© 1999 American Heart Association, Inc.
Original Contribution |
Soluble Transforming Growth Factor-ß Type II Receptor Inhibits Negative Remodeling, Fibroblast Transdifferentiation, and Intimal Lesion Formation But Not Endothelial Growth
From the Center for Molecular Medicine (J.D.S., S.R.B., L.L.C., C.P.H.V., V.L.), Maine Medical Center Research Institute, South Portland, Me, and Biogen, Inc (P.J.G., V.E.K.), Cambridge, Mass.
Correspondence to Volkhard Lindner, Center for Molecular Medicine, Maine Medical Center Research Institute, 125 John Roberts Rd, Suite 12, South Portland, ME 04106. E-mail lindnv{at}poa.mmc.org
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Abstract |
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Abstract—Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-ß (TGF-ß) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-ß1, TGF-ß2, TGF-ß3, and lower levels of TGF-ß receptor II (TGF-ßRII) mRNA. Compared with normal endothelium, TGF-ß1 and TGF-ß2, as well as TGF-ßRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-ßRII (TGF-ßR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-ßR:Fc treatment almost completely inhibited the induction of smooth muscle
-actin expression in adventitial cells. In the
vessel wall, TGF-ßR:Fc caused a marked reduction in mRNA levels for
collagens type I and III. TGF-ßR:Fc had no effect on
endothelial proliferation as determined by
reendothelialization of the denuded rat aorta.
Together, these findings identify the TGF-ß isoforms as major factors
mediating adventitial fibrosis and negative remodeling after vascular
injury, a major cause of restenosis after angioplasty.
Key Words: intimal hyperplasia • collagen • fibrosis • smooth muscle -actin • myofibroblast
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Introduction |
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Vascular remodeling is a response of blood vessels to both physiological and pathological stimuli, leading to either vessel enlargement (positive remodeling) or shrinking (negative remodeling). Several studies found that neointimal proliferation or intimal mass after angioplasty showed little correlation with restenosis because of permanent changes in vascular geometry.1 2 3 In humans, intravascular ultrasound has failed to show a correlation between lumen area and neointimal area, and vascular remodeling, as measured by a decrease in vessel area, was shown to account for most of the restenosis process.4 5 These observations clearly demonstrate that a successful therapeutic approach to restenosis would have to target negative vascular remodeling.
The transforming growth factor-ß (TGF-ß) family of
cytokines can have a variety of effects on vascular cells, but
very little is known about its role in vascular remodeling. TGF-ß
affects many functions, including proliferation of smooth muscle cells
(SMCs).6 7 8 Grainger et al9 have reported
inhibition of SMC proliferation by TGF-ß1 via
extension of the G2 phase of the cell cycle,
whereas others have shown that this inhibition is due to arrest in the
late G1 phase.10 SMCs derived from
atherosclerotic lesions responded to TGF-ß1
with an increase in proliferation, and lower levels of TGF-ß receptor
II (TGF-ßRII) have been implicated in the lack of inhibition by
TGF-ß1 in these cells.8 Several
other groups have reported stimulation of SMC proliferation by
TGF-ß1 in vitro. Low doses of
TGF-ß1 were reported to stimulate SMC
proliferation via platelet-derived growth factor
(PDGF)-AA–dependent and PDGF-AA–independent
mechanisms,11 12 whereas higher doses were
inhibitory. Bifunctional effects of
TGF-ß1 were also seen in migration assays with
SMCs.7 13 In one of the first studies on the role of
TGF-ß1 in intimal lesion formation, we reported
a 5- to 7-fold induction of TGF-ß1 mRNA in the
ballooned rat carotid artery with elevated levels of
TGF-ß1 mRNA persisting for 2
weeks.14 During this period, elevated
TGF-ß1 mRNA levels correlated with increases in
mRNA expression of fibronectin and the collagens
2 (I) and 1 (III). We also found that
infusion of recombinant TGF-ß1 caused an
increase in intimal SMC proliferation in vivo.14
Of clinical relevance is the study by Nikol et al,15 who reported increased TGF-ß1 mRNA expression in restenotic lesions compared with primary atherosclerotic lesions. Additional functional data were provided by studies that used neutralizing antibodies to TGF-ß1 to reduce16 or gene transfer to increase17 expression levels of TGF-ß1 in the vessel wall. In the rat balloon injury model, treatment with TGF-ß1 antibodies caused a small but significant reduction in neointima formation.16 Overexpression of TGF-ß1 in the rat carotid artery by adenoviral gene transfer led to transient neointima formation with cartilaginous metaplasia that almost completely resolved within 8 weeks.17 TGF-ß1 may also have effects on vascular tone, because the factor was found to suppress nitric oxide synthase expression18 while inducing the vasoconstrictor endothelin in SMCs in vitro.19 In addition, antiapoptotic effects for SMCs have been demonstrated for TGF-ß1.20
Studies examining the expression of TGF-ß ligand and receptor mRNAs
by reverse transcriptase–polymerase chain reaction (PCR) revealed that
TGF-ß1, TGF-ß3, and
TGF-ßRII mRNA levels were increased in the media of the injured rat
carotid artery,21 and expression of
TGF-ß2 and TGF-ß3 was
also reported in SMCs of the lung vasculature.22 23
Reduced levels of TGF-ßRII, however, were reported in human
atherosclerotic lesions by McCaffrey et al.8 With regard
to SMCs, the 3 TGF-ß ligands have overlapping functions, and all of
them induce expression of the 1 (I), 2 (I),
and 1 (III) chains of collagens.24
Endothelial cells are growth inhibited by TGF-ß in vitro,25 but little is known about the function and expression of TGF-ß ligands in endothelial cells in vivo.
Because expression of all 3 TGF-ß ligands has been reported in the vessel wall, the present study used a dimeric recombinant soluble TGF-ßRII (TGF-ßR:Fc) to determine the importance of TGF-ß ligands in the rat balloon injury model. We studied the effects of this reagent on SMC proliferation, neointima formation, remodeling, endothelial regeneration, and expression of matrix proteins. In addition, using an en face approach, we determined the time course of mRNA expression for the TGF-ß1, TGF-ß2, TGF-ß3, and TGF-ßRII in endothelium and SMCs after wounding by in situ hybridization.
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Materials and Methods |
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Arterial Injury Model
All animal studies were approved by the Institutional Animal Care and Use Committee. Sprague Dawley rats (400 g, 3 to 4 months old) were bred from animals purchased from Taconic (Germantown, NY). All surgical procedures were carried out with the animals under general anesthesia by intraperitoneal injection of xylazine (2.2 mg/kg, AnaSed, Lloyd Laboratories) and ketamine (50 mg/kg body weight, Ketalar, Parke-Davis). The left carotid artery and the aorta were denuded with a 2F balloon catheter, as recently described.26 Starting at the day of surgery, TGF-ßR:Fc (see below) was injected into groups of rats (n=5) via the tail vein every other day at a concentration of 0.5, 2.0, and 5 mg/kg body weight in 0.2 mL of PBS. The control group (n=5) received 5 mg/kg of recombinant human IgG1 in an equal volume of PBS. Two hours before the rats were killed, 6 mg of 5-bromo-2'-deoxyuridine (BrdU; Boehringer Mannheim) was administered subcutaneously. Deendothelialized segments of arteries were identified by intravenous injection of Evans blue (0.5 mL of 5% solution in saline) 5 minutes before harvesting of the vessels. The animals were killed 14 days after balloon injury (7 injections of TGF-ßR:Fc), and perfusion fixation was carried out under physiological pressure with phosphate-buffered (0.1 mol/L, pH 7.4) 4% paraformaldehyde. Reproducibility of the data was further verified in a separate experiment by injecting groups of rats (n=5) with TGF-ßR:Fc (5 mg/kg) and vehicle following the same protocol as described above.
The effects of TGF-ßR:Fc on proliferation of medial SMCs and mRNA expression of matrix molecules were assessed in an additional experiment in which groups of rats (n=5) had balloon catheter denudation performed on both common carotid arteries. The treatment group received TGF-ßR:Fc (2 mg/kg) intravenously immediately after balloon injury, with an additional injection given 2 days later, whereas the control group received recombinant human IgG. Two hours before killing at 4 days after denudation, 6 mg of BrdU was injected subcutaneously. The right carotid artery was excised and snap frozen in liquid nitrogen for RNA extraction. After perfusion fixation with 4% paraformaldehyde, the left carotid artery was then harvested for histology and determination of medial SMC proliferation.
Production of Soluble TGF-ßRII (TGFßR:Fc)
The recombinant rabbit TGF-ßR:Fc fusion gene, comprising the
extracellular domain of the rabbit type II TGF-ß receptor fused to
the Fc portion of human IgG1 (Figure 1A
and 1B), was constructed as follows. The extracellular domain of the
rabbit type II TGF-ß receptor was amplified from clone
MIS-3f1127 by conventional PCR. Amplified sequences were
flanked with a 5' NotI and a 3' SalI restriction
site and subsequently digested with these restriction enzymes. The
human IgG1 H chain C region containing the hinge region and the CH2 and
the CH3 sequences was isolated with H chain–specific primers by PCR
amplification, as described.28 Amplified sequences
were flanked by a 5' SalI and a 3' NotI
restriction enzyme recognition site and were subsequently digested with
these restriction enzymes. The receptor and IgG DNA fragments were
cloned into the NotI restriction enzyme site in plasmid
SAB13228 and transformed into competent bacteria.
Plasmids recovered from the transformants were analyzed for the
correct orientation of assembled fragments, and the entire coding
sequence was confirmed by DNA sequencing.
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The recombinant rabbit TGF-ßR:Fc fusion gene was transfected into Chinese hamster ovary cells. After the initial transfection, cells were selected in 250 nmol/L methotrexate. The resulting colonies were expanded and analyzed for TGF-ßR:Fc fusion gene expression. Clones expressing the highest levels of TGF-ßR:Fc were selected for production of the fusion protein. TGF-ßR:Fc, which is dimeric, was purified from culture supernatant by protein A–Sepharose affinity chromatography under sterile and endotoxin-free conditions.
The activity of this reagent was tested in mink lung epithelial cells (Mv1Lu, American Type Culture Collection). These cells were seeded in 96-well plates at a density of 1.8x103 cells per well in DMEM containing 10% FBS (Atlanta Biologicals). One nanogram of TGF-ß1 (R&D Systems) was added in the absence or presence of various concentrations of soluble TGF-ßRII and incubated for 72 hours at 37°C with 5% CO2. DNA synthesis was measured by adding 1 µCi per well of [3H]thymidine (Amersham) during the last 6 hours of the incubation. Subsequently, cells were harvested with a mechanical cell harvester, and [3H]thymidine incorporation was determined with a scintillation counter. The mean value±SEM of 6 replicates is shown.
SMC Proliferation, Morphometry, and Immunostaining
The denuded and therefore Evans blue–stained part of the left
carotid artery was divided into 3 segments that were embedded together
in paraffin in 1 block. Sections (5 µm) were cut, and 3 or more
sections spanning the length of the vessel were analyzed.
Immunostaining with an antibody against BrdU (Cappel)
was performed on the 4-day and the 2-week balloon-injured vessels as
described previously.29 The number of total cells and the
number of BrdU-positive cells were separately determined for the tunica
media, neointima, and adventitia. The proliferation index
was calculated as the percentage of BrdU-positive cells.
Masson trichrome staining was used to assess the composition and amount
of extracellular matrix production. To localize the injected
TGF-ßR:Fc and the control recombinant human IgG within the vessel
wall, sections were stained with a biotin-labeled antibody against
human IgG (DAKO) used at a dilution of 1:200. Subsequent steps followed
previously published protocols.29 Expression of smooth
muscle -actin was examined by immunostaining on
cross-sectioned carotid arteries 4 days after balloon injury and
treatment with TGF-ßR:Fc (2 mg/kg) or vehicle (5 rats per group). Two
injections were given, 1 immediately after surgery and the second 2
days later. A mouse monoclonal antibody against smooth muscle -actin
(Sigma, clone 1A4) was used at a 1:2500 dilution as
described,30 and 3 or more sections from each animal were
analyzed.
Morphometric analyses were carried out on the 2-week balloon-injured common carotid arteries in a blind manner. For each animal, 3 sections originating from the proximal, middle, and distal segments of the denuded vessel were analyzed for the morphometric studies. Digitized images of these vessels were analyzed using image analysis software for Apple Macintosh computers (NIH Image, version 1.60). The circumference (length) of the lumen and the lengths of the internal elastic lamina (IEL) and the external elastic lamina (EEL) were determined by tracing along the luminal surface, the perimeter of the neointima (IEL), and the perimeter of the tunica media (EEL). Very small folds were not included in the IEL and EEL data, and therefore the measurements more accurately reflect the perimeter of the neointima and media. Assuming a circular structure, these measurements were used to calculate the lumen area. The medial area was calculated by subtracting the area defined by the IEL from the area defined by the EEL, and intimal area was determined as the area defined by the luminal surface and IEL.
Endothelial Regrowth
After balloon denudation of the rat thoracic aorta,
reendothelialization occurs by
endothelial outgrowth from intercostal arteries.
Reendothelialization was determined by injection of
Evans blue as described above. The percentage of luminal surface
covered by endothelium was determined 2 weeks after
balloon denudation and IV injection of TGF-ßR:Fc every other day (5
mg/kg, n=5 animals) or control IgG (n=4 animals). Digitized images of
the vessel surface were analyzed with NIH Image software.
Northern Blotting and cDNA Probes
The right common carotid artery was harvested from rats 4 days
after balloon injury and injection of TGF-ßR:Fc or control IgG.
Vessels were stripped of periadventitial fat and then snap frozen in
liquid nitrogen. Frozen arterial tissue was ground to a
fine powder under liquid nitrogen, and total cellular RNA was prepared
by acid guanidinium thiocyanate extraction.31 Agarose gel
electrophoresis of RNA (15 µg total RNA per lane) and transfer to
nylon membranes (Zeta Probe, BioRad Laboratories) were carried out as
previously described.32 After transfer, RNA blots were
exposed to short-wave UV light both to cross-link RNA to the membrane
and to visualize the major ribosomal RNA bands stained with ethidium
bromide. The blot was hybridized using cDNA probes labeled with
[32P]dCTP by random primer extension
(Amersham), washed at 65°C in 2 changes of 0.015 mol/L NaCl-0.0015
mol/L sodium citrate (pH 7.0)-0.1% SDS for 30 minutes each, and then
exposed to Kodak X-AR5 film at -70°C.
All probes used for Northern blotting were rat-derived cDNAs and
included 2 type I collagen,
1 type III collagen, collagen XV,
tropoelastin, osteopontin, and fibronectin. The identity of these cDNAs
was verified by DNA sequence analysis.
In Situ Hybridization
In situ hybridization was carried out on en face preparations of
vessel segments as recently described.29 Segments from
normal rat carotid arteries and aortae were used to determine gene
expression in normal, quiescent endothelium. Gene
expression in migrating and proliferating endothelium
(at the wound edge) was examined in the thoracic aorta 8 days after
denudation during active reendothelialization from
intercostal arteries. mRNA expression in proliferating and migrating
SMCs was studied in 8-day-denuded carotid arteries and aortae. In the
rat balloon injury model, SMCs return to a quiescent state within
several weeks after denudation, and we examined mRNA expression in
quiescent luminal SMCs of carotid arteries and aortae at 4 weeks after
injury. A minimum of 4 specimens from 3 different animals was
hybridized with antisense probes, and 2 specimens were hybridized with
sense probes, to assess background hybridization. A cDNA for rat
TGF-ßRII was kindly provided by Dr W.W. Vale (Salk Institute,
La Jolla, CA),33 and a 700-bp PstI fragment was
subcloned into Bluescript II (Stratagene). cDNAs for rat
TGF-ß1, rat TGF-ß2 and
mouse TGF-ß3 were kindly provided by Dr Anita
Roberts (NIH, Bethesda, MD). 35S-labeled
UTP-labeled sense and antisense riboprobes were synthesized with T3 and
T7 RNA polymerases from linearized plasmids. Following the published in
situ hybridization procedure for en face
preparations,29 the slides were coated with
autoradiographic emulsion (NTB2, Kodak), exposed for 3
weeks, and developed (D-19, Kodak). Preparations were observed under
the light microscope using dark-field and bright-field
illumination.
Statistical Analysis
ANOVA was used to determine whether significant differences
between the means of treatment groups were present
(P
0.05). Multiple comparisons between groups were then
performed using the Scheffé test. The Student t test
was used to compare the means between 2 groups, and differences were
considered significant if P0.05. All data are shown as
mean±SEM.
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Results |
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Similarly to native TGF-ßRII, TGF-ßR:Fc binds active TGF-ß1 and TGF-ß3 but not TGF-ß2, which in addition requires betaglycan (data not shown).34 35 The ability of the recombinant dimeric soluble TGF-ßR:Fc to inhibit TGF-ß–mediated responses was tested in the mink lung epithelial cell line. [3H]Thymidine incorporation in these cells is inhibited by TGF-ß1, and as shown in Figure 2
, the TGF-ßR:Fc neutralized this
inhibitory effect in a dose-dependent manner, with 50 ng/mL
of TGF-ßR:Fc completely neutralizing the effects of 1 ng/mL of
TGF-ß1. TGF-ßR:Fc had no
inhibitory effect on proliferation mediated by PDGF-BB (10
ng/mL) or fibroblast growth factor-2 (10 ng/mL) in 3T3 cells (data not
shown).
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Expression of TGF-ß Ligands and TGF-ßRII in the Vessel
Wall
We have used in situ hybridization to determine the
expression of mRNA for TGF-ß1,
TGF-ß2, TGF-ß3, and
TGF-ßRII on en face preparations. We have previously demonstrated
that this is a very sensitive and semiquantitative method for the
detection of mRNA expression in cells on the luminal surface of rat
carotid arteries and aortae.29 36 Normal, quiescent
endothelium revealed low levels of mRNA for
TGF-ß1, TGF-ß3, and
TGF-ßRII, whereas hybridization with the
TGF-ß2 antisense probe was similar to
background (Figure 3). Proliferating and
migrating endothelium at the wound edge in the thoracic
aorta 8 days after denudation showed upregulated expression of
TGF-ß1, TGF-ß2, and
TGF-ßRII, but expression of TGF-ß3 was
similar to that in normal endothelium (Figure 3). Unlike TGF-ß1 and TGF-ßRII, the
increased TGF-ß2 mRNA expression at the wound
edge was restricted to only a few cells into the
endothelial monolayer. Proliferating SMCs on the
luminal surface of carotid arteries and aortae at 8 days after injury
showed high levels of expression for all 3 TGF-ß isoforms, with
TGF-ß1 being the most abundant mRNA (Figure 3). These proliferating SMCs expressed TGF-ßRII mRNA, but the
levels were lower than in wound-edge endothelium. At 4
weeks after injury, SMCs on the surface of these
deendothelialized vessels have returned to
quiescence, and at this time point, mRNA expression levels for
TGF-ß1, TGF-ß2,
TGF-ß3, and TGF-ßRII were similar to those at
8 days (Figure 3).
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Soluble TGF-ßRII and the Balloon-Injured Carotid Artery
The left common carotid artery was denuded with a balloon
catheter, and groups of animals received TGF-ßR:Fc at concentrations
of 0.5, 2, and 5 mg/kg by intravenous injection every other
day over a period of 2 weeks. Representative sections
of the vessels from the treatment group and control group injected with
control IgG harvested after 2 weeks are shown in Figure 4. Control vessels developed an extensive
neointima and adventitial fibrosis with abundant collagen
production seen in blue on the Masson trichrome–stained
sections, and some contraction of the elastic laminae was evident
(Figure 4A and 4C). Vessels from animals injected with
TGF-ßR:Fc at 2 mg/kg revealed only little neointima
formation and much less collagen deposition in the adventitia (Figure 4B and 4D). To localize the injected TGF-ßR:Fc within the
vessel wall, sections were stained with an antibody recognizing the
human IgG domain of the recombinant fusion protein. Immunoreactivity
was concentrated in the neointima and adventitia (Figure 4F), suggesting the presence of active TGF-ß ligands at these
sites. Control vessels showed the presence of the control IgG in a very
diffuse distribution throughout the vessel wall (Figure 4E).
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TGF-ß has been implicated in myofibroblastic
transdifferentiation,37 38 39 causing fibroblasts to
transiently express smooth muscle -actin.40 We
therefore examined the expression of smooth muscle -actin in the
carotid artery by immunostaining at 4 days after
balloon denudation, when proliferation of adventitial fibroblasts is
rapid. Immunoreactive smooth muscle -actin was either completely
absent or markedly reduced in the outer adventitia of vessels from rats
treated with TGF-ßR:Fc (2 mg/kg, Figure 4G
) compared with
controls (Figure 4H).
Morphometric analysis of the carotid arteries showed
significant increases in lumen area in all rats treated with
TGF-ßR:Fc (Figure 5A), with an 
88%
increase in the 2 mg/kg group. Intimal lesion formation and ratios of
intimal/medial areas were significantly inhibited in the 2 mg/kg
(65%) and 5 mg/kg groups when compared with control animals (Figure 5B and 5C).
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Loss of lumen area as a result of negative remodeling in the
balloon-injured carotid arteries was assessed by measuring the length
of the IEL and EEL. All treatment groups revealed significantly less
shrinking of IEL and EEL compared with control animals, with an 18.3%
increase in the IEL length in the 2 mg/kg group (Figure 6). Overall, the 2 mg/kg dose had the
most pronounced effects on all of the parameters measured
in this study.
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Subsequent experiments sought to address the mechanism by which intimal
lesion formation was inhibited in response to TGF-ßR:Fc
administration. To determine whether the reduced intimal lesion was the
result of decreased intimal SMC proliferation, we performed BrdU
labeling to identify the percentage of replicating cells. The
proliferation index of intimal SMCs at 2 weeks after injury showed no
significant differences between groups (Figure 7A). As expected from the intimal area
data, however, the number of intimal cells was significantly lower in
the 2 and 5 mg/kg treatment groups (Figure 7B). We then examined
whether reductions in the intimal area were also affected by
differences in extracellular matrix accumulation. This was done
indirectly by determining the intimal cell density as nuclear profiles
per mm2. The differences between groups were
not statistically significant, even though cell density was increased
up to 30% in the 2 mg/kg treatment group (Figure 7C). In an
additional experiment, we assessed whether medial SMC proliferation was
inhibited by TGF-ßR:Fc within the first 4 days after balloon injury,
but no significant difference was found between groups (Figure 8A). In the same experiment, however,
significantly fewer cells (44% reduction) had accumulated within the
adventitia (Figure 8B).
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Expression of Matrix Molecules
Masson trichrome staining of sections indicated that adventitial
collagen deposition was reduced in response to TGF-ßR:Fc treatment.
Northern blot analysis of total RNA isolated from
balloon-injured carotid arteries 4 days after treatment with
TGF-ßR:Fc was performed to determine expression of matrix molecules
that are thought to be under transcriptional control of TGF-ß. Marked
reductions in mRNA levels of fibrillar collagens, ie,
2 chain of type I and 1 chain of type III
collagen, were seen with TGF-ßR:Fc treatment, whereas levels of
collagen XV were not affected (Figure 9).
No obvious differences in mRNA levels were seen for osteopontin,
tropoelastin, and fibronectin (Figure 9), genes of which the
expression is induced during vascular injury.14 41 The
Northern blot shown in Figure 9 is
representative of 2 independent experiments.
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Endothelial Growth in the Aorta
The in situ hybridization data demonstrate that
TGF-ß1 and TGF-ß2, as
well as the TGF-ßRII, show increased expression in proliferating and
migrating endothelium compared with normal
endothelium. The effects of TGF-ß signaling on
reendothelialization of the denuded rat aorta were
therefore examined. The surface area covered with
endothelium 2 weeks after denudation and treatment with
TGF-ßR:Fc or control IgG was measured, and in both groups 53.5% of
the surface area was reendothelialized (Figure 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study sought to determine the role of TGF-ß in arterial repair processes after balloon catheter injury. Our expression analysis by en face in situ hybridization revealed that all 3 TGF-ß isoforms were abundantly expressed in intimal SMCs, and all of these isoforms were also present in endothelium with dramatically upregulated expression of TGF-ß1 and TGF-ß2 in migrating and proliferating endothelium. Among the TGF-ß isoforms, mRNA for TGF-ß1 showed the highest expression levels in intimal SMCs, suggesting that this isoform might be the most relevant. Since the TGF-ß1, TGF-ß2, and TGF-ß3 isoforms have distinct and overlapping functions, our approach was to interfere with receptor-mediated signaling that is shared by the different isoforms, thereby inhibiting the biological responses of several TGF-ß ligands. Recombinant human soluble TGF-ßRII has been shown to inhibit responses mediated by TGF-ß1, TGF-ß3, and TGF-ß5, but not TGF-ß2.42 Similarly, TGF-ßR:Fc used here did not bind TGF-ß2 in an ELISA (data not shown), which is consistent with the notion that this isoform requires presentation by betaglycan for binding to the TGF-ßRII.34 35 After verifying the efficacy of the TGF-ßR:Fc to inhibit TGF-ß1–mediated responses in the mink lung epithelial cell assay, we tested the role of TGF-ß signaling in the rat carotid artery balloon injury model. Even the lowest dose used (0.5 mg/kg) caused nearly a 60% increase in lumen area despite the fact that intimal lesion formation was not affected by this dose. This indicates that loss of lumen area is in large part due to negative remodeling (vessel shrinking) and measurements of the perimeter of the neointima (IEL) and media (EEL) demonstrate that all doses of TGF-ßR:Fc used in this study significantly inhibited the reduction in IEL and EEL. We envision the following mechanism for this phenomenon. Assuming that IEL and EEL are elastic structures that can be stretched and shortened in a reversible manner and, further, that in a normal vessel under physiological blood pressure the elastic laminae are stretched to a certain extent (not maximally shortened), one would measure a shorter IEL and EEL in a more relaxed state. We believe that the more relaxed state is forced on the elastic laminae by the constriction resulting from the fibrosis in the adventitia.
The effect of TGF-ßR:Fc on remodeling is highly relevant to the clinical situation in which this is the major cause for restenosis after angioplasty.4 5 Immunostaining with an anti-human IgG antibody demonstrated that the TGF-ßR:Fc primarily localized to the adventitia and neointima, suggesting that these are the predominant sites of TGF-ß activity. One obvious effect of soluble TGF-ßRII was on collagen synthesis, and this was particularly striking in the adventitia of Masson trichrome–stained sections. We further investigated the effects of TGF-ßR:Fc on collagen expression by Northern blot analysis of RNA isolated from carotid arteries 4 days after injury. A marked reduction in mRNA levels was apparent for collagen types I and III, despite the differences in RNA loading. Interestingly, expression of other genes that are thought to be regulated by TGF-ß, including tropoelastin and fibronectin,43 44 were not affected, and expression of the nonfibrillar collagen XV and osteopontin was also not changed. The increased cell density within the neointima of TGF-ßR:Fc–treated rats suggests that extracellular matrix production may also be reduced in the intimal lesion. Therefore, TGF-ßR:Fc is a very potent inhibitor of negative vascular remodeling by inhibiting collagen production, particularly in the adventitia.
Intimal lesion formation is the result of cell migration, cell
proliferation, and matrix accumulation. The studies performed here did
not reveal a statistically significant effect of TGF-ßR:Fc on
proliferation of intimal SMCs, despite the fact that intimal cell
numbers were significantly lower in the treated rats. In addition, the
increase in intimal cell density as an indirect measure of
extracellular matrix synthesis in the TGF-ßR:Fc group also did not
reach statistical significance. A potential explanation for the
differences in intimal SMC number could be via an effect of TGF-ßR:Fc
on cell death, but staining for apoptotic cells with the
terminal deoxynucleotidyl transferase–mediated
nick end labeling (TUNEL) technique revealed no differences, with very
few TUNEL-positive cells present in all groups (data not shown).
Proliferation of SMCs after balloon injury of the rat carotid artery
begins with medial SMC proliferation, and we therefore asked whether
early proliferative events are inhibited by TGF-ßR:Fc, by measuring
medial SMC replication at 4 days after injury. The difference in the
replication between the control and treatment groups was not
significant, and in both groups only very few TUNEL-positive cells were
detected at this time point (data not shown). Interestingly, the number
of fibroblasts in the adventitia was significantly lower in the
TGF-ßR:Fc group, suggesting inhibition of fibroblast proliferation;
however, the adventitial cell replication index at 4 days was not
significantly different between groups (data not shown). An additional
experiment measuring medial SMC proliferation at 4 days with a longer
BrdU labeling period (3 injections over 24 hours) also showed no
significant difference, although 30% more BrdU-positive cells were
present in the media compared with the experiment with a single
BrdU injection (data not shown). In both experiments, however, there
was a trend with a decreased average replication index in the soluble
TGF-ßRII group. Therefore, it is possible that a larger number of
animals per group may have revealed a significant difference. In the
present study we did not examine time points earlier than 4 days
after injury. Because the first wave of medial SMC and adventitial
fibroblast replication occurs within 48 hours45 and
maximal SMC apoptosis is seen within hours after balloon
injury,46 it still is possible that TGF-ßR:Fc treatment
was affecting early cell proliferation, apoptosis, or both.
Migration of SMCs from the media into the intima is difficult to assess
in this in vivo model, particularly because virtually all SMCs arriving
in the intima are also replicating.47
TGF-ß1 has been reported to prolong the
duration of the cell cycle.9 10 Although this remains a
possibility, it is difficult to reconcile this potential mechanism with
the present findings of reduced neointima formation in
response to TGF-ßR:Fc treatment. In a previous study, using a high
dose of TGF-ß1, we observed a significant
increase in intimal SMC proliferation.14 Together, these
data suggest that the net effect of TGF-ß signaling in SMCs and
fibroblasts in vivo is stimulatory. Although in the experiments
performed here we could not detect a significant effect of TGF-ßR:Fc
on SMC proliferation, it should be emphasized that loss of lumen area
is predominantly a result of negative remodeling, which is dramatically
inhibited by TGF-ßR:Fc. This is particularly evident by the
observation that even the lowest dose of TGF-ßR:Fc (0.5 mg/kg), which
had absolutely no effect on intimal lesion formation, caused nearly a
60% increase in lumen area. Thus, regardless of its effects on SMC
proliferation, TGF-ßR:Fc is a potent inhibitor of
negative vascular remodeling. Another potential mechanism that could
explain the TGF-ßR:Fc effects on remodeling is related to the ability
of TGF-ß to induce myofibroblastic
transdifferentiation,37 39 causing fibroblasts to express
smooth muscle -actin.38 48 The reduction in adventitial
smooth muscle -actin expression after treatment with TGF-ßR:Fc was
striking, indicating that the induction of smooth muscle -actin
expression by adventitial fibroblasts is mediated by TGF-ß. Whether
expression of smooth muscle -actin by adventitial myofibroblasts is
directly affecting negative remodeling by exerting an active
constrictive effect remains to be determined.
Unlike SMCs and fibroblasts, growth of endothelial cells in the aorta was not affected at all by the TGF-ßR:Fc treatment. This result is surprising, because numerous studies have demonstrated that at least in vitro, TGF-ß inhibits endothelial cell proliferation.25 It was also interesting to note that, compared with normal endothelium, much higher levels of mRNA for TGF-ß1, TGF-ß2, and TGF-ßRII were expressed by endothelial cells near the wound edge, where active migration and proliferation occur.49 To our knowledge, there is currently no information as to the effects of TGF-ß on large vessel endothelium in vivo, and the expression patterns for TGF-ß ligands and TGF-ßRII observed here do not support the concept of TGF-ß as an inhibitor of endothelial cell growth in vivo. One issue that the present study did not address relates to activation of TGF-ß, which is synthesized by most cells in an inactive form that does not bind to cellular receptors. Both plasmin50 51 and thrombospondin52 53 have been identified as physiologically relevant activators of TGF-ß1. Plasmin generation occurs in the arterial wall,54 and thrombospondin is expressed by many cell types, including SMCs in the balloon-injured vessel wall.55 56 57 It is therefore likely that active TGF-ß is present in injured arteries. The TGF-ßR:Fc binds only active TGF-ß, and given that TGF-ßR:Fc localized preferentially to the neointima and adventitia, it seems reasonable to assume that active TGF-ß is present in these locations. The differential effects of TGF-ßR:Fc on adventitia and neointima versus endothelium are interesting, because they indicate that the effects seen in SMCs and fibroblasts are specific or that localization of active TGF-ß is not near endothelial cells. One difference between endothelium and SMCs was that wound-edge endothelium expressed much higher levels of TGF-ßRII mRNA than SMCs, and thus there is the possibility that the doses of TGF-ßR:Fc used in the experiments were sufficient to block TGF-ß signaling in SMCs and fibroblast but not in endothelium.
In summary, the present study demonstrates that TGF-ß signaling is responsible for negative remodeling with adventitial fibrosis and neointima formation in an arterial balloon injury model. These TGF-ß–mediated effects can be inhibited with a soluble TGF-ßRII without affecting reendothelialization of the denuded vessels. This reagent may be useful in a clinical setting for targeting restenosis after angioplasty.
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Acknowledgments |
|---|
This work was supported by the American Heart Association in the form of Established Investigator Grant 9640015N (to V.L.). We thank Paul Carmillo, Rich Cate, Michelle Sanicola, Konrad Miatkowski, and Werner Meier for production of the TGF-ßR:Fc.
Received December 3, 1998; accepted March 16, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Kakuta T, Currier JW, Haudenschild CC, Ryan TJ, Faxon DP. Differences in compensatory vessel enlargement, not intimal formation, account for restenosis after angioplasty in the hypercholesterolemic rabbit model. Circulation. 1994;89:2809–2815.
2. Lafont AM, Chisolm GM, Whitlow PL, Goormastic M, Cornhill JF. Post-angioplasty restenosis in the atherosclerotic rabbit: proliferative response or chronic constriction? Circulation. 1993;88(suppl I):I-521.
3.
Nunes GL, Sgoutas DS, Redden RA, Sigman SR, Gravanis
MB. Combination of vitamins C and E alters the response to
coronary balloon injury in the pig. Arterioscler Thromb
Vasc Biol. 1995;15:156–165.
4. Mintz GS, Kovach JA, Javier SP, Ditrano CJ, Leon MB. Geometric remodeling is the predominant mechanism of late lumen loss after coronary angioplasty. Circulation. 1993;88(suppl I):I-654.
5. Mintz G, Popma J, Pichard A, Kent K, Satler L. Mechanisms of late arterial responses to transcatheter therapy: a serial quantitative angiographic and intravascular ultrasound study. Circulation. 1994;90(suppl I):I-24.
6. Halloran BG, Prorok GD, So BJ, Baxter BT. Transforming growth factor-ß1 inhibits human arterial smooth-muscle cell proliferation in a growth-rate-dependent manner. Am J Surg. 1995;170:193–197.[Medline] [Order article via Infotrieve]
7. Mii S, Ware JA, Kent KC. Transforming growth factor-beta inhibits human vascular smooth muscle cell growth and migration. Surgery. 1993;114:464–470.[Medline] [Order article via Infotrieve]
8. McCaffrey TA, Consigli S, Du B, Falcone DJ, Sanborn TA, Spokojny AM, Bush HL, Jr. Decreased type II/type I TGF-ß receptor ratio in cells derived from human atherosclerotic lesions: conversion from an antiproliferative to profibrotic response to TGF-ß1. J Clin Invest. 1995;96:2667–2675.
9. Grainger DJ, Kemp PR, Witchell CM, Weissberg PL, Metcalfe JC. Transforming growth factor ß decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase. Biochem J. 1994;299:227–235.
10. Reddy KB, Howe PH. Transforming growth factor beta 1-mediated inhibition of smooth muscle cell proliferation is associated with a late G1 cell cycle arrest. J Cell Physiol. 1993;156:48–55.[Medline] [Order article via Infotrieve]
11. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross R. TGF-ß induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell. 1990;63:515–524.[Medline] [Order article via Infotrieve]
12. Stouffer GA, Owens GK. TGF-ß promotes proliferation of cultured SMC via both PDGF-AA-dependent and PDGF-AA-independent mechanisms. J Clin Invest. 1994;93:2048–2055.
13. Koyama N, Koshikawa T, Morisaki N, Saito Y, Yoshida S. Bifunctional effects of transforming growth factor-ß on migration of cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1990;169:725–729.[Medline] [Order article via Infotrieve]
14. 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.
15. 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.
16. Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transforming growth factor-ß1 suppress intimal hyperplasia in a rat model. J Clin Invest. 1994;93:1172–1178.
17.
Schulick AH, Taylor AJ, Zuo W, Qiu CB, Dong G, Woodward
RN, Agah R, Roberts AB, Virmani R, Dichek DA. Overexpression of
transforming growth factor beta 1 in arterial
endothelium causes hyperplasia, apoptosis, and
cartilaginous metaplasia. Proc Natl Acad Sci U S A. 1998;95:6983–6988.
18.
Perrella MA, Patterson C, Tan L, Yet SF, Hsieh CM,
Yoshizumi M, Lee ME. Suppression of interleukin-1beta-induced
nitric-oxide synthase promoter/enhancer activity by transforming growth
factor-beta 1 in vascular smooth muscle cells: evidence for mechanisms
other than NF-
B. J Biol Chem. 1996;271:13776–13780.
19. Kurihara H, Yoshizumi M, Sugiyama T, Takaku F, Yanagisawa M, Masaki T, Hamaoki M, Kato H, Yazaki Y. Transforming growth factor-beta stimulates the expression of endothelin mRNA by vascular endothelial cells. Biochem Biophys Res Commun. 1989;159:1435–1440.[Medline] [Order article via Infotrieve]
20. Herbert JM, Carmeliet P. Involvement of u-PA in the anti-apoptotic activity of TGF-ß for vascular smooth muscle cells. FEBS Lett. 1997;413:401–404.[Medline] [Order article via Infotrieve]
21.
Ward MR, Agrotis A, Kanellakis P, Dilley R, Jennings G,
Bobik A. Inhibition of protein tyrosine kinases attenuates increases in
expression of transforming growth factor-beta isoforms and their
receptors following arterial injury. Arterioscler
Thromb Vasc Biol. 1997;17:2461–2470.
22. Khalil N, O'Connor RN, Flanders KC, Unruh H. TGF-ß1, but not TGF-ß2 or TGF-ß3, is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. Am J Respir Cell Mol Biol. 1996;14:131–138.[Abstract]
23. Pelton RW, Johnson MD, Perkett EA, Gold LI, Moses HL. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 mRNA and protein in the murine lung. Am J Respir Cell Mol Biol. 1991;5:522–530.
24.
Bray P, Agrotis A, Bobik A. Transforming growth
factor-beta and receptor tyrosine kinase-activating growth factors
negatively regulate collagen genes in smooth muscle of hypertensive
rats. Hypertension. 1998;31:986–994.
25.
Heimark RL, Twardzik DR, Schwartz SM. Inhibition of
endothelial regeneration by type-beta transforming
growth factor from platelets. Science. 1986;233:1078–1080.
26. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
27.
di Clemente N, Wilson C, Faure E, Boussin L, Carmillo
P, Tizard R, Picard JY, Vigier B, Josso N, Cate R. Cloning, expression,
and alternative splicing of the receptor for anti-mullerian hormone.
Mol Endocrinol. 1994;8:1006–1020.
28.
Miller GT, Hochman PS, Meier W, Tizard R, Bixler SA,
Rosa MD, Wallner BP. Specific interaction of lymphocyte
function-associated antigen 3 with CD2 can inhibit T cell responses.
J Exp Med. 1993;178:211–222.
29.
Lindner V, Reidy MA. Expression of basic fibroblast
growth factor and its receptor by smooth muscle cells and
endothelium in injured rat arteries: an en face study.
Circ Res. 1993;73:589–595.
30.
Lindner V, Fingerle J, Reidy MA. Mouse model of
arterial injury. Circ Res. 1993;73:792–796.
31. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
32.
Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox
JN, Schwartz SM. PDGF ligand and receptor gene expression during repair
of arterial injury. J Cell Biol. 1990;111:2149–2158.
33. Tsuchida K, Lewis KA, Mathews LS, Vale WW. Molecular characterization of rat transforming growth factor-ß type II receptor. Biochem Biophys Res Commun. 1993;191:790–795.[Medline] [Order article via Infotrieve]
34.
Lopez-Casillas F, Payne HM, Andres JL, Massagué
J. Betaglycan can act as a dual modulator of TGF-ß access to
signaling receptors: mapping of ligand binding and GAG attachment
sites. J Cell Biol. 1994;124:557–568.
35.
Cheifetz S, Hernandez H, Laiho M, Ten Dijke P, Iwata
KK, Massagué J. Distinct transforming growth factor-ß
(TGF-ß) receptor subsets as determinants of cellular responsiveness
to three TGF-ß isoforms. J Biol Chem. 1990;265:20533–20538.
36. Taguchi J, Murry CE, Herren BI, Pech M, Schwartz SM, Lindner V. A quantitative method for determination of endothelial mRNA expression in vivo: induction of platelet-derived growth factor by endotoxin. Am J Pathol. 1998;152:903–912.[Abstract]
37.
Orlandi A, Ropraz P, Gabbiani G. Proliferative activity
and -smooth muscle actin expression in cultured rat aortic
smooth muscle cells are differently modulated by transforming growth
factor-ß1 and heparin. Exp Cell Res. 1994;214:528–536.[Medline]
[Order article via Infotrieve]
38.
Desmoulière A, Geinoz A, Gabbiani F, Gabbiani G.
Transforming growth factor-ß1 induces -smooth muscle actin
expression in granulation tissue myofibroblasts and in quiescent and
growing cultured fibroblasts. J Cell Biol. 1993;122:103–111.
39.
Verbeek MM, Otte-Holler I, Wesseling P, Ruiter DJ, de
Waal RM. Induction of -smooth muscle actin expression in
cultured human brain pericytes by transforming growth factor-ß1.
Am J Pathol. 1994;144:372–382.[Abstract]
40.
Darby I, Skalli O, Gabbiani G. -Smooth muscle
actin is transiently expressed by myofibroblasts during experimental
wound healing. Lab Invest. 1990;63:21–29.[Medline]
[Order article via Infotrieve]
41. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993;92:1686–1696.
42. Tsang ML, Zhou L, Zheng BL, Wenker J, Fransen G, Humphrey J, Smith JM, O'Connor-McCourt M, Lucas R, Weatherbee JA. Characterization of recombinant soluble human transforming growth factor-beta receptor type II (rhTGF-ß sRII). Cytokine. 1995;7:389–397.[Medline] [Order article via Infotrieve]
43. Marigo V, Volpin D, Bressan GM. Regulation of the human elastin promoter in chick embryo cells: tissue-specific effect of TGF-ß. Biochim Biophys Acta. 1993;1172:31–36.[Medline] [Order article via Infotrieve]
44.
Yamamoto H, Ueno H, Ooshima A, Takeshita A.
Adenovirus-mediated transfer of a truncated transforming growth
factor-beta (TGF-ß) type II receptor completely and specifically
abolishes diverse signaling by TGF-ß in vascular wall cells in
primary culture. J Biol Chem. 1996;271:16253–16259.
45.
Majesky MW, Schwartz SM, Clowes MM, Clowes AW. Heparin
regulates smooth muscle S phase entry in the injured rat carotid
artery. Circ Res. 1987;61:296–300.
46.
Perlman H, Maillard L, Krasinski K, Walsh K. Evidence
for the rapid onset of apoptosis in medial smooth muscle cells
after balloon injury. Circulation. 1997;95:981–987.
47. Lindner V. Role of basic fibroblast growth factor and platelet-derived growth factor (B-chain) in neointima formation after arterial injury. Z Kardiol. 1995;84(suppl 4):137–144.
48. Matzner Y, Vlodavsky I, Michaeli RI, Eldor A. Selective inhibition of neutrophil activation by the subendothelial extracellular matrix: possible role in protection of the vessel wall during diapedesis. Exp Cell Res. 1990;189:233–240.[Medline] [Order article via Infotrieve]
49.
Lindner V, Collins T. Expression of NF-B and
IB- by aortic endothelium in an
arterial injury model. Am J Pathol. 1996;148:427–438.[Abstract]
50.
Sato Y, Rifkin DB. Inhibition of
endothelial cell movement by pericytes and smooth
muscle cells: activation of a latent transforming growth factor-beta
1-like molecule by plasmin during co-culture. J Cell
Biol. 1989;109:309–315.
51.
Gleizes PE, Munger JS, Nunes I, Harpel JG, Mazzieri R,
Noguera I, Rifkin DB. TGF-ß latency: biological significance and
mechanisms of activation. Stem Cells. 1997;15:190–197.
52.
Schultz-Cherry S, Murphy-Ullrich JE. Thrombospondin
causes activation of latent transforming growth factor-ß
secreted by endothelial cells by a novel mechanism.
J Cell Biol. 1993;122:923–932.
53. Crawford SE, Stellmach V, Murphy-Ullrich JE, Ribeiro SM, Lawler J, Hynes RO, Boivin GP, Bouck N. Thrombospondin-1 is a major activator of TGF-ß1 in vivo. Cell. 1998;93:1159–1170.[Medline] [Order article via Infotrieve]
54. Jackson CL, Reidy MA. The role of plasminogen activation in smooth muscle cell migration after arterial injury. Ann N Y Acad Sci. 1992;667:141–150.[Medline] [Order article via Infotrieve]
55.
Majack RA, Cook SC, Bornstein P. Platelet-derived
growth factor and heparin-like glycosaminoglycans
regulate thrombospondin synthesis and deposition in the matrix by
smooth muscle cells. J Cell Biol. 1985;101:1059–1070.
56. Raugi GJ, Mullen JS, Bark DH, Okada T, Mayberg MR. Thrombospondin deposition in rat carotid artery injury. Am J Pathol. 1990;137:179–185.[Abstract]
57.
Miano JM, Vlasic N, Tota RR, Stemerman MB. Smooth
muscle cell immediate-early gene and growth factor activation follows
vascular injury: a putative in vivo mechanism for autocrine growth.
Arterioscler Thromb. 1993;13:211–219.
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|
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 L. Mandinov, A. Mandinova, S. Kyurkchiev, D. Kyurkchiev, I. Kehayov, V. Kolev, R. Soldi, C. Bagala, E. D. de Muinck, V. Lindner, et al. Copper chelation represses the vascular response to injury PNAS, May 27, 2003; 100(11): 6700 - 6705. [Abstract] [Full Text] [PDF]
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J. Ibrahim, J. K. Miyashiro, and B. C. Berk Shear Stress Is Differentially Regulated Among Inbred Rat Strains Circ. Res., May 16, 2003; 92(9): 1001 - 1009. [Abstract] [Full Text] [PDF]
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 H. Zhang, H. O. Akman, E. L. P. Smith, J. Zhao, J. E. Murphy-Ullrich, and O. A. Batuman Cellular response to hypoxia involves signaling via Smad proteins Blood, March 15, 2003; 101(6): 2253 - 2260. [Abstract] [Full Text] [PDF]
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U. Kintscher, C. Lyon, S. Wakino, D. Bruemmer, X. Feng, S. Goetze, K. Graf, A. Moustakas, B. Staels, E. Fleck, et al. PPAR{alpha} Inhibits TGF-{beta}-Induced {beta}5 Integrin Transcription in Vascular Smooth Muscle Cells by Interacting With Smad4 Circ. Res., November 29, 2002; 91 (11): e35 - e44. [Abstract] [Full Text] [PDF]
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M. R. Ward, A. Agrotis, P. Kanellakis, J. Hall, G. Jennings, and A. Bobik Tranilast Prevents Activation of Transforming Growth Factor-{beta} System, Leukocyte Accumulation, and Neointimal Growth in Porcine Coronary Arteries After Stenting Arterioscler. Thromb. Vasc. Biol., June 1, 2002; 22(6): 940 - 948. [Abstract] [Full Text] [PDF]
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E. Lutgens, M. Gijbels, M. Smook, P. Heeringa, P. Gotwals, V. E. Koteliansky, and M. J.A.P. Daemen Transforming Growth Factor-{beta} Mediates Balance Between Inflammation and Fibrosis During Plaque Progression Arterioscler. Thromb. Vasc. Biol., June 1, 2002; 22(6): 975 - 982. [Abstract] [Full Text] [PDF]
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 D. Sheppard Uses of Expression Microarrays in Studies of Pulmonary Fibrosis, Asthma, Acute Lung Injury, and Emphysema : Roger S. Mitchell Lecture Chest, March 1, 2002; 121(2007): 21S - 25S. [Abstract] [Full Text] [PDF]
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S. Sartore, A. Chiavegato, E. Faggin, R. Franch, M. Puato, S. Ausoni, and P. Pauletto Contribution of Adventitial Fibroblasts to Neointima Formation and Vascular Remodeling: From Innocent Bystander to Active Participant Circ. Res., December 7, 2001; 89(12): 1111 - 1121. [Abstract] [Full Text] [PDF]
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H. O. Akman, H. Zhang, M. A. Q. Siddiqui, W. Solomon, E. L. P. Smith, and O. A. Batuman Response to hypoxia involves transforming growth factor-beta 2 and Smad proteins in human endothelial cells Blood, December 1, 2001; 98(12): 3324 - 3331. [Abstract] [Full Text] [PDF]
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P. A. Kingston, S. Sinha, A. David, M. G. Castro, P. R. Lowenstein, and A. M. Heagerty Adenovirus-Mediated Gene Transfer of a Secreted Transforming Growth Factor-{beta} Type II Receptor Inhibits Luminal Loss and Constrictive Remodeling After Coronary Angioplasty and Enhances Adventitial Collagen Deposition Circulation, November 20, 2001; 104(21): 2595 - 2601. [Abstract] [Full Text] [PDF]
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E. Lutgens and M. J.A.P. Daemen Transforming Growth Factor-{beta}: A Local or Systemic Mediator of Plaque Stability? Circ. Res., November 9, 2001; 89(10): 853 - 855. [Full Text] [PDF]
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G. Paranya, S. Vineberg, E. Dvorin, S. Kaushal, S. J. Roth, E. Rabkin, F. J. Schoen, and J. Bischoff Aortic Valve Endothelial Cells Undergo Transforming Growth Factor-{beta}-Mediated and Non-Transforming Growth Factor-{beta}-Mediated Transdifferentiation in Vitro Am. J. Pathol., October 1, 2001; 159(4): 1335 - 1343. [Abstract] [Full Text]
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 C. G. Lee, R. J. Homer, Z. Zhu, S. Lanone, X. Wang, V. Koteliansky, J. M. Shipley, P. Gotwals, P. Noble, Q. Chen, et al. Interleukin-13 Induces Tissue Fibrosis by Selectively Stimulating and Activating Transforming Growth Factor {beta}1 J. Exp. Med., September 17, 2001; 194(6): 809 - 822. [Abstract] [Full Text] [PDF]
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M. Negishi, D. Lu, Y.-Q. Zhang, Y. Sawada, T. Sasaki, T. Kayo, J. Ando, T. Izumi, M. Kurabayashi, I. Kojima, et al. Upregulatory Expression of Furin and Transforming Growth Factor-{beta} by Fluid Shear Stress in Vascular Endothelial Cells Arterioscler. Thromb. Vasc. Biol., May 1, 2001; 21(5): 785 - 790. [Abstract] [Full Text] [PDF]
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W. Durante, L. Liao, S. V. Reyna, K. J. Peyton, and A. I. Schafer Transforming Growth Factor-{beta}1 Stimulates L-Arginine Transport and Metabolism in Vascular Smooth Muscle Cells : Role in Polyamine and Collagen Synthesis Circulation, February 27, 2001; 103(8): 1121 - 1127. [Abstract] [Full Text] [PDF]
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M. R. Ward, P. S. Tsao, A. Agrotis, R. J. Dilley, G. L. Jennings, and A. Bobik Low Blood Flow After Angioplasty Augments Mechanisms of Restenosis : Inward Vessel Remodeling, Cell Migration, and Activity of Genes Regulating Migration Arterioscler. Thromb. Vasc. Biol., February 1, 2001; 21(2): 208 - 213. [Abstract] [Full Text] [PDF]
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C. D. Douillet, V. Velarde, J. T. Christopher, R. K. Mayfield, M. E. Trojanowska, and A. A. Jaffa Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells: role of TGF-beta and MAPK Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2829 - H2837. [Abstract] [Full Text] [PDF]
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M. Sajid, M. Lele, and G. A. Stouffer Autocrine thrombospondin partially mediates TGF-beta 1- induced proliferation of vascular smooth muscle cells Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2159 - H2165. [Abstract] [Full Text] [PDF]
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D. Cosgrove, K. Rodgers, D. Meehan, C. Miller, K. Bovard, A. Gilroy, H. Gardner, V. Kotelianski, P. Gotwals, A. Amatucci, et al. Integrin {alpha}1{beta}1 and Transforming Growth Factor-{beta}1 Play Distinct Roles in Alport Glomerular Pathogenesis and Serve as Dual Targets for Metabolic Therapy Am. J. Pathol., November 1, 2000; 157(5): 1649 - 1659. [Abstract] [Full Text] [PDF]
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S.-i. Hayashi, N. Watanabe, K. Nakazawa, J. Suzuki, K. Tsushima, T. Tamatani, S. Sakamoto, and M. Isobe Roles of P-Selectin in Inflammation, Neointimal Formation, and Vascular Remodeling in Balloon-Injured Rat Carotid Arteries Circulation, October 3, 2000; 102(14): 1710 - 1717. [Abstract] [Full Text] [PDF]
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 P. J. Gallagher, Y. Jin, G. Killough, E. K. Blue, and V. Lindner Alterations in expression of myosin and myosin light chain kinases in response to vascular injury Am J Physiol Cell Physiol, October 1, 2000; 279(4): C1078 - C1087. [Abstract] [Full Text] [PDF]
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K. Yamamoto, R. Morishita, N. Tomita, T. Shimozato, H. Nakagami, A. Kikuchi, M. Aoki, J. Higaki, Y. Kaneda, and T. Ogihara Ribozyme Oligonucleotides Against Transforming Growth Factor-{beta} Inhibited Neointimal Formation After Vascular Injury in Rat Model : Potential Application of Ribozyme Strategy to Treat Cardiovascular Disease Circulation, September 12, 2000; 102(11): 1308 - 1314. [Abstract] [Full Text] [PDF]
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E. Lutgens, K. B. J. M. Cleutjens, S. Heeneman, V. E. Koteliansky, L. C. Burkly, and M. J. A. P. Daemen Both early and delayed anti-CD40L antibody treatment induces a stable plaque phenotype PNAS, June 20, 2000; 97(13): 7464 - 7469. [Abstract] [Full Text] [PDF]
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R. Agah, K. S. S. Prasad, R. Linnemann, M. T. Firpo, T. Quertermous, and D. A. Dichek Cardiovascular Overexpression of Transforming Growth Factor-{beta}1 Causes Abnormal Yolk Sac Vasculogenesis and Early Embryonic Death Circ. Res., May 26, 2000; 86(10): 1024 - 1030. [Abstract] [Full Text] [PDF]
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M. O. Boluyt and O. H.L. Bing Matrix gene expression and decompensated heart failure: The aged SHR model Cardiovasc Res, May 1, 2000; 46(2): 239 - 249. [Abstract] [Full Text] [PDF]
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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]
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