(Circulation. 2001;103:562.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Adenoviral Expression of a Urokinase Receptor–Targeted Protease Inhibitor Inhibits Neointima Formation in Murine and Human Blood Vessels
From the Gaubius Laboratory TNO-PG (P.H.A.Q., M.L.M.L., J.H.P.L., J.M.G., M.R.d.V, J.S., J.H.V., V.W.M.v.H.), and Department of Anatomy and Embryology, LUMC (M.C.d.R.), Leiden, and Department of Physiology, Institute for Cardiovascular Research, Vrije Universiteit, Amsterdam (V.W.M.v.H.), Netherlands; and the Department of Pathology, AZ Middelheim, Antwerp, Belgium (M.M.K.).
Correspondence to Dr P.H.A. Quax, Gaubius Laboratory TNO-PG, PO Box 2215, 2301CE Leiden, Netherlands. E-mail pha.quax{at}pg.tno.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin–mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface–bound plasmin, which cannot be inhibited by its natural inhibitors, such as
2-antiplasmin. This hybrid protein,
consisting of the receptor-binding amino-terminal fragment of uPA
(ATF), linked to the potent protease inhibitor bovine pancreas trypsin
inhibitor (BPTI), can inhibit plasmin activity at the cell
surface. Methods and Results—The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5x109 pfu/mL) resulted in 87.5±3.8% (mean±SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9±2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect.
Conclusions—These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.
Key Words: plasminogen • restenosis • gene therapy • urokinase • receptors
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Smooth muscle cell (SMC) proliferation and migration play a major role in neointima formation and restenosis.1 2 It has been suggested that the role of SMC migration is strongly underestimated and that SMC migration from media and adventitia to the (neo)intima is essential in neointima formation.3 4
Cell migration requires coordinated detachment and renewal
of cell-matrix interaction sites, a process in which proteolytic
enzymes are involved. Involvement of the plasminogen activation/plasmin
system in the regulation of SMC migration in vitro and neointima
formation in vivo has been demonstrated in several
studies.5 6 7 8 9
Inhibition of protease activity, either by knocking out specific
proteases or by overexpressing protease inhibitors such as plasminogen
activator inhibitor (PAI)-1 or tissue inhibitors of metalloproteinases
(TIMPs), has resulted in inhibition of neointima formation, but the
observed inhibition was either 
50% or 79% for
TIMP-2,8 10 11 12 13 14
or was only temporary (uPA-/-,
Plg-/-).7 9
Binding to specific cell surface receptors facilitates local activation
of plasminogen at the cell
surface15 and restricts the
activity of plasminogen activators and plasmin to the direct
pericellular environment. Inhibition of plasmin activity to prevent SMC
migration should occur directly at the cell surface to be most
effective. However, the accessibility of receptor-bound plasmin to its
natural inhibitors, such as 2-antiplasmin and
2-macroglobulin, is very
low.16
This study describes the use of an adenoviral vector encoding a newly constructed hybrid protein designed to inhibit plasmin activity directly at the cell surface as a new approach to preventing neointima formation. This hybrid protein consists of the amino-terminal fragment (ATF) of human urokinase plasminogen activator (uPA), which binds to the uPA receptor, linked to bovine pancreas trypsin inhibitor (BPTI), also known as aprotinin, a very potent inhibitor of plasmin. For application within a murine system, a variant of ATF.BPTI was constructed, mATF.BPTI, in which amino acid residues 22, 27, 29, and 30 of human uPA are replaced by their murine counterparts,17 which can bind to the mouse uPA receptor (uPAR).
The effect of adenovirus-mediated expression of ATF.BPTI on neointima formation was studied in organ cultures of human saphenous vein. Furthermore, to test the efficacy of ATF.BPTI gene transfer in vivo, neointima formation was induced in the mouse femoral arteries as described by Moroi et al,18 and mice were infected with Ad.CMV.mATF.BPTI.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Construction of ATF.BPTI Expression Plasmid and Adenoviral Vectors
pCMV.ATF.BPTI, an adenoviral shuttle vector encoding the ATF.BPTI hybrid protein, was constructed by deleting the DNA sequences encoding amino acids 139 to 401 from a uPA cDNA–containing plasmid,17 resulting in a plasmid encoding the ATF and the 11 C-terminal amino acid residues of uPA, including the stop codon. Subsequently, a DNA fragment encoding amino acid residues 36 to 93 of BPTI, isolated by polymerase chain reaction (PCR) on genomic DNA from bovine aortic endothelial cells, was cloned into this vector. The ATF.BPTI-encoding DNA fragment was cloned in the adenoviral shuttle vector pCMV. For the generation of the recombinant adenovirus, pCMV.ATF.BPTI and pJM17 were cotransfected in HER9.11 cells19 by standard procedures. Ad.CMV.mATF.BPTI was constructed by an identical procedure, with the (Tyr22, Arg27, 29, 30) uPA mutant cDNA17 used as starting material. To construct AdCMV.BPTI, pCMV.BPTI was constructed by deleting the sequences encoding amino acids 2 to 137 from pCMV.ATF.BPTI by use of PCR. PCMV.ATF and p.CMV.mATF were used to construct Ad.CMV.ATF and Ad.CMV.mATF. An "empty" control adenoviral vector (Ad.Control) was constructed by use of the pCMV vector without insert. For all adenoviral preparations, the particle-to-pfu ratios were determined and shown to be between 15 and 20.
An ATF.BPTI ELISA was set up with antibodies against ATF20 as a catching antibody and against aprotinin (gift from Dr Emeis, Leiden, Netherlands) as a detecting antibody. With this ELISA, both ATF.BPTI and mATF.BPTI can be detected. For quantification, a standard of ATF.BPTI was prepared by infection of Chinese hamster ovary (CHO) cells, of which the ATF concentration was determined with a uPA ELISA.20
Plasmin activity was analyzed by use of the chromogenic
substrate S2251 (Chromogenix). Diluted samples of the conditioned CHO
medium (2 µg/mL ATF.BPTI, or 0.1 µmol/L) were incubated for 15
minutes with 500 pmol/L plasmin. Then S2251 was added, and after a
24-hour incubation at 37°C, the A405 was
measured. As a control, plasmin was incubated with buffer or with 10 or
100 pmol/L aprotinin (Trasylol, Bayer). For detection of plasmin
activity in vascular tissue, extracts of 4 murine aortas were prepared
as previously described21
and pooled. After 15 minutes of incubation at room temperature with 100
pmol/L plasmin, S2251 was added, and the activity was measured in the
absence and presence of aprotinin-inhibiting antibodies to discriminate
between intrinsic 2-antiplasmin
activity22 and BPTI-mediated
plasmin inhibition.
Saphenous Vein Organ Cultures and Analysis of
Neointima Formation
Segments of saphenous vein were obtained from
patients undergoing CABG surgery, according to the guidelines of the
Review Board of the Leiden University Medical Center. The veins were
cultured as described
previously.23 24
From every patient, 1 segment was infected for 1 hour with
5x109 pfu Ad.CMV.ATF.BPTI in 1 mL at 37°C
with gentle shaking, and 1 was mock-transfected. From 3 patients,
segments were infected with 5x109 pfu
Ad.CMV.ATF, Ad.CMV.BPTI, or Ad.Control or mock-transfected. Vein
segments were cultured for 5 weeks and analyzed histochemically. The
viability of cultures during 5 weeks was demonstrated by an unchanged
ATP production during the culture period (not shown). Neointima
formation in the treated segments was always compared with their
untreated counterparts and quantified on multiple sections (n=6) of the
segments with the QWin image analysis system
(Leica).
Immunohistochemistry
Immunohistochemistry was performed on
paraffin-embedded sections with monoclonal antibodies against -SMC
actin (Sigma), uPA receptor (H2, gift from Dr Weidle, Penzberg,
Germany), von Willebrand factor (DAKO A/S), the macrophage-specific
monoclonal antibody AIA31240 (Accurate Chemical), and polyclonal
antibodies against uPA25 and
aprotinin.
Bound antibodies were detected with horseradish peroxidase–conjugated rabbit anti-mouse antibodies or swine anti-rabbit antibodies (Dako A/S). Sections were counterstained with hematoxylin. In situ hybridization was performed as described26 with 0.7-kB 35S-labeled riboprobes for human uPA.
Femoral Artery Cuff Placement
All animal experiments were approved by the Animal
Welfare Committee of TNO. C57BL/6 mice (18 to 25 g) were
anesthetized with Hypnorm (Bayer) and Dormicum (Roche) (25 mg/kg each).
The left femoral artery was isolated from surrounding tissues, loosely
sheathed with a 1.0-mm polyethylene cuff (PE-50 tubing; ID, 0.4 mm; OD,
0.8 mm; Becton Dickinson) as described
previously.18 The right
femoral vein was dissected and used for intravenous injection with
viral vectors (2x109 pfu in 200 µL).
Animals were euthanized after 19 days. After perfusion fixation, tissue
segments were embedded in paraffin. Neointima formation was quantified
by image analysis in 6 representative serial sections per vessel
segment.
Statistical Analysis
Data are presented as mean±SEM. Statistical analysis
of neointima formation in organ cultures was performed with 1-way ANOVA
followed by Fisher’s least significant difference test. For the in
vivo experiments, overall comparisons between groups were performed
with the Kruskal-Wallis test. If a significant difference was found,
groups were compared with their control by Mann-Whitney rank sum tests.
Probability values of P<0.05
were regarded as significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In Vitro Characterization of ATF-BPTI
An adenoviral vector (Ad.CMV.ATF.BPTI) was constructed that was capable of directing the expression and secretion of the hybrid protein ATF.BPTI, consisting of ATF and BPTI (Figure 1A
). In parallel, Ad.CMV.mATF.BPTI, encoding the
murine residues Tyr22, Arg27, Arg29, and Arg30 in the human ATF domain,
was constructed. Northern blot analysis of Ad.CMV.ATF.BPTI-infected CHO
cells revealed a hybridization signal for both uPA and BPTI cDNA probes
with an RNA of 1100 nucleotides, the expected size of ATF.BPTI mRNA
(Figure 1B). By Western blot analysis of conditioned medium
of Ad.CMV.ATF.BPTI-infected CHO cells with antibodies against human uPA
to detect the ATF domain and antibodies against aprotinin to detect the
BPTI domain, the presence of the 2 domains in ATF.BPTI was confirmed
(Figure 1C). ATF.BPTI protein production in
Ad.CMV.ATF.BPTI-infected CHO cells was also demonstrated by ELISA (data
not shown).
|
Inhibition of plasmin activity at the surface of human saphenous vein SMCs was achieved by incubating SMCs for 1 hour with ATF.BPTI-containing CHO cell culture medium. After extensive washing, extracts were prepared and the plasmin inhibitory capacity was determined. Plasmin activity was inhibited by 85.2±3.9% with a 1:4 dilution of CHO medium. Incubation with increasing concentrations of a uPAR-blocking antibody (H2) reduced plasmin inhibition dose-dependently (not shown). Incubation with anti-aprotinin antibodies resulted in a total reduction of the plasmin inhibition. No plasmin inhibitory activity could be detected in lysates of cells that underwent a mild acid treatment (2 minutes in pH 3.0 glycine buffer), demonstrating that the inhibitory activity can be removed from the uPAR. Similarly, 94.7±1.5% inhibition of plasmin activity at the surface of murine endothelioma cells by mATF.BPTI was demonstrated. These results indicate that ATF.BPTI binds to the uPAR and can inhibit plasmin activity at the cell surface.
Effect of Ad.CMV.ATF.BPTI Infection on
Neointima Formation in Human Saphenous Vein Organ Cultures
In human saphenous vein organ cultures, a multilayer
neointima is formed in 5 weeks that consists mainly of -SMC
actin–positive cells, either SMCs or myofibroblasts. These cells
originate from the media and adventitia
(Figure 2A and 2C). uPAR expression was detected in cells of
the media (also before culture
[Figure 2B]) and neointima
(Figure 2D), as well as in the adventitia. Staining for uPAR
in media and neointima indicates that the receptor, to which ATF.BPTI
is expected to bind, is present in the target cells in the saphenous
vein.
|
Efficient transduction of saphenous vein segments with
Ad.CMV.ATF.BPTI and subsequent expression of ATF.BPTI were monitored by
reverse transcription (RT)-PCR, in situ hybridization, and ELISA
(Figure 3).
|
To assess whether adenovirus-mediated overexpression
of ATF.BPTI in saphenous vein segments would inhibit neointima
formation, segments were infected with Ad.CMV.ATF.BPTI and cultured for
5 weeks. A nearly complete reduction in neointima size was observed in
all the sections studied. Representative histological sections are
shown in
Figure 4A. In the control section, after 5 weeks, a
multilayer neointima can be observed, whereas in the
Ad.CMV.ATF.BPTI-transduced section of the same patient, virtually no
neointima is present, comparable to a segment harvested at the start of
the culture
(Figure 4A). Neointima formation was quantified by image
analysis in multiple sections of the segments, pairwise in treated and
untreated counterparts, and the rate of inhibition was calculated. The
mean neointimal area was 11.2±1.6 mm2
(no virus) compared with 1.2±0.3 mm2
(Ad.CMV.ATF.BPTI virus). The mean inhibition of neointima formation is
87.5±3.8% (mean±SEM, n=10,
P<0.001)
(Figure 4B). After transduction with
5x109 pfu Ad.CMV.Control, Ad.CMV.ATF, or
Ad.CMV.BPTI, the inhibition of neointima formation was not
significantly different from the mock-transfected control segments,
except for Ad.CMV.mATF
(P=0.018). After transduction
with 5x109 pfu AD.CMV.LacZ, intense blue
staining of the neointima can be observed
(Figure 4C), indicating that efficient transduction of the
segments can be obtained.
|
Effect of Ad.CMV.mATF.BPTI Infection on
Neointima Formation In Vivo
Neointima formation was induced in vivo in murine
femoral arteries by placement of a 0.4-mm polyethylene cuff. Cuff
placement resulted in profound neointima formation in 3 weeks,
consisting of 4 to 6 layers of SMCs, as described
previously18
(Figure 5A). The cuff-induced neointima consisted primarily
of SMCs
(Figure 5B) on top of the internal elastic lamina
(Figure 5C), beneath a layer of von Willebrand
factor–positive endothelial cells
(Figure 5D). Macrophage infiltrates were not detected in the
neointimal area but were present in the granulation tissue within the
cuff, surrounding the vessel
(Figure 5E).
|
) were detected only in granulation tissue surrounding vessel within cuff, but not in neointima (E).
Mice were infected with Ad.CMV.mATF.BPTI
(109 pfu in 200 µL) in the femoral vein
and were killed 5 days later. ATF.BPTI was detected by
immunohistochemistry with anti-aprotinin antibodies in liver
parenchymal cells
(Figure 6A) and in the cuffed arteries of
Ad.CMV.mATF.BPTI-infected mice. The most prominent staining was near
the luminal side, but positive cells could also be detected in the
deeper layers of the vessel wall
(Figure 6B). After 5 days, plasma levels of 40 ng/mL ATF.BPTI
were reached. In aorta tissue extracts, 1 ng/mg ATF.BPTI was present.
Furthermore, in the vessel wall, extracts of Ad.CMV.mATF.BPTI-infected
mice, in addition to the ubiquitously present
2-antiplasmin
activity22 (able to inhibit
65.3±3.5 µ-casein units (CU) plasmin [mean±SEM, n=3]), an
aprotinin-specific plasmin inhibitory activity was detected, able to
inhibit 11.2±2.9 µCU (equivalent to 0.45 ng active plasmin) per
milligram of tissue extract. This inhibitory activity was blocked by
antibodies against aprotinin. In extracts of control mice, only
2-antiplasmin activity, and no BPTI-mediated
plasmin inhibition, could be detected.
|
Next, mice were infected with Ad.CMV.mATF.BPTI,
Ad.CMV.mATF, Ad.CMV.BPTI, or Ad.Control in the contralateral femoral
vein. ATF.BPTI expression was monitored by ELISA
(Figure 6C
), and 19 days after infection, Ad.CMV.mATF.BPTI
plasma levels of 136±19 ng/mL ATF.BPTI, 118±11 ng/mL ATF, and 65±13
ng/mL BPTI were detectable. Mice were killed on day 19, and cuffed
vessel segments were analyzed for neointima formation. In
Ad.CMV.mATF.BPTI-treated animals, the neointima is maximally 1 or 2
cell layers thick, whereas in untreated control mice, Ad.Control-
(Figure 7), Ad.CMV.mATF-, or Ad.CMV.BPTI-infected mice (not
shown), the neointima is 4 to 6 cell layers thick. Quantification
revealed no significant difference in neointima formation in either
mock-infected, Ad.Control-, Ad.CMV.mATF-, or Ad.CMV.BPTI-infected mice,
whereas in Ad.CMV.mATF.BPTI-infected mice, a dose-dependent reduction
of neointima formation can be observed
(Figure 7B), with a mean neointima area of 333±127
µm2 (mean±SEM, n=6) after infection with
109 pfu. No decrease in media size could be
observed, only a small but significant increase in media area in the
Ad.CMV.mATF-infected mice. The mean percentage of luminal stenosis was
reduced from 25% to 2% in the Ad.CMV.mATF.BPTI-infected
mice.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In this report, adenoviral gene transfer of a novel hybrid protein, ATF.BPTI, was analyzed in 2 independent models for neointima formation, a human in vitro model for vein graft neointima formation and a murine in vivo model for neointima formation. It was demonstrated that this secreted, cell surface–binding serine protease inhibitor strongly reduces SMC migration and neointima formation in both human and murine blood vessels. Whereas the
2-antiplasmin activity ubiquitously present
in the vessel wall seems to have no effect on neointima formation, the
cell surface–targeted ATF.BPTI does so very efficiently. ATF and BPTI
had no significant effect on neointima formation. This indicates that
cell surface targeting of BPTI largely improves the efficacy of plasmin
inhibition in preventing neointima formation. Several gene transfer approaches have been used to inhibit intimal hyperplasia and SMC accumulation. Most of these studies are directed at inhibiting SMC proliferation after vessel wall injury. Recently, several studies reported partial or temporal inhibition of neointima formation by use of protease inhibitors, TIMP, or PAI-1.8 10 The genes used thus far, such as HSV-tk, p21, hirudin, TIMP1, and PAI-1,1 2 8 10 27 are all existing genes, in contrast to the new recombinant gene we describe here.
In this study, we first focused on inhibition of SMC
migration in human vein graft neointima formation. Saphenous vein organ
cultures were used as a human model forneointima formation
because in these cultures, a neointima develops, most likely in
response to the mechanical injury, that is very similar to early
lesions observed in vein grafts in
vivo.24 28 29 30 31
After Ad.CMV.ATF.BPTI infection, virtually no neointimal structures
were formed in these organ cultures, whereas infection with Ad.CMV.BPTI
had no effect on neointima formation
(Figure 4). ATF.BPTI is a secreted protein, and therefore,
because of the bystander effect, noninfected cells expressing uPAR may
also be affected. It was reported recently that adenovirus-mediated
TIMP-1 or TIMP-2 overexpression in saphenous vein organ cultures
results in inhibition of SMC migration and neointima
formation.12 13
The effect of ATF.BPTI on neointima formation was
confirmed in a second, independent model for neointima formation, the
cuff-induced neointima formation in murine femoral artery in vivo,
previously described by Moroi et
al.18 Although the exact
mechanism of cuff-induced neointima formation is not
clear,32 a SMC-rich
neointima, almost devoid of macrophages, can be observed in the mice,
comparable to cuff-induced neointima in the rabbit carotid
artery.32 Intravenous
infection with Ad.CMV.mATF.BPTI resulted in a reproducible expression
of mATF.BPTI, a variant of ATF.BPTI that can bind to the murine uPAR.
This was demonstrated by expression of ATF.BPTI in the liver, the
presence of ATF.BPTI in plasma detectable even after 3 weeks,
BPTI-specific plasmin inhibition in the vessel wall, and the presence
of ATF.BPTI in cuffed arteries. Furthermore, a profound inhibitory
effect on cuff-induced neointima formation in the femoral artery was
observed
(Figure 7). Infection with an "empty" control virus or a
vector encoding either ATF or BPTI had little or no effect on neointima
formation. Although BPTI is a potent inhibitor of plasmin, and plasmin
is involved in neointima formation after vascular injury, it cannot be
excluded that other serine proteases, eg, trypsin, are also inhibited
by ATF.BPTI. However, little is known about the presence of other
serine proteases in the vessel wall and their possible involvement in
intimal thickening.
Minimal or nonsignificant effects of Ad.CMV.ATF.BPTI infection on SMC proliferation occurred (our unpublished data). This suggests that the inhibitory effect of ATF.BPTI on neointima formation is due to inhibition of SMC migration; however, a contribution of an effect of ATF.BPTI on SMC proliferation cannot be excluded.
We think that the ATF.BPTI-expressing adenoviral vector represents a powerful approach to preventing SMC migration after vascular wounding and thus might contribute to the reduction of neointima formation and restenosis.
|
Acknowledgments |
|---|
This study was supported by the Netherlands Heart Foundation, grants M93.001 and 95.126. We would like to thank Marjanka Aalders, Erik Offerman, Ria van den Hoogen, and Hans van de Boom for their technical assistance.
Received June 26, 2000; revision received August 30, 2000; accepted August 31, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Yang ZY, Simari RD, Perkins ND, et al. Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc Natl Acad Sci U S A. 1996;93:7905–7910.
2. Rade JJ, Schulick AH, Virmani R, et al. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nat Med. 1996;2:293–298.[Medline] [Order article via Infotrieve]
3. Schwartz SM. Smooth muscle migration in atherosclerosis and restenosis. J Clin Invest. 1997;100:S87–S89.
4.
Shi Y,
O’Brien JE, Fard A, et al. Adventitial myofibroblasts contribute to
neointimal formation in injured porcine coronary arteries.
Circulation. 1996;94:1655–1664.
5.
Wijnberg MJ,
Quax PHA, Nieuwenbroek NME, et al. The migration of human smooth muscle
cells in vitro is mediated by plasminogen activation and can be
inhibited by (2)-macroglobulin receptor associated protein.
Thromb Haemost. 1997;78:880–886.[Medline]
[Order article via Infotrieve]
6.
Clowes AW,
Clowes MM, Au YPT, et al. Smooth muscle cells express urokinase during
mitogenesis and tissue-type plasminogen activator during migration in
injured rat carotid artery. Circ
Res. 1990;67:61–67.
7.
Carmeliet P,
Moons L, Herbert JM, et al. Urokinase but not tissue plasminogen
activator mediates arterial neointima formation in mice.
Circ Res. 1997;81:829–839.
8.
Carmeliet P,
Moons L, Lijnen R, et al. Inhibitory role of plasminogen activator
inhibitor-1 in arterial wound healing and neointima formation: a gene
targeting and gene transfer study in mice.
Circulation. 1997;96:3180–3191.
9. Carmeliet P, Moons L, Ploplis V, et al. Impaired arterial neointima formation in mice with disruption of the plasminogen gene. J Clin Invest. 1997;99:200–208.[Medline] [Order article via Infotrieve]
10.
Forough R,
Koyama N, Hasenstab D, et al. Overexpression of tissue inhibitor of
matrix metalloproteinase-1 inhibits vascular smooth muscle cell
functions in vitro and in vivo. Circ
Res. 1996;79:812–820.
11.
Dollery CM,
Humphries SE, Mcclelland A, et al. Expression of tissue inhibitor of
matrix metalloproteinases 1 by use of an adenoviral vector inhibits
smooth muscle cell migration and reduces neointimal hyperplasia in the
rat model of vascular balloon injury.
Circulation. 1999;99:3199–3205.
12. George SJ, Baker AH, Angelini GD, et al. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther. 1998;5:1552–1560.[Medline] [Order article via Infotrieve]
13. George SJ, Johnson JL, Angelini GD, et al. Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein. Hum Gene Ther. 1998;9:867–877.[Medline] [Order article via Infotrieve]
14.
Rouis M,
Adamy C, Duverger N, et al. Adenovirus-mediated overexpression of
tissue inhibitor of metalloproteinase-1 reduces atherosclerotic lesions
in apolipoprotein E–deficient mice.
Circulation. 1999;100:533–540.
15. Quax PHA, Pedersen N, Masucci MT, et al. Complementation between urokinase-producing and receptor- producing cells in extracellular matrix degradation. Cell Regul. 1991;2:793–803.[Medline] [Order article via Infotrieve]
16.
Hall SW,
Humphries JE, Gonias SL. Inhibition of cell surface receptor-bound
plasmin by 2-antiplasmin and 2-macroglobulin.
J Biol Chem. 1991;266:12329–12336.
17.
Quax PHA,
Grimbergen JM, Lansink M, et al. Binding of human urokinase-type
plasminogen activator to its receptor: residues involved in species
specificity and binding. Arterioscler
Thromb Vasc Biol. 1998;18:693–701.
18. Moroi M, Zhang L, Yasuda T, et al. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest. 1998;101:1225–1232.[Medline] [Order article via Infotrieve]
19. Fallaux FJ, Kranenburg O, Cramer SJ, et al. Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gen Ther. 1996;7:215–222.[Medline] [Order article via Infotrieve]
20.
Koolwijk P,
van Erck MGM, Devree WJA, et al. Cooperative effect of TNF, bFGF,
and VEGF on the formation of tubular structures of human microvascular
endothelial cells in a fibrin matrix: role of urokinase activity.
J Cell Biol. 1996;132:1177–1188.
21.
Quax PHA,
van den Hoogen CM, Verheijen JH, et al. Endotoxin induction of
plasminogen activator and plasminogen activator inhibitor type1 mRNA in
rat tissues in vivo. J Biol
Chem. 1990;265:15560–15563.
22.
Robbie LA,
Booth NA, Brown AJ, et al. Inhibitors of fibrinolysis are elevated in
atherosclerotic plaque. Arterioscler
Thromb Vasc Biol. 1996;16:539–545.
23. Soyombo AA, Angelini GD, Bryan AJ, et al. Intimal proliferation in an organ culture of human saphenous vein. Am J Pathol. 1990;137:1401–1410.[Abstract]
24. Slomp J, Gittenberger-deGroot AC, van Munsteren JC, et al. Nature and origin of the neointima in whole vessel wall organ culture of the human saphenous vein. Virchows Arch. 1996;428:59–67.[Medline] [Order article via Infotrieve]
25. Binnema DJ, Vaniersel JJL, Dooijewaard G. Quantitation of urokinase antigen in plasma and culture media by use of an ELISA. Thromb Res. 1986;43:569–577.[Medline] [Order article via Infotrieve]
26.
Hierck BP,
Poelmann RE, Vaniperen L, et al. Differential expression of -6 and
other subunits of laminin binding integrins during development of the
murine heart. Dev Dyn. 1996;206:100–111.[Medline]
[Order article via Infotrieve]
27.
Ohno T,
Gordon D, San H, et al. Gene therapy for vascular smooth muscle cell
proliferation after arterial injury.
Science. 1994;265:781–784.
28.
Motwani JG,
Topol EJ. Aortocoronary saphenous vein graft disease: pathogenesis,
predisposition, and prevention.
Circulation. 1998;97:916–931.
29. Kockx MM, Cambier BA, Bortier H, et al. The modulation of smooth muscle cell phenotype is an early event in human aorto-coronary saphenous vein grafts. Virchows Arch. 1992;420:155–162.
30.
Shi Y,
O’Brien JEJ, Mannion JD, et al. Remodeling of autologous saphenous
vein grafts: the role of perivascular myofibroblasts.
Circulation. 1997;95:2684–2693.
31.
O’Brien
JE, Shi Y, Fard A, et al. Wound healing around and within saphenous
vein bypass grafts. J Thorac
Cardiovasc Surg. 1997;114:38–45.
32.
Demeyer GR,
Vanput DJ, Kockx MM, et al. Possible mechanisms of collar-induced
intimal thickening. Arterioscler Thromb
Vasc Biol. 1997;17:1924–1930.
This article has been cited by other articles:
 |
|
 |
|
  J. Wu, L. Peng, G. A. McMahon, D. A. Lawrence, and W. P. Fay Recombinant Plasminogen Activator Inhibitor-1 Inhibits Intimal Hyperplasia Arterioscler Thromb Vasc Biol, October 1, 2009; 29(10): 1565 - 1570. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. M M Pires, D. Eefting, M. R de Vries, P. H A Quax, and J W. Jukema Sirolimus and paclitaxel provoke different vascular pathological responses after local delivery in a murine model for restenosis on underlying atherosclerotic arteries Heart, August 1, 2007; 93(8): 922 - 927. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. P. Fay, N. Garg, and M. Sunkar Vascular Functions of the Plasminogen Activation System Arterioscler Thromb Vasc Biol, June 1, 2007; 27(6): 1231 - 1237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. M.M. Pires, T. W.H. Pols, M. R. de Vries, C. M. van Tiel, P. I. Bonta, M. Vos, E. K. Arkenbout, H. Pannekoek, J. W. Jukema, P. H.A. Quax, et al. Activation of Nuclear Receptor Nur77 by 6-Mercaptopurine Protects Against Neointima Formation Circulation, January 30, 2007; 115(4): 493 - 500. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. D. Krom, N. M.M. Pires, J. W. Jukema, M. R. de Vries, R. R. Frants, L. M. Havekes, K. W. van Dijk, and P. H.A. Quax Inhibition of neointima formation by local delivery of estrogen receptor alpha and beta specific agonists Cardiovasc Res, January 1, 2007; 73(1): 217 - 226. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. W.M. van Hinsbergh, M. A. Engelse, and P. H.A. Quax Pericellular Proteases in Angiogenesis and Vasculogenesis Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 716 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M.M. Pires, A. Schepers, B. L. van der Hoeven, M. R. de Vries, L. S.M. Boesten, J. W. Jukema, and P. H.A. Quax Histopathologic alterations following local delivery of dexamethasone to inhibit restenosis in murine arteries Cardiovasc Res, December 1, 2005; 68(3): 415 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. Monraats, J. S. Rana, M. C. Nierman, N. M.M. Pires, A. H. Zwinderman, J. J.P. Kastelein, J. A. Kuivenhoven, M. P.M. de Maat, S. Z.H. Rittersma, A. Schepers, et al. Lipoprotein Lipase Gene Polymorphisms and the Risk of Target Vessel Revascularization After Percutaneous Coronary Intervention J. Am. Coll. Cardiol., September 20, 2005; 46(6): 1093 - 1100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Chandiwal, V. Balasubramanian, Z. K. Baldwin, M. S. Conte, and L. B. Schwartz Gene Therapy for the Extension of Vein Graft Patency: A Review Vascular and Endovascular Surgery, January 1, 2005; 39(1): 1 - 14. [Abstract] [PDF]
|
|
|
|
 |
|
 L. Holterman, R. Vogels, R. van der Vlugt, M. Sieuwerts, J. Grimbergen, J. Kaspers, E. Geelen, E. van der Helm, A. Lemckert, G. Gillissen, et al. Novel Replication-Incompetent Vector Derived from Adenovirus Type 11 (Ad11) for Vaccination and Gene Therapy: Low Seroprevalence and Non-Cross-Reactivity with Ad5 J. Virol., December 1, 2004; 78(23): 13207 - 13215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Plaisier, K. Kapiteijn, P. Koolwijk, C. Fijten, R. Hanemaaijer, J. M. Grimbergen, A. Mulder-Stapel, P. H. A. Quax, F. M. Helmerhorst, and V. W. M. van Hinsbergh Involvement of Membrane-Type Matrix Metalloproteinases (MT-MMPs) in Capillary Tube Formation by Human Endometrial Microvascular Endothelial Cells: Role of MT3-MMP J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5828 - 5836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. van Weel, M. M.L. Deckers, J. M. Grimbergen, K. J.M. van Leuven, J. H.P. Lardenoye, R. O. Schlingemann, G. P. van Nieuw Amerongen, J. H. van Bockel, V. W.M. van Hinsbergh, and P. H.A. Quax Vascular Endothelial Growth Factor Overexpression in Ischemic Skeletal Muscle Enhances Myoglobin Expression In Vivo Circ. Res., July 9, 2004; 95(1): 58 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Collen, R. Hanemaaijer, F. Lupu, P. H. A. Quax, N. van Lent, J. Grimbergen, E. Peters, P. Koolwijk, and V. W. M. van Hinsbergh Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix Blood, March 1, 2003; 101(5): 1810 - 1817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.L.M. Lamfers, J.M. Grimbergen, M.C. Aalders, M.J. Havenga, M.R. de Vries, L.G.M. Huisman, V.W.M. van Hinsbergh, and P.H.A. Quax Gene Transfer of the Urokinase-Type Plasminogen Activator Receptor-Targeted Matrix Metalloproteinase Inhibitor TIMP-1.ATF Suppresses Neointima Formation More Efficiently Than Tissue Inhibitor of Metalloproteinase-1 Circ. Res., November 15, 2002; 91(10): 945 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Shuhaiber, A. N. Evans, M. G. Massad, and A. S. Geha Mechanisms and future directions for prevention of vein graft failure in coronary bypass surgery Eur. J. Cardiothorac. Surg., September 1, 2002; 22(3): 387 - 396. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.H.P. Lardenoye, M.R. de Vries, J.M. Grimbergen, L.M. Havekes, M.W.M. Knaapen, M.M. Kockx, V.W.M. van Hinsbergh, J.H. van Bockel, and P.H.A. Quax Inhibition of Accelerated Atherosclerosis in Vein Grafts by Placement of External Stent in ApoE*3-Leiden Transgenic Mice Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1433 - 1438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Engelse, J. H.P. Lardenoye, J. M. Neele, J. M. Grimbergen, M. R. de Vries, M. L.M. Lamfers, H. Pannekoek, P. H.A. Quax, and C. J.M. de Vries Adenoviral Activin A Expression Prevents Intimal Hyperplasia in Human and Murine Blood Vessels by Maintaining the Contractile Smooth Muscle Cell Phenotype Circ. Res., May 31, 2002; 90(10): 1128 - 1134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. E. Havenga, A. A. C. Lemckert, J. M. Grimbergen, R. Vogels, L. G. M. Huisman, D. Valerio, A. Bout, and P. H. A. Quax Improved Adenovirus Vectors for Infection of Cardiovascular Tissues J. Virol., April 1, 2001; 75(7): 3335 - 3342. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||