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Departments of 1 Cell Biology and 2 Internal Medicine, Faculty of Health Science, University of Linköping, S-58185 Linköping, Sweden
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The interaction
of ANG II with platelet-derived growth factor (PDGF)-BB-induced DNA
synthesis was studied in cultured rat aortic smooth muscle cells.
PDGF-BB-induced DNA synthesis was delayed (~6-8 h) by ANG II as
shown by a time-course experiment. Losartan, an
AT1-receptor antagonist, blocked
the transient inhibitory effect of ANG II, whereas the
AT2-receptor antagonist PD-123319 had no effect. Autocrine- or paracrine-acting transforming growth factor-
1 (TGF-1), believed to be a mediator of ANG II-induced inhibitory effects, was not responsible for the delay of
PDGF-BB-induced DNA synthesis, because a potent TGF-1 neutralizing
antibody could not reverse this effect of ANG II, nor was the delay of
the PDGF-BB effect caused by inhibition of PDGF--receptor
phosphorylation as shown by Western blot analysis of immunoprecipitated
PDGF- receptor. In conclusion, our results show that ANG II can
exert a transient inhibitory effect on PDGF-BB-induced proliferation via the AT1 receptor.
rat; angiotensin AT1 receptor; transforming growth factor-1
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ANGIOTENSIN II (ANG II), a potent vasoconstrictor, is
also a regulator of growth of vascular smooth muscle cells (VSMC) (9, 27). ANG II may regulate VSMC growth directly through induction of
intracellular signaling pathways or act indirectly via the release of
growth factors such as platelet-derived growth factor (PDGF), basic
fibroblast growth factor (bFGF), or transforming growth factor-1
(TGF-1), as demonstrated for rat VSMC (15, 17). Different receptors
for ANG II have been characterized (4). Most known effects of ANG II
are mediated by the AT1 receptor (4). Several steps of ANG II-induced signal transduction are common to
those elicited by strong mitogens such as PDGF. ANG II and PDGF, acting
through a G protein-coupled receptor and a tyrosine kinase receptor,
respectively, both induce phosphorylation of phospholipase C (PLC),
activate protein kinase C (PKC) and mitogen-activated protein kinase
(MAP kinase), and induce immediate early genes in VSMC (7). Although
there are similarities in the signaling pathways evoked by ANG II and
PDGF, they also diverge in several ways. Induction of PKC isoenzymes
(12), protein tyrosine phosphorylation (24), activation of MAP kinase
phosphatase 1 (MKP-1) (6), and the time course for induction of PLC,
D-myo-inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3], and
Ca2+ (20), triggered by PDGF and
ANG II, differ in VSMC. There are indications for cross talk between
the PDGF- and ANG II-induced signaling pathways (19). To understand the
role of PDGF-BB and ANG II in vivo, it is of great interest to
determine whether they interact to modulate the growth response of
VSMC.
In the present study we examined the interaction of ANG II- with PDGF-BB-induced DNA synthesis. We found that ANG II, by binding to the AT1 receptor, delays PDGF-BB-stimulated VSMC growth.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cell culture. VSMC were isolated from aorta of male Sprague-Dawley rats weighing ~250 g according to a modified method of Nilsson et al. (26). The aortas were prepared free from fat, and the endothelium was removed by gently scraping the luminal surface. Aortas were then cut into small pieces that were subsequently digested with 0.1% collagenase in Ham's medium containing 50 µg/ml ascorbic acid, 50 µg/ml gentamicin sulfate, 2 µg/ml Fungizone, and 10% newborn calf serum in F-12 medium (pH 7.4) at 37°C for 1 h. The medium was then changed to remove any remaining endothelial cells digested free by collagenase. Cells were incubated under the same conditions overnight. Thereafter, the cell suspension was filtered through a nylon filter (pore size 48 µm) and washed in Ham's medium. Cells were cultured in Ham's medium in 75-cm2 culture flasks at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed twice a week, and the cells were harvested for passaging at confluence with a trypsin-EDTA (0.05% trypsin, 0.02% EDTA) solution. Cultured cells were characterized as smooth muscle cells by morphological criteria. Experiments were performed with cells at passages 3-10. Before experiments, cells were made quiescent by a 24-h incubation in serum-free medium containing 50 µg/ml ascorbic acid and 50 µg/ml gentamicin sulfate in F-12 medium.
[3H]thymidine incorporation. VSMC were seeded at 8,000 cells/well in 96-well plates and cultured in Ham's medium until nearly confluent. Before each experiment the wells were visually screened, and wells with deviating confluence were excluded. Quiescent cells were exposed to the substances indicated with the addition of 1 µCi/ml [3H]thymidine. After 24 h, cells were washed three times with ice-cold F-12 medium, incubated for 20 min in ice-cold 5% TCA at 4°C, rinsed three times with ice-cold 5% TCA, and lysed with 0.1 M KOH for 1 h at room temperature. Radioactivity was then measured in a liquid scintillation counter (1214 Rackbeta; LKB). For time-course experiments, cells were pulsed with [3H]thymidine 8 h before harvest.
Measurements of mRNA by solution hybridization. VSMC were plated on petri dishes and grown until nearly confluent. Quiescent cells were incubated with substances indicated. Twenty hours later, the cells were solubilized with 1× SET buffer (1% SDS, 20 mM Tris, 10 mM EDTA, pH 7.5) and then homogenized with a polytron (Ultra turrax T25, Labassco). Nucleic acids were extracted essentially as described by Durnam and Palmiter (8). Samples were digested with proteinase K and extracted with phenol and chloroform. Nucleic acids were precipitated with ethanol. Total nucleic acids (TNA) were measured by spectrophotometry. DNA content was measured by fluorimetry according to the method of Labarca and Paigen (18).
The mRNA level of TGF-1 was determined by a solution hybridization
assay (8) using a 35S-UTP RNA
probe complementary to a 1-kb fragment spanning the major coding region
of the TGF-1 precursor (28). The probe was prepared according to the
method of Melton et al. (23) and was hybridized to TNA samples at
70°C for 20 h. Hybridization was performed in 40 µl of a solution
of 0.6 M NaCl, 20 mM Tris · HCl (pH 7.5), 4 mM EDTA,
0.1% SDS, 0.75 mM dithiothreitol, 25% formamide, and 20,000 cpm
35S-UTP probe per incubation. The
samples were exposed to RNases, the hybrids were precipitated with 100 µl TCA (6 M) and collected on glass microfiber filters, and
radioactivity was measured in a liquid scintillation counter (1214 Rackbeta, LKB). The radioactivity of each sample was then compared with
a standard curve constructed from a sample with a known amount of in
vitro-synthesized TGF-1 sense RNA complementary to the probe. A
standard curve was included in each assay, and samples were analyzed in
triplicate. Tubes including only hybridization buffer and probe served
as blanks. In control experiments the TGF-1 probe was found to bind
specifically to in vitro-synthesized TGF-1 mRNA sense, and
hybridization products were checked by gel analysis.
Bioassay for TGF-1.
Inhibition of
[3H]thymidine
incorporation into DNA of mink lung epithelial cells (CCL-64) was used
as a sensitive bioassay for TGF-1 (21). Cells were grown in DMEM
supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin,
nonessential amino acids, and 10% FCS. At subconfluence cells were
trypsinated and plated at 4 × 104 cells per well in 24-well
plates for 8 h. Cells were washed in serum-free medium and incubated
with human recombinant TGF-1 or conditioned medium in serum-free
medium for 20 h. During the last 4 h, cells were pulsed with 1 µCi
[3H]thymidine. Cells
were harvested and radioactivity was measured as described in
[3H]thymidine
incorporation. Conditioned medium was
obtained from VSMC 24 h after treatment with PDGF-BB + ANG II and was
added to the mink lung epithelial cells in a dilution of 1:4.
1 antibody to recognize endogenously produced
TGF-1 in conditioned medium was tested. Conditioned media or human
recombinant TGF-1 (10
9
M) were incubated for 1 h at 37°C together with the TGF-1
antibody (10 µg/ml) before being added to the mink lung epithelial
cells at a dilution of 1:2.
Immunoprecipitation of the PDGF receptor.
Subconfluent, quiescent VSMC in
75-cm2 culture flasks were washed
in cold F-12-BSA medium (1 mg BSA/ml F-12) for 5 min and incubated 30 min on ice with a 50 µM
Na3VO4
solution diluted in F-12-BSA medium. The cultures were incubated on ice
for 1 h with the substances indicated, followed by a 5-min incubation
in a 37°C water bath. After treatment the cells were lysed for 15 min at 0°C with a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 1 % Trasylol, and 1 mM
Na3VO4. The lysates were centrifuged at 0°C for 15 min at 13,000 rpm. To
immunoprecipitate the PDGF receptor we incubated the supernatants with
PDGF- receptor antibody (5) at 4°C for 2 h. Immunocomplexes were
collected at 4°C for 30 min with a protein A Sepharose. The immunoprecipitates were washed three times with ice-cold lysis buffer
and diluted in 50 µl of reducing sample buffer.
Western blot analysis. Immunoprecipitated samples were boiled for 2 min. After centrifugation, proteins in the supernatant were separated on a 7.5% SDS-PAGE. The separated proteins were electrotransferred onto a nitrocellulose membrane and blocked overnight with 5% BSA in PBS at room temperature. The membrane was immunoblotted using a 1:2,500 dilution of an anti-phosphotyrosin antibody (PY20) for 2 h at room temperature. The proteins were detected with a horseradish peroxidase-linked anti-mouse IgG and enhanced chemiluminescence detection system. An autoradiograph was obtained by exposure to Amersham hyperfilm.
Chemicals and solutions.
Collagenase type 1 and ANG II were from Sigma (St. Louis, MO), and
losartan was from DuPont Merck Pharmaceutical (Wilmington, DE).
Recombinant human TGF-1, TGF-1 neutralizing antibody, and control
antibody were purchased from R & D Systems (Abingdon, UK). PDGF-BB and
PDGF-receptor antibody were kind gifts of Prof. C.-H. Heldin (Uppsala,
Sweden). Anti-phosphotyrosine antibody (PY20) was from Affiniti
(Exeter, UK) and protein A Sepharose was from Pharmacia-Upjohn
(Uppsala, Sweden).
[3H]thymidine and
35S-UTP were from Amersham
International (Amersham, UK), and chemicals for probe synthesis were
obtained from Promega (Madison, WI). RNases, proteinase K, and herring
sperm DNA were purchased from Boehringer Mannheim (Mannheim, Germany),
and phenol was from Fisher Scientific (Fair Lawn, NJ). Chemicals and
solutions for cell culture were from GIBCO BRL Life Technologies
(Täby, Sweden).
Statistics. Values are given as means ± SE. Statistical analysis of the data was performed using the ANOVA Scheffé's F-test or Fisher's protected least significant difference. A P of <0.05 was considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of ANG II on PDGF-BB-induced DNA synthesis over 24 h.
Administration of ANG II
(1013-105
M) to VSMC inhibited PDGF-BB
(109 M)-induced
[3H]thymidine
incorporation into DNA when measured with
[3H]thymidine present
in the medium during the whole incubation period of 24 h (Fig.
1). The inhibition was dose dependent, with an IC50 of 5.4 × 109 M ANG II. At the
highest concentration of ANG II
(105 M), DNA synthesis was
inhibited almost to the control level. In this experiment ANG II
(106 M) alone had no
significant effect on DNA synthesis (data not shown).
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Time course for effect of ANG II on PDGF-BB induced DNA synthesis. To study the duration of the ANG II-induced inhibitory effect, [3H]thymidine incorporation into DNA was measured up to 48 h in 8-h intervals (Fig. 2). The inhibitory effect of ANG II was followed by a stimulatory effect and seemed to be caused by a delay (~6-8 h) of the PDGF-BB effect. To check that the late stimulation was not caused by degradation of ANG II, two control experiments were made. At 24 h, either media were changed for fresh media with ANG II and/or PDGF-BB or ANG II and PDGF-BB were re-added to conditioned media. In both these control experiments PDGF-BB-induced DNA synthesis was stimulated by ANG II during the 24- to 48-h period (data not shown). ANG II-induced DNA synthesis showed a time course similar to that of PDGF-BB + ANG II. ANG II alone caused no or only weak stimulation of DNA synthesis at 0-24 h (57 ± 7% of control) but clearly increased DNA synthesis at 24-48 h (208 ± 14% of control) (data not shown in figure).
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ANG II and PDGF-BB stimulate TGF-1 mRNA expression
in VSMC.
We investigated whether ANG II
(106 M), PDGF-BB
(109 M), and a combination
of ANG II and PDGF-BB stimulated the mRNA expression of TGF-1, a
possible mediator of the ANG II-induced inhibitory effect. Experiments
were performed to detect TGF-1 mRNA levels in VSMC 20 h after
stimulation with growth factors. In previous reports the induction of
TGF-1 mRNA by ANG II was most pronounced at 20 or 24 h (11, 32). The
basal level of TGF-1 mRNA in near-confluent, quiescent rat aortic
VSMC was 2.8 ± 0.2 amol/µg DNA (Fig.
3). ANG II
(106 M) or PDGF-BB
(109 M) treatment for 20 h
induced a threefold increase in TGF-1 mRNA, whereas the combination
of ANG II and PDGF-BB caused a four- to fivefold increase.
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Neutralizing antibody directed against TGF-1 fails
to block initial inhibitory effect of ANG II on PDGF-BB-induced DNA
synthesis.
To assess the role of TGF-1 as a possible mediator of the ANG II
transient inhibitory effect on PDGF-BB-induced DNA synthesis, we
investigated the effect of a neutralizing TGF-1 antibody on ANG
II-inhibited DNA synthesis (Fig. 4).
TGF-1 neutralizing antibody (10 µg/ml) administered together with
ANG II (106 M) and PDGF-BB
(109 M) failed to reverse
the inhibitory effect of ANG II. This was not caused by inappropriate
blocking of active TGF-1, because inactivation of TGF-1
(1011 M) was confirmed by
reversing the inhibitory effect of TGF-1 on PDGF-BB-induced DNA
synthesis. A control antibody was tested but had no effect on
TGF-1-induced inhibition. In addition, the ability of the TGF-1
neutralizing antibody to recognize endogenously produced rat TGF-1
in PDGF + ANG II-conditioned medium was confirmed by measurement of
active TGF-1 with a bioassay for TGF-1 (Fig. 5). The TGF-1 antibody reversed the
growth inhibitory effect of both human recombinant TGF-1
(109 M) and conditioned
media (dilution 1:2) without affecting basal thymidine incorporation.
Thus the results suggest that TGF-1, acting in an autocrine or
paracrine manner, does not account for the inhibitory effect of ANG II
on PDGF-BB-induced DNA synthesis seen at 24 h. When TGF-1
neutralizing antibody was added together with ANG II, DNA synthesis at
0-24 h increased from 
18 ± 2 to 25 ± 3% of control
and at 24-48 h from 34 ± 7 to 84 ± 4% of control without
affecting basal thymidine incorporation (data not shown in Fig. 5).
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AT1-receptor antagonist losartan blocks
inhibitory effect of ANG II.
To study which receptor mediates ANG II-induced inhibition, the effect
of losartan, an AT1- receptor
antagonist, and PD-123319, an
AT2-receptor antagonist, was
investigated. Losartan reversed the inhibitory effect of ANG II
(106 M) on PDGF-BB-induced
(109 M) DNA synthesis (Fig.
6). A significant effect was found at 1 µM of losartan. Losartan alone had no effect on DNA synthesis (data
not shown). Administration of PD-123319
(106 M) did not reverse ANG
II-induced inhibition and had no effect on basal thymidine
incorporation (data not shown).
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Effect of ANG II on PDGF-BB-induced tyrosine phosphorylation of
PDGF- receptor.
PDGF- receptor tyrosine phosphorylation was measured in VSMC treated
with ANG II (106 M),
PDGF-BB (109 M), or both
(Fig. 7). In control and ANG II-treated
cells there was no tyrosine phosphorylation of the PDGF- receptor.
PDGF-BB stimulated PDGF- receptor tyrosine phosphorylation. ANG II
added together with PDGF-BB did not cause a significant change in
PDGF- receptor activation.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In the present study we have shown that ANG II, in a dose-dependent manner, inhibits DNA synthesis stimulated by PDGF-BB in near-confluent rat aortic smooth muscle cells during the first 24 h and that this inhibition is followed by a stimulatory effect. These differential effects of ANG II on PDGF-BB-induced DNA synthesis might explain some conflicting reports. Under similar conditions, studies on rat aortic smooth muscle cells showed either no (3) or moderate stimulatory (16, 30) effect of ANG II in combination with PDGF-BB at 24-h incubation. Commonly, measurements of DNA synthesis are performed by addition of [3H]thymidine to the culture medium only a few hours before harvesting. Consequently, a transient effect on DNA synthesis might fail to be detected under these conditions. Because [3H]thymidine was added to the cells 4 h before harvest at 24 h in the studies of Ko and colleagues (16) and Sachinidis and colleagues (30), it is likely that they missed the initial inhibitory effect of ANG II. Bobik and co-workers (3) did not define when [3H]thymidine was added to the cells.
ANG II stimulates expression of growth factors that act in an autocrine
or paracrine manner to modulate the growth response to ANG II in VSMC
(11, 15). One of these, TGF-1, is believed to mediate a key
antiproliferative growth response to ANG II (15, 17). In agreement with
previous reports (1, 25), we have shown that TGF-1 inhibits
PDGF-BB-induced DNA synthesis in cultured VSMC. We investigated the
hypothesis that TGF-1 might mediate the transient inhibitory effect
of ANG II on PDGF-BB-induced DNA synthesis. Consistent with other
reports (1, 11, 32), we found that ANG II and PDGF-BB stimulated the
gene expression of TGF-1 in VSMC. Coadministration of ANG II and
PDGF-BB caused a moderate additional increase in TGF-1 mRNA
expression compared with induction by PDGF-BB or ANG II alone, which
suggests that the induction of TGF-1 mRNA by ANG II and PDGF-BB
might be triggered through partly different signaling pathways. It has
been reported that induction of TGF-1 mRNA by ANG II peaks at
20-24 h (11, 32). This suggests that induction of TGF-1 mRNA
might actually occur too late to be the factor mediating the ANG
II-induced delay of the PDGF-BB effect. To study the role of autocrine-
or paracrine-acting TGF-1 in ANG II-induced transient inhibition of
the growth response to PDGF-BB, we evaluated the effect of a
neutralizing antibody against TGF-1. The ability of the antibody to
neutralize endogenously produced TGF-1 in conditioned media from rat
VSMC was confirmed by use of a mink lung epithelial cell bioassay for
TGF-1. In addition, the antibody was shown to reverse the inhibitory
effect of TGF-1 on PDGF-BB-induced DNA synthesis in VSMC. Addition
of the TGF-1 neutralizing antibody did not, however, reverse the initial inhibitory effect of ANG II on PDGF-BB-induced DNA synthesis. In agreement with previous reports (11, 15, 17), TGF-1 neutralizing
antibody slightly increased ANG II-induced DNA synthesis. Although
these results show that autocrine action of TGF-1 can inhibit ANG
II-induced DNA synthesis, this inhibitory effect was far too weak to be
the factor responsible for the ANG II-induced inhibition of the PDGF-BB
effect. Taken together, these results suggest that autocrine or
paracrine action of ANG II-induced TGF-1 does not account for the
transient inhibitory effect of ANG II.
To investigate which receptor mediates the transient inhibitory effect of ANG II, the role of the angiotensin AT1 and AT2 receptors was studied. We found that the initial inhibitory effect of ANG II on PDGF-BB-induced DNA synthesis was mediated by the AT1 receptor. Addition of losartan, a specific AT1-receptor antagonist, reversed the inhibition of DNA synthesis, whereas the AT2-receptor antagonist PD-123319 had no effect. ANG II-induced inhibition of mitogenic growth has recently been found in two other cell types, coronary endothelial cells (31) and pheochromocytoma-derived cells (PC12W; Ref. 22). The inhibitory effect (at 24 h, [3H]thymidine pulsed for 4 h) of ANG II on these two cell types was shown to be mediated by the AT2 receptor and not by the AT1 receptor. It was suggested that the AT1 receptor mediates growth stimulatory actions of ANG II, whereas the AT2 receptor mediates inhibition of growth (31). In essence, this is not contradictory to our results. However, it is interesting to note that the AT1 receptor can trigger transient inhibitory pathways capable of delaying the growth response of a strong mitogen. ANG II has been shown to induce transient inhibitory effects in AT1A receptor-transfected Chinese hamster ovary cells (2). In these cells ANG II was shown to delay both its own and cytokine-induced activation of the signal transducers and activators of transcription (STAT) pathway during a 2-h period. The authors suggested that ANG II, through the AT1A receptor, might initially induce an inhibitory pathway that is responsible for the delayed induction of STAT. The role of the STAT pathway in mediating proliferative responses has not yet been elucidated (14). Our finding that PDGF-BB-induced DNA synthesis is delayed by ANG II acting through the AT1 receptor is consistent with the idea that ANG II initially might induce an inhibitory pathway(s). A transient growth inhibitory signaling pathway may also explain the delay of ANG II-induced DNA synthesis in rat VSMC found by Weber and colleagues (32) and in the present study.
PDGF- receptor phosphorylation is one of the first crucial events in
the PDGF-BB-induced signaling pathway, and it is a potent action site for inhibition of PDGF-BB-induced effects (13). We have shown that PDGF-BB-induced tyrosine phosphorylation of the
PDGF- receptor is not considerably attenuated by coincubation with
ANG II. This is in agreement with a report suggesting that PDGF
receptor tyrosyl phosphorylation in rat aortic smooth muscle cells is
not affected by ANG II (10). The major point(s) of ANG II-induced
inhibition is therefore probably located downstream of PDGF-
receptor activation.
Linseman and co-workers (19) reported that ANG II and PDGF induce
similar signaling events in the first few minutes after stimulation,
including tyrosine phosphorylation of Shc proteins and subsequent
complex formation between Shc and growth factor receptor binding
protein-2. Although the signaling events induced by PDGF and ANG II
resemble each other in these respects, other signaling events such as
induction of PKC isoenzymes (12), protein tyrosine phosphorylation
(24), and time course for induction of PLC,
Ins(1,4,5)P3 and
Ca2+ (20) diverge. In addition,
other possible mechanisms for the delayed PDGF effect by ANG II can be
envisaged. PDGF-BB-induced DNA synthesis is shown to be dependent on
activation of PI3-kinase (29), as also confirmed by us (G. Dahlfors,
unpublished data). In a report by Folli and colleagues (10), ANG II was
found to inhibit phosphatidylinositol 3-kinase activation induced by
PDGF in VSMC from rat aorta without affecting PDGF- receptor tyrosyl phosphorylation. MKP-1 may also be involved in the transient inhibitory effect of ANG II. MKP-1 is suggested to play an important role in ANG
II-mediated effects on VSMC proliferation (7). ANG II has
been shown to induce MKP-1 to a greater extent than PDGF in rat VSMC,
and a connection between MKP-1 and inhibition of proliferation of VSMC
has clearly been demonstrated (7). It is possible that ANG II initially
might induce an alternative inhibitory signaling pathway that regulates
the growth stimulatory signaling pathway of ANG II and PDGF-BB.
Although the mechanism remains incompletely understood, the finding that ANG II can delay PDGF-BB-induced DNA synthesis in rat VSMC might be of great importance in the understanding of ANG II-induced growth effects. Drugs that interfere with the ANG II effect, angiotensin-converting enzyme inhibitors and AT1-receptor antagonists, are today widely used in the treatment of hypertension and heart failure. From our present results, it might be speculated that drugs with short- or long-term duration because of pharmacokinetic properties can have different effects depending on a transient inhibitory effect of ANG II.
Taken together, our results show that ANG II transiently inhibits DNA
synthesis induced by PDGF-BB in rat VSMC by delaying the PDGF-BB
response. This transient inhibitory effect of ANG II is mediated by the
AT1 receptor. Inhibition of the
PDGF-BB effect seems to occur at a point(s) downstream of PDGF-
receptor tyrosine phosphorylation and does not involve action of
autocrine or paracrine TGF-1.
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ACKNOWLEDGEMENTS |
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The authors thank Prof. C.-H. Heldin for kindly providing PDGF-
receptor antibody and PDGF-BB for this study.
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FOOTNOTES |
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Financial support was obtained from the Swedish Medical Research Council (19x-4952), Swedish Diabetes Association, and Nordisk Insulinfond.
Address for reprint requests: G. Dahlfors, Dept. of Cellbiology, Faculty of Health Sciences, Linköping Univ., S-58185 Linköping, Sweden.
Received 22 July 1997; accepted in final form 9 February 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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