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5ß1 with the Central Cell-Binding Domain of Fibronectin
From the Department of Medicine/Cancer Center,*
Cellular
and Molecular Medicine East, University of California San Diego, La
Jolla, California; and Research and Development
Cardiovascular,
DuPont Pharmaceuticals,
Wilmington, Delaware
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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v integrins have been shown to play
critical roles in angiogenesis, recent studies in v-null
mice suggest that other adhesion receptors and their ligands also
regulate this process. Evidence is now provided that the integrin
5ß1 and its ligand fibronectin are coordinately up-regulated on
blood vessels in human tumor biopsies and play critical roles in
angiogenesis, resulting in tumor growth in vivo.
Angiogenesis induced by multiple growth factors in chick embryos was
blocked by monoclonal antibodies to the cell-binding domain of
fibronectin. Furthermore, application of fibronectin or a
proteolytic fragment of fibronectin containing the central cell-binding
domain to the chick chorioallantoic membrane enhanced angiogenesis in
an integrin 5ß1-dependent manner. Importantly,
antibody, peptide, and novel nonpeptide antagonists of
integrin 5ß1 blocked angiogenesis induced by several growth
factors but had little effect on angiogenesis induced by vascular
endothelial growth factor (VEGF) in both chick embryo and murine
models. In fact, these 5ß1 antagonists inhibited tumor
angiogenesis, thereby causing regression of human tumors in
animal models. Thus, fibronectin and integrin 5ß1,
like integrin vß3, contribute to an angiogenesis pathway
that is distinct from VEGF-mediated angiogenesis, yet important
for the growth of tumors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood vessels arise during embryogenesis by two processes:
vasculogenesis and angiogenesis.5
The roles of growth
factors in both processes are well established. For example, vascular
endothelial growth factor (VEGF)11
and its
receptors12-15
and basic fibroblast growth factor
(bFGF)16,17
promote not only the initial development of
the embryonic vascular network but also the formation of new
blood vessels from pre-existing vessels during development, wound
healing and the female reproductive cycle. VEGF,18-20
bFGF,19,21-23
interleukin-8 (IL-8),20,24-31
and tumor necrosis factor- (TNF-)20
are some of the
growth factors with roles in the pathological angiogenesis that is
associated with solid tumors, diabetic retinopathy, and rheumatoid
arthritis.
Although growth factors stimulate new blood vessel
growth, adhesion to the extracellular matrix (ECM) regulates
endothelial cell survival, proliferation, and motility during new blood
vessel growth.6,7
Recent studies suggest that specific
integrins or their ligands influence vascular development and
angiogenesis. For example, the v integrins participate in
angiogenesis by providing survival signals to activated endothelial
cells.10,11,32-37
However, recent studies
demonstrate that, in the absence of v integrins, some aspects of
angiogenesis can proceed normally,11
suggesting that other
molecules may compensate for the absence of v integrins during
development. In fact, the ß1 integrin family has recently been shown
to play a role in angiogenesis.10,38
Although these studies identify active roles for integrins in the promotion of angiogenesis, the cognate ECM ligands for integrins during in vivo angiogenesis have rarely been identified. One extracellular matrix protein, fibronectin, is expressed in provisional vascular matrices and provides proliferative signals to vascular cells during wound healing, atherosclerosis, and hypertension.39 Fibronectin expression is up-regulated on blood vessels in granulation tissues during wound healing.40 In fact, one isoform of fibronectin, the ED-B splice variant, is preferentially expressed on blood vessels in fetal and tumor tissues, but not on normal quiescent adult blood vessels.41-43 As fibronectin has been shown to regulate cell proliferation,44 these observations suggest a possible role for fibronectin in angiogenesis. Animals lacking fibronectin die early in development from a collection of defects, including missing notochord and somites as well as an improperly formed vasculature.7 However, a functional role for fibronectin in vasculogenesis or in angiogenesis has never been directly established. As fibronectin may have a direct role in promoting angiogenesis, we sought to evaluate its functional role in angiogenesis and to identify the integrin receptor(s) with which it interacts.
One candidate receptor for some of the biological roles of fibronectin
is the integrin 5ß1. Although several integrins bind to
fibronectin,45
integrin 5ß1 is generally
selective for fibronectin46
as it requires peptide
sequences on the ninth (PHSRN) and tenth (RGDS) type III repeats
of fibronectin for ligand recognition.47
Loss of the gene
encoding the integrin 5 subunit is embryonic lethal and is
associated with a complete absence of the posterior somites, as
well as some vascular and cardiac defects.8,48
From these
studies, however, it is unclear whether integrin 5ß1 directly
plays a role in the regulation of vascular development or of
angiogenesis in particular.
Evidence is provided in this report that both fibronectin and its
receptor integrin 5ß1 directly regulate angiogenesis. Moreover,
interaction of fibronectin and 5ß1 is central to the contribution
of these two molecules to angiogenesis. In addition, evidence is
provided that integrin 5ß1 and integrin vß3 participate in
the same pathways of angiogenesis, which are distinct from those
involving integrin vß5. Finally, these studies reveal that
antagonists of the interaction between vascular cell integrin 5ß1
and fibronectin may be useful for the therapy of solid tumor cancers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture media and reagents were from Irvine Scientific (Irvine,
CA). HT29 integrin 5ß1-positive and integrin 5ß1-negative
colon carcinoma cells,49
as well as chick embryo
fibroblasts, were maintained in DMEM high glucose
supplemented with 10% fetal bovine serum and gentamicin.
Human umbilical vein endothelial cells (HUVECs) were maintained in M199
medium containing sodium bicarbonate, HEPES, heparin, endothelial cell
growth supplement, 20% fetal bovine serum, and gentamicin.
Vitronectin, LM609, and P1F6 were the kind gifts of Dr. David Cheresh.
Fibronectin and collagen were from Collaborative Biomedical Products
(Bedford, MA). Human 120-kd and 40-kd chymotryptic fragments
were purchased from Chemicon, Inc. (Temecula, CA). Murine anti-human
CD31 (PECAM; MA-3100) was purchased from Endogen (Woburn, MA). Rabbit
anti-von Willebrand factor (vWF; 016P) was purchased from Biogenex (San
Ramon, CA). Anti-5ß1 cytoplasmic tail polyclonal antibody
(AB1928P), anti-5ß1 function-blocking antibodies (NKI-SAM-1 and
JBS5), anti-5ß1 non-function-blocking antibody (HA5),
anti-fibronectin cell-binding peptide monoclonal antibody (784A2A6),
and anti-fibronectin N-terminal peptide monoclonal antibody were the
kind gifts of Chemicon. Anti-5ß1 function-blocking antibody (IIA1)
and anti-5ß1 non-function-blocking antibody (VC5) were purchased
from Pharmingen (San Diego, CA). Cross-absorbed secondary antibodies
were purchased from Biosource International (Camarillo, CA). OCT
embedding medium was obtained from Baxter (McGraw Park, IL).
Fluoromount-G was purchased from Southern Biotechnology Associates
(Birmingham, AL). Six-week-old CB17 female SCID mice were purchased
from Charles River (Wilmington, MA). Fresh human neonatal foreskins
were obtained from the Cooperative Human Tissue Network of the National
Institutes of Health and were stored in RPMI-1640 medium (Irvine
Scientific, Irvine, CA) supplemented with 2% fetal bovine serum
and 1% gentamicin. Growth factor-depleted matrigel was purchased from
Becton Dickinson (Bedford, MA). Ten-day-old chicken eggs were purchased
from McIntyre Poultry (Ramona, CA). bFGF, vascular endothelial growth
factor, IL-8, and TNF- were purchased from Genzyme, Inc. (Cambridge,
MA). Cyclic peptides were synthesized as
described.50,51
Integrin 5ß1 nonpeptide small
molecule antagonist SJ749 had the following structure:
(S)-2-[(2,4,6-trimethylphenyl) sulfonyl]
amino-3-[7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-(4,4)-non-2-en-3-yl]
car-bonylamino] propionic acid. Control nonpeptide small molecule
XU065 had the following structure:
3-[[3-[(4-Amidinophenyl)oxy]isoxazol-5-yl]carboxamido]-2(S)(butoxycarbonylamino)
propionic acid methyl ester.
Immunohistochemical Analysis of Blood Vessels
Five-micron frozen sections of human normal breast and colon,
colon carcinoma, breast carcinoma, human tumor xenotransplants in SCID
mice, and breast tumors from transgenic mice expressing the
polyoma virus (PyV) middle T antigen under control of the mouse
mammary tumor virus (Mtag) were fixed for 1 minute in acetone, air
dried, and rehydrated for 5 minutes in phosphate buffered saline (PBS).
Sections were then blocked for 2 hours in 8% normal goat serum in PBS
and incubated with 5 µg/ml anti-5ß1 cytoplasmic tail polyclonal
antibody and 5 µg/ml anti-CD31 monoclonal antibody, with 5 µg/ml
anti-5ß1 monoclonal antibody and 5 µg/ml anti-vWF antibody, or
with 5 µg/ml anti-fibronectin cell-binding peptide monoclonal
antibody and 5 µg/ml anti-vWF antibody in 2% bovine serum albumin in
PBS for 2 hours at room temperature. Sections were washed by dipping in
six fresh changes of PBS and incubated in 1:400–1:600 dilutions of
goat anti-rabbit-fluorescein isothiocyanate (FITC) and in 1:400–1:600
goat anti-mouse-rhodamine for 1 hour at room temperature. Slides were
well washed, and coverslips were mounted in one drop of Fluoromount
before digital image analysis under fluorescent illumination using a
supercooled CCD camera.
Cell Adhesion Assays
The wells of 48-well culture dishes (Costar, Inc., Cambridge, MA)
were coated with 1 µg/ml vitronectin, 2 µg/ml fibronectin (chick
embryo fibroblasts and human umbilical vein endothelial cells), or 10
µg/ml fibronectin (HT29-5-positive cells) for 1 hour at 37°C and
blocked with 2% heat denatured bovine serum albumin in PBS for 1 hour.
Fifty thousand cells in 250 µl of adhesion buffer were added to
triplicate wells containing 250 µl of a solution of 50 µg/ml of an
anti-5ß1 function-blocking antibody (NKI-SAM-1, JBS5, or IIA1), 50
µg/ml of an anti-5ß1 non-function-blocking antibody (HA5 or
VC5), 10 µmol/L cyclic peptides, 0–10 µmol/L SJ749, 50 µg/ml of
LM609, an anti-vß3 function-blocking antibody, 50 µg/ml P4C10,
an anti-ß1 function-blocking antibody, 50 µg/ml of an
anti-fibronectin cell-binding domain monoclonal antibody, or 50 µg/ml
of an anti-fibronectin N-terminus monoclonal antibody in adhesion
buffer (HEPES-buffered Hanks’ balanced salt solution containing 1%
bovine serum albumin, 2 mmol/L MgCl2, 2 mmol/L
CaCl2, and 0.2 mmol/L
MnCl2). Cells were allowed to adhere to dishes
for 20 minutes at 37°C. Nonadherent cells were removed by washing
each well four times with 500 µl of warm adhesion buffer. Adherent
cells were then fixed for 15 minutes with 3.7% paraformaldehyde in PBS
and stained with a 2% crystal violet solution. After extensive water
washing to remove excess crystal violet, plates were dried overnight.
Crystal violet was extracted by incubation for 15 minutes in 10%
acetic acid and absorbance at 562 nm determined as an indicator of
number of cells bound. Each experiment was performed in triplicate,
with triplicate samples per condition. The data are presented as
percentage of adhesion exhibited by the positive control (adhesion
medium alone) ± SEM.
Migration Assays
The lower side of 8-µm pore transwell inserts (Costar, Inc.)
were coated with 2 µg/ml of fibronectin, collagen, or no protein for
1 hour and were blocked with 2% bovine serum albumin in PBS for 1
hour. The inserts were then placed into 24-well culture dishes
containing 500 µl migration buffer in the lower chamber. Twenty-five
thousand HUVECs in 50 µl of migration buffer were added to the upper
chamber of duplicate inserts containing 50 µl of a solution of 50
µg/ml of an anti-5ß1 function-blocking antibody (NKI-SAM-1, JBS5
or IIA1), 50 µg/ml of an anti-5ß1 non-function-blocking antibody
(HA5 or VC5), or 50 µg/ml of LM609, an anti-vß3
function-blocking antibody in migration buffer (Hepes-buffered M199
medium containing 1% BSA, 2 mmol/L MgCl2, 2
mmol/L CaCl2, and 0.2 mmol/L
MnCl2) or migration buffer alone. Cells were
allowed to migrate from the upper to the lower chamber for 4 hours at
37°C. Nonmigratory cells were removed from the upper surface by
wiping the upper side with an absorbant tip. Cells that had migrated to
the lower side of the transwell insert were then fixed for 15 minutes
with 3.7% paraformaldehyde in PBS and stained with a 2% crystal
violet solution. After extensive water washing to remove excess crystal
violet, the number of cells that had migrated were counted in three
representative high power (200x) fields per insert. The data are
presented as number of cells migrating ± SEM.
Integrin Receptor Ligand Binding Assays
Integrin vß3 and 5ß1 receptors purified from human
placenta were obtained from Chemicon International. Platelet integrin
IIbß3 was purified from platelets according to established
procedures. Receptors were coated (100 µl/well) on Costar (3590) high
capacity binding plates overnight at 4°C. Coating solution was
discarded and plates were washed once with blocking/binding (B/B)
buffer (50 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl, 2 mmol/L
CaCl2, 1 mmol/L MgCl2, 1
mmol/L MnCl2, and 1% BSA). One hundred ten
microliters of B/B buffer was applied for 60 minutes at room
temperature. Thirty microliters of biotinylated extracellular matrix
protein ligand (fibronectin for integrin 5ß1, vitronectin for
integrin vß3, and fibrinogen for integrin IIbß3) plus 50 µl
of either SJ749 in B/B buffer or B/B buffer alone were added to each
well, and incubated for 25 minutes at room temperature. Plates were
washed twice with B/B buffer and incubated 1 hour at room temperature,
with anti-biotin alkaline phosphatase (100 µl/well) in B/B buffer.
Finally, plates were washed twice with B/B followed by the addition of
100 µl of phosphatase substrate (1.5 mg/ml). Reaction was stopped by
adding 2 N NaOH (25 µl/well), and developed color was read at 405 nm.
In Ovo Chick Chorioallantoic Membrane Angiogenesis Assays
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 is identified. The CAM was dropped away from the eggshell in
this area by grinding a small hole in the mineralized shell and
applying pressure to the underlying inner shell membrane. This caused
an air pocket to shift from the wide end of the egg to the identified
area and forced a circular region of the CAM approximately 2 cm in
diameter to drop away from the shell. A window was cut in the egg shell
and a cortisone acetate pretreated filter disk 5 mm in diameter that
had been saturated in 1 µg/ml bFGF, VEGF, TNF-, IL-8, or saline
was placed on the CAM. The window in the shell was sealed with adhesive
tape and the egg was incubated for 4 days. A range of 0 to 25 µg in
25 µl of function-blocking anti-5ß1 or a control
non-function-blocking anti-5ß1, 0 to 25 µmol/L in 25 µl cyclic
peptide (CRRETAWAC) or scrambled control peptide (CATAERWRC), 0 to 25
µmol/L in 25 µl of a small molecule antagonist of integrin 5ß1
(SJ749), an inactive control small molecule (XU065), or 25 µl of
saline were applied to the growth factor-saturated filter 24 hours
later. Anti-fibronectin antibodies (25 µg in 25 µl) were also
applied topically to the CAM. Fibronectin, vitronectin, and fibronectin
fragments (59 pmoles in a final volume of 25 µl) were applied to
stimulated or unstimulated CAMs. Peptide or small molecule antagonists
of 5ß1 (at a final serum concentration of 0 to 25 µmol/L) were
also injected intravenously into the chick circulation 24 hours later.
CAMs were harvested on the fourth day of stimulation by fixation with a
drop of 3% paraformaldehyde in PBS before excision of the stimulated
area. Blood vessel branch points in the 5-mm filter disk area were
counted at 30x magnification under fiber optic illumination in a
blinded fashion as a size-independent quantitative indicator of
vascular sprouting in response to growth factors. As angiogenesis
is characterized by the sprouting of new vessels in response to growth
factors, counting blood vessel branch points is a useful quantitative
means of obtaining an angiogenic index.52
At least 10
embryos were used per treatment group. Each experiment was performed a
minimum of three times. Data were evaluated in terms of average number
of blood vessel branch points per treatment group ± SEM.
Statistical analyses were performed using Student’s
t-test. Representative CAMS from each treatment group were
photographed at 10x magnification.
In some cases, CAM tissue excised from the egg was frozen in OCT in liquid nitrogen, cut into 5-µm sections, air dried, and processed as described in immunohistochemistry methods, without fixation.
Chick Chorioallantoic Membrane Tumor Assays
Ten million tumor cells were placed on the surface of each CAM and
cultured for 1 week. The resulting tumors were excised and cut into
50-mg fragments. These fragments were placed on additional CAMs and
treated topically the following day with 25 µg in 25 µl of
anti-5ß1 or a control non-function-blocking anti-5ß1, or
systemically by intravenous injection with a final serum concentration
of 25 µmol/L cyclic peptide CRRETAWAC or 25 µmol/L small molecule
antagonist of integrin 5ß1 (SJ749) and 25 µmol/L scrambled
control peptide CATAERWRC or 25 µmol/L inactive small molecule
(XU065) or 25 µl of saline. Forty-eight hours later, CAMs were
excised from the egg and the number of blood vessels entering the
tumors were counted (as vessel branch points). The data are presented
as mean blood vessel number per treatment group (± SEM). Each
treatment group incorporated at least 10 tumors per experiment.
Representative tumors were photographed at 10x magnification. Tumors
were then excised from the egg and weighed. The data are presented as
mean tumor weight per treatment group (± SEM). Statistical analyses
were performed using Student’s t-test. In some cases,
excised tumors were fixed in 3% paraformaldehyde, embedded in
paraffin, and sectioned before immunohistochemical analysis for the
presence of blood vessels.
SCID Mouse Model of Human Angiogenesis
Engraftment of SCID mice with human skins was performed as
previously described.53
SCID mice were engrafted with an 8
mm x 13 mm piece of human neonatal foreskin. Four weeks later,
after the skin had completely healed, 50 µl of growth factor
depleted matrigel reconstituted with 1 µg/ml bFGF, with 1 µg/ml
bFGF containing 25 µg/ml anti-5ß1 function-blocking monoclonal
antibody or with 1 µg/ml bFGF containing 25 µg/ml
non-function-blocking anti-5ß1 monoclonal antibody was injected
intradermally in the center of each engrafted skin. Three days later,
the human skin was excised from the mouse. Boundaries were easily
observed because the human skin was pink and hairless; the mouse skin
was covered with white fur. The human skin was embedded in freezing
medium, frozen, and sectioned. Sections were stained for the presence
of human blood vessels with human specific anti-CD31, as described in
immunohistochemical analyses of blood vessel densities. The data are
presented as mean CD31-positive blood vessel numbers per 100x
microscopic field, ± SEM. Statistical analyses were performed using
Student’s t-test.
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Results |
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5ß1 on Tumor-Associated Blood Vessels
Although previous reports have implicated v integrins in
angiogenesis,9,32-37
recent studies suggest that
alternative adhesion proteins regulate angiogenesis in the absence of
v expression.10
In addition, studies of integrin
5ß1 null mice8,48
and fibronectin null
mice7
suggest that the integrin 5ß1 and its ligand
fibronectin may be required for the proper formation of the vasculature
during development. However, it is unclear from these studies whether
fibronectin and integrin 5ß1 play direct roles in angiogenesis. To
determine whether fibronectin and its receptor, integrin 5ß1, are
expressed during angiogenesis, we evaluated their expression patterns
on the vasculature in human normal and tumor tissues (Figure 1)
and in response to growth factor
stimulation in animal models of angiogenesis (Figure 2)
.
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5ß1 by two-color immunohistochemistry indicated that
CD31-positive tumor vessels (red) were also positive for integrin
5ß1 expression (green). Vessels positive for both molecules are
shown in yellow (Figure 1, A and C)
. Large vessels with lumens, as well
as large and small vessels without apparent lumens, stain positively
for integrin 5ß1 and CD31. Sections of ovarian and pancreatic
carcinoma showed similar patterns of integrin 5ß1 expression on
blood vessels (not shown).
In contrast, CD31-positive blood vessels (red) routinely present in
sections of normal human colon and breast were negative for integrin
5ß1 (Figure 1, B and D)
. Blood vessels in other normal adult
tissues, including skin, were also negative for integrin 5ß1 (data
not shown). These results indicate that integrin 5ß1
expres- sion is up-regulated on tumor vasculature and that the
majority of blood vessels in these tumor sections are integrin 5ß1
positive. Furthermore, these studies indicate that integrin 5ß1 is
not significantly expressed on blood vessels in normal adult tissues.
We next stained tumor tissues with antibodies directed against
fibronectin (red) and vWF (green), another marker of blood vessels.
Examination of frozen sections of breast carcinoma (Figure 1E)
and
colon carcinoma (data not shown) as well as normal human breast (Figure 1F)
and colon (data not shown) indicated that the extracellular
matrix surrounding tumor vessels was positive for fibronectin
expression (arrows). In contrast, blood vessels in normal tissues
expressed little, if any, fibronectin. Sections of ovarian and
pancreatic carcinoma showed similar patterns of fibronectin expression
on blood vessels (not shown).
Notably, the expression of integrin 5ß1 (green) and its ligand,
fibronectin (red), were coordinately up-regulated on many of the same
blood vessels (yellow) within human tumor sections (Figure 1G)
,
suggesting a possible functional interaction between these two
proteins. Expression of integrin 5ß1 and fibronectin were also
observed on tumor vasculature in animal models of neoplasia,
including human M21L melanoma tumor xenotransplants in SCID mice
(Figure 1H)
and spontaneous mammary tumors (data not shown) in Mtag
transgenic mice expressing the polyoma virus (PyV) middle T antigen
under control of the mouse mammary tumor virus.54
Thus,
significantly elevated expression of integrin 5ß1 and fibronectin
is associated with the vasculature in spontaneous as well as
experimentally induced human and murine tumors compared to normal
tissues.
In Vivo Up-Regulation of Fibronectin and Integrin
5ß1 Expression in Response to Angiogenic Growth Factors
To determine whether fibronectin and integrin 5ß1 are
functionally involved in angiogenesis, chick chorioallantoic membranes
(CAMs) were stimulated with angiogenic growth factors and cryostat
sections of these tissues were stained with antibodies to fibronectin
and 5ß1. Integrin 5ß1 expression on the pre-existing
vasculature of unstimulated CAMs was minimal (Figure 2A)
but was
significantly up-regulated 24 hours after exposure to bFGF (Figure 2C)
,
TNF-, or IL-8 (data not shown). In contrast, VEGF did not induce
5ß1 expression (Figure 2E)
. Integrin 5ß1 was not noticeably
expressed on other cell types in the CAM.
Fibronectin expression in the extracellular matrix surrounding blood
vessels was also minimal on unstimulated CAM tissue (Figure 2B)
and,
like 5ß1, was significantly enhanced after bFGF (Figure 2D)
,
TNF-, and IL-8 (data not shown) stimulation. Fibronectin expression
in the extracellular matrix surrounding blood vessels was also
up-regulated after VEGF stimulation (Figure 2F)
. Fibronectin expression
was principally found in association with blood vessels and minimally
on other cell types in these tissues. These results indicate that
integrin 5ß1 and fibronectin expression are both up-regulated
during angiogenesis and that 5ß1 and fibronectin are found closely
associated with each other on growth factor-stimulated blood vessels.
Inhibition of Angiogenesis by Antibody Antagonists of Fibronectin
Since fibronectin was localized to 5ß1-expressing blood
vessels in tumors and growth factor-treated tissues, the effects of
function-blocking anti-fibronectin antibodies on angiogenesis were
evaluated. An antibody directed against the central cell-binding domain
peptide (CBP) of human and chicken fibronectin was first tested for its
ability to inhibit cell adhesion to fibronectin in vitro.
This antibody significantly inhibited the adhesion to fibronectin of
integrin 5ß1-positive cells, including 5ß1-positive HT29
colon carcinoma cells, chick embryo fibroblasts (CEF), and HUVECs.
HUVEC adhesion was blocked 70 ± 3% by the anti-CBP antibody
(Figure 3A)
. In contrast, antibodies
directed against the N-terminal domain (NT) of fibronectin were
ineffective in blocking cell adhesion to fibronectin (Figure 3A)
.
|
. Two days later, CAMs were
excised and blood vessels were quantified by counting vessel branch
points, as described.52
The counting of blood vessel
branch points provides a size-independent measure of the sprouting of
new vessels that occurs during angiogenesis. The anti-CBP antibody was
able to inhibit the growth of new blood vessels induced by bFGF by
75 ± 10% (P = 0.002), whereas the anti-NT
antibody had a minimal effect on angiogenesis (34 ± 15%
inhibition, P = 0.02) as shown in Figure 3B
. The
anti-CBP antibody also inhibited VEGF angiogenesis by 71 ± 7%
(P = 0.02), as did the anti-NT antibody (89
± 17% inhibition, P = 0.035; Figure 3C
). In contrast
to anti-fibronectin antibodies, function-blocking antibodies directed
against vitronectin failed to block angiogenesis significantly (not
shown). These results indicate that the cell-binding domain of
fibronectin plays a critical role in angiogenesis. The N-terminal
domain of fibronectin may also contribute to some angiogenesis. Enhancement of Growth Factor-Induced Angiogenesis by Fibronectin or Its 120-kd Cell-Binding Domain
To demonstrate further if there is a specific functional
association between fibronectin and angiogenesis stimulation,
fibronectin and vitronectin were directly applied to the CAMs of
10-day-old embryos in the presence or absence of growth factors.
Neither fibronectin nor vitronectin applied to CAMs in the absence of
growth factors promoted angiogenesis, as we have previously
documented.55
Equimolar amounts of intact human
fibronectin, a 120-kd fragment of fibronectin with the RGD
containing cell-binding domain or a 40-kd C-terminal chymotryptic
fibronectin fragment, which lacks the RGD containing cell-binding
domain56,57
were applied to bFGF-stimulated CAMs. As shown
in Figure 3C
, intact fibronectin enhanced growth factor-stimulated
angiogenesis at least 46 ± 11% (P =
0.04). The 120-kd cell-binding fragment of fibronectin also
significantly enhanced angiogenesis (65 ± 20%; P
= 0.05), whereas the 40-kd fragment of fibronectin had no significant
effect. This fibronectin-enhanced angiogenesis was dependent on
integrin 5ß1 activity, since anti-integrin 5ß1 antibodies
reversed this process (Figure 3C)
. Application of vitronectin to bFGF
stimulated CAMs had no effect on vessel number (data not shown).
Application of either fibronectin or vitronectin to VEGF-stimulated
CAMs did not potentiate the angiogenic effect of VEGF (data not shown).
These results suggest that fibronectin and the endothelial cell
integrin 5ß1 play critical functional roles in growth
factor-induced angiogenesis.
Antibody, Peptide, and Nonpeptide Antagonists of Integrin 5ß1
Selectively Block Adhesion and Migration on Fibronectin
Because integrin 5ß1 is one of the primary receptors for
fibronectin on endothelial cells and colocalizes with fibronectin on
blood vessels in tumors and in growth factor-stimulated tissues,
experiments were designed to evaluate the effects of monoclonal
antibody, peptide, and nonpeptide antagonists of integrin 5ß1 on
angiogenesis in vivo. To demonstrate the efficacy of these
three classes of inhibitors, we first tested these 5ß1 antagonists
for their abilities to interfere with the attachment and migration of
three types of integrin 5ß1-positive cells: HT29 colon carcinoma
integrin 5 transfectants, CEF, and HUVEC. Function-blocking
monoclonal antibody antagonists of integrin 5ß1, but not control
(non-function-blocking) anti-integrin 5ß1 monoclonal antibodies,
selectively inhibited HT29 5+ (100 ± 6%), CEF (89.7 ±
3.4%), and HUVEC (72 ± 2.5%) adhesion to fibronectin (Figure 4A)
. Function-blocking monoclonal
antibody antagonists of integrin 5ß1, did not block attachment of
HUVECs (Figure 4A)
, HT29 or CEF cells to vitronectin, although LM609,
an anti-vß3 specific antibody, did (Figure 4A)
. These results
demonstrate that 5ß1 antagonists selectively block human and chick
5ß1-mediated cell adhesion to fibronectin, as well as endothelial
cell 5ß1-mediated adhesion to fibronectin.
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5ß1 also potently
inhibit cell attachment to fibronectin. A selective cyclic peptide
antagonist of integrin 5ß1, CRRETAWAC,56,57
also
significantly inhibited 5+ HT29 colon carcinoma, CEF and HUVEC cell
adhesion to fibronectin (Figure 4B)
but not to vitronectin (not shown).
A scrambled control peptide (CATAERWRC) had little impact on cell
adhesion to either fibronectin (Figure 4B)
or vitronectin (not
shown). Furthermore, a selective nonpeptide antagonist of integrin
5ß1, SJ749
{(S)-2-[(2,4,6-trimethylphenyl) sulfonyl]amino-3-[7-benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7]-diazaspiro-(4,4)-non-2-en-3-yl]
carbonylamino] propionic acid}, blocked the adhesion of each of
these cell types to fibronectin in a concentration-dependent manner
with a half maximal inhibitory concentration of 0.8 µM for 5+ HT29
cells (Figure 4C)
. SJ749 was ineffective in blocking cell
attachment to vitronectin or other extracellular matrix ligands (Table 1)
. This compound selectively inhibited
ligand binding to integrin 5ß1 and was substantially less
effective in blocking ligand binding to integrin vß3 and other
integrins (Table 1)
. These results demonstrate that all three classes
of 5ß1 antagonists significantly and selectively inhibit human and
chick 5ß1 functions.
|
5ß1 to block HUVEC migration was evaluated. Migration on
fibronectin was significantly inhibited (87 ± 2%) by
function-blocking antibodies directed against integrin 5ß1 (Figure 4D)
. In contrast, this antibody did not affect endothelial cell
migration on other matrix proteins, including collagen (Figure 4D)
.
Peptide (not shown) and nonpeptide (Table 1)
inhibitors of integrin
5ß1 were also highly effective in blocking endothelial cell
migration on fibronectin but not on other matrix protein such as
collagen.
Antagonists of Integrin 5ß1 Block Angiogenesis in
Vivo
To establish whether integrin 5ß1 might contribute to
angiogenesis, we evaluated the abilities of these same integrin
5ß1 antagonists to impact growth factor-induced angiogenesis on
the chick CAM. Twenty-four hours after stimulating angiogenesis on the
CAM with bFGF, antagonists of integrin 5ß1 were applied directly
to the growth factor-saturated filter disk or were injected
intravenously into the embryonic circulation.
As shown in Figure 5, A, B, and E
,
antibody antagonists of integrin 5ß1 applied topically (Figure 5, A and B)
or intravenously (Figure 5E)
blocked bFGF-induced angiogenesis
on the CAM by at least 88 ± 6% (P = 0.01)
whereas control non-function-blocking anti-5ß1 antibodies had no
significant effect. Applications of function-blocking or control
anti-5ß1 antibodies to unstimulated CAMs had no effect on the
number or integrity of blood vessels present within the application
area (data not shown). Similar to antibody antagonists of 5ß1 and
as predicted by our previous studies,36
antibody
antagonists of vß3 also blocked angiogenesis induced by bFGF by
65 ± 10% (P = 0.008).
|
antagonists of integrin 5ß1 also
significantly blocked bFGF-induced angiogenesis by 90 ± 6%
(P < 0.0001), whereas control peptides did
not inhibit angiogenesis. Nonpeptide antagonists blocked
bFGF-induced angiogenesis (Figure 5D)
in a dose-dependent manner
when applied either topically or systemically; control nonpeptide
molecules did not inhibit angiogenesis, even at the highest dose.
Cyclic peptide and SJ749 antagonists of integrin 5ß1 were equally
effective in inhibiting angiogenesis when applied systemically by
intravenous injection (Figure 5F)
. The cyclic peptide CRRETAWAC
inhibited angiogenesis by 86 ± 13%, whereas SJ749 inhibited
angiogenesis by 81 ± 3%.
In summary, antibody, peptide, and nonpeptide small molecule
antagonists inhibited growth factor-induced angiogenesis with
IC50 values of approximately 5 µg, 120 pmoles,
and 15 pmoles, respectively. These results indicate that the
fibronectin receptor integrin 5ß1 contributes to growth
factor-induced angiogenesis on the CAM.
To extend these findings, we evaluated the ability of these integrin
5ß1 antagonists to block angiogenesis in an animal model of human
angiogenesis. Human neonatal foreskin engrafted onto SCID mice was
injected intradermally with growth factor depleted basement membrane
impregnated with bFGF in the presence or absence of the
function-blocking and control anti-5ß1 antibodies. Analysis of the
human skin after 3 days for the presence of human CD31-positive blood
vessels revealed that the function-blocking 5ß1 antibody
selectively blocked angiogenesis induced by the growth factor (Figure 6, A and B)
, reducing the number of
CD31-positive blood vessels per high power field by 94 ± 4.7%
(P = 0.006). These results indicate that
integrin 5ß1 has a functional role in the angiogenic response to
growth factors of human blood vessels.
|
5ß1 and vß3 Regulate the Same Pathways of
Angiogenesis
Distinct growth factors can induce selective pathways of
angiogenesis that activate and/or use distinct
integrins.36
For example, integrin vß3 participates
in the bFGF and TNF- pathways of angiogenesis, whereas vß5
participates in the VEGF and transforming growth factor- (TGF-)
pathways.36
Therefore, the effects of antagonists of
integrin 5ß1 on angiogenesis induced by additional growth factors
were examined. When angiogenesis was stimulated with TNF- or IL-8,
antibody antagonists of integrin 5ß1 blocked angiogenesis by up to
70.4 ± 12% (P = 0.04) and 85 ±
4.8% (P < 0.0001), respectively (Figure 7, A and B)
. In some experiments
anti-5ß1 inhibited TNF- and IL-8 angiogenesis by up to 99
± 5% (P = 0.005). Similarly, antibody
antagonists of integrin vß3 also blocked TNF and IL-8
angiogenesis by 93.6 ± 6.2% (P = 0.004)
and 77 ± 5.2% (P = 0.0001), respectively.
However, when angiogenesis was induced with VEGF (Figure 7C)
, antibody
antagonists of integrin 5ß1 failed to block angiogenesis, although
an antibody antagonists of integrin vß5 did block angiogenesis by
99 ± 0.1% (P = 0.004), as we previously
reported.36
Peptide and nonpeptide antagonists of integrin
5ß1 also failed to block VEGF angiogenesis. These results suggest
that integrin 5ß1 regulates the same pathway of angiogenesis as
does integrin vß3 and that this pathway is distinct from that
regulated by integrin vß5. When anti-integrin 5ß1 and
anti-integrin vß3 antibodies were applied to bFGF-stimulated CAMs
either alone or together, no additive or synergistic
inhibitory effects were observed (data not shown). These results
suggest that these integrins vß3 and 5ß1 participate in the
same angiogenic pathway.
|
5ß1 Is Required for Human Tumor Angiogenesis
The enhanced expression of 5ß1 on tumor-associated blood
vessels and its functional role in growth factor-stimulated
angiogenesis prompted us to examine its role in tumor angiogenesis and
growth. HT29 colon carcinoma cells, which lack 5ß1 expression,
were grown on the CAMs of 10-day-old embryos. These tumor cells have
been shown to secrete several angiogenic growth factors that
include VEGF, TGF-, TGF-ß, TNF-, and IL-8.49,58,59
Integrin 5ß1-negative tumor cells were used to distinguish
the potential anti-tumor effects from anti-vasculature effects of
integrin 5ß1 antagonists. The tumor-bearing embryos were treated
with doses of either function-blocking or non-function-blocking
antibodies directed to integrin 5ß1. Tumors were excised after
several days of treatment and the number of tumor-associated blood
vessels was assessed under a stereo microscope.
Treatment with anti-5ß1 function-blocking, but not control,
antibodies resulted in significant reduction (70 ± 10%,
P = 0.02) of the number of tumor-associated blood
vessels as measured by quantification of blood vessels entering tumors
(Figure 8, A and B)
or blood vessel
density present in tumors (Figure 8, F and G)
. No significant
differences were observed between saline and control antibody treated
tumors or their associated blood vessels. Importantly, treatment with
function-blocking anti-5ß1 antibodies resulted in tumor
regression. Anti-5ß1 treated tumors were 32% smaller than control
treated tumors (P = 0.02; Figure 8C
). Control
antibody treated tumors increased in size by 25% whereas anti-5ß1
antibody treated tumors decreased in size by 15%. In support of these
findings, systemic (intravenous) administration of cyclic peptide
inhibitors of integrin 5ß1 (Figure 8D)
and nonpeptide small
molecule inhibitors of integrin 5ß1 (Figure 8E)
also induced tumor
regression on the CAM while control peptide and control nonpeptide
treated tumors continued to increase in size. Tumors treated with
peptide inhibitors were 31% smaller than control treated tumors
(P = 0.003). Control peptide treated tumors
increased in size by 20% whereas the 5ß1 peptide antagonist
treated tumors decreased in size by 17%. Tumors treated with
nonpeptide (SJ749) inhibitors were 51% smaller than control-treated
tumors (P = 0.003). Control organic molecule
treated tumors increased in size by 78% whereas the 5ß1 organic
antagonist treated tumors decreased in size by 13%. Tumor cells
remained integrin 5ß1 negative throughout the course of the
experiment (data not shown), suggesting the anti-tumor effects were
based on the targeting of the tumor associated blood vessels.
|
5ß1 treated tumors were mostly necrotic (Figure 8H)
with few
mitotic bodies visible (Figure 8I)
. In contrast, control
antibody-treated tumors were invasive, actively proliferating tumors
(Figure 8H)
with robust mitotic bodies visible (Figure 8I)
. Many
microvessels were associated with the invasive edges of control-treated
tumors, whereas only a few large vessels were present in
anti-5ß1-treated tumors (Figure 8H)
. These results demonstrate
that targeting vascular cell integrin 5ß1 can lead to inhibition
of tumor growth and tumor angiogenesis and that antagonists of integrin
5ß1 are potent inhibitors of tumor growth and tumor-induced
angiogenesis. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3 and
vß5 in angiogenesis have been well
described,9,10,32-37
recent evidence suggests that
certain ß1 integrins may play roles in
angiogenesis.9,10,38,60
In contrast to cell surface
molecules, little is known about the extracellular matrix requirements
for angiogenesis. Interestingly, genetic analyses of fibronectin- and
integrin 5ß1-deficient mice implicate fibronectin and integrin
5ß1 in vascular development and in a number of nonvascular
events.7,8,47
However, a direct functional role for either
of these molecules in angiogenesis has not been previously established.
In this report, several lines of evidence demonstrate the participation
of the central cell-binding domain of fibronectin and its receptor
5ß1 in angiogenesis. First, expression of both integrin 5ß1
and fibronectin were significantly enhanced on blood vessels of human
tumors and in growth factor stimulated tissues, whereas these molecules
were minimally expressed on normal human vessels and on unstimulated
tissues. Second, antibody antagonists of the central cell-binding
domain of fibronectin as well as three classes of integrin 5ß1
antagonists (antibody, peptide, and a novel nonpeptide antagonist)
blocked growth factor-stimulated angiogenesis. Antagonists of integrin
5ß1 blocked bFGF-, TNF--, and IL-8-stimulated angiogenesis, but
had a minimal effect on VEGF-induced angiogenesis. Interestingly,
antagonists of fibronectin function blocked both bFGF and VEGF
angiogenesis, suggesting that other fibronectin receptors may be
critical for VEGF-mediated angiogenesis. Evidence was also provided
that all three types of integrin 5ß1 antagonists inhibit tumor
angiogenesis and result in tumor regression in animal models.
Our results demonstrate that the roles of integrin 5ß1 and
fibronectin in angiogenesis are coordinated. When the expression of
each molecule is minimal (on unstimulated, quiescent blood vessels),
antagonists of each molecule and addition of fibronectin to CAMs have
little effect on quiescent blood vessels. In contrast, after
stimulation with growth factors, integrin 5ß1 and fibronectin
expression is enhanced and blood vessels become sensitive to
antagonists of either molecule and to the effects of extraneous
fibronectin. Importantly, VEGF stimulation does not increase 5ß1
expression, supporting our observation that VEGF angiogenesis is
refractory to antagonists of 5ß1. This is further substantiated by
Collo and Pepper,61
who found that in vitro
expression of integrin 5ß1 on endothelial cells was up-regulated
in response to bFGF, whereas Senger and colleagues60,62
found that VEGF failed to up-regulate 5ß1 expression. Thus, the
functional roles of integrin 5ß1 and fibronectin in angiogenesis
appear to be the direct consequence of their growth factor-induced
expression.
Antibodies directed against the central cell-binding fragment of
fibronectin, which contains the PHSRN and RGDS integrin-binding sites,
inhibited angiogenesis, suggesting that these antibodies inhibit
integrin ligation by fibronectin and possible downstream signal
transduction events in vivo. Stimulation of bFGF
angiogenesis by fibronectin and its cell-binding domain in an
5ß1-dependent manner suggests that integrin 5ß1 is the
integrin receptor for fibronectin during angiogenesis. In fact, the
absence of integrin 5ß1 expression in VEGF-stimulated angiogenesis
may account for the failure of fibronectin to enhance VEGF angiogenesis
even though antibodies directed against the cell-binding peptide of
fibronectin blocked VEGF angiogenesis. It is possible that other
fibronectin binding integrins, such as vß1 or
3ß1,63
support VEGF-induced angiogenesis. Thus, it is
possible that fibronectin may bind to or activate distinct integrin
receptors during VEGF versus bFGF angiogenesis. Our results
are the first demonstration of a direct in vivo
role for fibronectin in angiogenesis.
Our results are also the first to identify clearly a role for an
extracellular matrix protein in the promotion of angiogenesis. Although
collagens have been suggested to have roles in vascular
development,64,65
intact collagens do not support
endothelial cell outgrowth, survival, or
proliferation.66,67
In fact, inhibition of the collagen
receptor integrins 2ß1 and 1ß1 was shown to prevent the
formation of large blood vessels and to promote the formation of small
vessels.60
These results suggest that 2ß1, 1ß1,
and their ligand collagen play roles in blood vessel maturation rather
than the promotion of new blood vessel sprouts.
A functional role for integrin 5ß1 in angiogenesis similar to that
of integrin vß3 was clearly established when antagonists of
integrin 5ß1 blocked angiogenesis induced by growth factors and
tumor fragments. Interestingly, integrin vß3, like integrin
5ß1,68
can serve as a fibronectin receptor, although
endothelial cells use 5ß1 as the major fibronectin receptor when
both integrins are expressed (data not shown). The expression of both
integrins is regulated by similar growth factors. Both integrin
5ß1 and vß3 play significant roles in bFGF-, TNF--, IL-8-,
and tumor-induced angiogenesis, but not in VEGF-induced
angiogenesis.32,36,51
These two integrins appear to
influence the same angiogenesis pathways, in that combinations of their
antagonists in angiogenesis animal models are neither additive nor
synergistic. Such results suggest the possibility that one of these
integrins functions downstream of the other. Although integrin 5ß1
is clearly required for angiogenesis, it may interact with more than
one ligand during angiogenesis. Integrin 5ß1 can also serve as a
receptor for fibrinogen on endothelial cells in vitro,
though no such associations have been demonstrated in
vivo.69
Ligation of integrins by extracellular matrix proteins has been shown
to promote cell attachment, migration, invasion, survival, and
proliferation6
via integrin signal
transduction.70
Antagonists of integrin vß3 induce
apoptosis of proliferating endothelial cells in vitro and
in vivo by interrupting integrin signal
transduction.32,36,37
We have also observed that
antagonists of integrin 5ß1 induce apoptosis of growth factor
stimulated endothelial cells in vitro and in vivo
(unpublished data). Interestingly, some in vitro studies
suggest that integrins vß3 and 5ß1 influence each other
through cross-talk signaling events.71,72
Thus it is
possible that one of these integrins regulates the actions of the other
through signal transduction mechanisms during angiogenesis.
Antagonists of integrin 5ß1 blocked tumor angiogenesis and growth
as did antagonists of integrin vß3.32,51
The tumor
cell lines chosen for in vivo tumorigenicity and
angiogenesis studies were integrin 5ß1 negative to discount any
effect of the integrin antagonists on the tumor cells. The tumor cells
remained integrin 5ß1 negative through the course of their culture
on CAMs. HT29 tumors express a variety of growth factors, including
VEGF, TNF-, TGF-, TGF-ß, PDGF, and IL-8.49,58,59
It is not known if these cells also express bFGF. Most, if not all,
tumors, use multiple growth factors for angiogenesis, including IL-8,
bFGF, VEGF, and others of the 15 to 20 known angiogenic growth factors.
In fact, VEGF is most commonly associated with the hypoxic core of the
tumor as it is transcriptionally regulated by hypoxia, whereas bFGF and
other factors are associated with the growing edge of the
tumor.72-74
Thus, it is not unexpected that angiogenesis
induced by the complex mixture of growth factors expressed by the HT29
colon carcinoma can be inhibited by antagonists that do not impact VEGF
angiogenesis. As observed for growth factor-stimulated CAMs,
antagonists of integrin 5ß1 did not impact large pre-existing
vessels on the CAM that underlay the tumors. These results suggest that
inhibitors of integrin 5ß1, like inhibitors of integrin vß3,
may provide clinical benefits to patients with certain solid tumors.
|
Acknowledgements |
|---|
5ß1
monoclonal antibodies. |
Footnotes |
|---|
Supported by a grant from the Charles Stern and Anna Stern Foundation and grant R01 CA71619 from the National Cancer Institute to J. A. V.
Accepted for publication December 21, 1999.
|
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P. Khalili, A. Arakelian, G. Chen, M. L. Plunkett, I. Beck, G. C. Parry, F. Donate, D. E. Shaw, A. P. Mazar, and S. A. Rabbani A non-RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo. Mol. Cancer Ther., September 1, 2006; 5(9): 2271 - 2280. [Abstract] [Full Text] [PDF]
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B. Favier, A. Alam, P. Barron, J. Bonnin, P. Laboudie, P. Fons, M. Mandron, J.-P. Herault, G. Neufeld, P. Savi, et al. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration Blood, August 15, 2006; 108(4): 1243 - 1250. [Abstract] [Full Text] [PDF]
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A. Maglott, P. Bartik, S. Cosgun, P. Klotz, P. Ronde, G. Fuhrmann, K. Takeda, S. Martin, and M. Dontenwill The Small {alpha}5{beta}1 Integrin Antagonist, SJ749, Reduces Proliferation and Clonogenicity of Human Astrocytoma Cells. Cancer Res., June 15, 2006; 66(12): 6002 - 6007. [Abstract] [Full Text] [PDF]
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N. Umeda, S. Kachi, H. Akiyama, G. Zahn, D. Vossmeyer, R. Stragies, and P. A. Campochiaro Suppression and Regression of Choroidal Neovascularization by Systemic Administration of an {alpha}5beta1 Integrin Antagonist Mol. Pharmacol., June 1, 2006; 69(6): 1820 - 1828. [Abstract] [Full Text] [PDF]
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Y. Huang, H. Shi, H. Zhou, X. Song, S. Yuan, and Y. Luo The angiogenic function of nucleolin is mediated by vascular endothelial growth factor and nonmuscle myosin Blood, May 1, 2006; 107(9): 3564 - 3571. [Abstract] [Full Text] [PDF]
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 S. A. Mousa, L. O'Connor, F. B. Davis, and P. J. Davis Proangiogenesis Action of the Thyroid Hormone Analog 3,5-Diiodothyropropionic Acid (DITPA) Is Initiated at the Cell Surface and Is Integrin Mediated Endocrinology, April 1, 2006; 147(4): 1602 - 1607. [Abstract] [Full Text] [PDF]
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 E. Toschi, I. Bacigalupo, R. Strippoli, C. Chiozzini, A. Cereseto, M. Falchi, F. Nappi, C. Sgadari, G. Barillari, F. Mainiero, et al. HIV-1 Tat Regulates Endothelial Cell Cycle Progression via Activation of the Ras/ERK MAPK Signaling Pathway Mol. Biol. Cell, April 1, 2006; 17(4): 1985 - 1994. [Abstract] [Full Text] [PDF]
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V. J. Yao, M. G. Ozawa, A. S. Varner, I. M. Kasman, Y. H. Chanthery, R. Pasqualini, W. Arap, and D. M. McDonald Antiangiogenic therapy decreases integrin expression in normalized tumor blood vessels. Cancer Res., March 1, 2006; 66(5): 2639 - 2649. [Abstract] [Full Text] [PDF]
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H. Jin, J. Su, B. Garmy-Susini, J. Kleeman, and J. Varner Integrin {alpha}4{beta}1 Promotes Monocyte Trafficking and Angiogenesis in Tumors Cancer Res., February 15, 2006; 66(4): 2146 - 2152. [Abstract] [Full Text] [PDF]
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 A. P. Hall, F. R. Westwood, and P. F. Wadsworth Review of the Effects of Anti-Angiogenic Compounds on the Epiphyseal Growth Plate Toxicol Pathol, February 1, 2006; 34(2): 131 - 147. [Abstract] [Full Text] [PDF]
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S. Mitola, B. Brenchio, M. Piccinini, L. Tertoolen, L. Zammataro, G. Breier, M. T. Rinaudo, J. den Hertog, M. Arese, and F. Bussolino Type I Collagen Limits VEGFR-2 Signaling by a SHP2 Protein-Tyrosine Phosphatase-Dependent Mechanism 1 Circ. Res., January 6, 2006; 98(1): 45 - 54. [Abstract] [Full Text] [PDF]
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 D. M. McDonald and P. Baluk Imaging of Angiogenesis in Inflamed Airways and Tumors: Newly Formed Blood Vessels Are Not Alike and May Be Wildly Abnormal: Parker B. Francis Lecture Chest, December 1, 2005; 128(6_suppl): 602S - 608S. [Full Text] [PDF]
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M. Economopoulou, K. Bdeir, D. B. Cines, F. Fogt, Y. Bdeir, J. Lubkowski, W. Lu, K. T. Preissner, H.-P. Hammes, and T. Chavakis Inhibition of pathologic retinal neovascularization by {alpha}-defensins Blood, December 1, 2005; 106(12): 3831 - 3838. [Abstract] [Full Text] [PDF]
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 I. Cascone, L. Napione, F. Maniero, G. Serini, and F. Bussolino Stable interaction between {alpha}5{beta}1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1 J. Cell Biol., September 12, 2005; 170(6): 993 - 1004. [Abstract] [Full Text] [PDF]
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A. Abdollahi, D. W. Griggs, H. Zieher, A. Roth, K. E. Lipson, R. Saffrich, H.-J. Grone, D. E. Hallahan, R. A. Reisfeld, J. Debus, et al. Inhibition of {alpha}v{beta}3 Integrin Survival Signaling Enhances Antiangiogenic and Antitumor Effects of Radiotherapy Clin. Cancer Res., September 1, 2005; 11(17): 6270 - 6279. [Abstract] [Full Text] [PDF]
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P. Parsons-Wingerter, I. M. Kasman, S. Norberg, A. Magnussen, S. Zanivan, A. Rissone, P. Baluk, C. J. Favre, U. Jeffry, R. Murray, et al. Uniform Overexpression and Rapid Accessibility of {alpha}5{beta}1 Integrin on Blood Vessels in Tumors Am. J. Pathol., July 1, 2005; 167(1): 193 - 211. [Abstract] [Full Text] [PDF]
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A. Magnussen, I. M. Kasman, S. Norberg, P. Baluk, R. Murray, and D. M. McDonald Rapid Access of Antibodies to {alpha}5{beta}1 Integrin Overexpressed on the Luminal Surface of Tumor Blood Vessels Cancer Res., April 1, 2005; 65(7): 2712 - 2721. [Abstract] [Full Text] [PDF]
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 T. A. Rege, C. Y. Fears, and C. L. Gladson Endogenous inhibitors of angiogenesis in malignant gliomas: Nature'santiangiogenic therapy Neuro Oncology, April 1, 2005; 7(2): 106 - 121. [Abstract] [PDF]
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T. Wang, Y. Ward, L. Tian, R. Lake, L. Guedez, W. G. Stetler-Stevenson, and K. Kelly CD97, an adhesion receptor on inflammatory cells, stimulates angiogenesis through binding integrin counterreceptors on endothelial cells Blood, April 1, 2005; 105(7): 2836 - 2844. [Abstract] [Full Text] [PDF]
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S. M. Moghimi, A. C. Hunter, and J. C. Murray Nanomedicine: current status and future prospects FASEB J, March 1, 2005; 19(3): 311 - 330. [Abstract] [Full Text] [PDF]
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S. M. Short, A. Derrien, R. P. Narsimhan, J. Lawler, D. E. Ingber, and B. R. Zetter Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by {beta}1 integrins J. Cell Biol., February 14, 2005; 168(4): 643 - 653. [Abstract] [Full Text] [PDF]
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 M. E. Akerman, J. Pilch, D. Peters, and E. Ruoslahti Angiostatic peptides use plasma fibronectin to home to angiogenic vasculature PNAS, February 8, 2005; 102(6): 2040 - 2045. [Abstract] [Full Text] [PDF]
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C. Liu, C. Dickinson, J. Shobe, F. Donate, W. Ruf, and T. Edgington A hybrid fibronectin motif protein as an integrin targeting selective tumor vascular thrombogen Mol. Cancer Ther., July 1, 2004; 3(7): 793 - 801. [Abstract] [Full Text] [PDF]
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F. B. Davis, S. A. Mousa, L. O'Connor, S. Mohamed, H.-Y. Lin, H. J. Cao, and P. J. Davis Proangiogenic Action of Thyroid Hormone Is Fibroblast Growth Factor-Dependent and Is Initiated at the Cell Surface Circ. Res., June 11, 2004; 94(11): 1500 - 1506. [Abstract] [Full Text] [PDF]
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S. Swenson, F. Costa, R. Minea, R. P. Sherwin, W. Ernst, G. Fujii, D. Yang, and F. S. Markland Jr. Intravenous liposomal delivery of the snake venom disintegrin contortrostatin limits breast cancer progression Mol. Cancer Ther., April 1, 2004; 3(4): 499 - 511. [Abstract] [Full Text] [PDF]
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V. Trochon-Joseph, D. Martel-Renoir, L. M. Mir, A. Thomaidis, P. Opolon, E. Connault, H. Li, C. Grenet, F. Fauvel-Lafeve, J. Soria, et al. Evidence of Antiangiogenic and Antimetastatic Activities of the Recombinant Disintegrin Domain of Metargidin Cancer Res., March 15, 2004; 64(6): 2062 - 2069. [Abstract] [Full Text] [PDF]
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N. J. Boudreau and J. A. Varner The Homeobox Transcription Factor Hox D3 Promotes Integrin {alpha}5{beta}1 Expression and Function during Angiogenesis J. Biol. Chem., February 6, 2004; 279(6): 4862 - 4868. [Abstract] [Full Text] [PDF]
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P. Baluk, S. Morikawa, A. Haskell, M. Mancuso, and D. M. McDonald Abnormalities of Basement Membrane on Blood Vessels and Endothelial Sprouts in Tumors Am. J. Pathol., November 1, 2003; 163(5): 1801 - 1815. [Abstract] [Full Text] [PDF]
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M. Yi, T. Sakai, R. Fassler, and E. Ruoslahti Antiangiogenic proteins require plasma fibronectin or vitronectin for in vivo activity PNAS, September 30, 2003; 100(20): 11435 - 11438. [Abstract] [Full Text] [PDF]
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T. S. Hiran, J. E. Mazurkiewicz, P. Kreienberg, F. L. Rice, and S. E. LaFlamme Endothelial expression of the {alpha}6{beta}4 integrin is negatively regulated during angiogenesis J. Cell Sci., September 15, 2003; 116(18): 3771 - 3781. [Abstract] [Full Text] [PDF]
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A. Orecchia, P. M. Lacal, C. Schietroma, V. Morea, G. Zambruno, and C. M. Failla Vascular endothelial growth factor receptor-1 is deposited in the extracellular matrix by endothelial cells and is a ligand for the {alpha}5{beta}1 integrin J. Cell Sci., September 1, 2003; 116(17): 3479 - 3489. [Abstract] [Full Text] [PDF]
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K. Horiuchi, G. Weskamp, L. Lum, H.-P. Hammes, H. Cai, T. A. Brodie, T. Ludwig, R. Chiusaroli, R. Baron, K. T. Preissner, et al. Potential Role for ADAM15 in Pathological Neovascularization in Mice Mol. Cell. Biol., August 15, 2003; 23(16): 5614 - 5624. [Abstract] [Full Text] [PDF]
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D. GRATZINGER, S. CANOSA, B. ENGELHARDT, and J. A. MADRI Platelet endothelial cell adhesion molecule-1 modulates endothelial cell motility through the small G-protein Rho FASEB J, August 1, 2003; 17(11): 1458 - 1469. [Abstract] [Full Text] [PDF]
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K. A. Hotchkiss, A. W. Ashton, and E. L. Schwartz Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins {alpha}5{beta}1 and {alpha}V{beta}3 J. Biol. Chem., May 23, 2003; 278(21): 19272 - 19279. [Abstract] [Full Text] [PDF]
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S. A. Wickstrom, K. Alitalo, and J. Keski-Oja Endostatin Associates with Integrin {alpha}5{beta}1 and Caveolin-1, and Activates Src via a Tyrosyl Phosphatase-dependent Pathway in Human Endothelial Cells Cancer Res., October 1, 2002; 62(19): 5580 - 5589. [Abstract] [Full Text] [PDF]
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S. Klein, A. R. de Fougerolles, P. Blaikie, L. Khan, A. Pepe, C. D. Green, V. Koteliansky, and F. G. Giancotti {alpha}5{beta}1 Integrin Activates an NF-{kappa}B-Dependent Program of Gene Expression Important for Angiogenesis and Inflammation Mol. Cell. Biol., August 15, 2002; 22(16): 5912 - 5922. [Abstract] [Full Text] [PDF]
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E. A. Ojo-Amaize, E. J. Nchekwube, H. B. Cottam, R. Bai, P. Verdier-Pinard, V. N. Kakkanaiah, J. A. Varner, L. Leoni, J. I. Okogun, A. A. Adesomoju, et al. Hypoestoxide, a Natural Nonmutagenic Diterpenoid with Antiangiogenic and Antitumor Activity: Possible Mechanisms of Action Cancer Res., July 15, 2002; 62(14): 4007 - 4014. [Abstract] [Full Text] [PDF]
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S. E. Francis, K. L. Goh, K. Hodivala-Dilke, B. L. Bader, M. Stark, D. Davidson, and R. O. Hynes Central Roles of {alpha}5{beta}1 Integrin and Fibronectin in Vascular Development in Mouse Embryos and Embryoid Bodies Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 927 - 933. [Abstract] [Full Text] [PDF]
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M. R. Ritter, M. I. Dorrell, J. Edmonds, S. F. Friedlander, and M. Friedlander From the Cover: Insulin-like growth factor 2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis PNAS, May 28, 2002; 99(11): 7455 - 7460. [Abstract] [Full Text] [PDF]
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K. Porkka, P. Laakkonen, J. A. Hoffman, M. Bernasconi, and E. Ruoslahti A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo PNAS, May 28, 2002; 99(11): 7444 - 7449. [Abstract] [Full Text] [PDF]
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H. Zhang, C. Li, and P. C. Baciu Expression of Integrins and MMPs during Alkaline-Burn-Induced Corneal Angiogenesis Invest. Ophthalmol. Vis. Sci., April 1, 2002; 43(4): 955 - 962. [Abstract] [Full Text] [PDF]
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G. Taraboletti, S. D'Ascenzo, P. Borsotti, R. Giavazzi, A. Pavan, and V. Dolo Shedding of the Matrix Metalloproteinases MMP-2, MMP-9, and MT1-MMP as Membrane Vesicle-Associated Components by Endothelial Cells Am. J. Pathol., February 1, 2002; 160(2): 673 - 680. [Abstract] [Full Text] [PDF]
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 R.O. HYNES, J.C. LIVELY, J.H. MCCARTY, D. TAVERNA, S.E. FRANCIS, K. HODIVALA-DILKE, and Q. XIAO The Diverse Roles of Integrins and Their Ligands in Angiogenesis Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 143 - 154. [Abstract] [PDF]
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J.D. HOOD and D.A. CHERESH Targeted Delivery of Mutant Raf Kinase to Neovessels Causes Tumor Regression Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 285 - 292. [Abstract] [PDF]
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D. R. Senger, C. A. Perruzzi, M. Streit, V. E. Koteliansky, A. R. de Fougerolles, and M. Detmar The {alpha}1{beta}1 and {alpha}2{beta}1 Integrins Provide Critical Support for Vascular Endothelial Growth Factor Signaling, Endothelial Cell Migration, and Tumor Angiogenesis Am. J. Pathol., January 1, 2002; 160(1): 195 - 204. [Abstract] [Full Text] [PDF]
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 J. Zhang, R. E. Clatterbuck, D. Rigamonti, D. D. Chang, and H. C. Dietz Interaction between krit1 and icap1{alpha} infers perturbation of integrin {beta}1-mediated angiogenesis in the pathogenesis of cerebral cavernous malformation Hum. Mol. Genet., December 1, 2001; 10(25): 2953 - 2960. [Abstract] [Full Text] [PDF]
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 E. S. White, D. L. Livant, S. Markwart, and D. A. Arenberg Monocyte-Fibronectin Interactions, Via {alpha}5{beta}1 Integrin, Induce Expression of CXC Chemokine-Dependent Angiogenic Activity J. Immunol., November 1, 2001; 167(9): 5362 - 5366. [Abstract] [Full Text] [PDF]
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D. G. Stupack, X. S. Puente, S. Boutsaboualoy, C. M. Storgard, and D. A. Cheresh Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins J. Cell Biol., October 29, 2001; 155(3): 459 - 470. [Abstract] [Full Text] [PDF]
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P. A. Rupp and C. D. Little Integrins in Vascular Development Circ. Res., September 28, 2001; 89(7): 566 - 572. [Abstract] [Full Text] [PDF]
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D. Taverna and R. O. Hynes Reduced Blood Vessel Formation and Tumor Growth in {{alpha}}5-Integrin-negative Teratocarcinomas and Embryoid Bodies Cancer Res., July 1, 2001; 61(13): 5255 - 5261. [Abstract] [Full Text] [PDF]
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M. Rehn, T. Veikkola, E. Kukk-Valdre, H. Nakamura, M. Ilmonen, C. R. Lombardo, T. Pihlajaniemi, K. Alitalo, and K. Vuori Interaction of endostatin with integrins implicated in angiogenesis PNAS, January 23, 2001; (2001) 31564998. [Abstract] [Full Text]
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S. Kim, M. Harris, and J. A. Varner Regulation of Integrin alpha vbeta 3-mediated Endothelial Cell Migration and Angiogenesis by Integrin alpha 5beta 1 and Protein Kinase A J. Biol. Chem., October 20, 2000; 275(43): 33920 - 33928. [Abstract] [Full Text] [PDF]
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M. Rehn, T. Veikkola, E. Kukk-Valdre, H. Nakamura, M. Ilmonen, C. R. Lombardo, T. Pihlajaniemi, K. Alitalo, and K. Vuori Interaction of endostatin with integrins implicated in angiogenesis PNAS, January 30, 2001; 98(3): 1024 - 1029. [Abstract] [Full Text] [PDF]
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M. Papetti and I. M. Herman Mechanisms of normal and tumor-derived angiogenesis Am J Physiol Cell Physiol, May 1, 2002; 282(5): C947 - C970. [Abstract] [Full Text] [PDF]
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) or
non-function-blocking anti-integrin 
) expressed as
percent of cells adhering in adhesion buffer alone. The adhesion of
HUVECs to vitronectin in the presence of 25 µg/ml function-blocking
anti-
)
B: The adhesion of HT29 
).
