© 1999 American Heart Association, Inc.
Original Contributions |
Vitaxin, a Humanized Monoclonal Antibody to the Vitronectin Receptor (
vß3), Reduces Neointimal Hyperplasia and Total Vessel Area After Balloon Injury in Hypercholesterolemic Rabbits
From the Section of Cardiology, Department of Internal Medicine (K.R.C., G.A.B., D.C.S.), and Department of Pathology (M.C.W.), Wake Forest University School of Medicine, Winston-Salem, NC.
Correspondence to David C. Sane, Section of Cardiology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1045. E-mail dsane{at}wfubmc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—The vitronectin receptor (
vß3)
mediates several biological processes that are critical to the
formation of a neointima after coronary
interventions. Blockade of vß3 could
reduce neointima formation by inhibiting smooth muscle cell
migration, decreasing transforming growth factor-ß1
expression, enhancing apoptosis, or reducing neovasculature.
The effects of short-term administration of Vitaxin, a humanized
monoclonal antibody to vß3, on the
responses to balloon injury were tested in
hyperlipidemic rabbits. Balloon angioplasty was
performed on the iliac arteries of male New Zealand White rabbits that
were fed an atherogenic diet for 1 week before injury and until
euthanization at 4 weeks. Rabbits were given either saline (control) or
1 of 2 dosing regimens of Vitaxin (high dose, 5.0 mg/kg, and low dose,
0.5 mg/kg), which were administered intra-arterially before
injury and intramuscularly on days 2 and 3. High-dose and low-dose
Vitaxin were equally effective in decreasing neointima
formation even in the presence of
hypercholesterolemia, a stimulus to
vß3 expression. Vitaxin reduced
transforming growth factor-ß1 and enhanced
apoptosis in injured arteries. Despite these positive effects,
Vitaxin administration was associated with a reduction in artery size,
indicating a negative effect on remodeling. Vitaxin has a potential
role in preventing intimal hyperplasia, especially if the negative
effects on remodeling can be overcome, by dose adjustment or other
strategies.
Key Words: vß3 • Vitaxin • balloon angioplasty • hypercholesterolemia • remodeling
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The proliferation and migration of smooth muscle cells (SMCs) are major contributors to the formation of a neointima after the vascular injury that occurs during coronary interventions.1 2 Medial SMCs proliferate in response to growth factors elaborated from various cells, including platelets, endothelial cells, and SMCs.3 4 SMC proliferation is followed by a wave of migration across the internal elastic lamina into the adjacent intimal layer.3 4 In the intima, SMCs undergo phenotypic changes and secrete a variety of ECM (extracellular matrix) proteins, including vitronectin (VN) and osteopontin (OPN).5 6 Many of these ECM proteins are effective haptotactic factors that further augment SMC migration.7 8 The accumulation of SMC and ECM contributes to intimal lesion formation and restenosis.
SMC migration is largely dependent on the integrin family of receptors
interacting with ECM components.9 Integrins are
heterodimeric transmembrane receptors that mediate cell-cell and
cell-matrix interactions.10 ECM proteins attach to
integrins primarily via the RGD (arginine-glycine-aspartic acid)
tripeptide sequence, which is the principal motif through which binding
to integrins is mediated.11 Of the integrin receptors,
vß3 appears to be the
major mediator of migration. A variety of agents, including RGD
peptides, antibodies, and selective small molecule
inhibitors12 13 directed at
vß3, have been used to
reduce SMC migration in vitro. Furthermore, several of these agents
have inhibited intimal hyperplasia in normolipidemic,
nonatherosclerotic animal models.12 13 14 15 16 The increased
expression of vß3 in
atherosclerotic versus normal coronaries17 demonstrates
that this receptor could potentially contribute to plaque progression
or restenosis. Abciximab, a humanized monoclonal Fab fragment
that binds to the fibrinogen and VN receptors with equal
affinity,18 has a long-term favorable effect on the
outcomes after coronary interventions.19 This
finding has led to speculation that part of the beneficial effect of
abciximab could be mediated via
anti-vß3 effects
rather than solely through anti-platelet activity. To further
examine the importance of
vß3, we tested the
effect of Vitaxin, a humanized form of LM609,20 which is a
function-blocking antibody to
vß3, on intimal
hyperplasia in the balloon-injured hypercholesterolemic
rabbit.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vitaxin, a humanized IgG1
monoclonal antibody to
vß3 derived from the
parent antibody LM609,20 was obtained from IXSYS.
Pharmacokinetic studies performed at IXSYS demonstrated a mean maximum
plasma concentration of Vitaxin of 95.6 µg/mL and an elimination
half-life (t1/2ß) of 60.3 hours after a
dose of 5 mg/kg in New Zealand White (NZW) rabbits. The mean maximum
plasma concentration of Vitaxin after a dose of 1 mg/kg was 16.8
µg/mL, and the t1/2ß was 38.9 hours.
NZW rabbits were obtained from Robinson Company (Winston-Salem, NC). A
Cobra 4.0-mm angioplasty balloon catheter was obtained from SCIMED.
Serum-free DMEM was purchased from Life Technologies. Rabbit aortic
SMCs (Rb-1) were a kind gift from Dr Maurice Nachtigal, University of
South Carolina School of Medicine (Columbia,
SC).21 Wild-type recombinant human VN was
expressed and purified (N. Wajih, D.C. Sane, unpublished data, 1998).
Human IgG1 was purchased from Sigma, and affinity-purified goat
anti-human IgG/rhodamine conjugate was purchased from Jackson
ImmunoResearch Laboratories. The In Situ Cell Death Detection Kit, AP
was purchased from Boehringer Mannheim, and the Vectastain
Elite ABC Kit was purchased from Vector Laboratories. The antibody to
transforming growth factor-ß1
(TGF-ß1; AB-100-NA) was purchased from R&D
Systems.
Balloon Injury
Balloon angioplasty was performed on the iliac arteries of 20
NZW rabbits weighing 4 kg. These rabbits were fed a
high-cholesterol diet, composed of 0.5%
cholesterol, 2.5% peanut oil, and 97% rabbit chow, for 1
week before balloon injury and until euthanization at 1 month. The
rabbits were randomized to 3 groups, as follows: saline control (n=6),
low-dose Vitaxin (LDV; 0.5 mg/kg; n=6), and high-dose Vitaxin (HDV; 5.0
mg/kg; n=8). Vitaxin-treated rabbits received the first dose
intra-arterially immediately before balloon injury.
Subsequent doses of Vitaxin were given intramuscularly daily for 2 days
after balloon injury. Saline, LDV, and HDV infusions were administered
in equal volumes in syringes that were coded by a collaborator not
involved in the care of the rabbits. The investigators remained blinded
to the treatment groups until all analyses were completed.
Before balloon injury, the rabbits were anesthetized with an
intramuscular injection of ketamine hydrochloride (50 mg/kg),
acepromazine maleate (0.25 mg/kg), and buprenorphine hydrochloride
(0.025 mg/kg). A longitudinal incision was made in the ventral neck
region, 
0.5 inches to the right of the trachea. Approximately 2
inches of the common carotid artery was isolated. A 4–0 Ti-Cron
polyester tie was used to ligate the cranial end of the carotid and to
anchor the caudal end. Arterial puncture was performed with
a 20-gauge radial artery catheter through which a 0.014-inch floppy
guide wire was introduced. The guide wire, with a "J" tip, was
advanced into the abdominal aorta with fluoroscopic guidance. The
radial catheter was removed, and a 4.0-mm Cobra angioplasty balloon
catheter, preinfused with heparinized saline, was inserted over the
guide wire and advanced to the middle of the right iliac artery. The
guide wire was removed from the catheter, and drug treatment was
administered through the catheter into the iliac artery.
Arterial injury was performed by inflating the balloon 3
times for 30 seconds at 5 atm of pressure. The balloon was then removed
from the animal, and the caudal end of the exposed external carotid was
ligated. The skin incision was closed using a 2.0 black monofilament
nylon suture. A 2-g (IM) injection of cefazolin was given as a
prophylaxis for infection. All procedures on animals were performed
under a protocol approved by the Animal Care and Use Committee at Wake
Forest University School of Medicine (protocol A95-069).
Cholesterol Measurements
Blood was collected at time of injury and at time of
euthanization and centrifuged at 1400g for 10
minutes, and the serum was frozen at -20°C until it was assayed
within 2 months after collection. Total cholesterol levels
were measured by the Lipid Analytic Laboratory at the Wake Forest
University School of Medicine using an assay based on the
cholesterol esterase/cholesterol oxidase
colorimetric procedure.22 Total
cholesterol values are expressed as mg/dL (mean±SEM).
Harvesting Iliac Arteries
The iliac arteries were harvested 1 month after balloon injury.
Rabbits were anesthetized with ketamine hydrochloride
(100 mg/kg) and acepromazine maleate (0.50 mg/kg). A longitudinal
abdominal incision was made to expose the aorta, inferior
vena cava, and iliac arteries. A 22-gauge catheter (Angiocath) was
inserted into the abdominal aorta, and blood was collected for
cholesterol measurements. An 18-gauge catheter was then
inserted in the inferior vena cava to allow for adequate
drainage of the perfused paraformaldehyde (PFA). The
abdominal aorta was then perfused with saline for 15 minutes at 80
mm Hg. The rabbits were then euthanized with an injection of
pentobarbital sodium (Beuthanasia-D special) (390 mg/kg) into
the heart. The aortas were perfused with 4% PFA (in PBS, at 4°C) at
80 mm Hg for 15 minutes. After perfusion, the right and left
iliac arteries and aorta were dissected free and placed in 4% PFA for
24 to 48 hours.
Tissue Processing
The right iliac artery was extracted from the rabbit and then
cut into 1-inch sections and placed into specimen jars filled with 4%
PFA at 4°C. These sections were then cut into 4.0-mm sections and
processed (using a Sakura VIP 3000 tissue processor) with a dehydration
series of ethanol followed by xylene and then embedded in
paraffin. The paraffin-embedded arteries were cut into 5-µm sections
with a Leica 2025 microtome and placed in a warm gelatin water bath.
Sections were then placed on slides, dried in a 70°C to 80°C oven
for 30 minutes, and stained with Gill's hematoxylin and eosin on an
automatic slide stainer (Leica Autostainer XL).
Morphometric Analysis
Four arterial sections per rabbit were viewed using
an Olympus CK2 microscope and imported into Image Pro Plus (Media
Cybernetics) to perform computer-assisted digital planimetry.
Briefly, areas of interest were drawn around the lumen, media, and
neointima. The total intimal area (area within the internal
elastic lamina minus the lumen) and the medial area [area within the
external elastic lamina (EEL) minus the internal elastic lamina (IEL)]
were determined. Measurements were expressed as
mm2±SEM.
Cell Migration Assay
The cell migration assay was performed as previously
described.23 Briefly, modified Boyden chambers with a
6.5-mm diameter, 10-µm thickness, and 8.0-µm porous membrane
separating the upper and lower chambers were used. The lower surface of
the membrane was coated with VN (10 µg/mL) for 16 hours in PBS (pH
7.4) at 4°C. Excessive ligand was removed, and the lower chamber was
filled with 0.3 mL of serum-free DMEM/0.5% BSA. The chamber was
equilibrated for 2 hours at 37°C in a 5% CO2
incubator before the migration assay. Rb-1 cells were harvested, and
50 000 cells were added to each well. Vitaxin was added to the upper
chamber of the wells at varying concentrations (0 to 200 µg/mL).
Migration was allowed to proceed for 8 hours at 37°C in a 5%
CO2 incubator. At the end of the migration assay,
the upper surface of the membrane was wiped with a cotton-tipped
applicator to remove nonmigratory cells, and the cells that migrated to
the undersurface were fixed in 10% neutral buffered formalin and then
stained with Diff-Quick. The number of stained cells per high-powered
field (HPF; x200) were counted, and the average of 3 nonoverlapping
fields was determined. Random movement was evaluated using PBS-coated
membranes. Each determination represents the average of 3
individual wells, and error bars indicate ±SEM.
To control for nonspecific antibody effects, human IgG1 was added at
varying concentrations (200, 150, 100, and 0 µg/mL), and migration
assays were performed as described above.
Immunofluorescence Binding Studies
The binding of Vitaxin to Rb-1 cells was analyzed using
immunofluorescence. Cells were cultured in 35-mm
cell dishes and then rinsed with Dulbecco's PBS (D-PBS) at 4°C
followed by PBS containing 10 mg/mL BSA. Vitaxin (10 µg/mL) or the
nonimmune human IgG1 (10 µg/mL) was added to subconfluent cells
and incubated at 4°C for 1 hour. Cells were then rinsed 5 times in
D-PBS at 4°C, followed by an incubation with the secondary antibody,
affinity-purified goat anti-human IgG/rhodamine conjugate (25 µg/mL
in BSA/D-PBS), for 30 minutes at 4°C. Cells were rinsed 5 times with
PBS at 4°C, fixed with 3.7% formaldehyde in PBS at room temperature
for 10 minutes, and then rinsed with PBS and mounted under a No. 1,
25-mm circular coverslip in 90% glycerol/10% PBS. Cells were viewed
using a Zeiss Axioplan fluorescence microscope equipped with a
40x, numerical aperture 1.3 Plan-Neofluar objective, rhodamine
filters, and phase-contrast optics. Images were captured using a SPOT
digital charge-coupled device camera and printed using a Kodak 8650 dye
sublimation printer.
Immunohistochemistry
Immunohistochemical detection of TGF-ß1
was performed on paraffin-embedded iliac artery sections. Briefly,
slides were deparaffinized and hydrated through a series of xylenes and
graded alcohol series. Slides were then rinsed in water, incubated in
0.3% H2O2 for 30 minutes,
and blocked with 0.1% BSA for 30 minutes. The primary antibody for
TGF-ß1 was placed on slides for 30 minutes, at
which point slides were rinsed with PBS and then incubated with diluted
biotinylated secondary antibody for 30 minutes. After further washes in
PBS, staining was achieved using the ABC Vectastain Elite peroxidase
system kit (Vector Laboratories). 3,3'-Diaminobenzidine was used to
visualize the staining. Slides were then counterstained with
hematoxylin.
Apoptosis Detection
Paraffin-embedded iliac arteries were deparaffinized and
prepared for staining as mentioned above. Apoptotic nuclei were
detected using the In Situ Cell Death Detection Kit (Boehringer
Mannheim), essentially a TUNEL assay that detects 3'-OH groups of
fragmented DNA. Briefly, slides were treated with proteinase K for 30
minutes at 37°C. Sections were then rinsed with PBS, and 50 µL of
the TUNEL reaction mix was placed on the slides for 1 hour at 37°C.
Slides were rinsed with PBS and incubated with 50 µL of the
converter-alkaline phosphatase mixture for 30 minutes at 37°C.
Sections were then rinsed with PBS, and incubated with the nitroblue
tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) color
detection solution containing 0.17 mg/mL BCIP and 0.33 mg/mL NBT
[in mmol/L, Tris-HCL (pH 9.5) 100, NaCl 100, and
MgCl2 5]. Finally, slides were rinsed with
PBS.
Apoptotic nuclei were quantified by counting the number of stained nuclei per HPF. Three sections were used from each experimental group, and apoptotic nuclei were counted in 5 separate HPFs per artery section (a total of 15 HPFs were averaged).
Statistical Analysis
All results are expressed as mean±SEM. Statistical significance
between mean differences was determined using a 2-tailed Student
t test. A P value <0.05 denoted statistical
significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Total Cholesterol Levels
Cholesterol levels in rabbits fed a normal diet were 25.2±2.4, 403.5±100.0 after being fed a high-cholesterol diet for 1 week (at time of injury), and 563.3±325.2 mg/dL at the time of euthanization.
Systemic Administration of Vitaxin Significantly Reduces
Neointima Formation and Intima-Media Ratio (IMR) After
Balloon Injury
Vitaxin significantly reduced neointima formation
after balloon injury in both the LDV and the HDV groups as compared
with saline control (Figure 1
). The
difference between saline and Vitaxin treatment was statistically
significant (P<0.0005 for saline versus HDV and for saline
versus LDV), but the difference in the 2 dosing regimens was not
statistically significant (Figure 2). The
neointima area was 1.08±0.13
mm2 in the saline group, 0.30±0.05
mm2 in the LDV group, and 0.31±0.15
mm2 in the HDV group.
|
|
Likewise, the IMR was significantly less in the Vitaxin-treated groups
than in the saline control group (Figure 3). The IMR for the LDV group was
0.46±0.05, representing a significant reduction from the
saline control group, which had an IMR of 1.72±0.16
(P<0.0005). The IMR in the HDV group was 0.49±0.07, which
also represented a significant reduction from the saline
control (P<0.0005). There was not a significant difference
between the LDV and HDV groups.
|
Effect of Vitaxin on Arterial and Lumen Size
The Vitaxin-treated rabbits had a smaller total artery size (the
area encompassed by the EEL; Figure 4).
The total area in the saline group was 2.6±0.2
mm2. Systemic administration of Vitaxin was
associated with a significant decrease in artery size in both LDV and
HDV groups. The artery size in the LDV group is 1.94±0.17
mm2 and 1.42±0.1 mm2
in the HDV group; P=0.01 (saline versus LDV) and
P<0.0005 (saline versus HDV).
|
There was no significant difference in lumen size between the saline
and LDV groups; however, the HDV had a lumen size of 0.56±0.04
mm2, which was significantly smaller than the
lumen size in the saline group (lumen area of 0.95±0.08
mm2; P<0.0005, Figure 5).
|
Effect of Vitaxin on Rb-1 Cell Migration
Vitaxin dose-dependently inhibited Rb-1 SMC migration (Figure 6). This reduction was significant at a
concentration of 20 µg/mL. The IC50 of Vitaxin
was 70 µg/mL. The maximum inhibition of migration was achieved at
a concentration of 150 µg/mL, with a 60% decrease in migration as
compared with control (P<0.0001). As seen in the inset, IgG
controls had no effect on cellular migration.
|
Immunofluorescence Binding Studies
Human IgG1 was used to control for nonspecific antibody binding
(Figure 7A and 7A'). As seen in Figure 7A', no fluorescent label was apparent in cells treated with
the nonimmune IgG control. Rb-1 cells treated with Vitaxin (10 µg/mL,
Figure 7B') showed intense labeling on the cellular surface.
|
Effect of Vitaxin on TGF-ß1 Accumulation
Both LDV- and HDV-treated groups had significantly lower amounts
of TGF-ß1 accumulation within the artery wall
as compared with saline control (Figure 8).
|
Effect of Vitaxin on Levels of Apoptosis
Both LDV- and HDV-treated groups had significantly higher levels
of apoptotic nuclei within the intima and media of the artery
as compared with saline control (Figures 9 and 10). The number of apoptotic
nuclei in the LDV group was 139±9.46/HPF and 129.67±11.64/HPF in the
HDV group, compared with the saline control–treated group, which had
41.2±3.54 apoptotic nuclei/HPF (P<0.0005). There
was no significant difference in apoptotic levels between the
Vitaxin-treated groups.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recent attention has focused on a number of processes that contribute to lesion formation after arterial injury, including SMC migration,1 2 3 4 apoptosis,24 ECM production,25 and angiogenesis.26 27 The integrin receptor,
vß3, contributes to
all of these events and is therefore a potential target for therapies
aimed at inhibiting lesion formation after arterial
injury.
There are many reports of decreased lesion formation in response to
inhibition of ECM protein-integrin interactions.12 13 14 15 16
Emerging data using selective inhibitors support the
importance of vß3 as a
major contributor to lesion formation. Van der Zee et al16
showed that LM609, an
anti-vß3-specific
antibody and the precursor of Vitaxin, reduced neointimal
hyperplasia after injury to the iliac artery of male NZW rabbits.
Furthermore, recent data from Srivatsa et al13
demonstrated that a 28-day infusion of the selective
vß3 peptidomimetic
antagonist XJ735 resulted in a marked reduction in
neointimal area and lumen stenosis in a porcine
model of balloon injury.
In the present study, we report that systemic administration of
Vitaxin, a monoclonal antibody to the VN receptor
vß3 inhibits
neointimal formation after balloon injury. Equally
significant inhibition was seen with both low-dose (0.50 mg/kg) and
high-dose (5.0 mg/kg) administration of Vitaxin. Vitaxin administration
resulted in a 72% reduction in neointima area in the LDV
group and a 71% reduction in neointima size in the HDV
group as compared with saline control. The IMR in the LDV group was
73% less than in the saline control group and was 72% less in the
HDV. Vitaxin was administered to each rabbit with an initial
intra-arterial dose on the day of injury and subsequent
intramuscular injections on days 2 and 3 after injury. Short-term
administration (single daily doses for 3 days) of the antibody was
sufficient to sustain the reduction in neointimal
hyperplasia until vessel harvest at 1 month after balloon injury. Prior
studies have used continuous intravenous infusions of 3 to
28 days of the vß3
antagonist. Our study was also the first to examine the
effect of vß3 blockade
in hypercholesterolemic animals. Both Van der
Zee16 and Srivatsa et al13 studied
vß3 blockade in
normolipidemic models. In the present study, rabbits were fed a
high-cholesterol diet for 1 week before balloon injury and
after injury until euthanization. Vitaxin was successful in reducing
neointimal hyperplasia even in the presence of
hypercholesterolemia, which is a stimulus to
vß3
expression.28
There are many potential mechanisms by which Vitaxin could reduce neointima formation after balloon injury. These include inhibition of SMC migration, inhibition of TGF-ß1 production, potentiation of apoptosis, inhibition of angiogenesis, and modulation of the activation or the localization of matrix metalloproteinase (MMP)-2.
SMC migration from the media into the intima of an injured artery is a
fundamental step in the response to injury, and
vß3 is a major
mediator of this process.12 13 14 15 16 Choi et al12
demonstrated that vß3
was evenly distributed on the surface of nonmigrating SMCs but
redistributed to the leading edge after platelet-derived growth
factor-AB–induced migration. This migration was blocked with a
monoclonal antibody to
vß3 (LM609) and with
an RGD-containing peptide (GpenGRGDSPCA).12 Brown et
al7 also demonstrated that the same RGD-containing
peptide, as well as a polyclonal antibody to
vß3, inhibited SMC
migration. Yue et al29 demonstrated that OPN is a
haptotactic factor for rat SMC and that an antibody to the rat
ß3 integrin, but not the
ß1 integrin, inhibited OPN-induced migration.
This group suggested that
vß3 is the major
integrin involved in OPN-induced SMC migration. Clyman et
al30 demonstrated that the migration of lamb ductus
arteriosus SMCs over a variety of ECM substrates, including
fibronectin, laminin, type I collagen, type IV collagen, and VN, was
vß3 dependent.
We confirmed that Vitaxin inhibits rabbit SMC (Rb-1) migration (Figure 6
).
At a concentration of 150 µg/mL, we found a 60% decrease
in migration from baseline. However, at the maximum achievable
concentration in the LDV group (10 µg/mL), there would be minimal
inhibition of migration (Figure 6), despite intense cell binding
(Figure 7). In contrast, the maximum Vitaxin concentration in
the HDV group (96 µg/mL) would produce 50% inhibition of
migration (Figure 6). Since Vitaxin did not cause total
inhibition of SMC migration in this assay, and since LDV and HDV
decreased neointima formation by nearly equal amounts
despite different achievable plasma concentrations, there may be other
mechanisms for the Vitaxin-mediated inhibition of
neointimal hyperplasia. One possibility is that Vitaxin
reduced the rate of re-endothelialization after injury.
The extent of endothelial injury and the rate of
recovery may be important determinants of vascular healing after
arterial injury.31 This mechanism is unlikely,
however, since equal luminal endothelial coverage was
seen in all groups at 4 weeks (data not shown) and since Van der Zee et
al32 found no reduction of endothelial
regrowth by LM609 at 2 and 4 weeks after balloon injury.
We have also shown that in both Vitaxin treatment groups,
TGF-ß1 accumulation was less than in the
saline-treated group (Figure 8). Ribeiro et al33
have demonstrated that the denatured, heparin-binding form of VN,
binding to vß3,
induces TGF-ß1 expression in cultured bovine
aortic endothelial cells. This induction was dependent
on the VN-vß3
interaction, since both RGD peptides and a monoclonal antibody to
vß3 (LM609) were able
to block the induction of TGF-ß1.
TGF-ß1, which is upregulated in
restenosis,34 induces the expression of various
ECM proteins known to stimulate SMC migration and, at the same time,
upregulate the expression of inhibitors of matrix
degradation, including plasminogen activator
inhibitor-1 and tissue inhibitors of
metalloproteinases.35 Given that balloon injury and
hypercholesterolemia have been shown to induce
TGF-ß1 expression,34 36 37 the
inhibition of TGF-ß1 production by
Vitaxin could be an important mechanism for its effects in this
setting.
The integrin receptor
vß3 also regulates
cellular apoptosis, or programmed cell death. Using the TUNEL
technique, Isner et al38 reported high levels of
apoptotic cells in human atherosclerotic lesions and even
higher levels in restenotic lesions. Inhibiting the
vß3 receptor could
therefore result in an increase in apoptosis, reducing cell
number and, therefore, reducing neointimal area. This
induction of apoptosis after inhibition of
vß3 has also been
shown in the microvasculature within tumors.39 We have
shown that levels of apoptosis were significantly higher in
both Vitaxin-treated groups as compared with saline control (Figures 9 and 10).
Vitaxin-mediated enhancement of
apoptosis in the injured artery could result in a decrease in
plaque formation.
Finally, vß3 may be
involved in reducing neointimal hyperplasia after
arterial injury by regulating the activation and
localization of MMPs and by inhibiting angiogenesis. It has been shown
that MMP-2 binds directly to
vß3 on angiogenic
blood vessels and on melanoma tumor cells.40 Because of
the proteolytic activities of MMP-2, cellular migration through the
surrounding ECM of the media and intima is
stimulated.41 42 Blockade of
vß3 by Vitaxin could
prevent the localization and activation of MMP-2 on the
vß3 receptor,
therefore decreasing cellular migration. Because
vß3 also mediates
angiogenesis27 and because plaque neovascularization may
provide a nutrient blood supply, perhaps sustaining plaque growth,
inhibition of vß3
could reduce intimal growth by starving the plaque.
Although intimal hyperplasia contributes to restenosis after arterial injury, it is not the sole determinant of this process. The narrowing of the entire vessel wall after injury may also contribute to the narrowing of the lumen.43 Indeed, our study makes a strong case for the importance of vascular remodeling after arterial injury. Although it is possible that the control group had more extensive remodeling to compensate for the increased intimal hyperplasia (the Glagov phenomenon44 ), a more likely interpretation of our data is that Vitaxin caused a dose-dependent enhancement of vessel constriction that offset its inhibitory effect on intimal hyperplasia.
The contraction of the vessel wall after injury has been compared with
wound contracture, a process that involves cell-matrix interactions
mediated by integrin receptors.45 The VN receptor has a
defined role in mediating contraction of extracellular
matrices.46 47 Given its role in ECM contraction, an
antibody to vß3 might
be predicted to produce favorable remodeling or an increase in the
total vessel area. Our finding that vessel cross-sectional area
decreased suggests that other factors affecting the dimensions of the
vessel wall must be considered.
Vitaxin could enhance vessel constriction through several mechanisms.
By binding to vß3,
Vitaxin could prevent the vasodilatory responses that are mediated by
the VN receptor.48 49 50 Furthermore, antibodies to
vß3 could potentially
block the conversion of pro–MMP-2 to active MMP-2, an event that has
been reported to occur on melanoma cell VN receptors.39
Collagen fragments that are generated by MMP activity induce
vasodilation that is mediated via
vß3.48 51 52
Thus, an antibody to
vß3 could prevent both
the generation of dilatory collagen fragments as well as their
subsequent binding to
vß3.
Our results are distinct from those of some other studies that have
examined vß3
inhibitors. Srivatsa et al13 studied
expression of vß3
after balloon injury to coronary arteries of normolipidemic
pigs as opposed to the setting of
hypercholesterolemia. Also, there was no
significant effect of XJ735 on the arterial size, as
measured by the EEL area.13 A recent study of abciximab
administration to atherosclerotic cynomolgus monkeys showed a
significant decrease in arterial wall area in the
c7E3-treated animals, a finding that is similar to our
report.53 Differential effects of inhibitory
antibodies and small molecule inhibitors of
vß3 could be elicited
by the known differences in signal transduction of receptors depending
on whether the receptor is occupied by a simple ligand or both occupied
and aggregated by inhibitory antibodies.54
In summary, we have found that Vitaxin, a function-blocking antibody to
the vß3 receptor,
resulted in a marked reduction of neointimal area after
balloon injury. We have shown that possible mechanisms of the effect of
Vitaxin on decreasing neointimal hyperplasia could include
a decrease in TGF-ß1 accumulation and an
increase in cellular apoptosis. Previous studies have shown a
reduction in neointimal area after arterial
injury in the presence of integrin antagonists, but most of
these inhibitors were not selective for
vß3. Our data are also
novel in that a short, 3-day administration of Vitaxin was effective in
reducing lesion size after angioplasty, unlike the continuous 1-month
infusion of drug treatment as seen with XJ735. A short infusion of the
drug is a more practical clinical situation. A third important finding
is that Vitaxin was effective even in the presence of
hypercholesterolemia, a condition we have shown
markedly accentuates
vß3 accumulation.
Hypercholesterolemia is a known risk factor for
coronary artery disease and is therefore a clinically relevant
setting in which to study the efficacy of
vß3 blockade.
Unexpectedly, Vitaxin therapy was also associated with constriction of
the artery. It is possible that a dose of Vitaxin of <0.5 mg/kg would
have retained potency at inhibiting intimal hyperplasia, but with a
more favorable effect on remodeling. Further studies need to be
performed to fully understand the potential vasoconstrictory effects of
Vitaxin. However, given that vascular recoil and negative remodeling
are prevented by stent implantation,55 which is an
increasingly popular method of vascular intervention, antibody-mediated
vß3 inhibition with
Vitaxin (perhaps using lower doses) could be considered in this
setting.
|
Acknowledgments |
|---|
D.C.S. was supported by NIH Grants HL46993, GM23875, CA 81233, and DK55061. The assistance of Jennifer J. Walter in randomizing animals to the treatment groups and the generous gift of Vitaxin from IXSYS are greatly appreciated. The assistance of Dr Zishan A. Haroon in performing immunohistochemistry for TGF-ß1 is also greatly appreciated.
Received September 8, 1998; accepted March 30, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Casscells W. Migration of smooth muscle and endothelial cells: critical events in restenosis. Circulation. 1992;86:723–729.
2. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
3.
Schwartz SM, deBlois D, O'Brien EM. The Intima: soil
for atherosclerosis and restenosis. Circ
Res. 1995;77:445–465.
4. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton S. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation 1992;86(suppl):III-47–III-52.
5.
Dufourcq P, Louis H, Moreau C, Daret D, Boisseau MR,
Lamaziere J, Bonnet J. Vitronectin expression and
interaction with receptors in smooth muscle cells from human
atheromatous plaque. Arterioscler Thromb Vasc
Biol. 1998;18:168–176.
6. Giachelli CM, Liaw L, Murry CE, Schwartz SM, Almeida M. Osteopontin expression in cardiovascular disease. Ann N Y Acad Sci. 1995;760:109–126.[Medline] [Order article via Infotrieve]
7.
Brown SL, Lundgren CH, Nordt T, Fuji S. Stimulation of
migration of human aortic smooth muscle cells by
vitronectin: implications for
atherosclerosis. Cardiovasc Res. 1994;28:1815–1820.
8. Nelson PR, Yamamura S, Kent KC. Extracellular matrix proteins are potent agonists of human smooth muscle cell migration. J Vasc Surg. 1996;24:25–32.[Medline] [Order article via Infotrieve]
9. Luscinskas FW, Lawler J. Integrins as dynamic regulators of vascular function. FASEB J. 1994;8:929–938.[Abstract]
10. Hynes RO. Integrin: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11–25.[Medline] [Order article via Infotrieve]
11. Ruoslahti E. Integrins. J Clin Invest. 1991;87:1–5.
12.
Choi ET, Engel L, Callow AD, Sun S, Trachtenberg J,
Santoro S, Ryan ES. Inhibition of neointimal hyperplasia by
blocking vß3 integrin with a small peptide
antagonist GpenGRGDSPCA. J Vasc Surg. 1994;19:125–134.[Medline]
[Order article via Infotrieve]
13.
Srivatsa SS, Fitzpatrick LA, Tsoa PW, Reilly TM, Holmes
DR Jr, Schwartz RS, Mousa SA. Selective vß3 integrin blockade
potently limits neointimal hyperplasia and lumen
stenosis following deep coronary arterial
stent injury: evidence for the functional importance of integrin
vß3 and osteopontin expression during neointima
formation. Cardiovasc Res. 1997;36:408–428.
14.
Matsuno H, Stassen JM, Vermylen J, Deckmyn H.
Inhibition of integrin function by a cyclic RGD-containing peptide
prevents neointima formation. Circulation. 1994;90:2203–2206.
15. Slepian MJ, Massia SP. Preferential antagonism of B3 versus B1 integrin-matrix interactions in vivo limits neointimal hyperplasia following arterial balloon injury. Circulation. 1994;90 (suppl I):I-85. Abstract.
16.
Van der Zee R, Passeri J, Barry JJ, Cheresh DA, Isner
JM. A neutralizing antibody to the vß3 integrin reduces
neointimal thickening in a balloon-injured rabbit iliac
artery. Circulation. 1996;94 (suppl I):I-257. Abstract.
17.
Hoshiga M, Alpers CE, Smith LL, Giachelli CM, Schwartz
SM. vß3 integrin expression in normal and atherosclerotic
artery. Circ Res. 1995;77:1129–1135.
18.
Tam SH, Sassoli PM, Jordan RE, Nakda MT. Abciximab
(ReoPro, chimeric 7E3 Fab) demonstrates equivalent affinity and
functional blockade of glycoprotein IIb/IIIa and
vß3 integrins. Circulation. 1998;98:1085–1091.
19.
Topol EJ, Ferguson JJ, Weisman HF, Tcheng JE, Ellis SG,
Kleiman NS, Ivanhoe RJ, Wang AL, Miller DP, Anderson KM, Califf RM.
Long-term protection from myocardial ischemic events in a
randomized trial of brief integrin ß3
blockade with percutaneous coronary
intervention. EPIC Investigator Group. Evaluation of platelet
IIb/IIIa inhibition for prevention of ischemic complication.
JAMA. 1997;278:479–484.
20.
Cheresh DA, Spiro RC. Biosynthetic and functional
properties of a Arg-Gly-Asp directed receptor involved in human
melanoma cell attachment to Vitronectin, Fibrinogen, and
von Willebrand factor. J Biol Chem. 1987;262:17703–17711.
21. Nachtigal M, Nagpal ML, Greenspan P, Nachtigal SA, Legrand A. Characterization of a continuous smooth muscle cell line derived from rabbit aorta. In Vitro Cell Dev Biol. 1989;25:892–898.[Medline] [Order article via Infotrieve]
22. Allain CC, Poon LS, Chan CG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20:470–475.[Abstract]
23.
Yebra M, Parry GCN, Stromblad S, Mackman N, Rosenberg
S, Mueller BM, Cheresh DA. Requirement of receptor-bound urokinase-type
plasminogen activator for integrin
vß5-directed cell migration. J Biol Chem. 1996;271:29393–29399.
24. Pollman MJ, Hall JL, Mann MJ, Zhang L, Gibbons GH. Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease. Nat Med. 1998;4:222–227.[Medline] [Order article via Infotrieve]
25. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1983;49:208–215.[Medline] [Order article via Infotrieve]
26. Williams JK, Armstrong ML, Heistad DD. Endothelial denudation stimulates proliferation of vasa vasorum. J Vasc Med Biol. 1990;2:12–17.
27.
Brooks PC, Clark RA, Cheresh DA. Requirement of
vascular integrin vß3 for angiogenesis. Science. 1994;264:569–571.
28.
Coleman KR, Corjay MH, Stoltenborg JK, Diamond SM,
Braden GA, Sane DC. Balloon injury of the rabbit aorta induces
accumulation of the vitronectin and vitronectin
receptor (vß3), and
hypercholesterolemia augments this effect.
J Am Coll Cardiol. 1998;31:1007–104. Abstract.
29. Yue TL, McKenna PJ, Ohlstein EH, Farach-Carson MC, Butler WT, Johanson K, McDevitt P, Feuerstein GZ, Stadel JM. Osteopontin-stimulated vascular smooth muscle migration is mediated by B3 integrin. Exp Cell Res. 1994;214:459–464.[Medline] [Order article via Infotrieve]
30. Clyman RI, Mauray F, Kramer RH. ß1 and ß3 integrins have different roles in adhesion and migration of vascular smooth muscle cells on extracellular matrix. Exp Cell Res. 1992;200:272–284.[Medline] [Order article via Infotrieve]
31.
Rogers C, Parikh A, Seifert P, Edelman ER.
Endogenous cell seeding: remnant
endothelium after stenting enhances vascular repair.
Circulation. 1996;94:2909–2914.
32.
Van der Zee R, Murohara T, Passeri J, Kearney M,
Cheresh DA, Isner JM. Reduced intimal thickening following
vß3 blockade is associated with smooth muscle cell
apoptosis. Cell Adhes Commun. 1998;6:371–379.[Medline]
[Order article via Infotrieve]
33. Ribeiro SM, Schultz-Cherry S, Murphy-Ullrich JE. Heparin-binding vitronectin up-regulates latent TGF-ß production by bovine aortic endothelial cells. J Cell Sci. 1995;108(pt 4):1553–1561.
34. Nikol S, Isner JM, Pickering JG, Kearney M, Leclerc G, Weir L. Expression of transforming growth factor-ß1 is increased in human vascular restenosis lesions. J Clin Invest. 1992;90:1582–1592.
35. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor ß1 during repair of arterial injury. J Clin Invest. 1991;88:904–910.
36. Eddy AA. Interstitial inflammation and fibrosis in rats with diet-induced hypercholesterolemia. Kidney Int. 1996;50:1139–1149.[Medline] [Order article via Infotrieve]
37. Lopez-Candales A, Scott MJ, Wickline SA. Cholesterol feeding modulates spatial expression of TGF-ß1 and -ß2 in aortas of Watanabe rabbits. Cytokine. 1995;7:554–561.[Medline] [Order article via Infotrieve]
38.
Isner JM, Kearney M, Bortman S, Passeri J.
Apoptosis in human atherosclerosis and
restenosis. Circulation. 1995;91:2703–2711.
39.
Stromblad S, Becker J, Yebra M, Brooks P, Cheresh D.
Suppression of p53 activity and p21 WAF1/CIP1 expression by vascular
cell integrin vß3 during angiogenesis. J Clin
Invest. 1996;98:426–433.[Medline]
[Order article via Infotrieve]
40.
Brooks PC, Stromblad S, Sanders LC, von Schalscha TL,
Aimes R, Stetler- Stevenson WG, Quigley JP, Cheresh DA. Localization of
matrix metalloproteinase MMP-2 to the surface of invasive cells by
interaction with integrin vß3. Cell. 1996;85:683–693.[Medline]
[Order article via Infotrieve]
41. Matrisian LM. The matrix-degrading metalloproteinases. Bioessays. 1992;14:455–463.[Medline] [Order article via Infotrieve]
42.
Lee RT, Libby P. The unstable atheroma.
Arterioscler Thromb Vasc Biol. 1997;17:1859–1867.
43.
Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler
LF, Wong SC, Hong MK, Kovach JA, Leon MB. Arterial
remodeling after coronary angioplasty: a serial intravascular
ultrasound study. Circulation. 1996;94:35–43.
44. Glagov S, Weisenberg E, Zarins CK, Stankunavicius R, Kolettis GJ. Compensatory enlargement of human atherosclerotic coronary arteries. N Engl J Med. 1987;316:1371–1375.[Abstract]
45. Geary RL, Nikkari ST, Wagner WD, Williams JK, Adams MR, Dean RH. Wound healing: a paradigm for lumen narrowing after arterial reconstruction. J Vasc Surg. 1998;27:96–108.[Medline] [Order article via Infotrieve]
46. Ashizawa N, Graf K, Do YS, Nunohiro T, Giacheli CM, Meehan WP, Tuan TL, Hsueh WA. Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction. J Clin Invest. 1996;98:2218–2227.[Medline] [Order article via Infotrieve]
47.
Yee KO, Rooney MM, Giachelli CM, Lord St, Schwartz SM.
Role of ß1 and ß3 integrins in human smooth muscle cell
adhesion to and contraction of fibrin clots in vitro. Circ
Res. 1998;83:241–251.
48.
Mogford JE, Davis GE, Platts SH, Meininger GA. Vascular
smooth muscle vß3 integrin mediates arteriolar vasodilatation
in response to RGD peptides. Circ Res. 1996;79:821–826.
49. Lipke DW, Soltis EE, Fiscus RR, Yang L, Newman PS, Aziz SM. RGD-containing peptides induce endothelium-dependent and independent vasorelaxations of rat rings. Regul Pept. 1996;63:23–29.[Medline] [Order article via Infotrieve]
50.
Müller JM, Chilian WM, Davis MJ. Integrin
signaling transduces shear-stress-dependent vasodilation of
coronary arterioles. Circ Res. 1997;80:320–326.
51.
Davis GE. Affinity of integrins for damaged
extracellular matrix: vß3 binds to denatured collagen type I
through RGD sites. Biochem Biophys Res Commun. 1992;182:1025–1031.[Medline]
[Order article via Infotrieve]
52.
Montgomery AM, Reisfeld RA, Cheresh DA. Integrin
vß3 rescues melanoma cells from apoptosis in
three-dimensional dermal collagen. Proc Natl Acad Sci
U S A. 1994;91:8856–8860.
53.
Deitch JS, Williams JK, Adams MR, Fly CA,
Herrington DM, Jordan RE, Nakada MT, Jakubowski JA, Geary RL. Effects
of ß3 blockade (c7E3) on the response to angioplasty and
intra-arterial stenting in atherosclerotic non-human
primates. Arterioscler Thromb Vasc Biol. 1998;18:1730–1737.
54.
Miyamoto S, Akiyama SK, Yamada KM. Synergistic roles
for receptor occupancy and aggregation in integrin transmembrane
function. Science. 1995;267:883–885.
55.
Hoffmann R, Mintz GS, Dussaillant GR, Popma JJ, Pichard
AD, Satler LF, Kent KM, Griffin J, Leon MB. Patterns and mechanisms of
in-stent restenosis: a serial intravascular ultrasound study.
Circulation. 1996;94:1247–1254.
This article has been cited by other articles:
 |
|
 |
|
  J. L. Wilkinson-Berka, D. Jones, G. Taylor, K. Jaworski, D. J. Kelly, S. B. Ludbrook, R. N. Willette, S. Kumar, and R. E. Gilbert SB-267268, a Nonpeptidic Antagonist of {alpha}v{beta}3 and {alpha}v{beta}5 Integrins, Reduces Angiogenesis and VEGF Expression in a Mouse Model of Retinopathy of Prematurity. Invest. Ophthalmol. Vis. Sci., April 1, 2006; 47(4): 1600 - 1605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Asano, H. Ihn, K. Yamane, M. Jinnin, Y. Mimura, and K. Tamaki Increased Expression of Integrin {alpha}v{beta}3 Contributes to the Establishment of Autocrine TGF-{beta} Signaling in Scleroderma Fibroblasts J. Immunol., December 1, 2005; 175(11): 7708 - 7718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Honda, T. Kitano, F. Fukuya, Y. Sato, S. Iwama, T. Morie, and M. Notake A Novel {alpha}v{beta}3 Integrin Antagonist Suppresses Neointima Formation for More Than 4 Weeks After Balloon Injury in Rats Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1376 - 1382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Stawowy, H. Kallisch, J. P. Veinot, A. Kilimnik, W. Prichett, S. Goetze, N. G. Seidah, M. Chretien, E. Fleck, and K. Graf Endoproteolytic Activation of {alpha}v Integrin by Proprotein Convertase PC5 Is Required for Vascular Smooth Muscle Cell Adhesion to Vitronectin and Integrin-Dependent Signaling Circulation, February 17, 2004; 109(6): 770 - 776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kanzaki and M. Otabe Latent Transforming Growth Factor-{beta} Binding Protein-1, a Component of Latent Transforming Growth Factor-{beta} Complex, Accelerates the Migration of Aortic Smooth Muscle Cells in Diabetic Rats Through Integrin-{beta}3 Diabetes, March 1, 2003; 52(3): 824 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. P. Moiseeva Adhesion receptors of vascular smooth muscle cells and their functions Cardiovasc Res, December 1, 2001; 52(3): 372 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lele, M. Sajid, N. Wajih, and G. A. Stouffer Eptifibatide and 7E3, but Not Tirofiban, Inhibit {alpha}v{beta}3 Integrin-Mediated Binding of Smooth Muscle Cells to Thrombospondin and Prothrombin Circulation, July 31, 2001; 104(5): 582 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Bishop, J. A. McPherson, J. M. Sanders, S. E. Hesselbacher, M. J. Feldman, C. A. McNamara, L. W. Gimple, E. R. Powers, S. A. Mousa, and I. J. Sarembock Selective {{alpha}}v{beta}3-Receptor Blockade Reduces Macrophage Infiltration and Restenosis After Balloon Angioplasty in the Atherosclerotic Rabbit Circulation, April 10, 2001; 103(14): 1906 - 1911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. A. Chico, J. Chamberlain, J. Gunn, N. Arnold, S. L. Bullens, T. R. Gadek, S. E. Francis, S. Bunting, M. Horton, L. Shepherd, et al. Effect of Selective or Combined Inhibition of Integrins {{alpha}}IIb{beta}3 and {{alpha}}v{beta}3 on Thrombosis and Neointima After Oversized Porcine Coronary Angioplasty Circulation, February 27, 2001; 103(8): 1135 - 1141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Bendeck, C. Irvin, M. Reidy, L. Smith, D. Mulholland, M. Horton, and C. M. Giachelli Smooth Muscle Cell Matrix Metalloproteinase Production Is Stimulated via {alpha}v{beta}3 Integrin Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1467 - 1472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kanda, M. Kuzuya, M. A. Ramos, T. Koike, K. Yoshino, S. Ikeda, and A. Iguchi Matrix Metalloproteinase and {alpha}v{beta}3 Integrin-Dependent Vascular Smooth Muscle Cell Invasion Through a Type I Collagen Lattice Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 998 - 1005. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Bhatt, S. P. Marso, A. M. Lincoff, K. E. Wolski, S. G. Ellis, and E. J. Topol Abciximab reduces mortality in diabetics following percutaneous coronary intervention J. Am. Coll. Cardiol., March 15, 2000; 35(4): 922 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Pasterkamp, D. P.V de Kleijn, and C. Borst Arterial remodeling in atherosclerosis, restenosis and after alteration of blood flow: potential mechanisms and clinical implications Cardiovasc Res, March 1, 2000; 45(4): 843 - 852. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rezaee, K. Penta, and T. Quertermous Del1 mediates VSMC adhesion, migration, and proliferation through interaction with integrin alpha vbeta 3 Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1924 - H1932. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||