Endothelial cell - Journal of Cell Science - The Company of Biologists

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integrin subunit (Argraves et al., 1986) subcloned into the EcoRI site of pGEM1, and was kindly provided by Dr E. Ruoslahti (Cancer. Research Centre, La Jolla, ...
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Journal of Cell Science 112, 569-578 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS3870

Endothelial cell integrin α5β1 expression is modulated by cytokines and during migration in vitro Ginetta Collo and Michael S. Pepper* Department of Morphology, University Medical Center, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland *Author for correspondence (e-mail: [email protected])

Accepted 8 December 1998; published on WWW 25 January 1999

SUMMARY Alterations in endothelial cell-extracellular matrix interactions are central to the process of angiogenesis. We have investigated the effect of wound-induced twodimensional migration, basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF-β1) and leukemia inhibitory factor (LIF) on expression of the α5β1 integrin in endothelial cells. In multiple-wounded monolayers of bovine microvascular endothelial (BME) cells, an increase in mRNA and total protein for both α5 and β1 subunits was observed, and this could be correlated with a reduction in cell density but not proliferation, both of which are induced following wounding. Although as previously reported, the α5 subunit was increased when

cells were exposed to TGF-β1 alone, co-addition of bFGF and TGF-β1 resulted in a striking synergistic induction of α5, with no significant changes in the expression of β1. In contrast, the α5 subunit was decreased by LIF in bovine aortic endothelial but not in BME cells. These findings suggest that quantitative alterations in α5 and β1 integrin subunit expression modulate the adhesive and migratory properties of endothelial cells during angiogenesis.

INTRODUCTION

composed of two non-covalently associated transmembrane subunits, α and β, which connect adhesive proteins in the extracellular matrix to the cytoskeleton, and which are also involved in intracellular signal transduction. At present 17 different α and 8 different β subunits have been identified, which associate to form more than 20 receptors recognizing one or more extracellular ligands (reviewed by Hynes, 1992, 1996; Giancotti and Mainiero, 1994; Juliano, 1996; Yamada and Geiger, 1997). Endothelial cells express a number of different integrins (reviewed by Luscinskas and Lawler, 1994; Ruoslahti and Engvall, 1997), two of which, namely αvβ3 and α5β1, have been shown to be particularly important during angiogenesis. αvβ3 is a receptor for matrix proteins with an exposed Arg-GlyAsp (RGD) tripeptide moiety including vitronectin, fibronectin, fibrinogen, osteopontin, denatured collagen and von Willebrand factor. Despite this apparent promiscuity, αvβ3 is not widely expressed, and is most prominent on cytokine-activated endothelial cells during angiogenesis as well as on smooth muscle cells in post-angioplasty restenosis, in atherosclerotic plaques and in healing arterial wounds (reviewed by Varner et al., 1995). A significant body of experimental evidence has demonstrated that αvβ3 antagonists (antibodies, cyclic RGD peptides) inhibit angiogenesis during development, wound healing, retinal neovascularization, and in growing tumors (in which they also induce tumor regression) (Brooks et al., 1994a,b, 1995; Drake et al., 1995; Clark et al., 1996; Friedlander et al., 1996; Hammes et al., 1996). In addition,

The formation of new capillary blood vessels, or angiogenesis, is an absolute requirement for the growth and repair of normal tissues, and is the process by which new blood vessels are formed in hemangiomas, proliferative retinopathy and rheumatoid arthritis. Angiogenesis is also necessary for tumor growth, and is required for the hematogenous dissemination of tumor cells and the growth of metastasis (reviewed by Folkman, 1995; Pepper et al., 1997a). Angiogenesis proceedes through two phases. The first is the activation phase, which includes increased vascular permeability and extravascular fibrin deposition, basement membrane degradation, cell migration and extracellular matrix invasion, endothelial cell proliferation and capillary lumen formation. The second is the resolution phase, which includes inhibition of endothelial cell proliferation, cessation of migration, basement membrane reconstitution and junctional complex maturation (Pepper et al., 1996). Many of these processes are regulated by polypeptide growth factors or cytokines including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) (reviewed by Dvorak et al., 1995; Pepper et al., 1996, 1997b; Christofori, 1997). In addition, endothelial cell proliferation, invasion and capillary-like tube formation are inhibited by leukemia inhibitory factor (LIF) in vitro (Pepper et al., 1995). Integrins are heterodimeric cell surface glycoproteins

Key words: Extracellular matrix, Angiogenesis, Basic fibroblast growth factor, Transforming growth factor-β1, Leukemia inhibitory factor

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tumor growth is suppressed by cytokines (TNF-α, interferonγ), which decrease αvβ3 activation in endothelial cells without affecting levels of expression (Rüegg et al., 1998). Angiogenic cytokines including bFGF, TGF-β1 and VEGF increase expression of the αv and β3 subunits in endothelial cells (Basson et al., 1992; Swerlick et al., 1992; Sepp et al., 1994; Senger et al., 1996; Suzuma et al., 1998). The relevance of αvβ3 to angiogenesis and its potential as an important therapeutic target have therefore been clearly established. The second integrin expressed by endothelial cells that is important for vascular development and the maintenance of vascular integrity is α5β1 which, like αvβ3, binds to an RGD tripeptide in fibronectin (reviewed by Ruoslahti, 1996). Fibrinogen is also a ligand for α5β1 in endothelial cells, and this interaction can be completely inhibited by RGD peptides (Suehiro et al., 1997). During angiogenesis, endothelial cells migrate on a fibronectin-rich extracellular matrix which in part they synthesize themselves (Clark et al., 1982a,b; Sariola et al., 1984; Risau and Lemmon, 1988), and extravascular accumulation of fibrin is one of the hallmarks of angiogenesis (Dvorak et al., 1995). Functional studies in vivo using linear and cyclic RGD peptides, β1 inhibitory antibodies or antibodies to the cell-binding region of fibronectin have defined a role for endothelial cell-fibronectin interactions in developmental angiogenesis (Britsch et al., 1989; Drake et al., 1992). Linear RGD peptides also inhibit angiogenesis in a collagen gel/aortic explant model in vitro (Nicosia and Bonanno, 1991). Previous studies have demonstrated that bFGF and TGF-β1 increase α5 and β1 protein biosynthesis in endothelial cells (Basson et al., 1992; Enenstein et al., 1992; Klein et al., 1993), although VEGF has no effect on these integrin subunits (Senger et al., 1996; Suzuma et al., 1998). However, with the exception of the β1 inhibitory antibody studies, none of the functional studies performed to date have been able to discriminate between α5β1/fibronectin interactions and interactions between fibronectin and the other integrins, since at least eight integrins (including αvβ3) have been reported to bind fibronectin. The most compelling evidence for a role for α5β1 in angiogenesis comes from studies on the phenotype of α5 null mice (β1 is the only β subunit that to date has been reported to form heterodimers with α5). These mice display a defect in the development of both embryonic and extraembryonic vasculatures, which results in lethality at days 10-11 of gestation (Yang et al., 1993). Deletion of β1 integrins in mice was lethal during the early post-implantation period, prior to development of the vascular system (Stephens et al., 1995). A null mutation in the fibronectin gene is also lethal at day 9 of development, and this is due to a defect in both extraembryonic (blood island) and intraembryonic blood vessel formation (George et al., 1993), demonstrating that fibronectin plays an important role in the development and integrity of blood vessels. In view of the potential role of the α5β1 integrin in angiogenesis and in the manitenance of vessel wall integrity, the present study was undertaken to evaluate the effects of wound-induced two-dimensional migration as well as a variety of cytokines (bFGF, TGF-β1 and LIF) on expression of the α5 and β1 integrin subunits in endothelial cells. The choice of cytokines was based on previous observations on the regulation of endothelial cell migration and capillary-like tube formation (Montesano et al., 1986; Pepper et al., 1990, 1992a, 1993,

1995) in a three-dimensional in vitro model of angiogenesis (Montesano and Orci, 1985).

MATERIALS AND METHODS Cell culture and cytokines Adrenal cortex-derived bovine microvascular endothelial (BME) cells provided by Drs M. B. Furie and S. C. Silverstein (Columbia University, NY) (Furie et al., 1984) were grown in alpha-modified minimal essential medium (α-MEM; Gibco BRL, Life Technologies, Paisley, Scotland), supplemented with 15% heat-inactivated donor calf serum (DCS) (Gibco), penicillin (110 i.u./ml) and streptomycin (110 µg/ml). Bovine aortic endothelial (BAE) cells (Pepper et al., 1992b) were grown in Dulbecco’s modified MEM (Gibco) containing 10% DCS and antibiotics. BME and BAE cells were cultured in 1.5% gelatin-coated tissue culture dishes (Falcon Labware, BectonDickinson Company, Lincon Park, NJ). Recombinant human bFGF (a gift from Dr P. Sarmientos, Farmitalia, Milan, Italy), human plateletderived TGF-β1 (R&D Systems Europe, Oxon, UK) and recombinant human VEGF and LIF (PeproTech Inc., Rocky Hill, NJ) were used alone or in combination as indicated, and were added directly to the medium of confluent monolayers of endothelial cells. Antibodies, cell labeling and immunoprecipitation Rabbit polyclonal antisera recognizing the cytoplasmic domains of the α5 and β1 integrin subunits (Defilippi et al., 1991) were generously provided by Dr Guido Tarone (Department of Genetics, Biology and Medical Chemistry, University of Torino, Italy). For metabolic labeling, confluent BME or BAE cell monolayers were incubated in Met/Cys-free MEM (Gibco) supplemented with 5% DCS and 40 µCi/ml L-35S in vitro cell labeling mix (>1000 Ci/mM; Amersham International plc, Little Chalfont, Buckinghamshire, UK) in the presence or absence of cytokines. Labelled cells were washed with cold PBS, extracted for 20 minutes at 4°C with 0.5% Triton X100 (Merck, Darmstadt, Germany) in TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 1 mM CaCl2, 1 mM MgCl2, 4 mM PMSF, 10 µg/ml leupeptin, 4 µg/ml pepstatin (all from Sigma Chemical Company, St Louis, MO) and 200 KIU/ml Trasylol (Bayer AG, Zurich, Switzerland), and scraped from the dish with a rubber policeman. Cell lysates were centrifugated at 10,000 g for 15 minutes at 4°C and radioactivity was normalized for all lysates. Samples of labeled cell extracts were preadsorbed with protein A-Sepharose beads (Pharmacia Biotech, Uppsala Sweden) for 1 hour at 4°C. Specific antibodies and protein A-Sepharose beads were added to cell lysates and rotated overnight at 4°C. The beads were recovered by centrifugation, washed three times with TBS and boiled for 5 minutes in non-reducing SDS-polyacrylamide gel electrophoresis (SDSPAGE) sample buffer. Immunocomplexes were electrophoresed in a 6% SDS-polyacrylamide gel. Dried gels were soaked in Amplify (Amersham) and exposed to Kodak X-OMAT XAR-5 Scientific Imaging Film (Eastman Kodak Co., Rochester, NY). Films were scanned and quantitated using ImageQuant (Molecular Dynamics, Sunnyvale, CA). Multiple wounding and low density cultures 24 hours after the last medium change, confluent monolayers of BME cells in gelatin-coated 100 mm tissue culture dishes were multiplewounded with a pointed rubber policeman as previously described (Pepper et al., 1992c). In some experiments, detached cells were removed and wounded monolayers were washed prior to addition of fresh medium; in other experiments, detached cells were not removed, and medium was not changed. 5 mM hydroxyurea (Sigma) was added to some dishes 30 minutes prior to wounding to determine whether alterations in integrin subunit mRNA might be proliferationdependent; in these experiments, medium was not changed after

Endothelial cell α5β1 integrin wounding. For metabolic labeling, Met/Cys-free MEM (Gibco) supplemented with 5% DCS, and 40 µCi/ml L-35S in vitro cell labeling mix (>1000 Ci/mM; Amersham) was added. Cells were processed for immunoprecipitation as indicated above. For low density cultures, confluent BME monolayers were trypsinized, split approximately 1/6, and cultured for 28-36 hours in αMEM supplemented with 15% DCS. Extraction of total cellular RNA and northern blot hybridization were performed as described below. Plasmid construction and in vitro transcription pGEM1-P7 contains a 1.7 kilobase-pair cDNA for the human α5 integrin subunit (Argraves et al., 1986) subcloned into the EcoRI site of pGEM1, and was kindly provided by Dr E. Ruoslahti (Cancer Research Centre, La Jolla, California). pBSβ1 contains a partial 842bp cDNA for the human β1 integrin subunit (positions 1418-2260; Argraves et al., 1987) subcloned into the SmaI site of pBluescript after blunt-ending, and was kindly provided by Dr P. Cervella (University of Torino, Torino, Italy). pSP64βa is a partial 600-bp cDNA from the 3′-untranslated region of human β-actin (Ponte et al., 1983) subcloned into pSP64, and was kindly provided by Dr G. Gabbiani (Department of Pathology, University Medical Center, Geneva). pGEM1-P7, pBSβ1 and pSP64βa were linearised respectively with NcoI, EcoRI and EcoRI, and were used as templates for bacteriophage SP6 (pGEM1-P7, pSP64βa) and T3 (pBSβ1) RNA polymerases. Transcription was performed exactly as described (Pepper et al., 1990).

A

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RNA extraction and northern blot hybridisation Total cellular RNA was extracted from multiple-wounded or cytokinetreated endothelial cell monolayers as described previously (Pepper et al., 1990). RNA was denatured with glyoxal, electrophoresed in a 1.2% agarose gel (5 µg RNA per lane), and transferred overnight onto nylon membranes. Filters were exposed to UV light and stained with Methylene Blue to reveal 18S and 28S rRNAs. Prehybridization, hybridization and post-hybridization washes were as previously described (Pepper et al., 1990). Filters were exposed to Kodak XOMAT XAR-5 Scientific Imaging Film (Eastman Kodak Co.), and autoradiograms were scanned and quantitated using ImageQuant (Molecular Dynamics).

RESULTS Effect of wound-induced migration on α5β1 integrin expression in BME cells BME cells migrate spontaneously in wounded monolayers, and cover the denuded region within 24 hours (Pepper et al., 1992b,c). Northern blots of total cellular RNA from multiplewounded monolayers were hybridized with probes for the α5 and β1 integrin subunits, and were re-hybridized with a probe for β-actin as an internal control (Fig. 1A). Densitometric analysis revealed a rapid increase in α5 mRNA with a maximal

B

Fig. 1. Kinetics of induction of α5 and β1 integrin subunits in multiple-wounded BME cell monolayers. (A) Northern blots of total cellular RNA (5 µg/lane) isolated from control and multiple-wounded monolayers were hybridized with 32P-labeled cRNA probes for α5 or β1 integrin subunits, and were subsequently rehybridized with a probe to β-actin. Positions of 28S and 18S ribosomal RNAs are indicated. (B) Quantitation based on densitometric scanning (Absorbance) of the experiment shown in A. Integrin subunit values have been normalized to β-actin, and are expressed relative to values at time 0.

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2.7-fold increase after 8 hours and a return to baseline levels after 24 hours (Fig. 1B). β1 mRNA levels were also increased, albeit with relatively delayed kinetics, reaching a maximum of 2.4-fold after 12 hours (Fig. 1B). A single representative experiment is shown in Fig. 1; this experiment has been repeated at least 3 times for each subunit, in which similar results were obtained. The increase in α5 and β1 subunit mRNAs was also observed in multiple-wounded BAE monolayers, and occurred irrespective of whether or not detached cells were removed and the medium was changed after wounding (data not shown). Since wounding induces migration and proliferation in cells lining the wound edge, we assessed whether the wound-induced induction of the α5 and β1 integrin subunits was proliferation-dependent. To this end, BME cell monolayers preincubated in hydroxyurea (5 mM) were maintained in hydroxyurea for 4 hours after multiple wounding, and total cellular RNA was analyzed as described above. We have previously shown that addition of hydroxyurea (5 mM) to low density cultures of BME cells inhibits [3H]thymidine incorporation by 81% (Pepper et al., 1992c). Under these conditions, the multiple-wound-induced increase in α5 and β1 mRNAs was unaffected (data not shown). Since cells migrating in a wounded monolayer are moving to a state of low density (Pepper et al., 1992b,c), these results suggested that the increase in α5β1 occurs independently of proliferation and is instead the consequence of a reduction in cell density. Indeed, we have observed that both α5 and β1 mRNA levels are increased in low density BME cell cultures (2.34- and 1.50-fold respectively, n=2 experiments) relative to levels seen at confluence. To examine the effect of wounding on α5β1 integrin protein biosynthesis, extracts of BME cells metabolically labeled for 6 or 12 hours after multiple wounding were immunoprecipitated with specific polyclonal antibodies to the α5 and β1 subunits (Fig. 2). Under non-reducing conditions, the anti-α5 antibody precipitates the α5 subunit (160 kDa) together with the associated β1 subunit (120 kDa), while the antibody to β1 precipitates the β1 subunit (120 kDa) as well as the associated alpha subunits, as previously described (Defilippi et al., 1991). The additional band of 100 kDa observed using the antibody to β1 represents the immature form of β1 (pre-β1; Defilippi et al., 1991). Fig. 2 demonstrates that α5 subunit biosynthesis was increased 3.6-fold in BME cells 6 hours after multiple wounding (mean of duplicate samples), as was the coprecipitated β1 subunit. Immunoprecipitation with β1 specific antibody revealed a modest 1.4-fold increase in β1 synthesis, as well as an increase in co-precipitated α chains. The increases in α5 and β1 were no longer apparent after 12 hours (Fig. 2). Effect of bFGF or TGF-β1 on α5β1 expression in BME cells Previous studies have demonstrated that bFGF and TGF-β1 increase α5 and β1 in endothelial cells (Basson et al., 1992; Enenstein et al., 1992; Klein et al., 1993). Thus bFGF increased synthesis of both subunits in human dermal and bovine adrenal cortex-derived microvascular endothelial cells (MECs and BCE cells), and both subunits were increased following exposure of MEC and BAE cells to TGF-β1. β1 subunit mRNA was increased by bFGF in MECs and by TGF-β1 in BAE cells. α5 subunit mRNA was increased by TGF-β1 in BAE cells but not by bFGF in MECs. To establish a baseline from which to explore possible interactions between these and other

Fig. 2. Effect of multiple wounding on the expression of α5 and β1 integrin subunits in BME cells. Confluent monolayers of BME cells were labeled with [35S]methionine for 6 or 12 hours after multiple wounding. C, control untreated cells; MW, multiple-wounded monolayers. Extracts were immunoprecipitated with polyclonal antibodies to α5 and β1 cytoplasmic domains. Radioactive antigens were separated by 6% SDS-PAGE under non-reducing conditions and were visualised by fluorography. The experiment was performed in duplicate for multiple-wounded monolayers.

cytokines, dose-response and kinetic experiments were performed to determine the effect of bFGF and TGFβ1 on α5β1 mRNA levels in our BME cells. In the dose-response experiments, BME cells were exposed to bFGF and TGF-β1 for 15 and 12 hours, respectively. In the kinetic experiments, BME cells were cultured in the presence of TGF-β1 (1 ng/ml) or bFGF (3 ng/ml) and total cellular RNA was extracted after 1, 4, 12 and 24 hours. The results of the kinetic experiments are shown in Fig. 3. TGF-β1 increased α5 mRNA levels to a maximum of 3.2-fold after 12 hours. With respect to dose, the maximal effect on α5 was seen with 3 ng/ml TGF-β1 (data not shown). TGF-β1 had little effect on the expression of β1 (Fig. 3). bFGF induced a modest increase in both α5 and β1 mRNA levels: maximal effects were seen after 12 hours and with 30 ng/ml (Fig. 3 and data not shown). We next determined the effects of bFGF and TGF-β1 on the biosynthesis of the α5 and β1 subunits following 15 hours of exposure to either cytokine alone. Immunoprecipitation with the two specific antibodies that recognize α5 or β1 revealed a marked increase in α5 biosynthesis in response to TGF-β1 (Fig. 4A), confirming the results obtained by northern blot analysis (Fig. 3A); the increase in the β1 subunit which coprecipitated with α5 most likely reflects recruitment of constitutively expressed β1 previously associated with other alpha subunits. A minor increase was observed in the biosynthesis of β1 in response to TGF-β1 and of α5 in response to bFGF, and no significant modification occurred in β1 in response to bFGF. In a dose-response experiment, BME cells were cultured in the presence of 0.5, 1, 2 and 5 ng/ml TGF-β1 for 15 hours. Immunoprecipitation with the α5-specific antibody showed that TGF-β1 induced biosynthesis of α5 in a dose-dependent manner, first detectable at 0.5 ng/ml and with a maximal 3.6-fold increase at 1ng/ml (Fig. 4B). These findings demonstrate that the most significant effect

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Fig. 3. Kinetics of α5 and β1 integrin subunit induction by TGF-β1 and bFGF in BME cells. Confluent monolayers of BME cells were treated with bFGF (3 ng/ml) or TGF-β1 (1 ng/ml) for the times indicated. Northern blots of total cellular RNA (5 µg/lane) were hybridized with 32P-labeled cRNA probes for α5 or β1 integrin subunits. Levels of α5 (left) and β1 mRNA (right) expression were determined by densitometric scanning of autoradiograms (Absorbance).

on BME cells is an increase in α5 protein and mRNA by TGFβ1. The modest increases in α5 and β1 mRNA levels observed in the presence of bFGF were not associated with a significant increase in synthesis of the corresponding proteins. Effect of co-added bFGF and TGF-β1 on α5β1 expression in BME cells Since it is likely that endothelial cells are exposed to multiple cytokines during angiogenesis, and since important interactions between bFGF and TGF-β1 have been described during angiogenesis in vitro (reviewed by Pepper, 1996, 1997b), we next assessed whether interactions might exist between bFGF and TGF-β1 with respect to α5β1 integrin expression. Combination treatments were chosen according to the results of the experiments described above. As shown in Fig. 5, when bFGF (10 ng/ml) and TGF-β1 (5 ng/ml) were coadded to BME cells, a synergistic increase was observed in α5 mRNA expression, i.e. levels obtained in the presence of both cytokines exceeded the predicted additive increase seen when bFGF and TGF-β1 were added separately. The synergistic induction of α5 by co-added bFGF and TGF-β1 was clearly evident after 4 hours and was maintained at 12 hours. Quantitation by densitometric scanning revealed that after 4 Fig. 4. Effect of TGF-β1 and bFGF on α5β1 integrin biosynthesis in BME cells. Confluent monolayers of BME cells were incubated in medium containing [35S]methionine for 15 hours. (A) During this time period, cells were treated with 1 ng/ml TGF-β1 (T) or 10 ng/ml bFGF (F). Extracts were immunoprecipitated with the anti-α5 and β1 cytoplasmic domain polyclonal antibodies. C, control untreated cells. (B) Cells were treated with TGF-β1 at 0.5 ng/ml (T 0.5), 1 ng/ml (T 1), 2 ng/ml (T 2) or 5 ng/ml (T 5), or with 10 ng/ml bFGF (F). C, control untreated cells. Extracts were immunoprecipitated with the anti-α5 cytoplasmic domain antibody. Radioactive antigens were separated by 6% SDS-PAGE under non-reducing conditions and were visualised by fluorography.

hours, α5 mRNA was increased 1.3-, 2.8- and 6.6-fold in response to bFGF, TGF-β1 or both cytokines, respectively. At the 12 hour time point, α5 mRNA was increased 1.7-, 4.6- and 8.1-fold in response to bFGF, TGF-β1 or both cytokines

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Fig. 6. Kinetics of the synergistic induction of α5 mRNA by bFGF and TGF-β1 in BME cells. Histograms represent levels of α5 and β1 integrin subunit expression obtained by densitometric scanning of autoradiograms (Absorbance). Cells were treated with bFGF (10 ng/ml) (F), TGF-β1 (0.5 ng/ml) (T), or a combination of both cytokines (F/T) for 4, 15 or 48 hours. Bars represent values expressed relative to controls at each time point.

Fig. 5. Synergistic induction of the α5 subunit mRNA by TGF-β1 and bFGF. Confluent monolayers of BME cells were incubated with 10 ng/ml bFGF (F), 5 ng/ml TGF-β1 (T), or with a combination of both cytokines (F/T) for 4 or 12 hours. (A) Northern blots of total cellular RNA (5 µg/lane) were hybridized with 32P-labeled cRNA probes for α5 or β1 integrin subunits. Positions of 28S and 18S ribosomal RNAs are indicated. (B) Levels of α5 mRNA expression were determined by densitometric scanning of autoradiograms (Absorbance). Pred, predicted additive increase in α5 mRNA when bFGF and TGF-β1 are added alone.

respectively. No significant modification in the level of β1 mRNA was detected at either time point, confirming the findings shown in Fig. 3A,B. Fig. 6 shows the quantitative results of a kinetic experiment in which cells were exposed to bFGF (10 ng/ml) and/or TGF-β1 at a tenfold lower concentration (0.5 ng/ml) than used in Fig. 5. The synergistic induction was once again evident, and was most marked after 4 hours, while after 48 hours levels of α5 and β1 mRNAs had returned to control values. To determine whether the synergistic effect of bFGF and TGFβ1 on α5 mRNA levels was mirrored at the level of total protein, extracts of BME cells metabolically labelled for 15 hours were

immunoprecipitated with the α5- and β1-specific polyclonal antibodies. As shown in Fig. 7A, co-addition of bFGF (10 ng/ml) further increased the synthesis of α5 seen in response to 1 ng/ml TGF-β1 alone. A quantitative analysis of results pooled from two separate experiments (Fig. 7B) revealed that the increase in α5 in response to co-added bFGF and TGF-β1 was slightly greater than the predicted additive increase of bFGF plus TGF-β1 when added alone, indicative of a modest synergistic effect. Although in the experiment shown in Fig. 7A both TGF-β1 and bFGF increased the level of the β1 subunit (compare with Fig. 4 in which no increase is seen with TGF-β1 and a minor increase is seen with bFGF), the synergistic effect on biosynthesis was restricted to α5. Synergism was greater at the level of mRNA (Figs 5, 6) that at the level of total protein (Fig. 7B). Since TGF-β1 induces a biphasic effect on bFGF-induced angiogenesis in vitro (Pepper et al., 1990, 1993), we further investigated the effect of the combination of low or high concentrations of TGF-β1 together with bFGF on α5 and β1 mRNA expression. BME cells were exposed 10 ng/ml bFGF together with 0.5 or 5 ng/ml TGF-β1 for 4 or 15 hours. bFGF as well as low and high concentrations of TGF-β1 induced a modest increase in the expression of α5 mRNA, while a greater than additive (synergistic) increase was observed when both cytokines were present (Figs 5, 6, 8). Synergism was observed at early (4 hours) and late (12-15 hours) time points; however, while the effect of 5 ng/ml TGF-β1 was sustained after 12-15 hours, the effect of 0.5 ng/ml had begun to wane at this time point (Figs 5, 6, 8). Significant changes in the level of β1 subunit mRNA were not observed under these conditions (Figs 5, 6, 8). Effect of LIF on α5β1 expression in BME cells We have previously demonstrated that leukaemia inhibitory factor (LIF) inhibits angiogenesis in vitro, and that LIF inhibits BAE but not BME cell migration in a two-dimensional woundinduced migration assay (Pepper et al., 1995). Here we have examined the effect on α5 and β1 integrin subunit expression

Endothelial cell α5β1 integrin

Fig. 7. Synergic induction of α5 and β1 protein synthesis by TGF-β1 and bFGF in BME cells. Confluent BME cell monolayers were treated with TGF-β1 (1 ng/ml) and bFGF (10 ng/ml) either alone (T, F) or in combination (T/F) for 15 hours and labeled with [35S]methionine during this time period. C, control untreated cells. (A) Extracts were immunoprecipitated with anti-α5 and -β1 cytoplasmic domain antibodies. Radioactive antigens were separated by 6% SDS-PAGE under non-reducing conditions and visualised by fluorography. Pre-β1, immature form of β1. (B) Histograms represent levels of α5 and β1 integrin subunit expression obtained by densitometric scanning (Absorbance). Bars represent values obtained from two experiments (±s.d) expressed relative to controls. Pred, predicted additive increase in α5 and β1 when bFGF and TGF-β1 are added alone.

in BAE and BME cells cultured for 15 hours in the presence of LIF. Fig. 9A shows that in BAE cells, the expression of α5 but not β1 mRNA is reduced in a dose-dependent fashion by 0.01-100 ng/ml LIF, with a maximal 47% reduction at 100 ng/ml. LIF did not affect α5 or β1 mRNA expression in BME cells (data not shown). To determine whether LIF alters the biosynthesis of the α5 integrin subunit, immunoprecipitation was performed on extracts of cells cultured for 15 hours in the presence of 1, 10 or 100 ng/ml LIF. LIF reduced α5 biosynthesis in BAE cells in a dose-dependent manner, with a maximal 64% reduction at 10 ng/ml (Fig. 9B). No changes in α5 protein levels were detected in BME cells (data not shown). DISCUSSION Endothelial cell migration and invasion are influenced by various factors including cytokines and cell-extracellular matrix interactions, the latter of which are mediated by

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Fig. 8. The synergistic induction of α5 mRNA is dose- and timedependent. Confluent monolayers of BME cells were incubated with bFGF (F) at 10 ng/ml, TGF-β1 (T) at 5 or 0.5 ng/ml, or a combination of both cytokines (F/T) for 4 or 15 hours. Northern blots of total cellular RNA (5 µg/lane) were hybridized with 32P-labelled α5 or β1 integrin subunit cRNA probes. Positions of 28S and 18S ribosomal RNAs are indicated. C, control untreated cells.

adhesion receptors including integrins. The extent to which cells migrate on a particular matrix depends on several variables including integrin levels, ligand levels and integrinligand binding affinities mediated by integrin ‘activation’ (Huttenlocher et al., 1996; Palecek et al., 1997). In this paper, we have examined one of these variables, namely the levels of integrin α5β1 expression. α5β1 integrin mediates adhesion of endothelial cells to fibronectin (Albelda et al., 1989; Cheng and Kramer, 1989) and fibrinogen (Suehiro et al., 1997), both of which are abundantly present in the endothelial pericellular matrix during angiogenesis. Although several in vitro studies have shown that cytokines modulate integrin expression in endothelial cells, almost no information is available on the effects of combinations of cytokines. This is important in light of the emerging concept that the activity of angiogenesisregulating cytokines is contextual, i.e. that angiogenesis is regulated by synergistic or biphasic effects induced by combinations of cytokines present in the endothelial pericellular environment (reviewed by Pepper et al., 1996). bFGF and TGF-β1 have been extensively studied with respect to their role in the regulation of angiogenesis. Although the role of bFGF in the endogenous regulation of angiogenesis remains to be clarified (reviewed by Pepper et al., 1996; Christofori, 1997), TGF-β and TGF-β receptor gene deletion studies in mice have clearly defined a role for this receptor-ligand pair in vessel wall maturation and stabilization (reviewed by Pepper, 1997b). VEGF is perhaps the most important cytokine discovered to date that is required for the endogenous regulation of angiogenesis (reviewed by Dvorak et al., 1995;

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Fig. 9. Down-regulation of α5 integrin subunit in BAE cells by LIF. Confluent monolayers of BAE cells were incubated for 15 hours with increasing concentrations of LIF. (A) Northern blots of total cellular RNA (5 µg/lane) were hybridized with 32P-labelled α5 or β1 integrin subunit cRNA probes. Positions of 28S and 18S ribosomal RNAs are indicated. (B) Cells were labeled with [35S]methionine, and extracts immunoprecipitated with an anti-α5 cytoplasmic domain antibody. Radioactive antigens were separated by 6% SDS-PAGE under nonreducing conditions and were visualised by fluorography.

Nicosia, 1998). However, since VEGF does not alter α5 or β1 integrin subunit expression either alone (Senger et al., 1996; Suzuma et al., 1998) or in combination with other cytokines such as bFGF or TGF-β1 (our own unpublished observations), we have focussed our attention on bFGF and TGF-β1. The data we present show (1) that migration of endothelial cells is associated with a significant increase in α5β1, both at the level of mRNA and total protein; (2) basal levels of α5β1 are minimally increased by either bFGF or TGF-β1 alone, with TGF-β1 being the more active, particularly with respect to α5. Co-addition of these two cytokines induces a dose- and timedependent synergistic increase in α5 with very little effect on β1. At a constant dose of bFGF, induction is greater with more prolonged kinetics at 5 ng/ml than at 500 pg/ml TGF-β1; (3) LIF decreases α5 but not β1 expression in BAE but not BME cells. Although it is well established that for many integrins adhesion is predominantly determined by modulation of integrin function rather than expression, changes in the level of α5β1 expression have nonetheless been shown to contribute to alterations in cell adhesion, migration and survival. For example, overexpression of α5β1 is associated with a reduction in migration and proliferation (Giancotti and Ruoslahti, 1990), and α5β1 supports survival of cells on fibronectin through upregulation of Bcl-2 (Zhang et al., 1995). These and other observations have led to the hypothesis that optimal adhesion to a fully organized extracellular matrix is required for cell migration, growth and survival, while weak or strong adhesion

to extracellular matrix has a negative effect on these cell functions (Giancotti and Mainiero, 1994; Frisch and Ruoslahti, 1997). In this paper, we report that a moderate increase in α5β1 occurs after multiple wounding or following treatment with bFGF, two conditions associated with the induction of endothelial cell proliferation and migration. These results suggest that upregulation of α5β1 is associated with the activation phase of angiogenesis. We have previously assessed the effect of a wide range of concentrations of TGF-β1 on bFGF-induced angiogenesis (Pepper et al., 1990, 1993) in a three-dimensional in vitro model (Montesano and Orci, 1985). bFGF-induced invasion was increased at 200-500 pg/ml TGF-β1 and decreased at 510 ng/ml TGF-β1. Similar findings in three-dimensional collagen gels have been reported by others (Gajdusek et al., 1993), and the biphasic effect of TGF-β1 is in accord with previous studies in which endothelial cell wound-induced migration (Heimark et al., 1986; Müller et al., 1987) and invasion of three-dimensional collagen gels (Müller et al., 1987) or the explanted amnion (Mignatti et al., 1989) were inhibited at relatively high concentrations of TGF-β1 (1-10 ng/ml), whereas 500 pg/ml TGF-β1 potentiated twodimensional wound-induced migration (Gajdusek et al., 1993; our own unpublished observations). We have also observed that lumen formation in the resulting tube-like structures is progressively reduced with increasing concentrations of TGFβ1. Thus in the absence of TGF-β1, bFGF-induced cell cords within fibrin gels contained widely patent lumina. Co-addition of TGF-β1 at 500 pg/ml reduced lumen diameter to a more physiological size (Pepper et al., 1993), whereas addition of TGF-β1 at 5 ng/ml completely inhibited lumen formation (Pepper et al., 1990). How do our present observations relate to the biphasic effects of TGF-β1 described above? Since, in the presence of bFGF, levels of α5β1 integrin expression are differentially affected at the potentiating (500 pg/ml) and inhibitory (5 ng/ml) concentrations of TGF-β1, we hypothesize that maximal invasion which occurs at the potentiating dose results from an optimal degree of α5β1-mediated cell adhesion. Submaximal invasion occurs when adhesion is either less than or greater than that achieved with the potentiating dose of TGFβ1, such as would occur with bFGF alone or with bFGF plus 5 ng/ml TGF-β1, respectively. In particular, high concentrations of TGF-β1, which markedly upregulate the expression of α5β1 in the presence of bFGF, are likely to contribute to the inhibitory effect exerted by TGF-β1 on migration and proliferation by increasing anchorage to the extracellular matrix. In addition, prolonged exposure to high concentration of TGF-β1 would contribute to vessel maturation which occurs during the phase of resolution. Differential regulation of integrin expression may also contribute to the alterations in lumen size in fibrin gels seen with different concentrations of TGF-β1. We have previously reported that LIF inhibits BME and BAE cell in vitro angiogenesis (Pepper et al., 1995). Surprisingly, although LIF inhibited proliferation in both cell lines, migration was only inhibited in BAE cells. In the present report we demonstrate that LIF decreases α5 (but not β1) subunit expression in BAE but not in BME cells. This finding correlates well with the cell-type specificity of LIF on migration. With respect to angiogenesis in vitro, this is a

Endothelial cell α5β1 integrin complex process which requires endothelial cell proliferation and matrix degradation in addition to migration. In contrast to migration, which is only reduced in BAE cells by LIF, proliferation and extracellular proteolysis are inhibited in both BME and BAE cells by this cytokine, which is likely to contribute to the inhibitory effect of LIF on in vitro angiogenesis in these cell lines. Interestingly, we have also observed that retinoic acid (10−5 and 10−6 M) decreases basal and bFGF-stimulated α5 but not β1 mRNA levels in BME cells (our unpublished observations). This correlates with the inhibitory effect of retinoic acid on BME cell migration and in vitro angiogenesis (Pepper et al., 1994). For both LIF and retinoic acid, a tight correlation therefore exists between reduction in the levels of α5β1 integrin expression and inhibition of endothelial cell migration. In conclusion, a moderate increase in microvascular endothelial cell α5β1 integrin is associated with a migratory phenotype or occurs following exposure to bFGF, which correlates with the activation phase of angiogenesis. Levels of α5β1 are further increased by co-incubation with bFGF and low doses of TGF-β1. Under these conditions, endothelial cell migration and capillary-like tube formation are optimal. The highest levels of α5β1 are seen when bFGF is co-added with high concentrations of TGF-β1. Since TGF-β1 at these levels is known to exert an inhibitory effect on endothelial cell migration, we propose that high levels of α5β1 integrin on the endothelial cell surface are associated with arrest of bFGFinduced cell migration and proliferation, and that this contributes to the resolution phase of angiogenesis. We would like to thank Dr Roberto Montesano and Stefano Mandriota for critically reading the manuscript, and Roberto Montesano for his continuing support. We would also like to thank Drs M. Furie and S. Silverstein for the BME cells, Dr G. Tarone for antibodies to the α5 and β1 integrin subunits, Drs E. Ruoslahti and P. Cervella for cDNAs for the α5 and β1 subunits, respectively, Dr G. Gabbiani for the β-actin cDNA and Dr P. Sarmientos for recombinant human bFGF. Technical assistance was provided by C. Di Sanza, M. Guisolan-Vallotton and M. Quayzin, and photographic work was done by B. Favri and P.-A. Ruttimann. This work was supported by a grant from the Swiss National Science Foundation (no. 3100-043364.95).

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