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(Circulation Research. 1996;78:596-605.)
© 1996 American Heart Association, Inc.

Articles

Regulation of ß₁-Integrin Function in Cultured Human Vascular Smooth Muscle Cells

Jiro Seki, Noriyuki Koyama, Nicholas L. Kovach, Ted Yednock, Alexander W. Clowes, John M. Harlan

From the Division of Hematology (J.S., N.L.K., J.M.H.), Harborview Medical Center, Seattle, Wash; the Department of Surgery (N.K., A.W.C.), University of Washington School of Medicine, Seattle; and Athena Neurosciences Inc (T.Y.), South San Francisco, Calif.

Correspondence to Jiro Seki, PhD, Department of Pharmacology, New Drug Research Laboratories, Fujisawa Pharmaceutical Co, Ltd, 2-1-6 Kashima, Yodogawa-Ku, Osaka 532, Japan.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Avidity modulation and function of ß₁-integrin receptors in cultured human vascular smooth muscle cells (SMCs) were investigated using monoclonal antibody (mAb) 8A2, which binds to the ß₁ subunit of integrin heterodimers and induces a high avidity state. The adhesion of SMCs to extracellular matrix proteins, but not to poly-L-lysine, was enhanced by pretreatment with mAb 8A2. A qualitative alteration of ß₁ integrin was assessed with mAb 15/7, which binds to an activation-dependent epitope on the ß₁ subunit. Binding of mAb 15/7 was enhanced by mAb 8A2 in a dose-dependent manner. Arg-Gly-Asp peptide and soluble fibronectin also enhanced expression of the 15/7 epitope, suggesting that the 15/7 epitope is closely related to the ligand-occupied state of ß₁ integrin. Platelet-derived growth factor (PDGF)-AA and -BB increased SMC adhesion to type I collagen but did not augment mAb 15/7 binding, suggesting that PDGFs increase binding avidity by a postreceptor mechanism. In addition, mAb 8A2 inhibited PDGF-BB–induced SMC migration through Matrigel-coated filters. These results suggest that avidity modulation of ß₁ integrin may play an important role in the function of SMCs.

Key Words: adhesion • migration • monoclonal antibody • extracellular matrix

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The proliferation and migration of SMCs are critical events in the pathogenesis of atherosclerosis and restenosis after PTCA.^{1 2} Growth factors, cytokines, proteoglycans, and ECM are believed to be important regulators of these events.^{3 4} SMC interactions with ECM proteins such as fibronectin, collagen, and laminin play an important role not only for the adhesion but also for the phenotypic change and migration of SMCs.^{5 6 7 8 9 10 11}

Integrins are a superfamily of noncovalently associated transmembrane ß heterodimers.^{12 13} The combination of these subunits defines the specificity of the integrin complex for ligands. To date, several ß₁ integrins and _vß₃ integrin have been identified in SMCs.^{14 15 16 17} The binding of SMCs to ECM proteins is largely mediated by integrin receptors of the ß₁ family.^{14 15 16 17}

Integrin-ligand interactions are not fixed but are dynamically regulated.^{18 19} First, integrin function is regulated through alterations in the cell surface expression. For example, transforming growth factor-ß upregulates the expression of ß₁ and several subunits of integrin receptors on WI-38 lung fibroblasts,²⁰ and tumor necrosis factor- and interferon gamma downregulate ß₃-integrin subunit synthesis at the transcriptional level in human endothelial cells.²¹ Expression of ß₂-integrin receptors is also regulated in leukocyte differentiation.²² Recently, Janat et al²³ and Basson et al¹¹ investigated the regulation of integrin expression in cultured vascular SMCs and found that transforming growth factor-ß and PDGF-BB upregulated mRNA and surface protein for ß₃ and ₅ subunits without modulating ß₁-integrin expression.

Another important mechanism of regulation of integrin function involves changes in the affinity/avidity of the receptor for ligands that occur without a change in surface expression.^{12 19} In this context, changes in affinity can be defined by the binding of soluble monovalent ligand; ie, a dissociation constant (K_d) can be determined. Alterations in apparent affinity may result from changes in receptor conformation. Avidity can be defined as binding to multivalent ligand, ie, adhesion, and can result from changes in affinity or from postreceptor events involving cytoskeletal components.²⁴ Avidity modulation of integrin receptors has been extensively studied for _IIbß₃ integrin in platelets and ß₁- and ß₂-integrin families in leukocytes and has been suggested to have an essential role in the pathogenesis of thrombosis, inflammation, and autoimmune diseases.^{12 19} For example, integrin _IIbß₃ on resting circulating platelet is in an inactive form and does not bind any of its soluble ligands but is functionally activated by ADP, thrombin, or other platelet agonists.²⁵ This activation is accompanied by a conformational change of the _IIbß₃ receptor that can be detected immunologically.²⁶ This regulation of the integrin receptor is mediated by several biochemical events, such as protein phosphorylation or phospholipid metabolism, inside the cell, ie, so-called "inside-out" signaling.^{12 27} On the other hand, several cytoplasmic events can be also triggered by ligand occupation of the receptors, ie, "outside-in" signaling.¹²

The ß₁-integrin–mediated adhesion of leukocytes to ECM proteins can be activated by divalent cations,²⁸ phorbol ester,^{29 30} cross-linking of cell surface receptors (eg, T-cell receptors),²⁹ and certain mAbs to ß₁ integrin^{31 32 33} without alterations in surface expression. However, avidity modulation of integrin receptors in vascular SMCs has not been demonstrated.

In the present study, we have used two novel mAbs to the ß₁ subunit of integrin to investigate the avidity regulation and function of ß₁ integrins in cultured human SMCs. One is mAb 8A2,³¹ which binds to the ß₁ subunit of integrins and activates ß₁-integrin–dependent cellular adherence. This mAb has been shown to enhance the binding of ligands by ß₁ integrins.²⁴ For example, the K_d for binding of soluble fibronectin to ₅ß₁ in K562 cells was enhanced 20-fold by this mAb.³⁴ The enhanced binding of ligand by mAb 8A2 was hypothesized to be due to a direct mAb-induced change in the receptor conformation, since it was observed in energy-depleted cells.³⁴

The second ß₁ probe used is mAb 15/7, which recognizes an activation-dependent epitope of ß₁ integrin.³⁵ This epitope has been shown to be absent or expressed only at a low level on resting leukocytes but is upregulated in parallel with ligand-binding capacity by extracellular Mn²⁺ or activating mAbs to the ß₁ integrin, such as mAb 8A2.^{36 37}

By using these mAbs, we have found that ß₁-integrin–mediated adhesion of cultured human SMCs is regulated by changes in receptor avidity and that this regulation is important for SMC migration.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies and Reagents
The production and characterization of mAb 8A2, an activating mAb to ß₁ integrin, was described previously.³¹ mAb 15/7, which recognizes an activation-dependent epitope of ß₁ integrin, was produced as described previously.³⁵ mAb P4C10, a blocking mAb to ß₁ integrin,³⁸ mAb P1E6, a nonblocking mAb to the ₂ integrin, mAb P1B5, a blocking mAb to ₃ integrin, and mAb P1D6, a blocking mAb to ₅ integrin were purchased from GIBCO BRL. mAb LM534, a nonactivating and nonblocking mAb to ß₁ integrin, and mAb LM609, a blocking mAb to _vß₃ integrin, were purchased from Chemicon International Inc. mAb G19, a blocking mAb to ₂ integrin, was purchased from Biodesign International. MOPC-21, which has no known antigen-binding activity and was used as a control IgG, and FITC-labeled goat anti-mouse IgG (Fc fragment) were purchased from Organon Teknika Corp.

Vitrogen was used as a type I collagen and was purchased from Celtrix Laboratories. Type IV collagen, fibronectin (from human plasma), and poly-L-lysine (molecular weight, >300 000) were purchased from Sigma Chemical Co. Laminin, GRGDSP, GRGESP, DME, sodium pyruvate, nonessential amino acid solution, penicillin-streptomycin, trypsin-EDTA, and BSA were purchased from GIBCO BRL. FBS was purchased from Hyclone. Matrigel was purchased from Collaborative Research Inc. Human -globulin was purchased from Calbiochem. [⁵¹Cr]Sodium chromate (250 to 500 mCi/mg chromium) was purchased from Amersham. Recombinant human PDGF-AA and PDGF-BB were kindly supplied by Dr Hart (Zymogenetics Inc). These PDGFs were produced in a yeast expression system and purified to >95% homogeneity.³⁹

Cell Culture
Human newborn (13-day) arterial SMCs were obtained from the thoracic aortas of infants after accidental death as described previously.⁴⁰ The cells were maintained in DME supplemented with 10% FBS, nonessential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin (FBS-DME). Cells were used between passages 9 and 12.

Adhesion Assay
ECM proteins (type I collagen, type IV collagen, laminin, and fibronectin), FBS, poly-L-lysine, or BSA in Dulbecco's PBS was coated on 48-well plates (Costar 3547, nontreated/sterile) for 24 hours at 4°C. Nonspecific adherence was blocked with 3% BSA in PBS for 1 hour at room temperature. Cultured SMCs at confluence were removed from culture plates by brief incubation (2 minutes) at room temperature with trypsin-EDTA. Trypsin was then inactivated by washing the cells with FBS-DME. The cells were washed two times with DME without serum and then incubated with [⁵¹Cr]sodium chromate (80 µCi/10⁷ cells) for 1 hour at 37°C.⁴¹ The cells were washed three times with DME supplemented with 1 mg/mL BSA (BSA-DME). The ⁵¹Cr-labeled cells were suspended in BSA-DME at 5x10⁵/mL, and 1x10⁵ cells were added to the wells coated with ECM protein after pretreatment with BSA-DME alone, control IgG, mAb 8A2, PDGF-AA, or PDGF-BB for 30 minutes at 37°C. The cells were allowed to adhere to the wells for 40 minutes at 37°C. Nonadherent cells were removed by washing once with BSA-DME. The adherent cells were lysed with 0.5 mL of 1N NH₄OH and counted with a gamma counter (Micromedic System, Inc). Percent adherence was calculated as follows:

Migration Assay
Migration of SMCs was assayed by a modified Boyden chamber method using microchemotaxis chambers (Neuro Probe Inc) with polycarbonate filters (Nucleopore Corp) with pores of 10-µm diameter.⁴² A polycarbonate filter coated with Matrigel solution (2.7 µg per well) was placed between the upper and lower chambers. Cultured SMCs were trypsinized and suspended at a concentration of 5x10⁵ cells per milliliter in serum-free medium. A volume of 50 µL of SMC suspension was placed in the upper chamber, and 25 µL of serum-free medium containing PDGF-BB (10 ng/mL) was placed in the lower chamber. To avoid the effect of antibodies on the initial cell attachment, the chamber was incubated at 37°C under 5% CO₂ in air for 2 hours, and then mAb 8A2 or control IgG was added in the upper chamber. Incubation was continued for 4 hours more. The filter was then removed and fixed in methanol and stained with Diff-Quick staining solution (Baxter). Nine filters were used for each treatment, and four fields were scored under a microscope (x100) for quantification of SMC migration. Data were expressed as the mean±SD number of cells that had migrated per high-power field.

Flow Cytometry
Confluent SMCs were trypsinized and suspended in BSA-DME at 2x10⁶ cells per milliliter. Before the incubation with mAb, cells were incubated with human -globulin (3 mg/mL) for 30 minutes on ice to block nonspecific binding. For determination of the expression of ß₁ integrin, 1x10⁵ cells were incubated with control IgG (20 µg/mL), mAb 15/7 (20 µg/mL), mAb P4C10 (1:50), or mAb 8A2 (20 µg/mL) for 30 minutes on ice. After they were washed twice with BSA-DME, cells were labeled with FITC-labeled anti-mouse IgG (1:50). For determination of the activation state of ß₁ integrin, cells were incubated with mAb 15/7 (20 µg/mL) after pretreatment with BSA-DME alone (control), Fab fragment of mAb 8A2 (0.01 to 10 µg/mL), PDGF-AA (10 ng/mL), PDGF-BB (10 ng/mL), GRGDSP (1 to 1000 µmol/L), GRGESP (1 to 1000 µmol/L), or fibronectin (400 µg/mL). After they were washed twice with BSA-DME, cells were labeled with FITC-labeled anti-mouse IgG (Fc fragment, 1:50). Fluorescence staining was analyzed on an EPICS XL flow cytometry system (Coulter Corp) after fixation with 1% paraformaldehyde in PBS. Analysis was based on data collected from 5000 events, and values were expressed as the MFI of the gated cell population.

Statistical Analysis
Differences between means were analyzed by ANOVA with Dunnett's test. Statistical significance was defined as P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enhancement of ß₁-Integrin–Dependent Adhesion of SMCs by mAb 8A2
To determine the effect of avidity modulation of ß₁ integrin in SMCs, we first studied unstimulated SMC adhesion to ECM proteins. Suspended human SMCs rapidly adhered to type I collagen– and poly-L-lysine–coated but not BSA-coated plates with maximal binding by 40 minutes. Adherence to type I collagen was almost completely blocked by EDTA or mAb P4C10, a blocking mAb to ß₁ integrin,³⁸ whereas the adherence to poly-L-lysine was not affected by these agents (data not shown). This result indicates that the binding of human SMCs to type I collagen, but not to poly-L-lysine, is primarily ß₁-integrin dependent. Adherence to type IV collagen, laminin, fibronectin, and serum proteins was also blocked by EDTA and mAb P4C10 (data not shown).

Recently, certain ß₁-integrin mAbs have been described to stimulate ß₁-integrin functions.^{31 32 33} We have shown that mAb 8A2 binds to the ß₁ subunit of the integrin receptor and enhances ß₁-integrin–dependent adherence of leukocytes to cellular and matrix ligands.³¹ We first tested the effect of this mAb on the adhesion of human SMCs to type I collagen– and poly-L-lysine–coated plates. In this experiment, plates were coated with limited concentrations of proteins that allowed approximately half of total seeded cells to adhere. mAb 8A2 enhanced the adhesion of SMCs to type I collagen in a dose-dependent manner, whereas the adhesion to poly-L-lysine was not affected by this mAb (Fig 1). Percent adherence of untreated SMCs to type I collagen was 40.6±4.8%, and it was enhanced to 79.7±2.8% by pretreatment with mAb 8A2 at 1 µg/mL. The effect of mAb 8A2 was significant even at 0.01 µg/mL. The monovalent Fab fragments of mAb 8A2 were as active as intact IgGs in stimulating SMC adhesion to type I collagen (data not shown), indicating that cross-linking of the receptor was not required. The effect of mAb 8A2 on the surface expression of the ₂-integrin subunit, a major subunit for type I collagen receptor, was determined by flow cytometry, and the expression was not altered by pretreatment with the Fab fragment of mAb 8A2 at concentrations up to 10 µg/mL (data not shown). These results indicate that ß₁ integrin in SMCs can be functionally activated by mAb 8A2.

View larger version (33K): [in this window] [in a new window]   Figure 1. Effect of mAb 8A2, an activating mAb to ß1 integrin, on the adhesion of cultured human SMCs to type I collagen– and poly-L-lysine–coated plates. 51Cr-labeled cells were allowed to adhere to the wells coated with type I collagen (0.3 µg/mL) or poly-L-lysine (0.1 µg/mL) after preincubation with medium alone (control) or with control IgG (1 µg/mL) or mAb 8A2 (0.01 to 1 µg/mL). Values are mean±SD of four wells in one experiment representative of three separate experiments. **P<.01 vs control.

The concentration-dependent curves for the adhesion of SMCs to wells coated with type I collagen, type IV collagen, laminin, and serum proteins were clearly shifted to the left by pretreatment with 1 µg/mL of mAb 8A2 (Fig 2, top and middle panels). Thus, several different ß₁-integrin receptors on SMCs can be activated by mAb 8A2. Surprisingly, the concentration-dependent curve for the adhesion to fibronectin, which was also primarily ß₁-integrin dependent, since this binding was almost completely blocked by mAb P4C10, was not affected by mAb 8A2 (Fig 2, bottom left). However, the rate of adhesion to fibronectin was slightly, but significantly, enhanced by this mAb (data not shown), indicating that mAb 8A2 also enhanced the avidity of fibronectin receptors in SMCs to some extent.

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of mAb 8A2 on the adhesion of cultured human SMCs to plates coated with type I collagen (top left), type IV collagen (top right), laminin (middle left), FBS (middle right), and fibronectin (bottom left). 51Cr-labeled cells were added to the wells coated with the indicated concentrations of proteins after preincubation with medium alone (), control IgG (), or mAb 8A2 (). Values are mean±SD of four wells in one experiment representative of three separate experiments.

Expression of Activation-Dependent Epitope of ß₁ Integrin in SMCs
Recently, Picker et al³⁵ reported a novel mAb to ß₁ integrin, designated 15/7, that recognizes an activation-dependent epitope of ß₁ integrin (15/7 epitope) in leukocytes. The expression of the 15/7 epitope correlates with the enhancement of adhesion in leukocytes.^{35 37} Therefore, the detection of 15/7 epitope can be used as a marker of an active state of ß₁ integrins.

To investigate the modulation of ß₁ integrin avidity in SMCs, we first analyzed the expression of the 15/7 epitope in untreated cultured human SMCs by flow cytometry (Fig 3). The ß₁ mAbs P4C10 and 8A2 were used as markers for total expression of ß₁ integrins. The 15/7 epitope was present at low levels in unstimulated SMCs. The MFI of cells labeled with mAb 15/7 was 4.5±0.3% (n=4) of the MFI of cells labeled with P4C10. The expression of 15/7 epitope in cultured human SMCs was markedly enhanced by the Fab fragment of mAb 8A2 in a dose-dependent manner (Fig 4). The MFI was increased 10-fold by 10 µg/mL of mAb 8A2 Fab fragment (Fig 5, top). To rule out the possibility of cross-reactivity of the secondary anti-mouse antibody with the Fab fragment of mAb 8A2, we also performed the experiment with directly FITC-conjugated 15/7. The result was the same (data not shown). This result indicates that mAb 8A2 activates ß₁-integrin adhesive function, inducing a neoepitope on the ß₁ subunit in cultured human SMCs. Interestingly, the 15/7 epitope was also induced by peptide GRGDSP (Fig 5, middle; Fig 6) and soluble fibronectin (Fig 5, bottom). Since the peptide GRGESP had no effect on the 15/7 epitope expression (Fig 6), the RGD sequence accounted for the induction of the 15/7 epitope on ß₁ integrins in cultured human SMCs. GRGDSP had no effect on the binding of the P4C10 mAb (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 3. Expression of ß1 integrin in cultured human SMCs. Cells were incubated with control (Cont) IgG (20 µg/mL), mAb P4C10 (x50), mAb 8A2 (20 µg/mL), or mAb 15/7 (20 µg/mL) for 30 minutes on ice. Cells were labeled with FITC-labeled anti-mouse IgG Fc fragment (1:50) and analyzed on a flow cytometer. Similar results were obtained in two additional experiments.

View larger version (18K): [in this window] [in a new window]   Figure 4. Dose-dependent enhancement of the expression of 15/7 epitope by Fab fragment of mAb 8A2 in cultured human SMCs. Cells were incubated with mAb 15/7 (20 µg/mL) after a 30-minute preincubation at 37°C with medium alone (Cont) or with 0.01 to 10 µg/mL of Fab fragment of mAb 8A2. After they were washed, cells were labeled with FITC-labeled anti-mouse IgG Fc fragment (1:50) and analyzed on a flow cytometer. mAb 15/7 binding was expressed as MFI. Similar results were obtained in two additional experiments.

View larger version (22K): [in this window] [in a new window]   Figure 5. Effect of Fab fragment of mAb 8A2 (top), RGD peptides (middle), and soluble fibronectin (bottom) on the expression of 15/7 epitope in cultured human SMCs. Cells were incubated with mAb 15/7 (20 µg/mL) after a 30-minute preincubation at 37°C with medium alone (control) or with 10 µg/mL of Fab fragment of mAb 8A2 (top), with 1 mmol/L of GRGDSP or GRGESP (middle), or with 400 µg/mL of soluble fibronectin (bottom). After they were washed, cells were labeled with FITC-labeled anti-mouse IgG Fc fragment (1:50) and analyzed on a flow cytometer. Similar results were obtained in two additional experiments.

View larger version (22K): [in this window] [in a new window]   Figure 6. Dose-dependent enhancement of the expression of 15/7 epitope by GRGDSP in cultured human SMCs. Cells were incubated with mAb 15/7 (20 µg/mL) after a 30-minute preincubation with medium alone (vehicle) or with 1 to 1000 µmol/L of GRGDSP (solid bar) or GRGESP (hatched bar) at 37°C. After they were washed, cells were labeled with FITC-labeled anti-mouse IgG Fc fragment (1:50) and analyzed on a flow cytometer. Similar results were obtained in two additional experiments.

Enhancement of ß₁-Integrin–Dependent Adhesion of SMCs by PDGF
Although mAb 8A2 represents a good tool for studying the functional modulation of ß₁ integrins, it is not a physiological stimulus. Therefore, we tested various agents for their effect on the adhesion of human SMCs to type I collagen–coated plates to determine whether physiological stimuli enhance ß₁-integrin–dependent adhesion in SMCs. A 30-minute pretreatment with 0.1 to 10 ng/mL of PDGF-AA and PDGF-BB increased the adhesion of human SMCs to type I collagen in a dose-dependent manner (Fig 7, left). In contrast, PDGF-AA and PDGF-BB at 10 ng/mL had no effect on SMC adhesion to poly-L-lysine (data not shown). Enhanced adhesion induced by the PDGFs was completely blocked by mAb P4C10 (data not shown). These results suggest that PDGFs activate ß₁-integrin function in SMCs. Therefore, we examined the effect of PDGFs on 15/7 epitope expression in SMCs. However, PDGF-AA or PDGF-BB at 10 ng/mL did not affect 15/7 epitope expression (Fig 7, right). Cell surface expression of the ß₁ (detected by mAb P4C10) and ₂ (detected by mAb P1E6) subunits of integrin was also not affected by 30 minutes of incubation with PDGF-AA or PDGF-BB (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 7. Effect of PDGF-AA and PDGF-BB on the adhesion to type I collagen–coated plates (left) and on the expression of 15/7 epitope (right) in cultured human SMCs. Left, 51Cr-labeled cells were added to the wells coated with type I collagen (0.3 µg/mL) after preincubation with medium alone (Cont), mAb 8A2 (1 µg/mL), PDGF-AA, or PDGF-BB. Values are mean±SD of four wells in one experiment representative of three separate experiments. **P<.01 vs Cont. Right, Cells were incubated with mAb 15/7 (20 µg/mL) after a 30-minute preincubation with medium alone (control) or with 10 ng/mL of PDGF-AA or PDGF-BB at 37°C. After they were washed, cells were labeled with FITC-labeled anti-mouse IgG Fc fragment (1:50) and analyzed on a flow cytometer. Similar results were obtained in two additional experiments.

Effect of mAb 8A2 on the Migration of SMCs
The adhesive interaction of SMCs to matrix proteins has been shown to regulate SMC functions such as migration. Therefore, we tested the effect of avidity modulation induced by mAb 8A2 on the migration of human SMCs.

The effect of mAb 8A2 on PDGF-BB–induced migration of SMCs through Matrigel-coated filters is shown in Fig 8. PDGF-BB at 10 ng/mL enhanced SMC migration up to 12-fold. mAb 8A2 at 1 µg/mL significantly inhibited SMC migration by 53%, whereas an isotype-matched control IgG was without effect.

View larger version (15K): [in this window] [in a new window]   Figure 8. Inhibitory effect of mAb 8A2 on PDGF-BB–induced migration of human SMCs through Matrigel-coated filters. Polycarbonate filters were coated with Matrigel. SMC suspension in serum-free medium was placed in the upper chamber of a Boyden chamber, and 10 ng/mL of PDGF-BB in serum-free medium was placed in the lower chamber. After 2 hours of incubation, control (Cont) IgG (10 µg/mL) or mAb 8A2 (1 µg/mL) was added into the upper chamber. Values were the mean (±SD) number of cells that had migrated per high-power field (HPF) (n=9). Similar results were obtained in two additional experiments. **P<.01 vs Cont.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we provide evidence for the avidity modulation of ß₁ integrins in cultured human SMCs. SMCs adhered to ECM protein–coated plates without any stimulation.¹⁵ However, the ligand concentration-dependent curves for the adherence of SMCs to wells coated with type I collagen, type IV collagen, laminin, and serum proteins, but not poly-L-lysine, were clearly shifted to lower concentration of ligand by pretreatment with mAb 8A2 (Fig 2, top and middle panels). This result indicates that ß₁ integrins of cultured human SMCs are at least partly constitutively active but are still able to be activated further by mAb 8A2. This conclusion is supported by flow cytometric analysis of the activated form of ß₁ integrin as detected by mAb 15/7. The activation-dependent epitope of ß₁ integrin (15/7 epitope) was present at low levels in SMCs without stimulation (Fig 3), and the expression of this epitope was clearly enhanced by mAb 8A2 (Fig 4). These results indicate that the function of ß₁ integrins on SMCs can be regulated through the qualitative changes of the ß₁ subunit, as previously suggested in other cells.^{43 44 45}

Interestingly, GRGDSP, but not GRGESP, clearly induced the expression of the 15/7 epitope. It is unlikely that GRGDSP itself directly activates the adhesive function of ß₁ integrins, since pretreatment with this peptide does not affect the adhesion of SMCs to type I collagen–coated plates (data not shown). The existence of several RGD-directed integrins has been reported in cultured SMCs.^{8 15} We observed that 0.5 mmol/L GRGDSP, but not GRGESP, almost completely inhibited the adhesion of SMCs to serum-coated plates. The adhesion to fibronectin was also partially inhibited by GRGDSP, whereas the binding to type I collagen, type IV collagen, laminin, and poly-L-lysine was not affected (data not shown). Thus, GRGDSP may bind to these RGD-directed ß₁ integrins and induce the change of their conformation, thereby producing the 15/7 epitope. This result suggests that ligand binding itself changes the conformation of some ß₁-integrin receptors, similar to a high avidity state in SMCs. In fact, soluble fibronectin also induced the expression of the 15/7 epitope. The conformation of the ligand-occupied state of ß₁ integrin thus may be closely related to that of the active state induced by mAb 8A2. As suggested by Faull et al,³⁴ mAb 8A2 may preferentially recognize the active form of ß₁ integrin and stabilize that state. Antibodies that selectively bind to the ligand-occupied state of the receptor are referred to as LIBS antibodies because they recognize the ligand-induced binding site.^{46 47} In this regard, mAb 15/7 may be designated as a LIBS antibody to ß₁ integrin.

Although the adhesion of SMCs to type I collagen, type IV collagen, laminin, and serum proteins was clearly enhanced by mAb 8A2, the adhesion to fibronectin, which was also primarily ß₁-integrin dependent since this binding was almost completely blocked by mAb P4C10, was minimally affected by this mAb. Thus, the fibronectin receptor in SMCs seemed to be resistant to mAb 8A2–induced activation. Recently, Weitzman et al⁴⁸ reported a similar phenomenon. They found that VLA-3 (₃ß₁)–mediated adhesion of erythroleukemia (K562) and rhabdomyosarcoma cells transfected with ₃ cDNA to ECM proteins was minimally stimulated by an activating anti–ß₁-integrin mAb, TS2/16, which could strongly activate VLA-2–, VLA-4–, VLA-5–, and VLA-6–mediated adherence.⁴⁸ However, the adhesion of SMCs to fibronectin was not blocked by the blocking mAb to ₃ (mAb P1B5,⁴⁹ ) but was strongly (70%) blocked by the blocking mAb to 5 (mAb P1D6,⁴⁹ ) (data not shown), indicating that the major receptor for fibronectin in SMCs was ₅ß₁ integrin. Moreover, mAb 8A2 has been proven to activate ß₁-integrin–dependent adhesion to fibronectin potently in leukocytes.^{24 31 34} These results indicate that the various ß₁ integrins are functionally regulated in a cell type–specific manner. Recently, using - and ß-subunit chimeras expressed in various cell types, O'Toole et al⁵⁰ demonstrated that (1) the affinity state of the ₅ß₁ receptor was regulated by cell type–specific factors, (2) cell type–specific signals modulating affinity were transmitted by the cytoplasmic domains of both and ß subunits, and (3) certain deletions in the conserved -subunit sequence GFFKR result in a high-affinity state of _IIbß₃. Notably, Leung-Hagesteijn et al⁵¹ found that leukocyte attachment to ECM ligands was found to be inhibited by antisense oligonucleotide-mediated downregulation of calreticulin, a cytoplasmic protein that binds to the conserved sequence KXGFFKR. Thus, the fibronectin receptor, probably ₅ß₁, but not collagen and laminin receptors, might be already "activated" or "locked" through the cytoplasmic domain of the subunit in human aortic SMCs. Since soluble fibronectin induced the expression of 15/7 epitope (Fig 5, bottom), fibronectin binding to its receptor might itself activate or lock ß₁-integrin function.

Flow cytometric analysis also indicates the existence of locked ß₁ integrins in SMCs. The conformational change of ß₁ integrins assessed by mAb 15/7 binding occurred only in 30% of total ß₁ integrins, even with saturating concentrations of mAb 8A2 (10 µg/mL). Since mAb 8A2 recognizes all forms of ß₁ integrins (Fig 3), this result suggests that large subsets of ß₁ integrins expressed on the surface of SMCs are resistant to the mAb 8A2–induced change in conformation that produces the 15/7 epitope. As has been reported for leukocytes,⁵² these findings might also suggest that only small subsets of ß₁ integrins are competent to mediate adhesion and function in SMCs.

Adhesion of SMCs to type I collagen was enhanced by the pretreatment with PDGF-AA and PDGF-BB. Since the adhesion to poly-L-lysine was not affected by these growth factors and enhanced adhesion was almost completely blocked by mAb P4C10, the effect was ß₁-integrin dependent. PDGF has been shown to modulate cell adhesion through cytoskeletal organization and the disruption of focal contact^{53 54 55 56} ; however, the direct evidence for the stimulatory effect of PDGF on the ß₁-integrin–dependent adhesion has not previously been reported. Recently, Janat et al²³ reported the increased expression of mRNA and polypeptides for the ß₃ and ₅ subunits, but not for the ß₁ subunit, in cultured rabbit SMCs at 2 and 6 hours after treatment with PDGF-BB. By using flow cytometry, we also observed that the surface expression of ß₁ and ₂ subunits was not affected by 30-minute incubation with PDGF-AA or PDGF-BB in human SMCs (data not shown). Therefore, the augmentation of adhesion to type I collagen by PDGF-AA and PDGF-BB seemed to be due to functional modulation of ß₁ integrins. However, a conformational change of ß₁ integrin, detected by mAb 15/7, was not induced by 10 ng/mL of PDGF-AA and PDGF-BB (Fig 7, right). This dose of PDGF-AA or PDGF-BB was as active as 0.1 µg/mL of the mAb 8A2 Fab fragment in stimulating SMC adhesion to type I collagen, and 0.1 µg/mL of mAb 8A2 Fab fragment clearly induced the 15/7 epitope. Therefore, the inability of PDGFs to induce the 15/7 epitope appeared not to be due to its reduced adhesion-promoting activity. Recent studies showed that increased avidity of leukocyte integrin receptors may result from two distinct mechanisms: an increased affinity (as measured by binding to soluble monovalent ligands) or postreceptor events (eg, cytoskeletal association).¹⁹ Faull et al²⁴ noted that mAb 8A2 enhanced the adhesion of leukocytes by increasing the affinity of ß₁ integrins, whereas phorbol ester enhanced the ß₁-integrin–dependent adhesion through postreceptor events without a change of affinity for ligand. Thus, PDGF-AA and PDGF-BB may enhance ß₁-integrin–dependent adhesion in SMCs through the modulation of postreceptor events, such as a cytoskeletal association to integrins. Binding of PDGF-AA and PDGF-BB to their receptors triggers tyrosine phosphorylation of the receptors themselves and cytoplasmic proteins.^{57 58} Since a high level of protein tyrosine phosphorylation in the focal adhesions has been demonstrated to be important for cell adhesion to ECM proteins,^{59 60} PDGF might regulate the avidity of ß₁ integrins through tyrosine phosphorylation of proteins in the focal adhesions or cytoplasmic domain of ß₁ integrins.⁶¹ Notably, Bartfeld et al⁶² demonstrated that PDGF-BB induced tyrosine phosphorylation of a 190-kD protein that was associated with _vß₃ integrin. Recently, we found that stem cell factor, which is known as a tyrosine kinase activator, modulates the avidity of ₄ß₁ and ₅ß₁ expressed on hematopoietic cell lines.³⁷ Taken together, tyrosine phosphorylation of certain cytoplasmic proteins seems to be important for the functional regulation of ß₁-integrin avidity. The cross talk between growth factor receptors and integrins promises to be a fruitful area of research. PDGF-BB is a well-known chemoattractant.⁶³ Since migration of SMCs is critically affected by its adhesive property,¹⁰ the effect of PDGF-BB on the adhesion of SMCs may be important for its function as a chemoattractant. Moreover, our group has shown that PDGF-AA suppresses the migration of SMCs.⁶⁴ Whether the effect of PDGF-AA on the adhesion of SMCs is also related to its inhibitory activity is now under investigation. Overall, these results suggest that as for leukocytes,²⁴ there are at least two mechanisms for the regulation of ß₁-integrin avidity in SMCs: (1) a change in receptor conformation associated with expression of the 15/7 epitope as induced by mAb 8A2 and (2) postreceptor events as induced by the PDGFs.

MAb 8A2 suppressed SMC migration. Recently, Kuijpers et al⁶⁵ reported that mAb 8A2 inhibited the migration of eosinophils and hypothesized that the inhibition was a result of "freezing" cells to the matrix ligands. We observed enhanced attachments of SMCs by mAb 8A2 to Matrigel-coated filters used for the migration assays (data not shown). Therefore, the inhibition of SMC migration by mAb 8A2 may also be due to its freezing activity. Recently, DiMilla et al¹⁰ reported that maximal migration of human SMCs on fibronectin and type IV collagen occurred at an intermediate attachment strength. Thus, appropriate strength of cell adhesion through ß₁ integrins is important for SMC migration. The importance of _vß₃ integrins for SMC migration has been previously reported.^{8 66 67} Our results indicate that not only _vß₃ integrin but also ß₁ integrins have important roles in SMC migration. Since the migration of SMCs is a critical event in the pathogenesis of atherosclerosis and restenosis after PTCA, activation of ß₁-integrin–mediated cell adhesion would represent a new approach to prevent these disorders.

In conclusion, ß₁ integrins on cultured human SMCs can be functionally regulated, and this modulation may play a crucial role in the migration of SMCs. The pathophysiological relevance of avidity regulation of ß₁ integrins in vivo remains to be determined.

	Selected Abbreviations and Acronyms

 

DME = Dulbecco's modified minimum essential medium ECM = extracellular matrix FITC = fluorescein isothiocyanate GRGDSP = Gly-Arg-Gly-Asp-Ser-Pro GRGESP = Gly-Arg-Gly-Glu-Ser-Pro LIBS = ligand-induced binding site(s) mAb = monoclonal antibody MFI = mean fluorescence intensity PDGF = platelet-derived growth factor PTCA = percutaneous transluminal coronary angioplasty RGD = Arg-Gly-Asp SMC = smooth muscle cell

	Acknowledgments

 
This study was supported by US Public Health Service grant HL-18645. We would like to acknowledge Dr Sam Sharar for his help in flow cytometric analysis.

Received August 14, 1995; accepted December 8, 1995.

	References

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