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Am J Physiol Cell Physiol 281: C1457-C1467, 2001;
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Vol. 281, Issue 5, C1457-C1467, November 2001

Upregulation of alpha 8beta 1-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-beta 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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: alpha 8beta 1, alpha 3beta 1, alpha 5beta 1, alpha vbeta 1, and alpha vbeta 3. Stimulation of cardiac fibroblasts by angiotensin II (ANG II) or transforming growth factor-beta 1 (TGF-beta 1) resulted in an increase of protein and heightening by 50% of the receptor density of alpha 8beta 1-integrin. The effect of ANG II was blocked by an AT1, but not an AT2, receptor antagonist, or by an anti-TGF-beta 1 antibody. ANG II and TGF-beta 1 increased fibronectin secretion, smooth muscle alpha -actin synthesis, and formation of actin stress fibers and enhanced attachment of fibroblasts to a fibronectin matrix. The alpha 8- and beta 1-subunits were colocalized by immunocytochemistry with vinculin or beta 3-integrin at focal adhesion sites. These results indicate that alpha 8beta 1-integrin is an abundant integrin on rat cardiac fibroblasts. Its positive modulation by ANG II and TGF-beta 1 in a myofibroblast-like phenotype suggests the involvement of alpha 8beta 1-integrin in extracellular matrix protein deposition and cardiac fibroblast adhesion.

adhesion molecules; vinculin; fibronectin; AT1 receptor; actin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTEGRINS ARE CELL-SURFACE receptors composed of one alpha - and one beta -subunit. More than 20 different integrins have been described on the basis of the identification of their 18 alpha - and 8 beta -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-beta 1 (TGF-beta 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 alpha -actin (SM alpha -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-beta 1 have been detected in fibrotic cardiac tissues (41). In vitro, TGF-beta 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, alpha 5beta 1, alpha 3beta 1, alpha vbeta 1, alpha vbeta 3, and alpha 8beta 1, distributed in three radioactive protein bands (40). The presence of alpha 8beta 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-beta 1 may have on cardiac remodeling, we examined whether the expression of alpha 8beta 1-integrin can be regulated by these agents and its cellular localization on primary cultures of rat cardiac fibroblasts.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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 alpha -actin (1A4, Sigma Chemical), anti-beta 3-integrin subunit (F11, Pharmingen Canada, Mississauga, ON, Canada), anti-beta 1-subunit (130L and 210, Dr. R. O. Hynes, Howard Hughes Medical Institute, Cambridge, MA), anti-alpha 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-beta 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 alpha 8-subunit and SM alpha -actin, the samples were boiled and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose sheet that was subsequently incubated with anti-alpha 8-antibody or anti-SM alpha -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Regulation of alpha 8beta 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-beta 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-beta 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-beta 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.

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 alpha 8beta 1-integrin and the 180-kDa band to alpha vbeta 3-integrin, whereas the middle 210-kDa band was heterogeneous and contained alpha 3beta 1-, alpha 5beta 1-, and alpha vbeta 1-integrins (40). Quantitative analysis of each radioactive band by PhosphorImager revealed a significant increase of alpha 8beta 1-integrin at >10-10 mol/l ANG II, whereas the intensity of the alpha vbeta 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-beta 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 alpha 8beta 1-integrin, a decrease in the intensity of the second band, and no change in alpha vbeta 3-integrin. The fact that the second band had a heterogeneous nature precluded any further analysis of the integrin subtype. TGF-beta 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-beta 1 (TGF-beta 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.

To evaluate more quantitatively the increase of expression of alpha 8beta 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-beta 1 enhanced the density of alpha 8beta 1-integrin by ~50% (in pmol/mg: 1.10 ± 0.07 and 1.11 ± 0.09 for ANG II and TGF-beta 1, respectively, vs. 0.74 ± 0.04 for control; both P <=  0.05 vs. control, n = 3), had no effect on alpha vbeta 3-integrin (0.31 ± 0.03 and 0.25 ± 0.03 for ANG II and TGF-beta 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-beta 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-beta 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 alpha 8beta 1-integrin. Bmax, maximal binding; n = 3.

To validate the echistatin-based assay and to confirm that ANG II and TGF-beta 1 effectively induced enhanced alpha 8beta 1-subunit expression, we performed Western blotting on boiled NP-40-solubilized fibroblast extracts with an anti-alpha 8-subunit antiserum (Fig. 4). Heating dissociated integrin heterodimers, and under denaturing SDS-PAGE the alpha 8-subunit behaved as a 170-kDa protein (32). Anti-alpha 8-antiserum effectively revealed a 170-kDa band in control and stimulated samples. Densitometric analysis of the bands demonstrated that ANG II and TGF-beta 1 significantly increased the amount of immunoreactive alpha 8-subunit (corrected per µg of protein).


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  		Fig. 4.   Analysis by Western blot of alpha 8-subunit expression after [Sar1]ANG II or TGF-beta 1 stimulation. Cardiac fibroblasts were stimulated for 48 h with 3 × 10-8 mol/l [Sar1]ANG II or 3 ng/ml TGF-beta 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-alpha 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.

To examine whether the effects of ANG II and TGF-beta 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 alpha 8beta 1-integrin level was not statistically different from that in the presence of TGF-beta 1 alone (results not shown), suggesting that the effects of ANG II and TGF-beta 1 are not synergistic or that alpha 8beta 1-integrin expression is already maximally stimulated by TGF-beta 1.

Involvement of the AT1 receptor. To determine which receptor subtype was involved in the alpha 8beta 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 alpha 8beta 1-integrin and to restore alpha 3/5/vbeta 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: alpha 8beta 1-integrin, B: alpha 3/5/vbeta 1-integrins, C: alpha vbeta 3-integrin. *P <=  0.05 vs. control; #P <=  0.05 vs. 3 ×10-8 mol/l [Sar1]ANG II; n = 4-6.

Anti-TGF-beta 1 antibody. Several reports have indicated that TGF-beta 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-beta 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-beta 1 secretion. Quiescent fibroblasts were, therefore, incubated with 3 × 10-8 mol/l ANG II or 3 ng/ml TGF-beta 1 in the presence of increasing concentrations of a blocking monoclonal anti-TGF-beta 1 antibody. At the end of stimulation, the 125I-echistatin-integrin complexes were analyzed by nondenaturing SDS-PAGE (Fig. 6). The anti-TGF-beta 1 antibody at 10 µg/ml completely attenuated the alpha 8beta 1-integrin response by TGF-beta 1, while the same antibody concentration had a small but nonsignificant effect on the ANG II response. Similar results were observed for the alpha 3/5/vbeta 1-subunit band, whereas there was no effect on alpha vbeta 3-integrin (results not shown). These observations were also confirmed by immunoblotting of the alpha 8-subunit: anti-TGF-beta 1 antibody blocked the TGF-beta 1-stimulated alpha 8-subunit, while it failed to attenuate the ANG II response (results not shown). This suggests that even though TGF-beta 1 expression can be augmented by ANG II, it did not play a significant role in alpha 8beta 1-integrin expression by ANG II under the conditions of this experiment.


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  		Fig. 6.   Effect of anti-TGF-beta 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-beta 1 in the presence of 2.5 or 10 µg/ml anti-TGF-beta 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: alpha 8beta 1-integrin, B: alpha 3/5/vbeta 1-integrins. Antibody alone had no effect on integrin expression. *P <=  0.05 vs. control; §P <=  0.05 vs. TGF-beta 1; n = 6.

Fibronectin secretion. We next examined the effects of ANG II and TGF-beta 1 addition on fibronectin secretion by cardiac fibroblasts (Fig. 7). ANG II and TGF-beta 1 increased significantly fibronectin concentrations in the medium by ~40% and 70%, respectively. The TGF-beta 1 effect was partially blunted by anti-TGF-beta 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-beta 1 stimulation. Cardiac fibroblasts were stimulated for 48 h with 3 × 10-8 mol/l [Sar1]ANG II or 3 ng/ml TGF-beta 1 in the presence of 2.5 or 10 µg/ml anti-TGF-beta 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-beta 1; n = 8-12.

Fibroblast attachment. To show that alpha 8beta 1-integrin upregulation may affect the adhesive properties of cardiac fibroblasts, ANG II- and TGF-beta 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-beta 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-beta 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.

Immunofluorescence studies. Differentiation of cardiac fibroblasts into myofibroblasts correlates with the expression of SM alpha -actin and reorganization of contractile microfilaments (35). Analysis of SM alpha -actin by Western blotting of stimulated fibroblast extracts revealed that ANG II and TGF-beta 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-beta 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 alpha -actin (SM alpha -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-beta 1, NP-40-solubilized cardiac fibroblasts were analyzed by Western blotting with anti-SM alpha -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.

Cellular localization of integrin alpha 8- and beta 1-subunits was performed by double labeling with anti-vinculin. In both cases, i.e., alpha 8- and beta 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-alpha 8- or anti-beta 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-beta 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-alpha 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-alpha 8-subunit antibody (B), anti-beta 1-subunit antibody (D), or anti-beta 3-subunit antibody (E). Arrowheads, focal adhesion sites. F: cellular footprint immunostained with anti-alpha 8-subunit antibody. Anti-vinculin and anti-beta 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-beta 1 stimulate synthesis of SM alpha -actin and formation of actin filaments and enhance alpha 8beta 1-integrin expression, we examined by fluorescence microscopy the colocalization of SM alpha -actin and alpha 8-subunit in basal and stimulated cardiac fibroblasts. Under the basal condition, while very few SM alpha -actin filaments could be visualized, it was qualitatively difficult to detect alpha 8-positive focal adhesion sites (Fig. 11, A and B). Conversely, after ANG II and TGF-beta 1 stimulation, several cells light up for SM alpha -actin distributed into microfilaments. Proportionally, numerous alpha 8-positive focal adhesion sites can now be observed (Fig. 11, C-F). A similar finding was seen with an anti-beta 1-subunit antibody (results not shown). Examination at higher magnification of the images indicates that the tip of SM alpha -actin filaments ends near the alpha 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 alpha -actin and alpha 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-beta 1 (E-H). After fixation and solubilization, cells were coincubated with anti-SM alpha -actin (A, C, E, and G) and anti-alpha 8-subunit (B, D, F, and H), and SM alpha -actin filaments and alpha 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using a novel echistatin-based pharmacological approach, we previously reported the presence of alpha 8beta 1-integrin on rat cardiac fibroblasts (40). We now extend this information by providing cell surface immunolocalization of alpha 8beta 1-integrin and demonstrating that ANG II and TGF-beta 1 increased its expression.

alpha 8beta 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 alpha 8beta 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 alpha 8- and beta 1-integrin subunits were immunolocalized on cellular structures of rat cardiac fibroblasts that stained positively for vinculin and the beta 3-integrin subunit and, thus, corresponded to focal adhesion complexes. A similar surface distribution of alpha 8beta 1-integrin has also been observed on vascular smooth muscle cells and glomerular mesangial cells (18, 32). This indicates that alpha 8beta 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 alpha -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 alpha 8beta 1-integrin and its recruitment at the level of adhesion contacts.

Because TGF-beta 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-beta 1 was used, from a comparative standpoint, to stimulate cardiac fibroblasts. It induced positive effects on protein synthesis, fibronectin expression, and alpha 8beta 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-beta 1 antibody, which completely blocked the TGF-beta 1 action itself, was unable to notably modify the ANG II responses. These results indicate that ANG II acts independently of TGF-beta 1 and/or that the level of TGF-beta 1 expression, via ANG II stimulation, is insufficient to trigger any significant effect.

Few studies have examined the relationship between ANG II or TGF-beta 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 beta 1-integrin (7) and/or beta 3- and beta 5-integrins (1). It was reported recently that ANG II can increase alpha v- and beta 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 alpha v- and beta 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 alpha vbeta 3-integrin expression (Figs. 1B and 2), but correction of the alpha vbeta 3-integrin level per unit of proteins brings these values back to the control level. Because our data showed upregulation of alpha 8beta 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-beta 1-stimulated cardiac fibroblasts demonstrated enhanced cell attachment to fibronectin, a protein ligand susceptible to interaction with alpha 8beta 1-integrin.

Our findings, taken together with those from the literature, demonstrate that ANG II and/or TGF-beta 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-beta 1 were able to upregulate the expression of functional alpha 8beta 1-integrin and relocalize it at focal adhesion sites in close association with SM alpha -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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Am J Physiol Cell Physiol 281(5):C1457-C1467
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