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(Hypertension. 2000;35:255.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Angiotensin II and PDGF-BB Stimulate ß₁-Integrin–Mediated Adhesion and Spreading in Human VSMCs

Kai Kappert; Gunther Schmidt; Gesine Doerr; Brigitte Wollert-Wulf; Eckart Fleck; Kristof Graf

From the Department of Medicine/Cardiology, Charité, Campus Virchow Klinikum, Humboldt Universität Berlin and Deutsches Herzzentrum Berlin (Germany).

Correspondence to Kristof Graf, MD, Department of Medicine/Cardiology, Charité, Campus Virchow Klinikum and Deutsches Herzzentrum Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany. E-mail kristof.graf{at}charite.de

	Abstract

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Abstract—ß₁-Integrins play an important role for adhesion and spreading of human smooth muscle cells. In the present study we examined the influence of angiotensin II and platelet-derived growth factor (PDGF)-BB on ß₁-integrin–dependent functions of human smooth muscle cells obtained from iliac arteries. Treatment of these cells with PDGF-BB (20 ng/mL) and Angiotensin II (1 µmol/L) did not change ß₁-integrin expression up to 48 hours as analyzed by flow cytometry and reverse transcription polymerase chain reaction. ß₁-integrins predominantly mediated adhesion of human smooth muscle cells to collagen I (79.7±4.4%, P<0.01) and fibronectin (66.6±2.4%, P<0.01). Treatment of smooth muscle cells with Angiotensin II (1 µmol/L) and PDGF-BB (20 ng/mL) significantly increased the adhesion to collagen I by 56.5% and 44.3%, respectively, and to fibronectin by 49.6% and 36.4%, respectively (all P<0.05). Angiotensin II–induced effects were mediated by the AT₁ receptor. The PDGF-BB mediated increase of adhesion was inhibited in the presence of genestein, a tyrosine-kinase inhibitor and by protein kinase C downregulation with phorbol 12-myristate 13-acetate. Spreading of smooth muscle cells also was ß₁-integrin dependent on collagen I and ₅ß₁-integrin dependent on fibronectin. Angiotensin II and PDGF-BB increased cell spreading on fibronectin up to 276% and 318%, respectively, and on collagen I up to 133% and 138% (all P<0.05). These increases were significantly inhibited by blocking antibodies against ß₁-integrin, ₅-integrin on fibronectin, the AT₁ receptor blocker irbesartan, and genestein. The present data demonstrate that angiotensin II and as well PDGF-BB enhance ß₁-integrin–dependent adhesion and spreading of human vascular smooth muscle cells. Furthermore, the experiments with PDGF suggest an involvement of protein kinase C activation leading to these enhanced effects.

Key Words: muscle, smooth, vascular • angiotensin II • platelet-derived growth factor

	Introduction

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The extracellular matrix is an important factor for the regulation of cell behavior and the functions of vascular smooth muscle cells (VSMCs) such as adhesion and spreading.¹ Cellular integrin receptors represent the linkage between the cytoskeleton, the extracellular matrix, and the intracellular signaling apparatus. The binding of VSMCs to extracellular matrix proteins (collagen type I and fibronectin) is mediated by members of the ß₁-integrin family. ₁ß₁- and ₂ß₁-integrin bind to collagen I and ₅ß₁-integrin bind to fibronectin.^{2 3} The interaction of extracellular matrix proteins and integrins are dynamically regulated by changes of the cell surface expression as well as by alterations of the affinity of the receptor for its ligands. This change in affinity results from alterations of receptor conformations or by events involving intracellular components such as association of integrins to the cytoskeletal proteins and proteins involved in the intracellular signaling cascades.^{4 5} Integrin expression is not only altered during embryogenesis, cell differentiation, and cell activation^{6 7} but was also described to be influenced by a variety of cytokines and growth factors,^{8 9} accompanying vascular diseases such as hypertension and atherosclerosis and during malignant transformation.¹⁰ Angiotensin II (AII) is a potent vasoconstrictor and has various effects on VSMCs such as proliferation, protein synthesis, induction of proto-oncogenes, activation of several intracellular pathways, and control of VSMC migration.^{11 12} Most effects are mediated by the AT₁ receptor. Platelet-derived growth factor (PDGF)-BB, another important growth factor, stimulates migration and proliferation of VSMCs and is involved in vascular remodeling as well.^{13 14} It was recently reported that a short-term incubation with PDGF increased the ability of cultured smooth muscle cells to adhere on collagen matrixes by an increase of the integrin receptor affinity.⁵ These data indicated that PDGF augments the matrix-cell interaction, leading to a higher responsiveness of VSMCs to adhesion-dependent mechanisms. So far, comparable effects of AII have not been determined in human VSMCs. Therefore we investigated the influence of AII and PDGF-BB on the expression and function of ß₁-integrins in VSMCs and studied their influence on ß₁-integrin–mediated adhesion and cell spreading.

	Methods

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Cell Culture
Human vascular smooth muscle cells (HSMCs) were prepared from aortic and iliac arteries according to the explant technique of Ross and Kariya.¹⁵ Tissues were derived from donors of the liver transplant program, which was approved by the local institutional review board. Human umbilical vein smooth muscle cells (HUVSMCs) were prepared from umbilical cords. The cells were cultured in DMEM containing 10% FCS, 15 mmol/L HEPES buffer, 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 mmol/L L-glutamine. Cells were identified as VSMC through their characteristic hill-and-valley growth pattern and by detection of -smooth muscle actin. Studies were performed with cells at passage 3 to 7.

Matrix Components, Peptides, Antibodies, and Blockers
Dermal human type I collagen was purchased by Vitrogen, human fibronectin by Gibco, and was solubilized in sterile water before use. The anti–ß₁-integrin P5D2¹⁶ and anti–₅-integrin BIIG2¹⁷ were from the Developmental Studies Hybridoma Bank, University of Iowa. As a control, an unspecific monoclonal mouse-IgG from Sigma was used. The synthetic peptides GRGESP and GRGDSP and AII were from Bachem, genestein, phorbol 12-myristate 13-acetate (PMA), and PD123319 from Sigma, irbesartan from the Bristol-Myers Squibb Pharmaceutical Research Institute, and recombinant PDGF-BB was from Biosource.

Flow Cytometry
The expression of integrins was evaluated by indirect immunofluorescence with the use of flow cytometric techniques, as shown previously.¹⁵ Cells were harvested by short trypsinization of subconfluent monolayers. After washing with cold PBS, cells were incubated in FACS-PBS (without Ca²⁺ and Mg²⁺) containing 5% BSA to suppress nonspecific binding for 20 minutes. Cells then were incubated with primary antibody for 20 minutes, washed with FACS-PBS, resuspended in the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody (goat anti-mouse, Sigma) for 20 minutes. Cells were fixed with 4% paraformaldehyde, washed, resuspended in FACS-PBS, and analyzed for fluorescence with a Becton-Dickinson FACScalibur flow cytometer. x and y axes represent log fluorescent intensity and cell number, respectively.

Reverse-transcription Polymerase Chain Reaction Assay
Total RNA was isolated from HSMCs through the use of RNAzol (BIOZOL). Reverse transcription polymerase chain reaction (RT-PCR) was performed following standard protocols. PCR was performed in a Perkins Elmer PE9700 thermal cycler as a hot-start PCR. After initial denaturation at 95°C for 5 minutes, PCR amplification was performed by using denaturation steps for 30 seconds at 95°C, annealing for 40 seconds at 58°C, primer extension for 30 seconds at 72°C, and a final extension for 10 minutes at 72°C. ß1 transcripts were amplified for 31 cycles, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 19 cycles. The PCR products were electrophoresed on 2% agarose gel. Primer sequences for ß1 were 5'-ACACGTCTCTCTCTGTCG-3' (position 11 to 28) and 5'-CAGTTGTTACGGCACTCT-3' (position 168 to 151) and for GAPDH 5'-GCAGGAGGCATTGCTGAT-3' (position 282 to 301) and 5'-CACCATCTTCCAGGAGCGAG-3' (position 516 to 499). Accession codes were M84237 for human ß1-integrin and M33197 for human GAPDH.

Adhesion Assay
The efficiency of cell adhesion was determined by measuring the number of cells that adhered to a substrate, as described previously.¹⁸ The test adhesive substrates (type I collagen, fibronectin) were diluted in sterile water. To determine the optimal collagen type I and fibronectin coating density, experiments with increasing concentrations of the proteins were performed. The concentration and cell number around the maximal calculated increase in adhesion was chosen for blocking experiments. Collagen type I (100 µL) or fibronectin (20 µg/mL) per well was added to 96-well plates (Falcon No. 35 3075) and was placed overnight at 4°C. Wells were blocked with 10 mg/mL BSA at 37°C for 1 hour. Cells (20 000) were placed in each well and allowed to adhere at 37°C for 60 minutes. Cells were preincubated with antibodies for 30 minutes at room temperature and gently mixed by permanent moving. Synthetic peptides were added immediately before cells. Nonadherent cells were rinsed off with PBS and the remaining cells were fixed with 4% paraformaldehyde for 5 minutes, then stained with 0.5% toluidine blue in 4% paraformaldehyde for 5 minutes and rinsed with water. Cells were solubilized by the addition of 100 µL of 1% SDS and quantified in a microtiter plate reader at 590 nm. Experiments described were performed in quadruplicate and repeated a minimum of 3 times.

Cell Spreading
The ability of cells to spread was determined by measuring the ratio of cells that spread after a defined time, as described previously.¹ Ninety-six–well plates (Falcon No. 35 3075) were coated with fibronectin or type I collagen (20 µg/mL) overnight at 4°C. To suppress nonspecific bindings plates were blocked with 1% BSA in PBS for 1 hour at 37°C. Cells were placed in 0.4% FCS medium at a concentration of 2000 cells per well in 100 µL. After the spreading period, cells were fixed with 2% paraformaldehyde for 20 minutes at room temperature and stained with 0.5% toluidine blue in 4% paraformaldehyde for 5 minutes. Spread cells were designated as cells having a nucleus recognizable by microscopy or a noncircular shape. Four high-power fields were counted per well (magnification x200). Experiments were performed in quadruplicate and at least 3 times.

Statistical Analysis
ANOVA and paired and unpaired t tests were performed for statistical analysis as appropriate. Values of P<0.05 were considered to be statistically significant.

	Results

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Integrin Expression on Cultured HSMCs Stimulated by Growth Factors
Flow cytometry demonstrated comparable expression of ß₁- and ₅-integrins on cultured HSMCs derived from aortic and iliac arteries and umbilical veins (data not shown). Treatment of HSMCs (from iliac arteries) and of HUVSMCs with growth factors AII (1 µmol/L) and PDGF-BB (20 ng/mL) for 24 and 48 hours revealed no significant changes of ß₁- and ₅-integrin expression (Figure 1A). Experiments were performed 3 times with separate sets of cells. Interestingly, the expression of both integrin subunits, ß₁- and ₅-integrins, were lower in HUVSMC than in adult arterial smooth muscle cells (Figure 1A).

View larger version (37K): [in this window] [in a new window]   Figure 1. A, Representative results of integrin receptor detection on cultured HSMCs and HUVSMCs by flow cytometry. Cells were incubated with an unspecific mouse IgG (gray area) or with anti–ß1-integrin (clone P5D2, 5 µg/mL) or anti-n5-integrin (clone BIIG2, 5 µg/mL). Cells were stimulated for 48 hours with AII (1 µmol/L) and PDGF-BB (20 ng/mL) in 0.4% FCS-containing medium. The x-axis corresponds to fluorescence intensity on a logarithmic scale; the y-axis corresponds to the cell number. There was no detectable change in ß1-integrin and 5-integrin surface expression. Experiments were repeated twice. B, Representative RT-PCR blot shows transcription levels for ß1-integrins in HSMCs. Cells were incubated with AII (1 µmol/L) and PDGF-BB (20 ng/mL) for 0, 6, and 16 hours. GAPDH, a ubiquitously nonregulated housekeeping gene, was used as an internal standard for RNA loading (n=3).

Analysis of RT-PCR Levels
RT-PCR assays were performed to investigate the expression of ß₁-integrin receptor mRNAs in stimulated and nonstimulated HSMCs. No remarkable differences could be observed in the expression of transcripts of AII-treated (1 µmol/L) and PDGF-treated (20 ng/mL) cells after 6 and 16 hours. GAPDH mRNA served as a control for RNA loading. (Figure 1B). Quantitative data revealed no significant changes when the ratio of ß₁-integrin/GAPDH mRNA levels were analyzed (control 0.85±0.06 ß₁/GAPDH, AII 6 hours 0.89±0.03 ß₁/GAPDH, AII 16 hours 0.90±0.1 ß₁/GAPDH, PDGF 6 hours 0.73±0.11 ß₁/GAPDH, PDGF 6 hours 0.88±0.09 ß₁/GAPDH). These experiments were repeated with different set of cells twice.

Cell Adhesion Studies
The functional importance of ß₁- and ₅-integrins was examined by an adhesion assay. Blocking antibodies against ß₁ (P5D2, 5 µg/mL) and ₅ (BIIG25, 5 µg/mL) were tested on collagen I and fibronectin layers. Results are demonstrated in Figure 2A. Adhesion to collagen I was predominantly inhibited by P5D2 and adhesion to fibronectin by both antibodies, P5D2 and BIIG2 (Figure 2A). RGD and RGE peptides and nonspecific IgG did not alter adhesion to both matrix proteins. Treatment of HSMCs for 24 to 72 hours with AII and PDGF significantly increased adhesion to collagen I and fibronectin. Data from a representative experiment with 48 hours of stimulation are shown in Figure 2B. Maximal effects were seen with AII concentrations of 1 µmol/L and PDGF-BB at 20 ng/mL (data not shown). Significant effects were already observed with 0.1 µmol/L AII. The effect of AII was completely inhibited in the presence of the AT₁-receptor blocker irbesartan (10 µmol/L) but not by the AT₂-receptor blocker PD 123319 (10 µmol/L). The effect of PDGF was completely inhibited either in the presence of the tyrosine kinase blocker genestein (10 µg/mL) or by 24-hour pretreatment of HSMCs with PMA (1 µg/mL), which downregulates protein kinase C (PKC). Studies of adhesive substrate concentrations to adherent cell numbers showed a shift to lower concentrations of matrix proteins (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 2. Adhesion of HSMCs (20000/well) to A, collagen type I–coated (20 µg/mL), and B, fibronectin-coated (20 µg/mL) plates. Cells were incubated with or without anti-ß1, anti-5, linear RGD, linear RDE peptides, or unspecific mouse-IgG and allowed to adhere for 1 hour at 37°C (*P<0.01 vs control [Co], mean±SEM). HSMCs were treated for 48 hours with AII (1 µmol/L) or PDGF-BB (20 ng/mL) with or without PD123319 (PD123, 10 µmol/L), irbesartan (Irb, 10 µmol/L), genestein (Gen, 10 µg/mL), and phorbol 12-myristate 13-acetate (PMA, 1 µg/mL), and adhesion was determined on C, collagen I–coated (20 µg/mL), and D, fibronectin-coated (20 µg/mL) plastic. Experiments were performed in quadruplicate, with 3 different sets of cells. (*P<0.05 vs untreated control cells [Co], #P<0.05 vs AII alone, and +P<0.05 vs PDGF alone, mean±SEM.)

Motility Studies
To investigate the influence of growth factors on the chemokinetic motility, spreading experiments were performed. Blocking antibodies against ß₁ (P5D2) and ₅ (BIIG2) were tested on collagen I and fibronectin layers. Spreading on collagen I was only partially inhibited by P5D2 for up to 30% (P<0.05, Figure 3A). Spreading on fibronectin was almost completely abolished by P5D2 and BIIG2 (Figure 3A). RGD and RGE peptides and nonspecific IgG did not influence spreading on both matrix proteins. Treatment of HSMCs for 48 hours with AII and PDGF significantly increased spreading on both proteins (Figure 3B). The effect of AII was completely inhibited in the presence of the AT₁-receptor blocker irbesartan (10 µmol/L) but not by the AT₂-receptor blocker PD 123319 (10 µmol/L). The effect of PDGF was completely inhibited either in the presence of the tyrosine kinase blocker genestein (10 µg/mL) or by PKC inhibition with PMA (1 µg/mL). We observed a significant effect of both growth factors on fibronectin-dependent spreading, which was increased by >3-fold. This effect was predominantly mediated by the ₅ß₁-integrin because coincubation with the antibodies P5D2 (5 µg/mL) and BIIG2 (5 µg/mL) significantly diminished the AII and PDGF effects on fibronectin (Figure 4). On collagen I, these effects were only inhibited by coincubation with P5D2 (Figure 4).

View larger version (36K): [in this window] [in a new window]   Figure 3. Spreading of HSMCs (2000/well) on A, collagen type I–coated (20 µg/mL), and B, fibronectin-coated (20 µg/mL) plates. Cells were allowed to spread for a total time of 25 minutes. For antibodies and peptides these were added after a minimal time of adhesion after 5 minutes. Rounding cells and spread cells were counted in 4 randomly chosen high-power fields, with 3 different sets of cells. HSMCs were treated for 48 hours with AII (1 µmol/L) or PDGF-BB (20 ng/mL) with or without PD123319 (PD123, 10 µmol/L), irbesartan (Irb, 10 µmol/L), genestein (Gen, 10 µg/mL), and phorbol 12-myristate 13-acetate (PMA, 1 µg/mL), and spreading was determined on C, collagen I–coated (20 µg/mL), and D, fibronectin-coated (20 µg/mL) plastic. Experiments were performed in quadruplicate, with 3 different sets of cells (*P<0.05 vs untreated control cells [Co], #P<0.05 vs AII alone, and +P<0.05 vs PDGF alone, mean±SEM).

View larger version (20K): [in this window] [in a new window]   Figure 4. Spreading of HSMCs after treatment with AII (1 µmol/L) and PDGF-BB (20 ng/mL) for 48 hours. Stimulated cells were coincubated with anti–ß1- and anti–5-integrin antibodies after an adhesion time of 5 minutes and were allowed to spread for additional 20 minutes on A, collagen type I (20 µg/mL), and B, fibronectin (20 µg/mL) (#P<0.05 vs AII alone; +P<0.05 vs PDGF alone, mean±SEM). Experiments were performed in quadruplicate, with 3 different sets of cells.

	Discussion

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In the present study we demonstrated that AII and PDGF increased ß₁-integrin–mediated adhesion on collagen type I and fibronectin. Furthermore, both growth factors significantly increased the spreading of HVSMCs, which involves a rearrangement of the cytoskeleton and increased contractility of the VSMC. Both cellular mechanisms are essentially important for the control of cell behavior during vascular remodeling and repair mechanisms, which are induced by increased shear stress or vascular damage. So far this is the first report that demonstrates that both AII and PDGF promote cell spreading. These effects were observed after incubation for 24 hours and were maintained up to 72 hours. The effect of both growth factor effects could not be related to an increase of integrin receptor numbers because we could show by flow cytometry and RT-PCR transcript levels for ß₁-integrin.

The effect of AII was mediated by an AT₁ receptor–dependent mechanism, whereas the AT₂ receptor blocker PD 123319 was without any effect on cell spreading or adhesion. The effect of PDGF was abolished by blocking the phosphorylation of tyrosine kinases. Although genestein is not very specific, it might indicate the possible involvement of the PDGF receptor kinase. The increases in spreading were also blocked by the specific competitive antibodies against the ß₁-integrin on collagen type I and ₅ß₁ integrin on fibronectin, which suggested that the ß₁-integrin receptor was directly activated and its post–receptor signaling pathway was modulated by growth factor treatment. The effect of PDGF on cell spreading and adhesion appeared to involve activation of PKC because the downregulation of this enzyme significantly reduced the PDGF-induced effects. PKC is activated before integrin-mediated cell spreading starts, and inhibition of PKC prevents cell spreading on fibronectin.¹⁹ Haller and coworkers²⁰ could demonstrate that spreading of rat VSMCs on fibronectin induced a rapid increase of diacylglycerol within 10 minutes and an activation of PKC isoforms and .

Dynamic interactions between cell surface receptors and extracellular matrix components play a key role for the control of cell behavior, which includes proliferation, adhesion, spreading, and migration.^{4 6 7} The deposition of matrix proteins by vascular cells contributes to these mechanisms. The principal interstitial matrix in the medial layer of arteries that surrounds smooth muscle cells completely is predominantly composed of types I and III collagen.²¹ Deposits of fibronectin are also found in the vascular wall.²¹ Adhesion, deadhesion, and spreading are integral parts to achieve the ability of cells to move and migrate. This is important for adaption and rearrangement of the vascular tissue in response to pathophysiological factors such as increased shear stress caused by hypertension.

Several integrins such as _vß₃-integrin mediate adhesion to matrix proteins through the classic Arg-Gly-Asp (RGD) sequence. This is not the case for adhesion to collagen I by ₁ß₁-integrin and to fibronectin by ₅ß₁-integrins, as we demonstrated by the adhesion experiments. This observation is in accordance with the studies reported by Clyman^{2 3} and Bilato et al,²² who did not observe an effect of RGD peptides on adhesion of VSMCs to collagen I and fibronectin.

An important mechanism of regulation of integrin receptors involves changes of the affinity/avidity of the receptor for ligands. Affinity is defined as binding of a monovalent soluble ligand. Alteration is mediated by changes of the receptor conformation.^{4 5} Avidity can be defined as binding of a multivalent ligand and can result from changes of the affinity or from postreceptor events following cytoskeletal components.²³ Whether growth factors directly influence these mechanisms has been investigated by Seki and coworkers,⁵ who reported that ß₁-integrin–dependent adhesion could be increased by short-term exposure to PDGF. Using a specific antibody mAB15/7, which binds to an activation-dependent epitope on the ß₁-integrin, they could demonstrate that this increase of adhesion by PDGF was induced by changes of the affinity/avidity of the ß₁-integrins. This is in accordance with our investigations, in which we did not observe an alteration of ß₁-integrin receptor expression after treatment with AII and PDGF but a stimulation of ß₁-integrin–mediated cellular functions. Therefore one of the possible mechanisms can be a change of binding affinity/avidity of the integrin receptors.

In our studies we have focused on 2 major issues. First, the effect of long-term treatment with growth factors, and second, the functional effect of these factors. Because different growth factors can activate VSMCs in the same way, we compared the effects of 2 physiologically relevant factors: AII and PDGF-BB on ß₁-integrin–mediated functions. Both growth factors are also known to be chemotactic for VSMCs.^{11 24} Interestingly, both growth factors showed comparable effects in this study with human cells. It demonstrates that AII is physiologically relevant in cultured human vascular smooth muscle cells, and its activity is comparable to PDGF. Both AII and PDGF increased the ability of cells to spread and to adhere on fibronectin and type I collagen after 24 hours of treatment. We did not study short-term exposure, which has been done by others in the case of PDGF,⁵ because we were also interested whether ß₁-integrin expression was affected in human cells. However, AII might have this effect also after shorter treatment periods. The present data demonstrate clearly that expression was not affected in human vascular cells, but ß₁-integrin–mediated functions were increased when we studied the time frame between 24 and 72 hours.

Integrin binding itself initiates signals that stimulate the tyrosine phosphorylation of several cellular proteins, Ras, Raf, FAK, and phospholipase C, MAPK, MEK, and JNK, which are associated with focal adhesions.^{4 7 25} The colocalization of these molecules together with receptor for growth factors is important for facilitating their interactions. Growth factors might modify the pathway of inside-out signaling and thereby induce a modification of integrin receptor avidity/affinity. However, it can only be speculated which intracellular components are involved. In the present study we demonstrated that the cell-matrix interaction is modified by AII and PDGF, leading to a higher affinity/avidity of VSMCs to collagen and fibronectin and increasing the spreading of human VSMCs on the matrixes. The effects of PDGF were dependent on the activation of PKC.

In summary, stimulation with the growth factors AII and PDGF increased the adhesion and spreading of human smooth muscle cells on fibronectin and collagen type I. These effects were mediated by ß₁-integrin receptors. Since the ß₁-integrin receptor numbers were not increased, this effect probably was due to a change of the receptor affinity/avidity and/or the modulation the postreceptor pathway involving PKC activation.

	Acknowledgments

 
This work was supported by a grant (to K.G.) from the Deutsche Forschungsgemeinschaft (GR 1368/2-1).

Received September 14, 1999; first decision October 11, 1999; accepted October 19, 1999.

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