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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3268-3276
Accutin, a New Disintegrin, Inhibits Angiogenesis In Vitro and In
Vivo by Acting as Integrin  v 3 Antagonist
and Inducing Apoptosis
By
Chia Hsin Yeh,
Hui-Chin Peng, and
Tur-Fu Huang
From the Department of Pharmacology, College of Medicine, National
Taiwan University, Taipei, Taiwan.
 |
ABSTRACT |
Endothelial integrins play an essential role in angiogenesis and
cell survival. Accutin, a new member of disintegrin family derived from
venom of Agkistrodon acutus, potently inhibited human platelet
aggregation caused by various agonists (eg, thrombin, collagen, and,
adenosine diphosphate [ADP]) through the blockade of
fibrinogen binding to platelet glycoprotein IIb/IIIa (ie, integrin  IIb 3). In this report, we describe that
accutin specifically inhibited the binding of monoclonal antibody
(MoAb) 7E3, which recognizes integrin v3,
to human umbilical vein endothelial cells (HUVECs), but not those of
other anti-integrin MoAbs such as 21,
31, and 51.
Moreover, accutin, but not the control peptide GRGES, dose-dependently
inhibited the 7E3 interaction with HUVECs. Both 7E3 and
GRGDS, but not GRGES or Integrelin, significantly blocked fluorescein
isothiocyanate-conjugated accutin binding to HUVEC. In functional
studies, accutin exhibited inhibitory effects on HUVEC adhesion to
immobilized fibrinogen, fibronectin and vitronectin, and the
capillary-like tube formation on Matrigel in a dose- and RGD-dependent
manner. In addition, it exhibited an effective antiangiogenic effect in
vivo when assayed by using the 10-day-old embryo chick CAM model.
Furthermore, it potently induced HUVEC apoptotic DNA fragmentation as
examined by electrophoretic and flow cytometric assays. In conclusion,
accutin inhibits angiogenesis in vivo and in vitro by blocking integrin
v3 of endothelial cells and by inducing
apoptosis. The antiangiogenic activity of disintegrins might be
explored as the target of developing the potential antimetastatic
agents.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ANGIOGENESIS, THE development of new
capillaries from preexisting blood vessels, facilitates the
physiological process of embryonic development, female reproduction,
and wound healing. However, undesirable angiogenesis plays a critical
role in a variety of pathological mechanisms such as tumor growth,
metastasis, diabetic retinopathy, and various inflammation
diseases.1 The process of angiogenesis is
complex,2 typically consisting of (1) enzymatic degradation
of the basement membrane, (2) vascular endothelial cell migration into
perivascular space, (3) proliferation and alignment to form tubular
structures, and (4) new vessel formation. Multiple factors are capable
of stimulating an angiogenic process. These factors include basic
fibroblast growth factor (bFGF), vascular endothelial cell growth
factor (VEGF), tumor necrosis factor- (TNF-), and
platelet-derived growth factor (PDGF). There are also many endogenous
inhibitors of angiogenesis such as thrombospondin, cartilage-derived
inhibitor, and tissue inhibitor of metalloprotease in
vivo.3
Interactions between vascular cells and extracellular matrices (ECMs)
are involved in the multiple steps of angiogenesis. To date, four
families of cell adhesion molecules have been described: integrins,
immunoglobulin superfamily members, cadherins, and selectins. Members
of each family have been detected in angiogenic blood
vessels.4 Integrins are a family of heterodimeric
transmembrane receptors that mediate cell-cell and cell-ECM
interaction.5 The function of integrin during angiogenesis
has been studied most extensively with
v3, which is not readily detectable in quiescent vessels but becomes highly expressed in angiogenic
vessels.6 The dependence of angiogenesis on vascular cell
adhesion events in vivo is evidenced by the observation that antibody
and peptide antagonists of v3 integrin
blocked angiogenesis on chick chorioallantoic membrane (CAM) induced by
bFGF and fragments of tumor.7,8 Furthermore,
v3 receptor blockade resulted in
unscheduled programmed cell death (apoptosis) of the proliferative
vascular cells but not the cells of preexisting
vessels.9,10 These findings indicate that
v3 integrin provides a survival signal to
the proliferative vascular cells during new blood vessel growth, and
the induction of vessel cell apoptosis is a major mechanism for
inhibition of angiogenesis.
Disintegrins, a family of low molecular weight, cysteine-rich,
Arg-Gly-Asp (RGD) containing peptides derived from snake venom, inhibit
platelet aggregation by antagonizing fibrinogen binding to platelet
glycoprotein IIb/IIIa.11 Disintegrins also block the
binding of other adhesive ligands, such as vitronectin, fibronectin, and von Willebrand factor to RGD-dependent integrins expressed on the
surface of other cells. Recently, disintegrins were found to inhibit
the adhesion of human umbilical vein endothelial cell (HUVEC) with
fibrin12 or to immobilized ECMs13 through the blockade of v3 integrin. Because the
endothelial v3 integrin plays a major
role in angiogenesis as described above, the antiadhesive activity of
disintegrins on endothelial cells toward ECM may contribute to their
antiangiogenic activity.14,15 However, the mechanisms of
the antiangiogenic activity of disintegrins are not fully understood.
Accutin, a RGD containing small peptide (5241 daltons), was recently
purified from the viper venom of Agkistrodon acutus, potently
inhibited human platelet aggregation triggered by various agonists. The
mechanism of its antiplatelet activity is through antagonizing
fibrinogen binding to integrin IIb3
(in preparation). In this study, we showed that accutin,
belonging to the member of disintegrin family, inhibited HUVEC adhesion
to immobilized ECMs and angiogenesis in vitro and in vitro in a
RGD-dependent manner. We also explored the possible working mechanisms
regarding its antiadhesive and antiangiogenic
activities.
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MATERIALS AND METHODS |
Materials.
Lyophilized powder of A acutus was purchased from local
merchant. Monoclonal antibody (MoAb) 7E3 raised against integrin
IIb3 and
v3,16 and 6F1 raised against
integrin 21 were kindly donated from Dr B. Coller (The Mount
Sinai Hospitol, New York, NY). Anti-integrin MoAbs raised against
2, 3, 4, and
5 were purchased from DAKO (Carpinteria,
CA). Anti-integrin 1 MoAb was purchased from
Transduction Lab (Lexington, KY). Two
anti-v3 integrin MoAbs, LM609 and 23C6,
were purchased from Chemicon (Temecula, CA) and Serotec (Oxford, UK),
respectively. Integrelin, a cyclic heptapeptide based on a Lys-Gly-Asp
(KGD) sequence that specifically antagonizes integrin
IIb3 but not integrin
v3,17 was kindly donated from
COR Therapeutics (South San Francisco, CA). Sephadex G-75 and
DEAE-Sephadex A50 were purchased from Pharmacia (Uppsala, Sweden), and
a reverse-phase C18 column for HPLC was from Merck Chemical Co
(Darmstadt, Germany). Fibrinogen, fibronectin, propidium iodide
(PI), collagenase, and bovine serum albumin (BSA) were purchased from
Sigma Chemical Co (St Louis, MO). Synthetic peptides GRGDS and GRGES
were from Peninsula Laboratories (Belmont, CA). Medium 199 (M199),
fetal bovine serum (FBS), agarose, human vitronectin, and all cultured
reagents were purchased from GIBCO-BRL (Grand Island, NY). Endothelial
cell growth supplement (ECGS) was from Upstate Biotech Inc (Lake
Placid, NY). 2 ,
7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM) and fluorescein isothiocyanate (FITC)
were from Molecular Probes (Eugene, OR).
Purificaiton of accutin.
Accutin was purified from A acutus venom as described
(in preparation). In brief, the purifying procedures were
achieved by three steps of liquid chromatography including gel
filtration on Sephadex G-75, anionic exchanger on
DEAE-Sephadex A-50, and finally high-pressure liquid chromatography
(HPLC) on a C18 reverse-phase column. The purity of the purified
accutin was examined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE; 20%) and mass spectrometry, and the stock
solution of accutin was prepared in phosphate-buffered saline (PBS) and
stored at  20°C. The amino acid analysis and sequencing
showed that accutin is a 47-residue polypeptide containing a RGD
sequence with a molecular mass of 5241 daltons.
Cell culture.
HUVECs were prepared by the method as previously
described.18 Umbilical cord veins were cannulated and
flushed with cord buffer to remove blood and then filled with 0.1%
collagenase (type I) for 10 minutes at 37°C. Isolated
endothelial cells, identified as positively immunofluorescent staining
for von Willebrand factor antigen (DAKO), were maintained in M199
containing 20% FBS, 30 µg/mL ECGS, 4 mmol/L L-glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin, and incubated at 37°C in
5% CO2. The cells were used between second to fourth
passages.
Adhesion assays.
Ninety-six-well plates (Costar, Cambridge, MA) were coated with
fibrinogen (40 µg/mL), fibronectin (30 µg/mL), vitronectin (10 µg/mL), or 1% BSA in PBS overnight in a laminar flow hood to air
dry. HUVECs grown to confluence in 75-cm2 flasks were
obtained by using EDTA/Trypsin, resuspended in M199 and labeled with
BCECF/AM (10 µg/mL), for 30 minutes at 37°C. Labeled cells were
washed and resuspened in M199 to a density of 5 × 105
cells/mL, then incubated with indicated concentrations of accutin or
PBS for 30 minutes at 37°C. Control or the pretreated HUVECs were
applied into ECM-precoated plates at a density of 5 × 104 cells/well and incubated for 90 minutes at 37°C
allowing to adhesion. After two-time washing with PBS, the nonadherent
cells were removed by aspiration and plates were subjected to a
CytoFlour 2300 fluorescence plate reader (Minipore, Bedford, MA).
Following detachment by trypsin, the adherent cells were also counted
by hemacytometer (Hausser Sci, Horsham, PA), and the
percent adhesion of HUVECs to ECMs was calculated. All experiments were
conducted in quadruplicate and repeated at least three times.
Matrigel-induced capillary tube formation.
This assay was performed by the method as previously
described.19 Matrigel, a bsaement membrane matrix extracted
from Engelbreth-Holm-Swarm mouse sacroma (Becton Dickinson, San Jose,
CA), was diluted to 4 mg/mL with cold PBS and added to 24-well plates
(Costar) in a total volume of 200 µL in each well. Plates were stood
at 37°C for 30 minutes to form a gel layer. After gel formation,
HUVECs (2 × 105 cells) in a medium containing 20%
FBS, in the presence of various concentrations of peptides or PBS, were
applied to each well, and plates were incubated at 37°C for 24 hours with 5% CO2. After incubation, cells were washed,
fixed in 2% glutaldehyde for 10 minutes, subjected to inverted
contrast-phase microscope (Nikon, Tokyo, Japan) for
observation, and photographed.
Chick CAM assays.
Eggs of 10-day-old chick embryos were opened into a
1.0-cm2 window that allowed direct access to underlying CAM
by the method as previously described.9 Various doses of
accutin or control peptide were applied to the top of CAM in a total
volume of 100 µL. The window was covered with sterile cellophane tape
and the embryos were incubated for a further 48 hours at 37°C with
60% humidity to induce spontaneous angiogenesis. After incubation, CAM
tissue was resected and analyzed with a stereomicroscope (Nikon). Photographs were taken at 10× magnification.
Binding assays of accutin toward HUVEC.
Flow cytometric studies were performed to quantify the expression of
the integrins and to assay the binding reactions of accutin to HUVECs.
HUVECs were suspended in PBS/1% BSA and fixed with 1%
paraglutaldehyde at 4°C for 30 minutes. Following washing with PBS/1%BSA, the cells were labeled with primary anti-integrin MoAbs or
nonimmune IgG (as negative control in a 1:50 dilution) at 4°C for 1 hour. Labeled cells were washed twice and then incubated with secondary
FITC-conjugated goat anti-mouse IgG (CALTAG Lab, Burlingame,
CA) for 30 minutes at room temperature with a continuous shaking. After incubation, cells were washed twice, resuspended in PBS,
and analyzed immediately by FACscan (Becton Dickinson) using excitation
and emmision wavelength at 488 and 525 nm, respectively. Fluorescence
signals from 10,000 cells were collected to calculate mean fluorescence
intensity of single cell and the percentage of positively staining
cells. To evaluate the binding of accutin to HUVEC, fixed cells were
incubated with accutin (0.2 µmol/L) for 30 minutes before the
addition of primary antibodies.
To assess the effect of 7E3 and GRGDS on accutin binding to HUVECs,
accutin and BSA were firstly conjugated with FITC following the
protocol described by Liu et al.20 The concentration of FITC-conjugated proteins was determined by BCA protein assay kit (Pierce, Rockford, IL). Fixed HUVECs were preincubated
either with MoAbs or peptides for 1 hour at 4°C. After incubation,
cells were washed twice and labeled with FITC-conjugated accutin (1.3 µmol/L). Nonspecific binding was determined by incubating cells with
FITC-conjugated BSA.
Apoptotic DNA fragmentation assays.
Both agarose gel electrophoretic and PI staining of flow cytometric
methods were used to examine apoptotic DNA fragmentation. Apoptosis of
HUVECs was induced by adding the indicated concentrations of accutin to
prevent cells from adherence. DNA fragmentation electrophoresis was
performed by the method as previously described.21 Control
or pretreated HUVECs were obtained, washed, and lysed with lysis buffer
(50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L EDTA, 0.5% SDS, and 0.5 mg/mL
protein K) at 50°C overnight. Following lysis, samples were
incubated with RNase A (500 µg/mL) at 50°C for 1 hour and
extracted with phenol/chloroform/isoamyl alcohol. After centrifugation,
the upper layer containing DNA was precipitated with ethanol and
electrophoresis was performed with a 1.8% agarose gel. DNA fragments
were stained with ethidium bromide and observed under ultraviolet
light.
For PI staining of flow cytometric analysis, HUVECs were adjusted to 2 × 106 cells/mL and fixed with absolute ethanol at
4°C for 30 minutes. After washing, DNA of cells was stained with PI
staining solution (100 µg/mL PI, 0.1% Triton-X, 5 mmol/L EDTA in
PBS) containing DNase-free RNase (100 µg/mL) and analyzed immediately
by FACScan.
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RESULTS |
Effect of accutin on HUVEC adhesion to immobilized ECMs.
Multiple integrins are expressed on endothelial cells, allowing
adhesion to extracellular RGD-containing matrices. The percent efficiency of HUVEC adhesion to immobilized fibrinogen (40 µg/mL), fibronectin (30 µg/mL), and vitronectin (10 µg/mL) was 30.2 ± 1.2%, 49.2 ± 2.2%, and 24.0 ± 0.7%, respectively (n = 3). As
shown in Fig 1, accutin dose dependently inhibited HUVEC
adhesion to these ECMs, including fibrinogen, fibronectin, and
vitronectin, but exhibited little effects on other matrices, such as
collagen type I (80 µg/mL) and laminin (15 µg/mL; data not shown).
Furthermore, the control peptides, both GRGES (1 mmol/L; Fig 1) and
Integrelin (50 µmol/L; data not shown), showed no significant
inhibition (<5%) on HUVEC adhesion to immobilized ECMs.
The inhibitory potencies of accutin on HUVEC adhesion to these
RGD-containing ECMs were varied. Accutin showed a more marked
inhibition on HUVEC adhesion to immobilized vitronectin (65%
to 95%) than to fibronectin (35% to 60%) or fibrinogen
(30% to 75%).
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| Fig 1.
Effect of accutin on HU VEC adhesion to immobilized ECMs.
HUVECs (5 × 104 cells/well) were subjected to 96-well
plate, which were precoated with fibrinogen (40 µg/mL), vitronectin
(10 µg/mL), or fibronectin (30 µg/mL), in the absence or presence
of indicated concentrations of accutin (0.25, 1, 2 µmol/L) or GRGES
(1 mmol/L). Results are expressed as percentage inhibition of adhesion
compared with control cells in the absence of accutin. All experiments
were conducted in quadruplicate and repeated at least three times. Data
are presented as mean ± SEM (n = 3 to 6).
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Effect of accutin on angiogenesis in vitro and in vivo.
Several different models of either in vitro or in vivo have been used
to study the role of cell-adhesion molecules in
angiogenesis.22 Matrigel is useful for the study of
attachment and differentiation of many anchorage-dependent cells. Human
cultured endothelial cells adhered on Matrigel within 18 hours
displayed high motility and cell-cell comunication and formed an
anastomosing network of capillary-like tube.23 As shown in
Fig 2A, when HUVECs were plated on Matrigel, they
aligned with one another and formed tube-like structures resembling a
capillary plexus within 18 hours. Accutin (0.25 and 2 µmol/L, Fig 2C
and 2D, respectively) significantly inhibited this tube formation on
Matrigel in a dose-dependent manner, in contrast to the little effect
of Integrelin (50 µmol/L; Fig 2B), a specific antagonist of integrin
IIb3. In addition, the effect of accutin
on angiogenesis in vivo was evaluated by using the in vivo model of
chick embryo CAM assay (Fig 3). Upon dissection of the
CAM of 12-day-old chick embryo, the spontaneous angiogenesis in CAM was
clearly observed (Fig 3A). After its topical application for 48 hours,
accutin inhibited the spontaneous angiogenesis in a dose-dependent
manner (Fig 3C through F). However, a control peptide GRGES (1 mmol/L)
showed little effect on the new vessel formation (Fig 3B).
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| Fig 2.
Effect of accutin on Matrigel-induced tube formation of
HUVECs in vitro. HUVECs (1 × 105 cells/well) were plated
on Matrigel in the presence of vehicle (A, control), Integrelin (B, 50 µmol/L), or accutin (C and D, 0.25 and 2 µmol/L, respectively) for
18 hours. After washing and fixation, cells were photographed under a
phase-contrast microscope at 40× magnification.
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| Fig 3.
Effect of accutin on spontaneous angiogenesis in vivo.
CAMs of 10-day-old chick embryos were incubated with vehicle
(A, control), GRGES (B, 1mmol/L), or indicated concentrations
of accutin (C through F, 1, 2, 5, 10 µmol/L, all in 100 µL) for 48 hours, and then resected, fixed, and photographed with a
stereomicroscope at 10× magnification.
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Identification of the binding receptors of accutin on HUVEC.
To identify which integrins expressed on HUVEC interact with accutin,
the effect of accutin on several anti-integrin MoAbs binding to HUVEC
was examined by flow cytometry as described in Materials and Methods.
Table 1 showed the quantitative results of multiple
integrin expression on HUVEC shown either by mean fluorescence
intensity or the mean number of positively staining cells. After the
pretreatment of accutin (0.2 µmol/L), HUVECs exhibited a
significantly reduced fluorescence and a decrease in the number of
positively staining cells (P < .001) as probed by 7E3, that
recognizes integrin v3. However, if
HUVECs were probed with MoAb raised against 2, , or
5 integrins, they did not show a significant reduction
of fluorescence intensity in response to accutin pretreatment. In
addition, accutin specifically inhibited 7E3 binding to HUVECs in a
dose-dependent manner, whereas GRGES (1 mmol/L) showed little effect
(Fig 4). We further conjugated accutin with FITC and
performed the binding studies of FITC-conjugated accutin with flow
cytomery by a direct staining protocol. Increment of fluorescence
intensity of HUVECs was dose-dependent and saturable (data not shown).
Incubation of HUVECs with 7E3 (20 µg/mL) or GRGDS (1 mmol/L) significantly inhibited FITC-conjugated accutin binding to
HUVEC, whereas the incubation of HUVECs with nonimmune IgG (1:50
dilution) and GRGES (1 mmol/L) showed little effect (Fig
5). Moreover, incubation of HUVECs with Integrelin (50 µmol/L), a
cyclic KGD derivative that recognizes integrin
IIb3, MoAbs LM609, 23C6 (anti-integrin
v3, 20 µg/mL), 6F1 (anti-integrin 21, 20 µg/mL), or anti-integrin
1, 5 MoAbs showed no significant inhibition on FITC-conjugated accutin binding to HUVECs (data not
shown).
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| Fig 4.
Percentage inhibition of accutin on 7E3 binding to
HUVEC. HUVECs were pretreated with GRGES (1 mmol/L) or indicated
concentrations of accutin (0.068, 0.25, 1, 2 µmol/L) and with the
primary antibody, 7E3 (20 µg/mL). After incubation with IgG-FITC (the
second antibody), the mean fluorescence intensity of cells was
determined by flow cytometry. Results are presented as percentage
inhibition of adhesion compared with control cells (in the absence of
accutin). Data are presented as mean ± SEM (n = 4).
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| Fig 5.
Effect of 7E3 and GRGDS on FITC-conjugated accutin
interaction with HUVEC. HUVECs pretreated with (A) antibodies, ie, 7E3
(b, 20 µg/mL), nonimmune IgG (c, 1:50 dilution) or (B) peptides (d,
GRGDS and e, GRGES, both at 1 mmol/L) were incubated with
FITC-conjugated accutin (1.3 µmol/L) and analyzed by flow cytomery.
Nonspecific binding was performed by incubating cells with
FITC-conjugated BSA (a in A and B). The tracing of PBS (control HUVECs)
and that of GRGES pretreated HUVECs was almost identical. Similar
results were obtained in at least four separate experiments. (C)
Quantitative analyses of FITC-accutin and FITC-BSA were presented as
mean fluorescence intensity and percentage of positively staining
cells. Data are presented as mean ± SEM (n = 4). *P < .05 as compared with control.
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Characterization of apoptosis of HUVECs induced by accutin.
After the incubation of HUVECs with accutin (2 µmol/L) for
18 hours, the detached HUVECs exhibited the typical apoptotic
morphology as previously described,24 including cell
shrinkage, plasma membrane blebbing, and highly condensed nuclei (data
not shown). The biochemical hallmark of apoptosis, DNA fragmentation as
well as the internucleosomal DNA degradation, was analyzed by agarose gel electrophoresis. As shown in Fig 6, DNA isolated
from vehicle or GRGES (1 mmol/L) pretreated HUVECs migrated
as a single band with high molecular mass (lanes 1 and 2). In contrast,
DNA isolated from accutin-pretreated HUVECs exhibited a considerable
degree of degradation (lane 3). Furthermore, the occurrence of DNA
fragmentation was also examined by flow cytometric measurement of the
percentage of nuclei with a hypodiploid DNA content. Normal HUVECs
(control, Fig 7A) as well as GRGES (1 mmol/L) pretreated
HUVECs (Fig 7B) showed a typical cell cycle stage, consisting of a
major diploid peak (G0/G1), a small
hyperdiploid region (S), and a minor tetraploid peak
(G2/M). However, accutin (0.1 to 0.8 µmol/L) pretreatment led, in a dose-dependent manner, to increase the percentage of hypodiploid cells (designated as A0, eg, 34.15%, in the
presence of 0.8 mmol/L accutin v 4.28%, 1 mmol/L GRGES),
reflecting that cells had undergone apoptosis-associated DNA
degradation (Fig 7C through F).
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| Fig 6.
Patterns of electrophoretic DNA fragmentation in
accutin-treated HUVECs. HUVECs were incubated with vehicle (lane 1),
GRGES (1 mmol/L, lane 2), or accutin (2 µmol/L, lane 3) for 18 hours
and lysed. The total cellular DNA was isolated and subjected to
electrophoretic separation by a 1.8% agarose gel. The internucleosomal
DNA fragmentation was represented as the oligonucleosomal banding at
lower molecular weight. A 100-bp ladder was shown in lane M. Similar
results were obtained in three separate experiments.
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| |
DISCUSSION |
Integrins expressed on the membrane surface of HUVEC have been well
established. These integrins, as shown in Table 1 and the previous
report,25 include 21,
31, 51, and
in particular, v3. The
v3 integrin plays a critical role in
mediating endothelial cell spreading and migration, in facilitating
angiogenesis, and in regulating apoptosis.26 Several lines
of experimental evidence presented in this study illustrate that
accutin, a member of disintegrins, interacted with integrin
v3 on HUVECs. Firstly, accutin showed a
significant inhibitory activity on HUVEC adhesion to immobilized v3 ligands including vitronectin,
fibronectin, and fibrinogen (Fig 1), but showed little effect on HUVEC
adhesion to laminin and collagen type I (data not shown). Therefore,
accutin exhibited a rather specific binding and higher affinity toward
integrin v3 on HUVEC. Moreover, accutin
specifically inhibited the binding of 7E3, a MoAb antagonizing integrin
v3 function in vivo and in
vitro,27 to HUVECs, but not the binding of other
anti-integrin MoAbs (eg, 2, 3, and
5 integrin) to HUVECs as analyzed by flow cytometry
(Table 1). In addition, our data showed that accutin inhibited 7E3
binding to HUVECs in a dose-dependent manner (Fig 4), and 7E3
significantly blocked FITC-conjugated accutin binding to HUVECs (Fig
5). However, Integrelin, a cyclic KGD-containing heptapeptide that
specifically recognizes integrin IIb3,
and other anti-integrin MoAbs used (ie, 5,
2, 1 integrin) showed little effect on
the accutin binding reaction (data not shown). It is noted that other
MoAbs raised against integrin v3 (eg, LM609 and 23C6) showed little effect on FITC-accutin binding to HUVECs,
indicating that accutin and 7E3 may bind to a common epitope of
integrin v3, whereas accutin and LM609
bind to different epitope. Therefore, accutin appeared to compete with
7E3 for the same or the overlapped binding site on the integrin
v3. Similar results were recently
reported that some disintegrins (ie, echistatin and kistrin) interacted
with integrin v3 on HUVECs and also showed little inhibitory effect on the binding of LM609 to the same
cells.13 In addition, GRGDS but not GRGES significantly blocked the FITC-conjugated accutin binding to HUVECs, indicating the
RGD motif within accutin molecule is essential for its binding activity.
Primary endothelial cells are anchorage dependent. As integrins are
essentially important for endothelial cell adhesion to ECM, it is not
surprising that they would be involved in anchorage dependence.28 Ligation of integrins induces a cascade of
intracellular signals, regulates gene expression, and contributes to
the mechanisms of proliferation, differentiation, and cell
survival.29 For example, ligation of integrin
v3 on HUVEC suppressed p53 activity, blocked p21WAF1/CIP1 expression, and increased the
bcl-2/bax ratio, thereby promoting cell survival.30 On the
other hand, prevention of cell attachment to matrix, thereby blocking
the ligation of integrin-induced signals and inducing apoptotic cell
death.31,32 Previous studies reported that human cultured
endothelial cells underwent apoptosis when the cells were detached from
matrix by an addition of RGD peptides.33,34 Here, we show
that accutin, a natural occurring small peptide containing a RGD
sequence, induced apoptosis as it was incubated with HUVECs for 18 hours. The biochemical characteristics of apoptosis were evaluated both
by agrose gel electrophoresis (Fig 6) and PI staining of flow cytometry
(Fig 7). In addition, the bcl-2/bax ratio of HUVECs, examined
by Western blot, was decreased after accutin (0.6 µmol/L)
pretreatment as compared to that of control HUVECs (data not shown).
Accutin is an effective inhibitor of angiogenesis in vitro (Fig
2) and in vivo (Fig 3). Our data suggest the mechanism of actions
of accutin in suppressing angiogenesis appear to be related to the
selective blockade of the receptor integrin
v3 on endothelial cells. Similar results
were recently reported with 7E3 that it inihibted angiogenesis in the
human-SCID mouse chimera model of human tumor growth via blocking the
function of integrin v3.35 Furthermore, accutin might exhibit its antiangiogenic activity through
inducing apoptosis of the angiogenic cells as other antagonists of
integrin v3 (ie, MoAb LM609 and cyclic
RGD peptide) did.9 The signal transduction between
blockade of integrin v3 and induction of
apoptotic death of angiogenic cells requires further investigation. In
addition, it remains to be elucidated regarding whether accutin affects
other steps in angiogenic process such as proliferation, tissue
proteolytic degradation, migration, and differentiation.
Tumor metastasis is the spread of tumor cells from a primary tumor to
colonize other sites of the body. The tumor metastastic process is a
complex cascade of events, including invasion, intravasation, and
extravasation from the circulatory system, colonization, and finally
angiogenesis at a distant site.36 The metastatic process has not been completed in many cancer patients at the stage of diagnosis and surgery. In melanoma, prostate, breast, and bladder cancers, more than 90% of the patients were free of distant metastases at the diagnostic and surgery stage. The development of an
antiangiogenic therapy could conceivably be useful in these patients.
For other types of cancer, the percentages of patients with detectable
distant metastases is higher but rarely exceed 50%, still leaving a
significant therapeutic window open for clinical
intevention.37 Therefore, suppression of the final
angiogenesis process is a reasonable strategy to prolong survival in
these cases. It is reported recently that two novel angiogenesis
inhibitors, angiostatin38 and endostatin,39were
active therapeutic agents in suppression of tumor growth and
metastasis. The potential use of these angiogenic inhibitors as
anticancer drugs is currently under clinical trial.40
Disintegrins, the potent platelet aggregation inhibitors, have been
shown to be useful tools for investigating cell-matrix and cell-cell
interaction.41 Disintegrins (ie, triflavin, trigramin, and
rhodostomin) were found to inhibit tumor-cell induced platelet
aggregation,42,43 a step crucial for the deposition of some
tumor cells onto the endothelium prior to extravasation. In this study,
we show a member of disintegrins (ie, accutin) inhibits the spontaneous
angiogenesis, an important process required de novo at metastatic sites
for the continuous tumor growth.44 Taken together,
disintegrins and their derivatives may be used as the lead compound for
developing the potential antimetastatic agents.
| |
FOOTNOTES |
Submitted February 24, 1998;
accepted June 25, 1998.
Supported by grants from National Health Research Institute
(DOH-87-HR-738) and National Science Council of Taiwan (NSC
87-2314-B002-302).
Address reprint requests to Tur-Fu Huang, PhD, Department of
Pharmacology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd, Taipei, Taiwan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We appreciate very much the generous supply of monoclonal antibody,
7E3, from Dr Barry S. Coller. We also thank Kuam-Ting for preparing
HUVECs.
| |
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Abstract
Introduction
Abstract
