Upregulation of
8
1-integrin in
cardiac fibroblast by angiotensin II and transforming growth
factor-
1
Gaétan
Thibault1,
Marie-Josée
Lacombe1,
Lynn M.
Schnapp2,
Alexandre
Lacasse1,
Fatiha
Bouzeghrane1, and
Geneviève
Lapalme1
1 Laboratory of Cell Biology of Hypertension,
Multidisciplinary Research Group in Hypertension of the Canadian
Institutes of Health Research, Institut de Recherches Cliniques de
Montréal and Université de Montréal, Montreal,
Quebec, Canada H2W 1R7; and 2 Pulmonary and Critical Care
Medicine, University of Washington, Harborview Medical Center, Seattle,
Washington 98104
 |
ABSTRACT |
Using a novel pharmacological tool with
125I-echistatin to detect integrins on the cell, we have
observed that cardiac fibroblasts harbor five different RGD-binding
integrins:
8
1,
3
1,
5
1,
v
1, and
v
3.
Stimulation of cardiac fibroblasts by angiotensin II (ANG II) or
transforming growth factor-
1 (TGF-
1) resulted in an increase of
protein and heightening by 50% of the receptor density of
8
1-integrin. The effect of ANG II was
blocked by an AT1, but not an AT2, receptor
antagonist, or by an anti-TGF-
1 antibody. ANG II and TGF-
1
increased fibronectin secretion, smooth muscle
-actin synthesis, and
formation of actin stress fibers and enhanced attachment of fibroblasts
to a fibronectin matrix. The
8- and
1-subunits were colocalized by immunocytochemistry with vinculin or
3-integrin at focal adhesion sites.
These results indicate that
8
1-integrin
is an abundant integrin on rat cardiac fibroblasts. Its positive
modulation by ANG II and TGF-
1 in a myofibroblast-like
phenotype suggests the involvement of
8
1-integrin in extracellular
matrix protein deposition and cardiac fibroblast adhesion.
adhesion molecules; vinculin; fibronectin; AT1
receptor; actin
 |
INTRODUCTION |
INTEGRINS ARE
CELL-SURFACE receptors composed of one
- and one
-subunit.
More than 20 different integrins have been described on the basis of
the identification of their 18
- and 8
-subunits (20). Among these integrins, several regulate cellular
functions via binding to the extracellular matrix (ECM). The strength
of integrin binding to proteins such as laminins, collagens, and fibronectin determines the extent to which the cells adhere, migrate, differentiate, and proliferate (16, 28). Any factors that will affect the expression and/or distribution of integrins may thus
have important consequences in cell behavior (22).
Among the agents that can cause pathological myocardial remodeling,
angiotensin II (ANG II) and transforming growth factor-
1 (TGF-
1)
are recognized as important humoral factors (5).
Peripheral ANG II administration has been shown to induce cardiac
enlargement due to myocyte hypertrophy along with the appearance of
myofibroblasts and collagen deposition (23, 34, 42).
Differentiation of fibroblasts into myofibroblasts elicits a synthetic
phenotype: proliferation, increased protein synthesis capacity, and the
presence of smooth muscle
-actin (SM
-actin) microfilaments
(35). Accumulation of ECM material, as evaluated by
interstitial collagen deposition, results in fibrosis with alteration
of myocardium stiffness and contractility. Drugs blocking ANG II
actions by inhibiting its generation, as with angiotensin-converting
enzyme inhibitors, or its receptor activation, as with AT1
receptor antagonists, have demonstrated potent ability in reducing
cardiac fibrosis in animal models and in humans (11, 29,
31).
High concentrations of TGF-
1 have been detected in fibrotic cardiac
tissues (41). In vitro, TGF-
1 can differentiate cardiac fibroblasts into myofibroblasts and, thus, produce increased ECM protein secretion (14). In addition, ANG II has been
reported to heighten the expression of this growth factor in cardiac
fibroblasts (8, 26).
We recently developed a novel approach to study integrins
(40). 125I-echistatin, a snake venom
disintegrin, forms SDS-stable complexes with RGD-dependent integrins
under nondenaturing SDS-PAGE. This makes it possible to detect directly
functional integrins on cell or tissue extracts. For instance, on
cultured adult cardiac fibroblasts, we previously described the
presence of five integrins, namely,
5
1,
3
1,
v
1,
v
3, and
8
1,
distributed in three radioactive protein bands (40). The
presence of
8
1-integrin on cardiac fibroblasts is a novel finding, and its relative abundance indicates that it may play a major role in mediating fibroblast adhesion to ECM
proteins. Considering the impact ANG II and TGF-
1 may have on
cardiac remodeling, we examined whether the expression of
8
1-integrin can be regulated by these
agents and its cellular localization on primary cultures of rat cardiac fibroblasts.
 |
MATERIALS AND METHODS |
Materials.
Echistatin and [Sar1]ANG II were purchased from Bachem
California (Torrance, CA). The [Sar1]ANG II agonist was
preferred to ANG II because of its higher stability to aminopeptidase
degradation in culture medium. TGF-
1 and TRITC-phalloidin were
purchased from Sigma Chemical (St. Louis, MO); culture media, reagents,
and plates from Life Technologies (Burlington, ON, Canada); and fetal
bovine serum (FBS) from Wisent (St-Bruno, QC, Canada). Rat
fibronectin was purified from rat plasma on a gelatin-Sepharose column
(44). All the antibodies used in immunocytochemistry were
documented to cross-react with the following rat antigens:
anti-vinculin (hVIN-1, Sigma Chemical), anti-SM
-actin (1A4, Sigma
Chemical), anti-
3-integrin subunit (F11, Pharmingen
Canada, Mississauga, ON, Canada), anti-
1-subunit (130L
and 210, Dr. R. O. Hynes, Howard Hughes Medical Institute, Cambridge, MA), anti-
8-subunit (Dr. Lynn M. Schnapp,
Pulmonary and Critical Care Medicine, University of Washington,
Seattle, WA), and antibody 2415 (Dr. Louis F. Reichardt, University of California, San Francisco, CA). Secondary fluorescein- or
rhodamine-labeled antibodies were obtained from Chemicon (Temecula,
CA). A blocking anti-TGF-
1 monoclonal antibody (MAb 1835) was
purchased from R & D Systems (Minneapolis, MN).
Rat cardiac fibroblast culture.
The animal protocol was approved by the Institut de Recherches
Cliniques de Montréal Animal Care Committee. Cells were prepared from the heart ventricles of 200- to 250-g male Sprague-Dawley rats
exactly as described previously (15, 40). Digested cells were diluted (1 heart/80 ml) and seeded in 12- or 24-well plates (0.25 ml/cm2). They were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 0.1% bovine serum albumin (BSA) and
10% FBS until they reached 60-80% confluence (4 days). The
subconfluent cells were then fasted in DMEM and 0.1% BSA in the
absence of serum for 24 h before stimulation. Quiescent cells were
used as control.
Cell growth analysis.
DNA synthesis was detected by [3H]thymidine
incorporation. [Methyl-3H]thymidine (Amersham
Pharmacia Biotech, Uppsala, Sweden) was added to the culture medium (37 MBq/l) in the last 24 h of cell stimulation. At the end of
incubation, the culture plates were kept on ice, and the cells were
washed three times with ice-cold 0.05 mol/l NaPO4 buffer,
pH 7.4, and 0.15 mol/l NaCl (PBS). Ice-cold 10 g/dl trichloroacetic
acid was then added for 15 min to allow precipitation. The precipitated
cells were washed twice with 95% methanol, air-dried, and solubilized
in 0.2 mol/l NaOH. After neutralization with 0.2 mol/l HCl and addition
of liquid scintillant to each sample, radioactivity was measured in a
beta counter.
The amount of proteins per well was used as an index of protein
synthesis and cellular hypertrophy. At the end of stimulation, cells in
24-well plates were washed twice with PBS. Proteins were then
solubilized with 100 µl of 1 mol/l NaOH. After neutralization with
100 µl of 1 mol/l HCl, they were measured by the Bradford assay
(Bio-Rad Laboratories, Hercules, CA) with BSA as standard.
Analysis of RGD-dependent integrins.
Nondenaturing SDS-PAGE was used to analyze RGD-dependent integrins. At
the end of the stimulation period, cells in 24-well plates were washed
twice with 0.05 mol/l HEPES and 5 mmol/l MnCl2, pH 7.4. The
cell membranes were then solubilized with 100 µl of lysis buffer
[0.05 mol/l HEPES, 1% Nonidet P-40 (NP-40), 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l CaCl2, 1 mmol/l
MgCl2, and 5 mmol/l MnCl2]. To 40 µl of the
protein samples, 500,000 counts/min of 125I-echistatin were
added. After 90 min of incubation at room temperature and the addition
of 5 µl of electrophoresis sample buffer (without thiol reagent and
no heating), the samples were immediately separated on 6% gel SDS-PAGE
according to Laemmli (25). Proteins were then stained, and
the gels were dried and exposed to X-OMAT AR5 film for visualization of
125I-echistatin-integrin complexes. The full methodology
has been described elsewhere (40).
For analysis of the immunoreactive
8-subunit and SM
-actin, the samples were boiled and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose sheet that was subsequently incubated with anti-
8-antibody or anti-SM
-actin.
Blots were developed by chemiluminescence with SuperSignal WestPico
(Pierce, Rockford, IL).
Fibronectin enzyme-linked immunosorbent assay.
The media were collected after cell stimulation and stored at
20°C.
Fibronectin was measured by enzyme-linked immunosorbent assay according
to Rennard et al. (30). Twenty-five microliters of medium
or 0.057-125 ng of rat plasma fibronectin were incubated with
rabbit anti-rat fibronectin (Calbiochem, San Diego, CA) for 16 h
at 4°C in a final volume of 500 µl of 0.02 mol/l NaPO4
buffer, pH 7.4, containing 0.15 mol/l NaCl, 0.05% Tween 20, and 30 g/l BSA. Each sample was then incubated for 30 min in rat
fibronectin-coated (200 ng/well) 96-well Maxisorp plates (Nunc,
Naperville, IL). After it was washed, each well was incubated for
2 h with goat anti-rabbit IgG coupled to horseradish peroxidase
(Bio-Rad Laboratories, Richmond, CA). The wells were washed again, and
200 µl of 2.2 mmol/l o-phenylenediamine in 0.1 mol/l
citrate phosphate buffer, pH 5.0, and 3 mmol/l
H2O2 were added for 30 min. The reaction was
stopped with 100 µl of 4 mol/l H2SO4, and
absorbency was read at 490 nm.
Attachment assay.
The plastic surface of 96-well plates was coated for 2 h at 37°C
with human fibronectin (Life Technologies) at 0, 0.25, 0.5, and 1 µg/well in PBS. Any remaining sites were blocked by addition of 200 µl of 3% BSA. After stimulation, cells were harvested by protease
digestion, counted, diluted in DMEM containing 0.1% BSA and 1 mmol/l
MgCl2 at a density of 60,000 cells/100 µl, and
distributed in fibronectin-coated 96-well plates. Plates were
immediately centrifuged at 1,500 g for 1 min to sediment the
cells. Attached cells were washed three times with DMEM, BSA, and
MgCl2 and measured by crystal violet staining
(24).
Fluorescence microcopy.
After stimulation, the cells were collected by trypsin digestion and
seeded at a density of 20,000 cells/cm2 in
fibronectin-coated (5 µg/cm2) Lab-Tek glass chamber
slides (Nunc). They were left to adhere and spread for 3 h at
37°C, then they were fixed in acetone-methanol (1:1) at
20°C or
in 4% formaldehyde at room temperature. After membrane
permeabilization in 0.2% Triton X-100, the cells were incubated
overnight with the appropriate antibody dilution (1:50-1:800). Binding of primary antibodies was revealed with rhodamine- or fluorescein-coupled anti-rabbit or anti-mouse immunoglobulin
antibodies. Immunofluorescence was observed with a Zeiss Axiovert 100M microscope.
Occasionally, cells can be observed to peel off from the slide surface
and leave behind cellular footprints. These footprints, corresponding
to focal adhesion protein complexes that remained attached to the
fibronectin matrix (2), were immunostained as their parent cells.
F-actin was detected by TRITC-phalloidin staining (19).
Cells in 12-well plates were fixed with 4% formaldehyde and
permeabilized as described previously. Cells were then incubated for
2 h with 250 nmol/l TRITC-phalloidin in PBS. After they were
washed, the cells were examined by fluorescence microscopy with a ×10
objective and a videocamera to capture digitized images. Quantification of phalloidin staining was evaluated by extraction of F-actin-bound phalloidin with methanol. To increase the sensitivity of the assay, the
methanol fraction was concentrated 10-fold by lyophilization. Fluorescence was measured in a luminescence spectrometer (model LS50B,
Perkin Elmer Instruments, Norwalk, CT; excitation 540 nm, emission 565 nm). Phalloidin fluorescence was normalized per cell number, as
evaluated by crystal violet staining (24).
Statistical analysis.
Results are expressed as means ± SD. Differences between groups
were analyzed by one-way analysis of variance followed by the pairwise
multiple comparison Student-Newman-Keuls method with P
0.05. All experiments were repeated three to four times, and representative data are shown.
 |
RESULTS |
Regulation of
8
1-integrin expression.
Stimulation of primary cultures of quiescent, adult cardiac fibroblasts
by ANG II for 48 h resulted in a significant increase of protein
synthesis without change in DNA synthesis. Similar results were
observed by others (6, 36, 37). A similar pattern was
obtained by TGF-
1 stimulation. By comparison, FBS induced protein
and DNA synthesis as well as cell count (results not shown). Additional
experiments indicated that stimulation of protein synthesis by ANG II
increased progressively over a 24- to 32-h period and then reached a
plateau (results not shown). During that time, no cell death was
apparent in unstimulated control cells. Consequently, all subsequent
stimulation protocols were conducted for 48 h.
ANG II increased protein content in a dose-related manner with a
plateau at >10
8 mol/l and an EC50 of
~10
9 mol/l (Fig.
1A). TGF-
1 produced an
optimal effect at >1 ng/ml. Consequently, all other stimulations were
performed with 3 × 10
8 mol/l ANG II or 3 ng/ml
TGF-
1. In the same experiment, we examined 125I-echistatin binding to ANG II-stimulated fibroblasts.
125I-echistatin binding increased in the same fashion as
protein content, indicating an elevation of RGD-binding integrins (Fig. 1A).

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Fig. 1.
Dose-response curve of [Sar1]ANG II on
RGD-dependent integrin expression on cardiac fibroblasts. Total
RGD-dependent integrins were directly quantitated by binding of
125I-echistatin in each well. After binding and washing,
cells were solubilized in NaOH for counting and for protein measurement
(A; n = 3 for each concentration). In a
parallel experiment, Nonidet P-40 (NP-40)-solubilized fibroblasts were
incubated with 125I-echistatin and separated by
nondenaturing SDS-PAGE, and each radioactive band was quantitated with
a PhosphorImager system (B; n = 3 for each concentration). Bottom: typical electrophoretic
pattern. *P 0.05 vs. 10 12 mol/l
[Sar1]ANG II.
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|
To examine which of the RGD-dependent integrins could be affected by
ANG II, NP-40-solubilized fibroblast extracts were incubated for 90 min
with 125I-echistatin, and proteins were analyzed by
nondenaturing SDS-PAGE. Under mild conditions,
125I-echistatin formed SDS-resistant complexes that could
be separated by gel electrophoresis and visualized on X-ray film. As
shown in Fig. 1B, three radioactive protein bands at 220, 210, and 180 kDa were detected. Previous identification by
immunoblotting, immunoneutralization, and RGD affinity chromatography
indicated that the 220-kDa band corresponded to
8
1-integrin and the 180-kDa band to
v
3-integrin, whereas the middle 210-kDa
band was heterogeneous and contained
3
1-,
5
1-, and
v
1-integrins (40).
Quantitative analysis of each radioactive band by PhosphorImager
revealed a significant increase of
8
1-integrin at >10
10 mol/l
ANG II, whereas the intensity of the
v
3-integrin band was enhanced only at
10
7 mol/l.
Figure 2 presents the results obtained
when cardiac fibroblasts were stimulated with optimal concentrations of
ANG II or TGF-
1 and the subsequent analysis of each radioactive
band. This time, however, each set of data was corrected for the same
amount of proteins. ANG II produced a significant increase in
8
1-integrin, a decrease in the intensity
of the second band, and no change in
v
3-integrin. The fact that the second
band had a heterogeneous nature precluded any further analysis of the
integrin subtype. TGF-
1 and FBS had similar effects on the pattern
of integrin expression.

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Fig. 2.
Analysis of RGD-dependent integrins after
[Sar1]ANG II, transforming growth factor- 1 (TGF- 1),
and fetal bovine serum (FBS) stimulation. After 48 h of
stimulation, NP-40-solubilized fibroblasts were incubated for 90 min
with 125I-echistatin. Protein samples were then analyzed by
nondenaturing SDS-PAGE, and radioactive bands were quantitated with a
PhosphorImager. Integrin levels were corrected per µg of protein.
*P 0.05 vs. control; n = 6 for each
concentration.
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To evaluate more quantitatively the increase of expression of
8
1-integrin on cardiac fibroblasts after
stimulation, we performed saturation binding experiments.
NP-40-solubilized fibroblast extracts were incubated in the presence of
increasing concentrations of 125I-echistatin (Fig.
3, A and B).
Integrin densities were then calculated from the Scatchard
transformation of the data. ANG II and TGF-
1 enhanced the density of
8
1-integrin by ~50% (in pmol/mg:
1.10 ± 0.07 and 1.11 ± 0.09 for ANG II and TGF-
1,
respectively, vs. 0.74 ± 0.04 for control; both P
0.05 vs. control, n = 3), had no effect on
v
3-integrin (0.31 ± 0.03 and
0.25 ± 0.03 for ANG II and TGF-
1, respectively, vs. 0.23 ± 0.03 for control, n = 3), and slightly decreased the
second radioactive integrin band (1.43 ± 0.07 and 1.58 ± 0.10 for ANG II and TGF-
1, respectively, vs. 1.65 ± 0.06 for
control; P
0.05, ANG II vs. control,
n = 3).

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Fig. 3.
Saturation binding analysis of RGD-dependent integrins by
125I-echistatin. Cardiac fibroblasts were stimulated for
48 h with 3 × 10 8 mol/l
[Sar1]ANG II or 3 ng/ml TGF- 1. After stimulation,
cells were solubilized in lysis buffer. Protein (15 µg) was incubated
with increasing concentrations of 125I-echistatin
(0-600 fmol). Samples were analyzed by nondenaturing SDS-PAGE, and
radioactive bands were quantitated with a PhosphorImager. A:
typical example of a saturation experiment. B: saturation
curves and Scatchard transformation for
8 1-integrin. Bmax, maximal
binding; n = 3.
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|
To validate the echistatin-based assay and to confirm that ANG II and
TGF-
1 effectively induced enhanced
8
1-subunit expression, we performed
Western blotting on boiled NP-40-solubilized fibroblast extracts with
an anti-
8-subunit antiserum (Fig.
4). Heating dissociated integrin
heterodimers, and under denaturing SDS-PAGE the
8-subunit behaved as a 170-kDa protein
(32). Anti-
8-antiserum effectively revealed a 170-kDa band in control and stimulated samples.
Densitometric analysis of the bands demonstrated that ANG II and
TGF-
1 significantly increased the amount of immunoreactive
8-subunit (corrected per µg of protein).

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Fig. 4.
Analysis by Western blot of 8-subunit
expression after [Sar1]ANG II or TGF- 1 stimulation.
Cardiac fibroblasts were stimulated for 48 h with 3 × 10 8 mol/l [Sar1]ANG II or 3 ng/ml TGF- 1.
After stimulation, cells were solubilized in lysis buffer, diluted in
electrophoresis sample buffer, heated, separated by SDS-PAGE, and
transferred to nitrocellulose. The sheet was then incubated with
anti- 8-subunit antiserum and detected by anti-rabbit
immunoglobulins coupled to peroxidase and chemiluminescence.
A: immunoblot. B: quantification of the bands.
*P 0.05 vs. control; n = 5.
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To examine whether the effects of ANG II and TGF-
1 were additive,
cells were stimulated by the concomitant addition of these two agents
for 48 h. The integrins were then analyzed by the echistatin-based assay. In the presence of both agents, the
8
1-integrin level was not statistically
different from that in the presence of TGF-
1 alone (results not
shown), suggesting that the effects of ANG II and TGF-
1 are not
synergistic or that
8
1-integrin
expression is already maximally stimulated by TGF-
1.
Involvement of the AT1 receptor.
To determine which receptor subtype was involved in the
8
1-integrin response to ANG II
stimulation, fibroblasts were stimulated with 3 × 10
8 mol/l ANG II in the presence of irbesartan, an
AT1-receptor antagonist, or PD-123319, an
AT2-receptor antagonist. As illustrated in Fig. 5, only irbesartan was able to dose
dependently and significantly inhibit the upregulation of
8
1-integrin and to restore
3/5/v
1 band intensity to its initial
level. This implied participation of the AT1 receptor.

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Fig. 5.
Inhibition of [Sar1]ANG II stimulation by
an AT1 antagonist. Cardiac fibroblasts were stimulated for
48 h with 3 × 10 8 mol/l
[Sar1]ANG II in the presence of increasing concentrations
of irbesartan, an AT1 antagonist, or PD-123319, an
AT2 antagonist. After solubilization, incubation with
125I-echistatin, and separation by nondenaturing SDS-PAGE,
each radioactive band was quantitated with a PhosphorImager and
corrected per µg of protein. A:
8 1-integrin, B:
3/5/v 1-integrins, C:
v 3-integrin. *P 0.05 vs. control; #P 0.05 vs. 3 ×10 8 mol/l [Sar1]ANG II; n = 4-6.
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Anti-TGF-
1 antibody.
Several reports have indicated that TGF-
1 expression is augmented in
cardiac fibroblasts after ANG II stimulation (8, 26).
Because the results observed in the presence of ANG II and TGF-
1 in
our experiments were qualitatively similar, we wondered whether the ANG
II-stimulated effects could be mediated totally or in part by increased
TGF-
1 secretion. Quiescent fibroblasts were, therefore, incubated
with 3 × 10
8 mol/l ANG II or 3 ng/ml TGF-
1 in
the presence of increasing concentrations of a blocking monoclonal
anti-TGF-
1 antibody. At the end of stimulation, the
125I-echistatin-integrin complexes were analyzed by
nondenaturing SDS-PAGE (Fig. 6). The
anti-TGF-
1 antibody at 10 µg/ml completely attenuated the
8
1-integrin response by TGF-
1, while
the same antibody concentration had a small but nonsignificant effect
on the ANG II response. Similar results were observed for the
3/5/v
1-subunit band, whereas there was no
effect on
v
3-integrin (results not shown). These observations were also confirmed by immunoblotting of the
8-subunit: anti-TGF-
1 antibody blocked the
TGF-
1-stimulated
8-subunit, while it failed to
attenuate the ANG II response (results not shown). This suggests that
even though TGF-
1 expression can be augmented by ANG II, it did not
play a significant role in
8
1-integrin
expression by ANG II under the conditions of this experiment.

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Fig. 6.
Effect of anti-TGF- 1 antibody on stimulated integrin
expression. Cardiac fibroblasts were stimulated for 48 h with
3 × 10 8 mol/l [Sar1]ANG II or 3 ng/ml
TGF- 1 in the presence of 2.5 or 10 µg/ml anti-TGF- 1 antibody.
After stimulation, cells were solubilized in lysis buffer, diluted in
electrophoresis sample buffer, and separated by nondenaturing SDS-PAGE.
Radioactive bands were quantitated with a PhosphorImager. A:
8 1-integrin, B:
3/5/v 1-integrins. Antibody alone had no
effect on integrin expression. *P 0.05 vs. control;
§P 0.05 vs. TGF- 1; n = 6.
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Fibronectin secretion.
We next examined the effects of ANG II and TGF-
1 addition on
fibronectin secretion by cardiac fibroblasts (Fig.
7). ANG II and TGF-
1 increased
significantly fibronectin concentrations in the medium by ~40% and
70%, respectively. The TGF-
1 effect was partially blunted by
anti-TGF-
1 antibody, but the same antibody failed to affect ANG
II-mediated fibronectin secretion.

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Fig. 7.
Fibronectin secretion expression after
[Sar1]ANG II or TGF- 1 stimulation. Cardiac fibroblasts
were stimulated for 48 h with 3 × 10 8 mol/l
[Sar1]ANG II or 3 ng/ml TGF- 1 in the presence of 2.5 or 10 µg/ml anti-TGF- 1 antibody. Media were collected, and
fibronectin concentrations were measured by ELISA. Antibody alone had
no effect on basal secretion. *P 0.05 vs. control;
§P 0.05 vs. TGF- 1; n = 8-12.
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Fibroblast attachment.
To show that
8
1-integrin upregulation may
affect the adhesive properties of cardiac fibroblasts, ANG II- and
TGF-
1-stimulated cells were harvested and seeded on
fibronectin-coated plastic for a short period of time (<3 min),
allowing the direct interaction of integrins with fibronectin and
avoiding cell spreading (Fig. 8). Under
these conditions, a higher number of ANG II- and TGF-
1-stimulated cells bound to the fibronectin matrix, suggesting that the increased density of RGD-dependent integrins resulted in a stronger adhesiveness of the cells.

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Fig. 8.
Attachment of cardiac fibroblasts to fibronectin. Cardiac
fibroblasts were stimulated for 48 h with 3 × 10 8 mol/l [Sar1]ANG II or 3 ng/ml TGF- 1.
Cells were harvested, and 60,000 cells were deposited in
fibronectin-coated wells. Cells were sedimented by centrifugation, and
unattached cells were washed. Attached cells were measured by crystal
violet staining. OD, optical density; n = 4 for each
concentration.
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Immunofluorescence studies.
Differentiation of cardiac fibroblasts into myofibroblasts correlates
with the expression of SM
-actin and reorganization of contractile
microfilaments (35). Analysis of SM
-actin by Western
blotting of stimulated fibroblast extracts revealed that ANG II and
TGF-
1 enhanced the expression of this contractile protein (Fig.
9A). Phalloidin staining of
stimulated cells also indicated an increased presence and
reorganization of microfilaments (Fig. 9B). In the basal
condition, only scarce actin filaments can be observed at the
fibroblast periphery. After ANG II stimulation, microfilaments
accumulated at the cell boundary, and some can be observed crossing the
cells. With TGF-
1, numerous filaments were visualized throughout the
long axis of the cells. Quantification of F-actin-bound
TRITC-phalloidin by methanol extraction (Fig. 9B) correlated
with the microscopic images.

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Fig. 9.
Smooth muscle -actin (SM -actin) expression and localization
in cardiac fibroblasts. A: after 48 h of stimulation
with 3 × 10 8 mol/l [Sar1]ANG II or 3 ng/ml TGF- 1, NP-40-solubilized cardiac fibroblasts were analyzed by
Western blotting with anti-SM -actin. Bands were quantified by
densitometry. *P 0.05 vs. control; n = 5. B: stimulated cardiac fibroblasts were fixed with
formaldehyde, solubilized, and incubated with TRITC-phalloidin.
Digitalized images were obtained by fluorescence microscopy. Phalloidin
staining was eluted with methanol and quantitated by
spectrofluorescence. *P 0.05 vs. control;
n = 6.
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Cellular localization of integrin
8- and
1-subunits was performed by double labeling with
anti-vinculin. In both cases, i.e.,
8- and
1-subunits, diffuse staining was observed throughout the
cell surface, but weak, punctuated, streaklike immunostaining could
also be seen at the periphery of the cells (Fig.
10, B and D).
Similar images were obtained with two different anti-
8-
or anti-
1-subunit antibodies. Such diffuse staining of
integrin is not unusual, as already reported (9, 12, 18, 32, 39, 45). The punctuated, streaklike sites were also positive by double immunostaining for vinculin, an important protein member of the
focal adhesion complex and, thus, corresponded to focal adhesions (Fig.
10, A and C) (10).
Anti-
3-subunit immunostaining was restricted to focal
adhesions (Fig. 10E). On some occasions, cells peeled from
the slide surface, leaving cellular footprints. These structures
correspond to focal contacts between the cells and the fibronectin
matrix (2). The footprints were positively stained when
incubated with the anti-
8-subunit antibody (Fig. 10F).

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Fig. 10.
Cellular immunolocalization of integrins. Quiescent cardiac
fibroblasts were seeded on fibronectin-coated slides for 3 h.
Cells were then fixed with formaldehyde or cold acetone, solubilized
with 0.2% Triton X-100, and incubated with anti-vinculin antibody
(A and C), anti- 8-subunit antibody
(B), anti- 1-subunit antibody (D),
or anti- 3-subunit antibody (E). Arrowheads,
focal adhesion sites. F: cellular footprint immunostained
with anti- 8-subunit antibody. Anti-vinculin and
anti- 3-antibodies were detected with a rhodamine
anti-mouse secondary antibody; the other antibodies were detected with
a fluorescein anti-rabbit secondary antibody.
|
|
Because ANG II and TGF-
1 stimulate synthesis of SM
-actin and
formation of actin filaments and enhance
8
1-integrin expression, we examined by
fluorescence microscopy the colocalization of SM
-actin and
8-subunit in basal and stimulated cardiac fibroblasts. Under the basal condition, while very few SM
-actin filaments could
be visualized, it was qualitatively difficult to detect
8-positive focal adhesion sites (Fig.
11, A and B).
Conversely, after ANG II and TGF-
1 stimulation, several cells light
up for SM
-actin distributed into microfilaments. Proportionally,
numerous
8-positive focal adhesion sites can now be
observed (Fig. 11, C-F). A similar finding was seen
with an anti-
1-subunit antibody (results not shown).
Examination at higher magnification of the images indicates that the
tip of SM
-actin filaments ends near the
8-positive
streaklike structures (Fig. 11, G and H),
indicating a close association of these two structures, in agreement
with the literature (10).

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Fig. 11.
Colocalization of SM -actin and 8-subunit.
Cardiac fibroblasts were seeded on fibronectin-coated slides, made
quiescent, and incubated for 48 h in serum-free medium
(A and B), with 3 × 10 8 mol/l
[Sar1]ANG II (C and D), or with 3 ng/ml TGF- 1 (E-H). After fixation and
solubilization, cells were coincubated with anti-SM -actin (A,
C, E, and G) and anti- 8-subunit
(B, D, F, and H), and SM -actin filaments and
8-positive adhesion sites were detected with a
fluorescein anti-mouse IgG antibody and a rhodamine anti-rabbit IgG
antibody, respectively. Arrowheads, focal adhesion sites.
*Fibroblasts with little or no F-actin.
|
|
 |
DISCUSSION |
Using a novel echistatin-based pharmacological approach, we
previously reported the presence of
8
1-integrin on rat cardiac fibroblasts
(40). We now extend this information by providing cell
surface immunolocalization of
8
1-integrin
and demonstrating that ANG II and TGF-
1 increased its expression.
8
1-Integrin is a recently discovered
integrin that was first described on neurons and epithelial cells
(3). This information was extended to vascular smooth
muscle cells (32), glomerular mesangial cells
(18), and lung and hepatic fibroblasts (32). Although the natural ligand of
8
1-integrin has not been elucidated, this
integrin has the capacity to bind proteins having an RGD motif and has
been reported to interact with fibronectin, vitronectin, tenascin, and
osteopontin (13, 33). More interestingly, its presence and
its upregulation in lung and hepatic fibroblasts were recently
associated with the differentiation of these cells into myofibroblasts
and tissue deposition of fibrotic material (27).
We showed that
8- and
1-integrin subunits
were immunolocalized on cellular structures of rat cardiac fibroblasts
that stained positively for vinculin and the
3-integrin
subunit and, thus, corresponded to focal adhesion complexes. A similar
surface distribution of
8
1-integrin has
also been observed on vascular smooth muscle cells and glomerular
mesangial cells (18, 32). This indicates that
8
1-integrin is implicated in the
formation of focal adhesion protein complexes and may participate in
the attachment and spreading of fibroblasts on matrix proteins
(18).
ANG II has been implicated as a causal agent in the development of
cardiac hypertrophy and accumulation of ECM proteins (5, 43). In the present study, ANG II, through the AT1
receptor, induced the differentiation of quiescent fibroblasts into
myofibroblasts, as judged by their synthetic phenotype: increased
protein and SM
-actin synthesis, accumulation of extracellular
fibronectin, and reorganization of contractile actin filaments. These
changes were accompanied by a 50% enhancement of cell surface
expression of
8
1-integrin and its
recruitment at the level of adhesion contacts.
Because TGF-
1 expression has been reported to be enhanced by ANG II
(8, 26) and because it has also been implicated in the
development of cardiac hypertrophy and fibrosis (4, 38),
TGF-
1 was used, from a comparative standpoint, to stimulate cardiac
fibroblasts. It induced positive effects on protein synthesis, fibronectin expression, and
8
1-integrin,
very similar to those observed with ANG II, suggesting that the action
of ANG II could be mediated totally or in part by this growth factor.
However, the addition of an anti-TGF-
1 antibody, which completely
blocked the TGF-
1 action itself, was unable to notably modify the
ANG II responses. These results indicate that ANG II acts independently of TGF-
1 and/or that the level of TGF-
1 expression, via ANG II
stimulation, is insufficient to trigger any significant effect.
Few studies have examined the relationship between ANG II or TGF-
1
and integrin expression and function in cardiac fibroblasts. ANG II has
been demonstrated to cause collagen gel contraction, presumably by
enhancing the secretion of ECM proteins, such as osteopontin and
collagen, that subsequently bind through
1-integrin (7) and/or
3- and
5-integrins (1). It was reported recently that ANG II can increase
v- and
3-subunit
integrin expression in cultured neonatal and adult cardiac fibroblasts,
resulting in augmented cell attachment to matrix proteins (17,
21). In these studies, detection of the
v- and
3-integrin subunits was assessed by mRNA analysis and
cytofluorometry. With our method of detecting functional integrins, the
results suggest that ANG II, at higher concentrations, can apparently
increase
v
3-integrin expression (Figs.
1B and 2), but correction of the
v
3-integrin level per unit of proteins
brings these values back to the control level. Because our data showed
upregulation of
8
1-integrin expression, this may also explain changes in fibroblast adhesion, as observed by
these authors (17). Indeed, in the present study, ANG II- and TGF-
1-stimulated cardiac fibroblasts demonstrated enhanced cell
attachment to fibronectin, a protein ligand susceptible to interaction
with
8
1-integrin.
Our findings, taken together with those from the literature,
demonstrate that ANG II and/or TGF-
1 cause profound modifications of
the phenotype of cardiac fibroblasts. Indeed, ANG II-treated fibroblasts possess several major characteristics of differentiated synthetic cells: enhanced protein synthesis, actin and actinin upregulation (21), and augmented secretion of matrix
proteins such as osteopontin (1, 21), collagen
(6), and fibronectin. In addition, ANG II and TGF-
1
were able to upregulate the expression of functional
8
1-integrin and relocalize it at focal
adhesion sites in close association with SM
-actin filaments. ANG II
can thus transform cardiac fibroblasts into a cell type that
demonstrates profibrotic capacity.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant to G. Thibault from the Canadian
Institutes of Health Research. F. Bouzeghrane is a fellow of the Heart
and Stroke Foundation of Canada.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: G. Thibault, Laboratory of Cell Biology of Hypertension, Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, PQ, Canada H2W 1R7
(E-mail: thibaug{at}ircm.qc.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 March 2001; accepted in final form 5 July 2001.
 |
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