CL-4B beads at 4 °C, washed 3 times with
ice-cold RIPA lysis buffer, boiled with 2 × Laemmli buffer for 5 min, resolved with 7.5% SDS-PAGE, and transferred to nitrocellulose
membranes. Immunoprecipitated tyrosine-phosphorylated proteins were
detected by incubating with anti-phosphotyrosine monoclonal antibody at
1:1000 dilution. At a similar dilution polyclonal
anti-p125FAK antibody was used to detect immunoprecipitated
p125FAK. To verify equivalent amounts of
p125FAK between samples, p125FAK
immunoprecipitations on Western blot were probed with
p125FAK antibody. All primary antibody incubations were
performed in blocking buffer overnight at 4 °C. Membranes were
extensively washed with TBST buffer followed by incubation with
horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit
antibody at 1:4000 dilution for 1 h at room temperature in
blocking buffer. After washing, immunoreactivity was detected by chemiluminescence.
For MAPK experiments, cell lysates were prepared in the MAPK lysis
buffer as described above in a total volume of 100 µl. Approximately
5-10% of the total lysates were resolved on a 12.5% SDS-PAGE and
subsequently subjected to Western blotting as above. An
anti-phospho-specific MAPK antibody that detects p42 and p44 MAPK only
when catalytically activated by phosphorylation at Tyr204
and Thr202 (New England Biolabs) was used as an
immunoprobe, followed by a secondary anti-rabbit horseradish
peroxidase-conjugated immunoglobulin. Total MAPK protein was evaluated
by probing the Western blots with a MAPK polyclonal antiserum. For
evaluation of phosphorylation of Shc, identical techniques were
employed as for p125FAK and MAPK, except
immunoprecipitation was performed with an anti-Shc antibody, and
evaluated on Western blot with antibodies to phosphotyrosine and Shc
(23).
In Ovo CAM Assays--
Recombinant Del1 protein expressed in
both mammalian cells and in insect cells was employed in in
ovo CAM assays according to well established methodology (24).
Ten-day-old embryonated chicken eggs were candled to illuminate blood
vessels under the shell and an area with a minimum of small blood
vessels thus identified. A window was cut in the egg shell and a filter
disc 5 mm in diameter saturated with equal volumes of bFGF at 59 nM (total of 30 ng/disc), Del1 proteins at 100 nM, or 1 µM (total of 0.2 and 2.0 µg/disk, respectively), or equal amounts of VN or FN at 1 µM were
placed on the CAM. The window was sealed with adhesive tape and the egg incubated for 4 days. In one group of experiments, Del1 or a buffer control were evaluated as putative inhibitors of bFGF induced angiogenesis by application to the filter disc or injection
intravenously 24 h later. In other experiments, the anti-
v
3
monoclonal antibody LM609 (75 µg of affinity purified antibody) or a
negative control antibody of the same isotype directed against an
myosin heavy chain class I marker (W632, 75 µg) were injected
intravenously at 24 h.
In all cases, CAMs were harvested on the fourth day of stimulation by
excising the region adherent to and surrounding the filter disc and
placed in a 30-mm culture dish. Vessels were then visualized in a
dissecting microscope and vessel branch points quantified within the
5-mm disc area. At least 10 embryos were used per treatment group. Data
was evaluated in terms of average number of branch points per treatment group.
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RESULTS |
Recombinant Del1 Mediates Endothelial Cell Attachment in an
RGD-dependent Manner--
Recombinant Del1 MJR, Del1 MNR,
and Del1 RAD proteins were expressed in Sf9 insect cells,
purified on a nickel column, and fractions were analyzed by SDS-PAGE
and Western blotting with polyclonal Del1 antisera. Both Del1 MJR and
Del1 RAD mutant proteins migrated at the expected size of 52 kDa, while
the MNR form resolved into multiple bands at approximately 25 kDa (Fig.
1, A and B). Multiple bands observed with electrophoresis of the MNR protein may
represent variation in glycosylation, other post-translational modification, or minor proteolytic degradation. Preimmune sera failed
to recognize any of the recombinant forms of Del1 protein (data not
shown).

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Fig. 1.
Recombinant Del1 protein mediates endothelial
cell attachment and this activity is RGD dependent. Insect cells
infected with high titer baculoviruses encoding recombinant His-tag
fusion proteins of Del1 major (Del1 MJR), Del1 minor (Del1 MNR), and
the RAD mutant of Del1 MJR (Del1 RAD) were harvested at 72 h
post-infection and expressed proteins were affinity purified. Eluted
fractions were analyzed with: A, 10% SDS-PAGE, and
B, Western blots probed with Del1 antisera. In both Del1 MJR
and Del1 RAD fractions, the antibody recognizes bands migrating at 52 kDa (arrowheads), the predicted size for Del1 MJR protein.
The Del1 MNR isoform of Del1 was resolved into multiple bands around 25 kDa (arrows), all recognized by the antibody. C,
the ability of recombinant baculovirus (BV) and bacterial
(BT) Del1 MJR protein, VN, and FN to mediate attachment of
BAEC was investigated. D, adhesion of BAEC to Del1 MNR, Del1
MJR, and Del1 RAD mutant protein was compared. Del1 MNR was less
effective as an attachment substrate. Most significant was the decrease
in cell attachment activity of Del1 MJR when the RGD motif was mutated
to RAD.
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Both BAEC and HUVEC adhered to wells coated with recombinant bacterial
and baculovirus forms of Del1, VN, and FN in a
dose-dependent manner (Fig. 1C and data not
shown). VN and FN were used as positive control adhesive substrates,
based on earlier reports of their ability to promote endothelial cell
adhesion (25). Dose-dependent adhesion was observed when
coating solutions containing up to 100 nM of either of the
two Del1 proteins were used, and half-maximal effect was observed using
solutions containing 7 nM Del1, 10 nM VN, and
10 nM FN. Cell adhesion to Del1 was thus comparable to that
observed with VN and FN substrates.
To investigate the role of the RGD motif in cell adhesion,
site-directed mutagenesis was employed to construct a cDNA encoding a form of Del1 MJR with an RAD motif in the B loop of EGF repeat 2, instead of RGD. This mutant, Del1 RAD, was compared with the wild-type
Del1 MJR, and the truncated protein that is encoded by the less common
alternative Del1 transcript, Del1 MNR. The Del1 MNR protein contains
the same signal sequence and three EGF-like regions as Del1 MJR, but
has only a small portion of the amino-terminal discoidin I-like domain
(8). High titer baculovirus encoding these three proteins were
generated and used to produce protein in Sf9 cells, and
recombinant proteins compared for their ability to mediate endothelial
cell attachment (Fig. 1, A and D). Adhesion of
BAEC to all the three forms of Del1 at 1, 10, 100, and 500 nM final coating concentrations were compared (Fig.
1D). Both Del1 MJR and Del1 MNR showed a
dose-dependent increase in attachment of BAEC, with a
half-maximal effect for Del1 MJR at 7 nM. In contrast, the
RAD mutant form of Del1 MJR supported cell attachment poorly, suggesting that the RGD motif in Del1 protein plays an important role
in endothelial cell adhesion.
Protein Competition Studies Suggest Adhesion to Del1 Is
Integrin-mediated--
The effect of soluble Del1 on the adhesion of
BAEC to extracellular matrix proteins was investigated. Microtiter
wells were coated overnight with the test ligands at a final
concentration of 100 nM, and cells were preincubated in
solutions containing 1, 10, 100, and 250 nM Del1 for 30 min
at 37 °C before applying them to the wells. Del1 protein was an
effective competitor of BAEC adhesion to VN, with 50% inhibition
obtained at 100 nM soluble Del1 protein (Fig.
2A).
Dose-dependent inhibition of BAEC adhesion to FN was also
observed, with a maximum of 40% at 250 nM final concentration of soluble Del1. These data suggest that Del1 is capable
of blocking a significant portion of the integrin-mediated endothelial
cell attachment to VN and FN, but that Del1 interacts with only a
subset of the receptors for each of these ligands.

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Fig. 2.
Soluble protein competition assays.
A, soluble Del1 MJR inhibits BAEC attachment to VN and FN in
a dose-dependent manner. B, in a similar
fashion, soluble VN and FN inhibit adhesion of BAEC to Del1.
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In a similar series of experiments, the ability of soluble VN and FN to
compete for cell adhesion to Del1 was investigated. The ability of BAEC
to adhere to microtiter wells coated with 100 nM final
coating concentration of Del1 in the presence of competing soluble
adhesive ligands 10, 100, and 250 nM soluble VN and FN. A
dose-dependent inhibition of BAEC adhesion to Del1 was
observed (Fig. 2B). Interestingly, an equivalent amount of inhibition was seen with both VN and FN, suggesting that Del1 may
employ integrin receptors that are recognized by both VN and FN.
RGD Peptide Competition Studies Confirm Adhesion to Del1 Is
Integrin-mediated--
In peptide blocking studies, BAEC adhesion to
Del1 at 10 nM coating concentration was inhibited with 100 µM GRGDSP hexamer peptide, with significant inhibition
seen with as low as 10 µM peptide (Fig.
3A). This inhibition of cell
attachment is similar to that seen with RGD peptides competing for
attachment to FN and VN substrates (Fig. 3B, and data not
shown). In contrast, even high concentration (500 µM
final) of the control GRGESP peptides showed no significant inhibition.
The concentrations of competing peptides used in these adhesion assays
are typical for RGD-containing peptides used to compete other
RGD-dependent binding activities (26). Synthetic peptides
corresponding to the RGD sequence in Del1 showed similar
dose-dependent inhibition of adhesion of BAEC to Del1
protein, with approximately 50% inhibition achieved with 100 µM peptide. The RGD-Del1 peptide could also compete for
attachment of BAEC to VN and FN substrates in a similar adhesion assay
(data not shown).

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Fig. 3.
RGD peptides inhibit vascular endothelial
cell adhesion to Del1. A, adhesion of BAEC to 10 nM Del1-coated wells was competed with increasing
concentrations (1-250 µM) of GRGDSP and Del1 peptides.
More than 50% inhibition was achieved at 100 µM peptide
concentration. Control GRGESP peptides had no significant effect on
adhesion at the concentrations tested. B, peptide
competition experiments employing Del1, VN, and FN proteins coated at a
concentration of 100 nM, and competing peptides at 250 µM concentration. The v subunit-specific peptide
GPenGRGDSPCA, containing the penicillamine motif, inhibited BAEC
adhesion to 100 nM Del1 coated wells to less than 20% of
the control value. Experiments with VN showed that competition with the
Pen RGD peptide, as well as the GRGDSP and GRGESP peptides was similar
for VN and Del1.
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Experiments were also conducted with a peptide containing the
penicillamine motif, Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala, which has
been shown to have some specificity for integrin receptors containing
the
v subunit (26). Del1, VN, and FN were evaluated at 100 nM coating concentration, with peptides at 250 µM. The penicillamine containing RGD peptide strongly
inhibited BAEC adhesion to both Del1 and VN, suggesting these cells use
similar
v receptors to attach to Del1 and VN (Fig. 3B).
RGD and control RGE peptides included in these experiments showed that
inhibition of endothelial cell attachment by RGD peptides was
equivalent for these two proteins.
Integrin Receptor Blocking Antibodies Suggest Del1 Binds
v
3--
To further define the integrin receptors involved in
endothelial cell adhesion, blocking studies were performed with HUVEC and well characterized monoclonal antibodies. The monoclonal antibody against
v
3 (LM609) routinely blocked 75% HUVEC adhesion to Del1, and this was comparable to results seen with VN (Fig.
4A). Interestingly, blocking
antibodies that recognize another VN receptor, anti-
v
5 (PIF6),
consistently showed less blocking activity for cells on Del1,
suggesting a lower affinity interaction with this receptor. Antibodies
to the
6 subunit had no blocking activity associated with Del1
adhesion, but inhibited adhesion to laminin by approximately 80% (data
not shown). Monoclonal antibodies to the
1 integrin subunit (clone
JB1a) also failed to show significant inhibition. Taken together, these
data suggest that Del1 mediates endothelial cell attachment primarily
through molecular interaction with the
v
3 receptor VN receptor,
and to a lesser extent with the
v
5 VN receptor. Additional
receptors, such as
v
1 might also be involved.

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Fig. 4.
Del1 mediates endothelial cell attachment and
migration through ligation of specific vitronectin receptors.
A, effects of integrin receptor blocking antibodies on HUVEC
adhesion to Del1 were investigated. Approximately 75% inhibition was
achieved with the v 3 receptor antibody. The antibody against the
v 5 receptor and specifically the v receptor subunit showed
approximately 25% inhibition of cell adhesion, which was somewhat less
than the 50% inhibition seen toward cell adhesion to VN. Antibodies to
the 1 and 6 receptor subunits failed to inhibit adhesion.
B, Del1 stimulates endothelial cell migration via a
chemotactic mechanism. Migration of BAEC was evaluated after 6 h
in a modified Boyden chamber assay, revealing the greatest migration
when protein was in the bottom chamber only, consistent with a
chemotactic mechanism. C, blocking antibodies were included
in migration assays with HUVEC, and migration evaluated for Del1 and
VN. Antibodies to v 3 blocked approximately 50% of the migration
of the endothelial cells on both Del1 and VN. Antibodies to 2 1
and v 5 failed to inhibit Del1-mediated endothelial cell
migration.
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Del1 Stimulates Endothelial Cell Migration--
The ability of
Del1 to mediate endothelial cell migration was evaluated with studies
using a Boyden-type chamber assay (19). Del1 stimulated a
dose-dependent migration of BAEC with half-maximal effect
observed at 75 nM (data not shown). To test whether the nature of this migration was chemotactic or chemokinetic, the location
of Del1 protein (100 nM final concentration) in the
migration chamber was varied. A chemotactic effect was observed, since
a gradient of Del1 (bottom only) was required for maximal migratory activity (Fig. 4B). VN at the same concentration was also
chemotactic for endothelial cells in this assay, in agreement with
earlier results (21). Specificity of the migratory effect on HUVEC was tested with blocking antibodies to integrin receptors (Fig.
4C). Approximately half of the chemotactic migration in
response to Del1 was inhibited by blocking antibodies against
v
3.
Antibodies to the
v
5 vitronectin receptor, and the
2
1
collagen/laminin receptor did not alter HUVEC migration.
Endothelial Cells Form Focal Contacts on Del1--
Endothelial
cells attached and spread on VN and Del1 in a similar fashion, although
the process was slower on Del1. Cells attached and spread as early as
30 min on VN, but required 60 min for this process on Del1. BAEC and
HUVEC in the BSA-coated wells remained rounded even after 120 min (data
not shown). Active protein synthesis was not required for the
attachment of either endothelial cell type to Del1 since pretreatment
of cells with 25 µg/ml cycloheximide for 2 h and maintenance of
the drug during the entire assay period did not decrease the total
number of attached cells.
To investigate whether cell attachment and spreading on Del1 is
associated with cytoskeletal and signaling events associated with
integrin activation, we performed a number of immunofluorescent studies
with cultured endothelial cells. BAEC were allowed to adhere and spread
on Del1-coated coverslips for 3 h, fixed, and stained with
fluorescein isothiocyanate-conjugated phallicidin or monoclonal
antibodies to vinculin, talin, or the
3 integrin subunit. Labeling
with phallicidin revealed actin polymerization and filament formation
in these cells plated on Del1, similar to that seen in endothelial
cells plated on VN (Fig. 5). Labeling with the vinculin and talin antibodies showed that the cells had formed
focal contacts on Del1, as demonstrated by the localization of vinculin
and talin in discrete contact areas within the cell. The attachment of
BAEC to Del1 also led to the clustering of integrin receptors
containing the
3 subunit, presumably
v
3, as demonstrated by a
punctate staining pattern similar to that seen with the vinculin and
talin antibodies (Fig. 5). Cells were also stained with normal mouse
IgG as a control or without the primary antibody and no specific
immunofluorescence was observed (data not shown). Also, no difference
in the staining was observed when cells were treated with
cycloheximide.

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Fig. 5.
Endothelial cells attach and spread on Del1
and form focal contacts that are similar to those formed on VN.
Immunofluorescent studies of BAEC plated on Del1 and VN. Labeling of
cells with fluorescein-conjugated phallicidin shows that actin
polymerization and stress fiber formation is similar on Del1 and VN.
Staining with antibodies to phosphotyrosine residues, vinculin and
talin, verify that focal complexes are formed, protein in the complexes
are phosphorylated, and that cytoplasmic proteins are recruited into
these structures. Punctate staining with antibodies to the 3
integrin subunit verified integrin clustering in endothelial cells on
Del1. (Photos at × 400 original magnification).
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Integrin signaling has been linked to phosphorylation of proteins in
the focal adhesions associated with integrin clustering. As a first
step to define the cellular response to ligation of integrin receptors
with Del1, immunofluorescent staining experiments were performed with
cultured BAEC and anti-phosphotyrosine antibodies. In these studies,
labeling was seen in the focal contacts in the endothelial cells
adhered to Del1, similar to the pattern that was detected with cells
plated on VN (Fig. 5).
Adhesion and Spreading of BAEC on Del1 Induces Tyrosine
Phosphorylation of p125FAK, MAPK, and
Shc--
p125FAK undergoes phosphorylation in focal
contacts, and has been implicated in integrin signaling pathways (27).
To investigate whether adhesion to Del1 is associated with
phosphorylation of p125FAK, immunoprecipitation and
blotting experiments were conducted. Serum-starved BAEC were allowed to
attach and spread on dishes coated with Del1 or VN, and harvested after
30, 60, or 120 min at 37 °C. Cells were lysed, immunoprecipitated,
and subjected to Western analysis with antibodies to phosphotyrosine
and p125FAK. A time-dependent increase in
tyrosine phosphorylation of one or more 120-kDa proteins occurred on
both the substrates, with a maximum level reached at 60 min for VN
(Fig. 6A, arrowhead). Phosphorylation in cells adherent to Del1 gave a similar but somewhat delayed increase. Stripping and reprobing the blot with
anti-p125FAK showed that one of the 120-kDa phosphorylated
proteins corresponded to p125FAK (Fig. 6B).
Total lysates immunoprecipitated with anti-p125FAK and
probed on Western blots with the same antibody showed that equal
amounts of this protein were present under all conditions of the
experiment (Fig. 6C).

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Fig. 6.
Adhesion and spreading of BAEC on Del1
induces phosphorylation of signaling proteins p125FAK,
MAPK, and Shc. Serum-starved BAEC were suspended in serum-free
media, incubated for 30 min at 37 °C in suspension, and then allowed
to attach and spread on Del1 or VN-coated dishes. Cells were then
harvested into RIPA buffer at 30, 60, and 120 min after spreading.
Attached cells, "A," were harvested directly into RIPA buffer on
the dish prior to trypsinization, and detached cells, "D," were
lysed in suspension before replating. A, when cellular
extracts were subjected to immunoprecipitation and Western blotting
with anti-phosphotyrosine antibody (4G10), increased phosphorylation of
a 120-kDa protein (arrowhead) was observed by 60 min on VN
and 120 min on Del1. B, the blot was stripped and re-probed
with a polyclonal antisera to p125FAK. A band representing
p125FAK was seen in lanes representing attached cells on VN
for 60 and 120 min, and cells on Del1 for 120 min. C,
immunoprecipitation and Western blotting with the same antisera to
p125FAK documented that equal amounts of
p125FAK protein were present under all conditions of the
experiment. D, identical experiments were performed and
lysates analyzed on Western blot with an antibody that recognizes the
phosphorylated forms of MAPK. Increased phosphorylation of MAPK was
detected by 30 min in cells on both Del1 and VN. E,
reprobing the blot with a polyclonal antibody to MAPK indicated that
equal amounts of protein were present in the various samples.
F, identical experiments were performed, and lysates
subjected to immunoprecipitation with an antibody to Shc. Precipitated
protein was analyzed on Western blot with a phosphotyrosine antibody,
revealing phosphorylation on Del1. G, protein
immunoprecipitated with the Shc antibody were analyzed on Western blot
with the same antibody, and results indicated that equal amounts of Shc
protein were present under the various conditions of the
experiment.
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To investigate MAPK activation, lysates were prepared from attached and
detached cells, as outlined for p125FAK experiments, and
analyzed by Western blotting with a phospho-specific MAPK antibody
(Fig. 6, D and E). Activation of p44 and p42 MAPK occurs through phosphorylation of specific threonine and tyrosine residues (202 and 204 of the human MAPK (ERK 1)) at the sequence T*EY*
by a single upstream MAPK kinase, MEK (28, 29). The phospho-specific
MAPK antibody detects p42 and p44 MAPK (ERK1 and ERK2) only when
activated by tyrosine phosphorylation on these residues. In this
experiment, MAPK was phosphorylated in the attached condition, and
revealed a rapid increase in phosphorylation after cells attached to
Del1 and VN. Increased phosphorylation was observed by 30 min after the
cells spread on either Del1 and VN substrates, and was sustained (Fig.
6D). Probing Western blots with a polyclonal anti-MAPK
antiserum showed that equal amounts of MAPK were present under all
conditions of the experiment (Fig. 6E).
The adapter protein Shc has been implicated in functions mediated by
v
3, thus additional experiments evaluated the phosphorylation of
Shc in endothelial cells on recombinant Del1 and VN (30). Experiments
were performed as for p125FAK and MAPK, with
immunoprecipitations from cell lysates being performed with an anti-Shc
antibody. Precipitated protein was evaluated on Western blot for level
of phosphorylation by probing with a phosphotyrosine antibody. These
data revealed a time-dependent increase in phosphorylation
of Shc in cells on VN and Del1, with a peak at approximately 60 min
(Fig. 6F). Equal amounts of Shc protein were present within
the experimental conditions (Fig. 6G).
Del1 Is Angiogenic in the in Ovo Chick CAM Assay--
Baculovirus
Del1 MJR was evaluated in the in ovo CAM assay at 100 nM and 1 µM, and found to stimulate
angiogenesis at both concentrations (Fig.
7, A, C, and
D). At the higher concentration baculovirus Del1 MJR
produced an angiogenic response similar to the maximal bFGF response.
Mammalian cell expressed Del1 MJR was also angiogenic, and was
significantly more potent, achieving maximal levels of angiogenesis at
100 nM (Fig. 7B). This difference in early
batches of protein most likely reflected technical difficulty with the
purification rather than differences in folding or glycosylation, since
subsequent purifications have yielded baculovirus protein with potency
similar to the mammalian expressed protein (data not shown). In
experiments where Del1 MJR was added to the same filter disc 1 day
after bFGF, it did not inhibit the angiogenic actions of this growth
factor, and also did not add to the angiogenesis that was observed with
bFGF alone (Fig. 7B). When applied to the CAM in an
identical fashion at 1 µM concentration, VN and FN were not
angiogenic (Fig. 7C).

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Fig. 7.
Del1 is angiogenic in the chick CAM assay,
and this activity requires integrin signaling. A,
baculovirus Del1 MJR (Del1) and Del1 MJR RGD RAD mutant (Del1 RAD)
proteins at 1 µM (2 µg total), bFGF at 59 nM (30 ng total), or phosphate-buffered saline
(PBS) were applied to the chick chorioallantoic membrane and
their effects on vascular development evaluated. In some experiments,
the v 3 blocking antibody LM609 (Del1+LM609) or an unrelated
control antibody W632 (Del1+W632) were added after 1 day, and their
ability to modify the angiogenic response evaluated. B,
quantitative comparison of angiogenic activity of mammalian murine Del1
protein at 100 nM and bFGF at 59 nM. Maximal
angiogenic response achieved with Del1 was similar to that seen with
the maximal bFGF dose employed, and Del1 did not augment or inhibit the
bFGF response. C, VN and FN at 1 µM were
compared with baculovirus Del1 MJR at 1 µM, and found to
not stimulate angiogenesis in the CAM assay. D, quantitation
of CAM data with baculovirus protein indicated that integrin signaling
is required for angiogenic activity. Recombinant baculovirus Del1 MJR
protein was inactive in the presence of the v 3 blocking antibody
LM609, but was not affected by the negative control antibody W632.
Also, Del1 RAD had significantly reduced angiogenic activity.
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The role of integrin binding in Del1-mediated angiogenesis was
evaluated in two ways. First, an
v
3 monoclonal antibody with blocking activity was introduced into the CAM assay along with Del1 MJR
protein, where it was able to abrogate completely the angiogenic
response to baculovirus Del1 protein (Fig. 7, A and D). A control antibody of the same isotype, antibody W632
directed against an myosin heavy chain class I antigen, had no effect
on angiogenesis. In a separate series of experiments, baculovirus Del1
MJR protein with an RGD
RAD mutation was compared with the
wild-type Del1 MJR protein in CAM assays (Fig. 7, A and
D). The RAD mutant protein had only minimal angiogenic
activity. These data strongly suggest that the angiogenic actions of
Del1 require endothelial cell integrin activation.
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DISCUSSION |
The pattern of expression of Del1 in embryogenesis, and the
limited functional data available from initial experiments have provided a complex picture regarding the role of Del1 in vascular development (8). The transient expression pattern of this protein in
the embryonic endothelium, and the microvascular angiogenic aspect of
the response observed in CAM assays employing tumor cells expressing
Del1, suggested an angiogenic or angiogenesis supporting role during
vascular formation. However, persistent expression of Del1 in
endothelial cells not associated with vascular formation, and
expression by nonendothelial cells known to produce inhibitors of blood
vessel formation, suggested an inhibitory function. The ability of Del1
to inhibit in vitro tube formation by cultured yolk sac
cells, and the apparent loss of large patterned vessels in the CAM was
consistent with an inhibitory function or a role in the complex process
of vascular remodeling (8). Regarding this second possibility, it was
difficult to understand how Del1 might mediate such a function through
its interactions with the
v
3 receptor. All evidence to date has
indicated that signaling through ligation of this receptor is essential
for supporting angiogenesis (6, 31, 32). To reconcile an inhibitory
functional profile with the ligation of
v
3, one would have to
postulate that Del1 is an antagonist, competitively inhibiting
productive activation of this integrin by matrix substrates. To address
these issues experimentally, we have taken advantage of recombinant Del1 proteins expressed in baculovirus to more completely define the
interactions of Del1 with endothelial cell integrin receptors, and the
cellular and molecular response to Del1 ligation of these receptors. In
addition, experiments in the CAM assay employing Del1 MJR protein from
two different sources have been employed to assess the overall vascular
response to this protein in vivo.
Experiments presented here strongly suggest that Del1 is a functional
integrin ligand. Cell adhesion to Del1 is comparable to adhesion on
similar concentrations of VN and FN, and Del1 mediates endothelial cell
migration similar to that seen on VN. These activities are mediated
primarily through Del1 binding the
v
3 VN receptor, and in part
through the
v
5 VN receptor. Endothelial cells attach and spread
on Del1, and this is associated with the formation of focal contacts
that include vinculin and talin. Proteins in the focal contacts,
including p125FAK, show increased phosphorylation when
endothelial cells attach and spread on Del1. Del1-mediated integrin
signaling is suggested by the time-dependent
phosphorylation of Shc and MAPK in endothelial cells on Del1. Thus, in
all aspects investigated thus far, Del1 appears to function as a
classical integrin ligand. Of course, one unique feature of
Del1-integrin interaction is the cell-specific and potential autocrine
nature of its function, which result from the temporal endothelial cell
restricted pattern of expression of Del1.
Given these data supporting a high affinity productive interaction of
Del1 with endothelial cell integrins, it was important to investigate
again the functional profile of Del1 proteins in the CAM assay. Also,
the use of recombinant affinity purified baculovirus protein, in
comparison to the transfected cell lines used as a source for Del1 in
the initial studies, was felt likely to give more easily interpreted
results. Surprisingly, in an in ovo model of the CAM assay
that has been employed extensively in the laboratory of one of the
investigators, the Del1 MJR isoform was found to be a potent angiogenic
factor (24). Indeed, quantitation of an average number of vessel branch
points indicated that Del1 MJR was equally active as bFGF in this
angiogenesis model. While the initial preparations of baculovirus
protein were less active on a molar basis than bFGF, such as in Fig. 7,
later preparations of protein were as active as FGF on a molar basis
(data not shown). There was no evidence of the loss of vascular pattern
or a decrease in the number of larger vessels, such as that observed in
the initial CAM assays employing tumor cells expressing Del1 MJR. To
further evaluate this difference in functional profile, to obviate the
possibility that the angiogenic activity of recombinant protein might
represent a contaminating baculovirus or insect cell protein, Del1 MJR
was expressed in eukaryotic cells. Human osteosarcoma cells transfected
with a Del1 MJR expression construct were employed in culture as a
source of Del1 MJR, and protein was purified from cultured supernatant
with a 7-step column purification strategy. This protein was also found
to be highly angiogenic in the in ovo chick CAM assay (Fig.
7).
There are multiple possible reasons for the differences observed in
these CAM assays employing purified protein versus those employing transfected tumor cells as a source of Del1. First, the
complex vascular reorganization observed in the CAM assay employing
tumor cells could represent the independent activity of factors
produced by the cultured cells, or the actions of such tumor
cell-derived factors in combination with Del1. Alternatively, the
cultured cells might be stimulated by Del1 to produce a factor or
factors that alone or in combination with Del1 produce the observed
vascular reorganization. It is also possible that the observed
differences simply represent differences in the amount of Del1 protein
provided to the CAM. It has been established that thrombospondin, for
instance, can produce differing effects on the developing vasculature,
depending on the concentration of protein that is provided to the
endothelial cells (33). However, this latter explanation would seem
unlikely since a dose-response experiment was performed with each batch
of purified protein.
The finding that Del1 is angiogenic is surprising and intriguing. While
significant evidence suggests that integrin signaling is required for
angiogenesis, by supplying survival signals that prevent apoptosis,
there is no evidence that integrin ligands can initiate the angiogenic
process. Indeed, experiments presented here show that vitronectin and
fibronectin are not angiogenic in the CAM assay. These data suggest two
hypotheses. One possibility is that Del1 can initiate angiogenesis
through activation of a unique molecular signaling pathway. The
requirement for
v
3 receptor activation indicates that integrin
signaling is a critical component of the angiogenic response, and is
consistent with this possibility. However, all data presented here
suggests that Del1 signaling is not different from that of other matrix
ligands such as vitronectin and fibronectin. Currently, little is known
regarding angiogenic signaling pathways, making further study of this
hypothesis difficult.
A second hypothesis is that Del1 stimulates angiogenesis through a
second signaling pathway. This activation pathway might promote
endothelial cell division as seen with angiogenic growth factors, or
inhibit the differentiation of endothelial cells so that they can
respond to local growth factor signals. The EGF repeats of Del1 have
amino acid similarity with the notch receptors and their ligands, and
interaction of these proteins are known to inhibit terminal
differentiation of a wide variety of cell types. There is an
endothelial cell-specific form of notch, Notch4, and preliminary
studies suggest that notch signaling can regulate the endothelial cell
phenotype (34, 35). Del1 signaling, through a notch-related receptor,
might function to promote an undifferentiated phenotype, with continued
cell division being a component of that program. These cellular
functions would be appropriate for the endothelial cell involved in
angiogenesis. In the context of this hypothesis, data presented here
indicating that Del1 angiogenic activity requires
v
3 activation
would suggest that the receptor mediating this second signaling pathway
must be coordinately activated in conjunction with
v
3 ligation.
While such interaction of an integrin receptor and a second cell
surface receptor has not been studied in detail,
v
3 has been
shown to interact with a thrombospondin-binding protein (36, 37).
Despite the potential complexity of its molecular actions, these data
provide the basis for a more complete understanding of the role of Del1
in vascular development. Through its interaction with integrin
receptors, Del1 may provide the endothelial cell an autocrine pathway
for regulating its own migration and response to growth and
differentiation factors. Endothelial cells in the embryo migrate
widely, and migration is a requisite feature of angiogenesis in the
adult (2, 38). The Del1 autocrine pathway might also have a critical
role in protecting the endothelial cell from apoptosis as it migrates
through growth factor-rich environments in the embryo. The
v
3
receptor has a unique role in supporting division of the endothelial
cell by protecting it from apoptosis, and this receptor has been found
to be essential for embryonic vascular formation (6, 39). In
embryogenesis and other situations where the endothelial cell expresses
Del1, it may be the primary ligand that supports the function of this receptor. If Del1 has the capacity to activate a second pathway that
initiates angiogenesis, in a fashion similar to angiogenic growth
factors, this would make Del1 a uniquely potent activator of vascular growth.