Journal of Cell Science 108, 797-809 (1995) 797 Printed in Great Britain © The Company of Biologists Limited 1995

Evidence that and thrombospondin-1 modulate sprouting of endothelial cells

Ann E. Canfield* and Ana M. Schor† CRC Department of Medical Oncology, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 9BX, UK *Present address: University of Manchester, School of Biological Sciences, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT, UK †Author for correspondence at present address: Department of Dental Surgery and Periodontology, The Dental School, University of Dundee, Park Place, Dundee, DD1 4HR, UK

SUMMARY

Cultured endothelial cells undergo a reversible transition matrices was then analysed. Inhibitory matrices contained from a resting (cobblestone) phenotype to an angiogenic reduced levels of tenascin and increased levels of throm- (sprouting) phenotype. This transition mimics the early bospondin-1 by comparison to the permissive matrices. In events of angiogenesis. We have previously reported that contrast, no differences were detected in the relative levels the addition of exogenous xylosides inhibits endothelial cell of . The roles of tenascin and thrombospondin-1 in sprouting and modifies the (ECM) endothelial sprouting were confirmed using specific anti- synthesised by the cells. We have now investigated whether bodies. Immunolocalisation studies revealed the presence endothelial sprouting is mediated by the nature of the of both in sprouting cells. Antibodies to tenascin extracellular matrix in contact with the cells. Accordingly, inhibited the formation of sprouting cells on permissive cell-free matrices deposited by bovine aortic endothelial matrices and on -coated dishes without affecting cell cells (BAEC) were isolated. These matrices were produced growth. Tenascin synthesis was increased when sprouting under conditions in which the formation of the sprouting cells were present in the cultures. Antibodies to throm- phenotype was permitted (controls) or inhibited (by the bospondin-1 stimulated sprouting on inhibitory matrices. addition of exogenous xylosides). BAEC were then plated These results suggest that the transition from a resting to on these matrices and grown under conditions which a sprouting phenotype is promoted by tenascin and promote sprouting. Sprouting proceeded normally on inhibited by thrombospondin-1. control matrices, whereas it was inhibited when the cells were grown on matrices deposited in the presence of Key words: endothelial cell, angiogenesis, laminin, thrombospondin-1, xylosides. The composition of the permissive and inhibitory tenascin

INTRODUCTION other matrix proteins and to the cell surface (for reviews see Frazier, 1991; Adams and Lawler, 1993). The role played by Matrix macromolecules are able to modulate cell adhesion, cell thrombospondin-1 in the regulation of angiogenesis is not shape and migration as well as the response of cells to clear. It has previously been shown that this may be cytokines. The extracellular matrix has therefore been impli- inhibitory for angiogenesis both in vivo and in vitro (Good et cated in the regulation of many developmental and morpho- al., 1990; Iruela-Arispe et al., 1991; Tolsma et al., 1993). In genetic processes in vivo (Bissell and Barcellos-Hoff, 1987; apparent contrast to these findings, a recent study by Nicosia Hay, 1991; Nathan and Sporn, 1991; Adams and Watt, 1993). and Tusznyski (1994) has suggested that matrix-bound throm- The role played by the extracellular matrix in the formation of bospondin-1 may indirectly promote angiogenesis in vitro. new blood vessels (angiogenesis) has previously been Furthermore, Ben Ezra et al. (1993) have shown that TSP-1 examined using either complex matrices or purified single enhanced the induction of angiogenesis by either bFGF or macromolecules (Madri and Williams, 1983; Montesano et al., bacterial endotoxin lipopolysaccharide in the rabbit cornea 1983; Schor et al., 1983; Ingber and Folkman, 1989a; Grant et assay. al., 1989; Vernon et al., 1992; Tolsma et al., 1993). In this Laminin is a large (Mr 800,000), multidomain, basement regard, thrombospondin-1 and laminin have received particu- membrane-specific glycoprotein (see recent review by Tryg- lar attention; tenascin has been implicated in morphogenetic gvason, 1993). A role for laminin in angiogenesis has been events in vivo, but its possible role in angiogenesis has not demonstrated in several in vivo and in vitro studies using intact been studied. laminin isolated from the EHS tumour and specific peptides The glycoprotein thrombospondin-1 (Mr 420,000) is a mul- derived from the A and B1 chains of this molecule (Grant et tifunctional extracellular matrix component which can bind to al., 1989, 1992; Sakamoto et al., 1991).

798 A. E. Canfield and A. M. Schor

Tenascin is a hexameric glycoprotein with a molecular mass Dr J. Lawler, Brigham and Women’s Hospital, Harvard Medical of more than one million daltons. This protein is particularly School, Boston, USA. Mouse anti-human platelet thrombospondin-1 associated with morphogenetic events during development (for (clone TSP-B7) and rabbit anti-EHS laminin were purchased from reviews see Erickson and Bourdon, 1989; Chiquet, 1992). In Sigma Chemical Co, Poole, UK. Peroxidase and FITC-conjugated the adult, tenascin expression is enhanced in tissues undergo- anti-rabbit and anti-mouse IgG were obtained from Dako Ltd, High ing angiogenesis (Mackie et al., 1988; Chiquet-Ehrismann et Wycombe, UK. al., 1986; Whitby et al., 1991). Cell culture In a mature blood vessel, the ‘resting’ endothelial cells Cells, culture media and substrata lining the lumen are attached to a complex extracellular matrix Bovine aortic endothelial cells were isolated as previously described (or ) which they are believed to have syn- (Schor et al., 1983). Cells were identified as endothelial on the basis thesised. The migration of luminal endothelial cells through the of their morphology and positive staining for Factor VIII-related vessel basement membrane into the underlying extracellular antigen and negative staining for α-smooth muscle actin. Stock matrix (where they adopt an elongated ‘sprouting’ cell mor- cultures were routinely maintained on gelatin-coated dishes in Eagle’s phology) is an early event of angiogenesis. We have developed minimum essential medium (MEM) supplemented with 20% donor an in vitro model system in which cultured endothelial cells calf serum, 50 µg ml−1 ascorbic acid, 2 mM glutamine, 1 mM sodium pyruvate, non-essential amino acids, penicillin (100 units ml−1) and are induced to change from a resting phenotype (resembling − cells in a mature vessel) to a sprouting or angiogenic phenotype streptomycin (0.1 mg ml 1). This growth medium will be referred to (resembling cells in a newly forming vessel) in a controlled as 20% DCS-MEM. Cultures were incubated at 37°C in a humidified manner (Schor et al., 1983; Schor and Schor, 1988). Previous atmosphere consisting of 5% CO2, 95% air. These studies were performed with one endothelial cell line (U10) used between passages studies have shown that cultured endothelial cells are able to 10 and 21. These cells were free from mycoplasmal contamination, synthesise many of the matrix macromolecules which have as assessed by fluorescent Hoescht 33256 stain (Schor and Schor, been identified in the vascular wall in vivo, including: throm- 1988). bospondin-1, laminin and tenascin (Canfield et al., 1986; Sage, Xylosides were prepared as described previously (Schor and Schor, 1986; Schor et al., 1991). 1988). Two xylosides were used: (a) p-nitrophenyl-beta-D-xylopyra- The purpose of this study was to investigate the role of noside (β-xyloside) and (b) p-nitrophenyl-alpha-D-xylopyranoside complex matrices on endothelial sprouting. To this effect we (α-xyloside). They were dissolved in dimethyl sulfoxide (DMSO) at have used matrices deposited by endothelial cells themselves a concentration of 500 mM and diluted in growth medium to the (Schor et al., 1984). The composition of matrices that were desired final concentration (either 1.5 mM or 2 mM). DMSO was found to exert differential effects on endothelial sprouting was added to the controls to give a final concentration equivalent to that in the xyloside-containing cultures (0.4%, v/v, DMSO). then analysed. Data are presented showing that the formation Gelatin-coated dishes were prepared by incubating plastic tissue of the sprouting phenotype can be inhibited by culturing endo- culture dishes with a gelatin solution (0.1%, v/v, in distilled water) thelial cells on modified matrices. We report that these for 1 hour at room temperature. The gelatin was removed and the modified (i.e. inhibitory) matrices differ from control (i.e. per- dishes washed twice with Hanks’ balanced salt solution. Native type missive) matrices in the relative levels of thrombospondin-1 I (isolated from rat tail tendons) was made into three-dimen- and tenascin. The involvement of these proteins in endothelial sional (3-D) gels as previously described (Schor et al., 1983). sprouting and morphogenesis was then confirmed in functional Cell numbers were determined using a Coulter counter as previ- assays using specific antibodies. Our results suggest a possible ously described (Schor et al., 1983). antagonistic role for tenascin and thrombospondin-1 in the reg- Preparation of cell-free matrices ulation of angiogenesis. Endothelial cells were plated on the surface of 3-D gels of native at a density of between 8×104 and 1.5×105 cells/8 cm2 dish. After 24 hours, the medium was changed to either control medium MATERIALS AND METHODS (containing 0.4%, v/v, DMSO) or medium containing α- or β- xylosides (either 1.5 mM or 2 mM). In some experiments, control Materials cultures without DMSO were also included. DMSO had no effect on Culture medium, donor calf serum, sodium pyruvate, glutamine, non- any of the parameters examined. The medium was changed every 2 essential amino acids, antibiotics and prestained protein standards to 3 days until the cells were (a) confluent (day 7) and (b) post- were obtained from Gibco BRL, Paisley, Scotland, UK. Ascorbic acid confluent (day 21). Cell-free matrices were then prepared by incu- was obtained from BDH Chemicals Ltd, Poole, UK. Xylosides (p- bating the cultures with 1 ml of 0.2 M ammonia for 1 hour at room nitrophenyl-alpha-D-xylopyranoside and p-nitrophenyl-beta-D- temperature (Schor et al., 1984). The matrices were washed exten- xylopyranoside), were purchased from NBS Biologicals, Hatfield, sively before use (at least 15 times with sterile distilled water over a UK. Bovine serum albumin (fraction V) was obtained from Sigma 24-48 hour period and then 5 times with growth medium). Chemical Co., Poole, UK. Highly purified bacterial collagenase (form In some experiments, the following control matrices were also III) was obtained from Advanced Biofactures Corporation, New York, included: (a) confluent cell-free matrices incubated for a further 14 USA. days in either control medium or medium containing α- or β- The enhanced chemiluminescence kit and Hyperfilm-ECL for xylosides, and (b) plain collagen gels incubated with and without developing immunoblots were purchased from Amersham PLC, xylosides for a total of 7 days or 21 days. During these incubations, Amersham, UK. Rabbit anti-chick tenascin (Tn-2) was generously the medium was changed every 2-3 days. The confluent matrices and provided by Dr R. Chiquet-Ehrismann, Friedrich Meischer Institute, the collagen gels were incubated with 0.2 M ammonia for 1 hour and Basel, Switzerland. Rabbit anti-human tenascin polyclonal antiserum then washed as described above. was purchased from Chemicon International, Temecula, CA 92590, USA. Two monoclonal antibodies raised against human platelet Experimental protocol thrombospondin-1 (MA-II and MA-IV) were generously provided by Endothelial cells were plated at a density of between 8×104 and

Endothelial sprouting 799

1.5×105 cells/8 cm2 dish on three types of substrata: (a) gelatin-coated protein deposited into the cell layer/matrix (Canfield et al., 1994). dishes, (b) on the surface of 3-D gels of native type I collagen, and Samples obtained from cells cultured on collagen gels were digested (c) on cell-free matrices. The cells were allowed to attach for 24 hours with highly purified bacterial collagenase (Canfield et al., 1986) in 20% DCS-MEM. Cells plated on gelatin-coated dishes or on before subsequent analyses. The protein content of the cell collagen gels were then incubated in either control medium (contain- layer/matrix was determined using Coomassie protein assay reagent ing 0.4%, v/v, DMSO) or medium containing α- or β-xylosides (either from Pierce (Rockford, Illinois, USA). 1.5 mM or 2 mM) for up to 21 days. These cultures were used for the preparation of cell-free matrices (as described above) and for the Characterisation of proteins by immunoblotting isolation and characterisation of newly synthesised proteins (see Proteins extracted from the cell layer/matrix (see above) were below) at two points: (a) when confluent (5-7 days after plating), and separated by electrophoresis under reducing conditions on 6.5% poly- (b) when post-confluent (19-21 days after plating). acrylamide slab gels and transferred to nitrocellulose membranes by Cells plated on newly isolated cell-free matrices were maintained semi-dry blotting (25 V for 1 hour, 25°C). An equal amount of protein in 20% DCS-MEM for up to 10 days, and the formation of the (5 µg) was loaded onto each track to permit comparison of the relative sprouting phenotype was monitored. The number of sprouting cells levels of specific proteins. Prestained Mr standards, i.e. myosin (Mr was quantified by determining the percentage of fields within a dish 200,000), phosphorylase b (Mr 97,400), bovine serum albumin (Mr containing networks of interconnecting sprouting cells. A network 68,000), ovalbumin (Mr 43,000) and carbonic anhydrase (Mr 29,000) was defined as a group of 3 or more sprouting cells self-associating were electrophoresed at the same time. Remaining active sites on the in a head-to-tail fashion. Approximately 50 fields were counted on nitrocellulose membranes were blocked by incubation either for 16 each dish. Student’s t-test was used to determine whether the differ- hours at 4°C or 1 hour at room temperature in phosphate buffered ences between control and test samples were significant. saline (PBS) containing bovine serum albumin (BSA) (5%, w/v) and In some experiments, matrices were incubated in 20% DCS-MEM Tween-20 (0.05%, v/v) and the membranes were washed (3 times for containing specific antibodies (added at a final v/v ratio of 1:50). at least 10 minutes) in PBS, Tween-20 (0.05%, v/v). The membranes These antibodies were added 24 hours before the cells were plated were then incubated for 2 hours at room temperature with either (A) and then every time the medium was changed. The antibodies used polyclonal antiserum raised against chick tenascin (Tn-2; 1:100), (B) were (a) polyclonal anti-tenascin serum (Chemicon), and (b) a polyclonal antiserum raised against EHS mouse laminin (1:100), (C) cocktail of monoclonal antibodies against thrombospondin-1 (MA-II, monoclonal antiserum raised against human platelet thrombospondin- MA-IV and TSP-B7). Normal rabbit serum was used as a control. The 1 (MA-IV; 1:1000) or (D) normal rabbit serum (1:100). Sera were polyclonal antibody raised against human tenascin was specific for diluted (as indicated in brackets) in PBS containing BSA (0.1%, w/v) tenascin and showed no reactivity against other plasma proteins and 10 mM sodium azide. The antisera used have previously been (Bourdon et al., 1983; Chemicon Int., personal communication). The shown to be specific for their respective antigens (Lawler et al., 1985; monoclonal antibodies (MA-II, MA-IV and TSP-B7) were raised Chiquet-Ehrismann et al., 1986). The membranes were then washed against human platelet thrombospondin-1 and were specific for this (3 times for 20 minutes) in PBS, Tween-20 (0.05%, v/v) before incu- protein (Lawler et al., 1985; Dardik and Lahav, 1991). In other exper- bation for 90 minutes at room temperature with peroxidase-conju- iments, cells plated on gelatin-coated dishes were incubated in the gated anti-rabbit IgG diluted 1:1000 in this buffer. After this time, the continuous presence of polyclonal anti-tenascin serum. The antiserum membranes were washed extensively in PBS, Tween-20 (0.05%, v/v) was added when the cells were sparse (2 experiments) or at conflu- for 1 hour and the immunoreactive proteins were detected by ence (2 experiments) at a final v/v ratio of between 1:50 and 1:1000. enhanced chemiluminescence. The films were then scanned using a At least duplicate dishes were used in every experiment. laser densitometer. This procedure enabled the relative levels of each protein in the different matrices to be determined. Samples from every Localisation of matrix macromolecules by indirect experiment were analysed at least twice by this procedure. immunofluorescence Confluent and post-confluent cultures of endothelial cells grown in control medium were fixed, permeabilised and stained by indirect RESULTS immunofluorescence according to standard techniques (Schor et al., 1991). The polyclonal antibody to tenascin (Tn-2) and normal rabbit Cell-free matrices produced by cells cultured in the serum were diluted 1:50 (v/v) in phosphate buffered saline contain- presence of xylosides can inhibit endothelial ing 0.5% (w/v) bovine serum albumin and incubated for 2 hours at sprouting room temperature. The monoclonal antibodies to thrombospondin-1 (MA-IV and TSP-B7) and normal mouse serum were used at a 1:50 We have previously shown that endothelial cells can display (v/v) dilution (as above) and incubated for 1 hour at room tempera- two distinct phenotypes in culture, cobblestone and sprouting ture. The second antiserum (fluorescein isothiocyanate conjugated) (Schor et al., 1983). The cobblestone phenotype is formed by was applied for 1 hour at room temperature. Photographs were taken confluent cells cultured on a 2-D substratum. If the cells are with an automatic camera; the exposure time was generally between maintained to post-confluence in the presence of growth 30 and 90 seconds, negative controls were stopped manually after 5 medium containing serum the sprouting phenotype appears as minutes exposure. a second layer of elongated (sprouting) cells underneath the Isolation and characterisation of proteins deposited into cobblestone monolayer. The formation of the sprouting the cell layer phenotype can be inhibited by culturing endothelial cells in the Isolation presence of exogenous xylosides under conditions in which cell adhesion, proliferation and migration are not affected Proteins present in the cell layer/matrix of confluent and post- α β confluent endothelial cells cultured in the presence or absence of (Schor and Schor, 1988). Both - and -xylosides also xylosides were extracted with 4 M guanidinium chloride/50 mM Tris- modulate matrix deposition by endothelial cells (Schor and HCl, pH 7.4, for 24 hours at 4°C. Guanidinium chloride-insoluble Schor, 1988; Canfield et al., 1994). Thus, β-xylosides but not material was removed by centrifugation (15,000 g for 20 minutes at α-xylosides, modulate the synthesis of proteoglycans and gly- 4°C) and the supernatant dialysed extensively against 0.5 M acetic cosaminoglycans by these cells. In addition, we have shown acid at 4°C. This procedure extracted between 90 and 95% of the total that both of these compounds inhibit total protein synthesis 800 A. E. Canfield and A. M. Schor and, interestingly, both compounds increase the relative levels of thrombospondin-1 secreted into the medium by these cells (Canfield et al., 1994). Based on these results, and in view of the possible role of thrombospondin-1 in angiogenesis, we have now investigated whether the nature of the insoluble matrix in contact with the cells can influence endothelial cell sprouting. To this effect, we have isolated matrices deposited by endothelial cells cultured in the presence and absence of xylosides. Accordingly, endothelial cells were plated at sub- confluent densities on the surface of 3-D collagen gels and grown for up to 21 days in the presence and absence of xylosides. At confluence (day 5-7 post-plating) all the cells displayed a compact, contact-inhibited monolayer of cobble- stone appearance. At post-confluence (day 19-21 post-plating) the control cultures contained a mixture of cobblestone and sprouting cells, whereas cultures incubated with xylosides (either α- or β-) contained cells displaying the cobblestone morphology only (Schor and Schor, 1988). Cell-free matrices were prepared from confluent and post-confluent cultures by hydrolysing the cells with ammonia as described in the Materials and Methods. Four types of matrices were therefore isolated in these studies, these will be referred to as: (a) control, confluent (control-c), (b) control, post-confluent (control-pc), (c) xyloside-modified, confluent (xyl-c), and (d) xyloside- modified, post-confluent (xyl-pc). As identical results were obtained using matrices deposited by cells cultured in the presence of α- and β-xylosides, the term ‘xyloside’ will be used to refer to either of these compounds. These matrices were then used as substrata to plate fresh endothelial cells. Cells were plated on these matrices at a density of between 8×104 and 1.5×105 cells/8 cm2 dish and grown in control medium for up to 10 days. The cells proliferated at the same rate, reached the same saturation density (approx. 1×106 cells/8 cm2 dish by day 5) and displayed an identical cobblestone morphology at confluence on all the matrices tested (not shown). However, when these cells were maintained to post-confluence, marked differences were observed depending on the nature of the matrix used as a substratum (Fig. 1). Cells plated on matrices isolated from confluent cultures incubated in the absence and presence of xylosides (i.e. control-c and xyl-c) and post- confluent cultures in control medium (i.e. control-pc) proceeded to form extensive networks of sprouting cells under- neath the cobblestone monolayer (Fig. 1A). These matrices Fig. 1. Morphology of endothelial cells grown on matrices deposited were therefore permissive for sprouting. In marked contrast, by cells in the presence and absence of xylosides. Cell-free matrices were prepared from post-confluent cultures as described in Materials cells plated on matrices deposited by post-confluent cells and Methods. Fresh endothelial cells were plated on the matrices cultured in the presence of xylosides (i.e. xyl-pc) remained (1×105 cells/8 cm2 dish) and maintained in control medium for 10 confluent and viable, but sprouting was markedly inhibited days. (A) Cells grown on control-pc matrices (i.e. deposited by post- (Fig. 1B and C). These matrices were therefore inhibitory for confluent cells in control medium) displayed both cobblestone and sprout formation. The number of sprouting cells formed on the sprouting morphologies. (B and C) The number of sprouting cells inhibitory matrices varied from experiment to experiment. In was markedly reduced when cells were grown on xyl-pc matrices some experiments no sprouting cells were seen (as in Fig. 1B (i.e. deposited by post-confluent cells in the presence of α-xyloside and C); in other experiments sprouting cell formation was (B) or β-xyloside (C)). Bar, 150 µm. reduced to approximately 50% of that seen on permissive matrices (see Table 2). These results suggest that in the presence of growth medium (which is permissive for two types of permissive matrices (xyl-c and control-c) as well sprouting), endothelial sprouting can be inhibited by the extra- as plain collagen gels were incubated with medium containing cellular matrix in contact with the cells. xylosides for up to 21 days, changing the medium every 2-3 Since the inhibitory matrices were prepared by incubating days during this period. The matrices were then washed exten- endothelial cultures with xylosides for a total of 21 days, we sively and cells were plated on these matrices and grown for investigated the possibility that xylosides were bound either to 10 days in control medium. The cells proliferated and sprouted these matrices or to the underlying collagen gels. To this effect, equally well on all the matrices tested (results not shown). Endothelial sprouting 801

These results demonstrate that the inhibitory nature of xyl-pc Table 1. Protein contents of permissive and inhibitory matrices was not due to the non-specific binding of xylosides matrices or serum components in long-term culture, and suggest that Protein content these inhibitory matrices must be different in some way from Effect on of cl/matrix the permissive matrices. Matrix sprouting (µg/dish) Control-c Permissive 290±14 Differences in the composition of matrices which α-xyl-c Permissive 255±20 are permissive and inhibitory for endothelial β-xyl-c Permissive 228±19 sprouting Control-pc Permissive 604±40 α-xyl-pc Inhibitory 232±10 Several factors are known to influence morphogenesis in vitro, β-xyl-pc Inhibitory 262±1 including the thickness of the matrix on which cells are cultured and the composition of that matrix (Vernon et al., Endothelial cells were plated on gelatin-coated dishes (9×104 cells/8 cm2 1992). We have therefore investigated the composition of dish) and allowed to attach for 24 hours. At this time the medium was changed to either control medium or medium containing 2 mM α- or β-xylosides. matrices which were permissive and inhibitory for endothelial Cultures were incubated until confluence (c; day 5) or post-confluence (pc; day sprouting. Firstly, the amount of protein present in the cell 21), changing the medium every 2-3 days during this period. The proteins layer/matrices of these cultures was compared. These values deposited into the cell layer/matrix were collected and assayed as described in were taken as an estimate of the apparent ‘thickness’ of the Materials and Methods. The results shown are from a typical experiment, and matrices, a higher protein content equating with a thicker represent the mean (± standard deviation) of duplicate dishes. matrix (Schor et al., 1984). Secondly, the presence of three 7) are shown in Fig. 2; the results in Fig. 3 are from cell specific matrix components which have been implicated in layer/matrices extracted at post-confluence (day 21). angiogenesis (thrombospondin-1, tenascin and laminin; see Matrices deposited by confluent endothelial cells were found Introduction) was determined in these cultures. to contain thrombospondin-1, tenascin and laminin (Fig. 2). To measure total protein synthesis, endothelial cells were The monoclonal antibody (MA-IV) raised against human plated on either gelatin-coated dishes or on 3-D collagen gels platelet thrombospondin-1 detected a single immunoreactive and incubated with and without xylosides until (a) confluence band of apparent M 180,000 (Fig. 2A). The polyclonal and (b) post-confluence. The proteins present in the cell r antibody (Tn-2) raised against chick tenascin (Chiquet- layer/matrix were then extracted as described in Materials Ehrismann et al., 1986) detected a single immunoreactive band and Methods. At confluence, the cultures contained between of apparent M approx. 220,000 (Fig. 2B). The polyclonal 220 and 290 µg protein/8 cm2 dish (see Table 1). When the r antibody raised against EHS laminin detected a single cells were maintained to post-confluence in control medium immunoreactive band of apparent M approx. 200,000, indi- the amount of protein in the cell layer/matrix increased r cating that endothelial cells secrete either the B1 or B2 chain approximately two-fold. In contrast, there was either no of laminin (Fig. 2C). A protein corresponding to the A chain change or a small increase (approx. 10%) in the protein of laminin was never detected in these cultures. The presence content of cultures maintained to post-confluence in the of xylosides in the cultures did not alter the electrophoretic presence of xylosides (Table 1). These data agree with our mobilities of the proteins analysed. Furthermore, scanning the earlier report showing that xylosides inhibit protein synthesis tracks using a laser densitometer revealed that xylosides had by endothelial cells (Canfield et al., 1994). No correlation no apparent effect on the relative levels of any of these proteins was found between the total protein content of a matrix and at confluence (Fig. 2). Interestingly, all of these matrices were its ability to either permit or inhibit sprouting (Table 1). permissive for sprouting. These results suggest that the total amount of protein in a The cell layer/matrices deposited by post-confluent endo- matrix or the thickness of this matrix are not critical factors thelial cells grown in control medium (i.e. control-pc; permis- in determining whether this matrix will be permissive or sive) and in the presence of xylosides (i.e. xyl-pc; inhibitory) inhibitory for sprouting. were then examined. Marked differences were observed in the The macromolecular composition of the cell layer/matrices relative levels of thrombospondin-1 and tenascin in the per- was investigated by immunoblotting as described in Materials missive and inhibitory matrices (Fig. 3). Inhibitory matrices and Methods. An equal amount of protein (5 µg) was applied were found to contain between 2- and 10-fold more throm- to each track of the polyacrylamide gel in order to determine bospondin-1 relative to other proteins than permissive matrices whether the overall composition of these matrices was altered. (Fig. 3A). This increase was consistently more marked in α- Staining of the nitrocellulose membranes with protogold xyloside treated cultures than β-xyloside treated cultures (Fig. (BioCell Research Laboratories, Cardiff, UK) revealed that the 3A, compare tracks 2 and 3). By comparison, inhibitory matrices contained a large number of proteins, but specific matrices contained between 1.5 and 13 times less tenascin than changes were not apparent (not shown). The membranes were permissive matrices (Fig. 3B). In this case, the decrease was then probed with antibodies to thrombospondin-1, tenascin and consistently more marked in the presence of β-xylosides than laminin. Normal rabbit serum was used as a control. Similar α-xylosides (Fig. 3B, compare tracks 2 and 3). Xylosides were results were obtained for cells plated on both gelatin and found to have no apparent effect on the relative levels of collagen substrata. Confluent and post-confluent matrices were laminin at post-confluence (Fig. 3C, compare tracks 1-3). We compared on the same blot for every antibody used. As matrix have previously reported that xylosides inhibit fibronectin biosynthesis is known to vary with cell density and time in synthesis by endothelial cells (Canfield et al., 1994). Accord- culture (Canfield et al., 1990a), we have compared those ingly, we have confirmed that the relative levels of fibronectin matrices which have been deposited by cells maintained for the were reduced in the inhibitory matrices by comparison with the same length of time. The results obtained at confluence (day 802 A. E. Canfield and A. M. Schor permissive matrices. This reduction was consistently less comparison the levels of laminin did not change. These data marked than that observed for tenascin (results not shown). suggest that whether a matrix is permissive or inhibitory for The presence of xylosides in the cultures had no effect on the sprouting may be determined by either: (i) the absolute levels electrophoretic mobilities of tenascin, thrombospondin-1, of a specific protein (e.g. thrombospondin-1), and/or (ii) the laminin or fibronectin extracted from the cell layer/matrices. relative levels of two or more proteins in that matrix (e.g. Taken together, the results presented above demonstrate that tenascin and thrombospondin-1). Of the four molecules inves- inhibitory matrices contained elevated levels of throm- tigated, thrombospondin-1, laminin and fibronectin have pre- bospondin-1 compared with permissive matrices (compare viously been reported to modulate angiogenesis in vivo and Figs 2A and 3A). The levels of tenascin in inhibitory matrices endothelial sprouting in vitro (Grant et al., 1989, 1992; Ingber were either similar (compare Figs 2B and 3B) or reduced (Fig. and Folkman, 1989a; Good et al., 1990; Iruela-Arispe et al., 3B, compare tracks 1-3) in comparison to those in permissive 1991; Tolsma et al., 1993; Ben Ezra et al., 1993; Nicosia et al., matrices. was also decreased in the inhibitory 1993; Nicosia and Tusznyski, 1994). Our results indicate that matrices, although to a much lesser extent than tenascin. By tenascin may also be involved in these processes. Thrombospondin-1 has been reported to both inhibit and promote angiogenesis in vivo (Tolsma et al., 1993; Ben Ezra et al., 1993) and endothelial sprouting in vitro (Iruela-Arispe et al., 1991; Nicosia and Tusznyski, 1994). We and others have previously shown that sprouting cells synthesise markedly less thrombospondin-1 than cobblestone cells (Canfield et al., 1990a; Iruela-Arispe et al., 1991). In contrast, data presented in Fig. 2B (track 1) and Fig. 3B (track 1) show that tenascin levels are increased significantly when sprouting cells are

Fig. 2. The identification of thrombospondin-1, tenascin and laminin in the cell layer/matrix of confluent endothelial cells. Endothelial cells were plated on gelatin-coated dishes (9×104 cells/8 cm2 dish) and allowed to attach for 24 hours. The medium was then changed to control medium or medium containing either 2 mM α- or β- xylosides and the incubation continued until the cells were confluent (day 7). The cell layer/matrices were extracted and the protein contents determined as described in Materials and Methods. Samples were separated by SDS-PAGE and transferred to nitrocellulose by semi-dry blotting. The same amount of protein (5 µg) was applied to each track of the polyacrylamide gel. Nitrocellulose sheets were Fig. 3. Permissive and inhibitory matrices contain different levels of incubated with either a monoclonal antibody (MA-IV) to thrombospondin-1 and tenascin. Cell layer/matrix extracts were thrombospondin-1 (A), a polyclonal antibody (Tn-2) to tenascin (B), prepared from post-confluent endothelial cells cultured in the or a polyclonal antibody to laminin (C), and the immunoreactive presence and absence of xylosides. Samples were then bands were detected by enhanced chemiluminescence. For each electrophoresed on polyacrylamide gels, transferred to nitrocellulose antibody tested, the samples were electrophoresed on the same gel, and incubated with antibodies to thrombospondin-1 (A), tenascin (B) transferred to the same filter and were exposed to Hyperfilm-ECL for and laminin (C) as described in the legend to Fig. 2. Samples in the same length of time. Samples in tracks 1, 2 and 3 are from cells tracks 1, 2 and 3 are from cells grown in control medium (1), in the grown in control medium (1), in the presence of β-xylosides (2) and presence of β-xylosides (2) and in the presence of α-xylosides (3). in the presence of α-xylosides (3). Densitometric scans of each track Densitometric scans of each track are shown next to the are shown next to the immunoblots. immunoblots. Endothelial sprouting 803 present in the cultures. Scanning the blots using a laser den- matrices and grown in control medium and in the continued sitometer revealed that post-confluent cultures (containing cob- presence of these antibodies for 10 days. blestone and sprouting cells) expressed between 2.3 and 4.8 Endothelial cells grew at the same rate and reached the same times more tenascin than confluent cultures (containing only saturation density under all the conditions tested. However, the cobblestone cells). Further experiments were therefore number of sprouting cells produced in these cultures was sig- conducted to ascertain the roles of both tenascin and throm- nificantly affected by the addition of the various antibodies. bospondin-1 in endothelial sprouting. Cells plated on permissive matrices (i.e. control-pc) in the presence of normal rabbit serum formed an extensive network Antibodies to tenascin and thrombospondin-1 of sprouting cells underneath the cobblestone monolayer (Fig. modulate endothelial sprouting 4A and Table 2). The addition of anti-tenascin serum to these We next investigated whether the addition of antibodies to cultures inhibited sprouting (Fig. 4C and Table 2). Cells plated tenascin and thrombospondin-1 could modulate sprouting by on inhibitory matrices (i.e. xyl-pc; Fig. 4B) contained signifi- cells cultured on permissive and inhibitory matrices (respec- cantly less sprouting cells than cells plated on permissive tively). These matrices were isolated from post-confluent endo- matrices (compare Fig. 4A and B). When present, these thelial cultures as described in Materials and Methods. Per- sprouting cells occurred predominantly as single cells rather missive matrices were incubated in the presence of polyclonal than as networks of cells. The addition of anti-throm- anti-tenascin serum or normal rabbit serum. Inhibitory matrices bospondin-1 sera to these cultures significantly increased both were incubated in the presence of a cocktail of monoclonal the number of sprouting cells present (Fig. 4D and E) and the anti-thrombospondin-1 sera or normal rabbit serum (see proportion of these cells in networks (Table 2). Materials and Methods). Cells were then plated on these These results demonstrate that anti-tenascin serum can

Fig. 4. Endothelial cells cultured on permissive and inhibitory matrices in the presence and absence of antibodies to tenascin and thrombospondin-1. Permissive and inhibitory matrices were prepared from post-confluent endothelial cultures as described in Materials and Methods. Permissive matrices (i.e. control-pc) were then incubated for 24 hours with normal rabbit serum or polyclonal anti-tenascin serum (Chemicon AB1906) in 20% DCS-MEM. Inhibitory matrices (i.e. xyl-pc) were incubated for 24 hours with normal rabbit serum or a combination of monoclonal antibodies to thrombospondin-1 (MA-II, MA-IV and TSP-B7) in 20% DCS-MEM. All antibodies were added to give a final v/v ratio of 1:50. Cells were then plated on these matrices (1.5×105 cells/8 cm2 dish) and grown for 10 days in control medium containing sera. The medium was changed every 2-3 days, at which time fresh antibody or normal rabbit serum was also added (as appropriate). (A) Cells grown on permissive matrices in normal rabbit serum. An extensive network of sprouting cells is seen underneath the cobblestone monolayer. (B) Cells grown on inhibitory matrices in the presence of normal rabbit serum; these cultures contain few sprouting cells. (C) Cells grown on permissive matrices in the presence of anti-tenascin serum. The formation of a sprouting cell network is inhibited by comparison to the corresponding controls (A). (D and E) Cells grown on inhibitory matrices in the presence of monoclonal anti- thrombospondin-1 sera. These photomicrographs show the range of sprouting observed in a single culture. The formation of sprouting cells is enhanced by comparison to the corresponding controls (B). Bar, 150 µm. 804 A. E. Canfield and A. M. Schor

Table 2. Modulation of endothelial sprouting on complex Table 3. Antibodies to tenascin inhibit endothelial matrices by antibodies to tenascin and thrombospondin-1 sprouting on gelatin-coated dishes in a dose-dependent Fields containing manner sprouting cell networks Number of fields Matrix Antibody (% of total) containing sprouting cells Permissive NRS 63±7 Sample (% of total fields counted) Permissive Tenascin 0 Control 89±4 Inhibitory NRS 35±3 +aTn (1:1000) 66±1 Inhibitory Thrombospondin-1 78±7 +aTn (1:100) 25±4 +aTn (1:50) 6±1 Permissive and inhibitory matrices were isolated from post-confluent endothelial cultures as described in Materials and Methods. The matrices Endothelial cells were plated on gelatin-coated dishes (1×105/8 cm2 dish) were then incubated with antibodies at a final v/v ratio of 1:50 as described. and allowed to attach for 24 hours. At this time, the medium was changed and Permissive matrices (i.e. control-pc) were incubated with either normal rabbit antibodies to tenascin were added at a final v/v ratio of between 1:50 and serum (NRS) or polyclonal anti-tenascin serum (Chemicon). Inhibitory 1:1000. Normal rabbit serum (1:50 v/v ratio) was added to control cultures. matrices (i.e. xyl-pc) were incubated with normal rabbit serum or a cocktail The medium was changed every 2-3 days, at which time fresh antibody was of monoclonal antibodies against thrombospondin-1. Cells were then plated added. Cells reached confluence 5 days after plating. The number of sprouting on these matrices (1.5×105 cells/8 cm2 dish) and grown for 7 days. The cells present in each culture was assessed 7 days after confluence was medium was changed every 2-3 days, at which time fresh antibody or NRS reached. A total of 50 fields on each dish were counted using a ×10 objective was added as appropriate. Cells reached confluency on day 3 on all the and the proportion of those fields which contained sprouting cells was matrices tested. The number of sprouting cells on duplicate dishes were then determined. The results are expressed as a percentage of the total number of assessed on day 7. Data are expressed in terms of the number of fields fields counted (± standard deviation). containing networks of sprouting cells (± standard deviation). Cells grown on inhibitory matrices showed a significant decrease in the number of sprouting cells forming networks by comparison to the permissive matrices (P<0.01). The addition of tenascin antibodies to permissive matrices abolished the formation of sprouting cells. The addition of antibodies to dose-dependent. Very few sprouting cells were observed when thrombospondin-1 to inhibitory matrices significantly increased the number antiserum to tenascin was added at a v/v ratio of 1:50; as the of sprouting networks by comparison to NRS controls (P<0.01). levels of tenascin antibodies were reduced, the number of sprouting cells in the cultures increased. Thus, when antibodies were added at a v/v ratio of 1:1000, there was only a small reverse the permissive nature of certain insoluble matrices and reduction in the number of fields containing sprouting cells by inhibit endothelial sprouting. Furthermore, antibodies to comparison to controls. thrombospondin-1 can neutralise the inhibitory nature of certain matrices and increase endothelial sprouting. Immunolocalisation of tenascin and It has previously been shown that antibodies to throm- thrombospondin-1 in endothelial cultures bospondin-1 can increase sprouting by endothelial cells We next used the technique of indirect immunofluorescence to cultured on plastic dishes (Iruela-Arispe et al., 1991). We have determine the localisation of tenascin and thrombospondin-1 now investigated whether anti-tenascin sera can also inhibit in confluent and post-confluent endothelial cells. The results sprouting on simple 2-D substrata. Cells were plated on obtained using polyclonal antiserum to tenascin (Tn-2) are gelatin-coated dishes at sub-confluent densities (approx. 1×105 shown in Fig. 6A-D. At confluence, a weak cytoplasmic cells/8 cm2 dish) and grown in 20% DCS-MEM. Polyclonal staining was detected in every cell (Fig. 6A and B). By com- antiserum to tenascin (Chemicon) was either added to the parison, in post-confluent cultures, tenascin was localised pref- culture medium 24 hours after plating or when the cells had erentially in the sprouting cells; in addition, a weak diffuse reached confluence. Fresh antiserum was added every time the extracellular staining was observed throughout (Fig. 6C and medium was changed (every 2-3 days) so that the cells were D). Endothelial cultures were also stained with two mono- cultured in the continual presence of antibodies to tenascin for clonal antibodies to thrombospondin-1 (MA-IV and TSP-B7; up to 12 days. The anti-tenascin serum had no effect on endo- Fig. 6E-H). Both antibodies stained all endothelial cells at con- thelial cell growth. The cells proliferated at the same rate and fluence (not shown). Marked differences were observed, reached the same saturation density (approx. 7.5×105 cells/8 however, when post-confluent cultures of endothelial cells cm2 dish) in the presence and absence of antibodies to tenascin. were stained with these two antibodies: antibody MA-IV, However, antibodies to tenascin markedly inhibited endo- which recognises the N-terminal heparin-binding domain of thelial cell sprouting when these cultures were maintained to thrombospondin-1 (Lawler et al., 1985), stained both cobble- post-confluence. This inhibition occurred irrespective of stone and sprouting cells (Fig. 6E and F). By comparison whether the antibodies were added when the cells were sub- antibody TSP-B7, which recognises the trypsin-resistant 70 confluent or confluent. Photomicrographs of post-confluent kDa core fragment of thrombospondin-1 (Dardik and Lahav, cells cultured with and without anti-tenascin serum are shown 1991), only stained the cobblestone cells and did not stain the in Fig. 5; data showing quantitation of the sprouting cells sprouting cells (Fig. 6G and H). These results suggest either present in these cultures are shown in Table 3. that the epitope for antibody TSP-B7 is masked in sprouting Fig. 5A shows that cells cultured in control medium endothelial cells, or that cobblestone and sprouting endothelial contained a network of sprouting cells underneath the cobble- cells synthesise different isoforms of thrombospondin-1. stone monolayer. By comparison, sprouting cells were only rarely detected in cultures maintained in the presence of anti- DISCUSSION bodies to tenascin (Fig. 5B). The data presented in Table 3 demonstrates that the inhibition of endothelial sprouting was The role of the extracellular matrix in angiogenesis has been Endothelial sprouting 805

CONTROL

Fig. 5. Antibodies to tenascin inhibit endothelial cell sprouting on gelatin-coated dishes. Endothelial cells were plated on gelatin-coated dishes (1×105 cells/8 cm2 dish) and allowed to attach for 24 hours. At this time either polyclonal antiserum to tenascin or normal rabbit serum was added to the anti-TENASCIN cultures (1:50 v/v ratio). Fresh antisera were added every time the medium was changed, and the cultures were maintained to 7 days post- confluence. (A) Cells grown in medium containing normal rabbit serum: a second layer of sprouting cells is seen under the cobblestone monolayer. (B) Cells grown in medium containing antibodies to tenascin: sprout formation is inhibited. Bar, 150 µm. investigated by several groups using two general approaches. position of which can also be modified. For example, we have The first approach has been to purify individual matrix macro- previously shown that exogenous xylosides inhibit the molecules and to study these proteins in isolation (Madri and formation of the sprouting phenotype (Schor and Schor, 1988) Williams, 1983; Schor et al., 1983; Montesano et al., 1983; and the synthesis of matrix proteins by endothelial cells Ingber and Folkman, 1989a; Good et al., 1990). In the second (Canfield et al., 1994). The results presented in this communi- approach more complex matrices have been used, including: cation indicate a causal relationship between these two effects. matrigel, the amnionic membrane and matrices produced by That is, the composition of the complex subendothelial matrix the cells themselves (this study; Madri and Williams, 1983; played a direct role in determining whether endothelial cells Grant et al., 1989; Vernon et al., 1992). These alternative would sprout or not. It is important to emphasise that inhibitory approaches have both advantages and disadvantages. The matrices prevented sprouting even though the cells were functions of specific proteins may be inferred by studying them cultured in growth medium containing permissive factors in isolation. However, matrix macromolecules do not exist as which promoted sprouting on permissive matrices. isolated proteins in vivo; instead they interact with each other The approach used in this study was to isolate matrices to form highly organised and complex structures (Yurchenco, deposited by endothelial cells themselves under conditions in 1990). Cell behaviour is now known to be influenced by both which sprouting was either allowed to proceed normally the overall composition of a complex matrix as well as the (control matrices) or inhibited by the presence of exogenous mechanical properties of that matrix (Ingber and Folkman, xylosides (xyl-matrices). We then used these matrices as 1989a,b; Vernon et al., 1992; this study). Studies using substrata and examined their effect on the sprouting of freshly- complex matrices may therefore be more relevant to the in vivo plated endothelial cells. Our results show that endothelial cells situation, but it is more difficult to determine the specific attached and proliferated equally well on all the matrices role(s) of individual components using this approach. tested. However, whereas sprouting occurred normally on Endothelial cells in vivo are attached to a complex extra- control matrices, this process was markedly inhibited on cellular matrix (or basement membrane). The precise compo- matrices deposited by post-confluent cells cultured in the sition of this matrix depends on the size and location of the presence of xylosides. We demonstrate that this inhibition was vessel and may be affected by various pathological conditions not due to the presence of xylosides in the inhibitory matrices (Wight et al., 1985; Barnes, 1988; Engvall et al., 1990; Hedin nor to the total amount of protein present in these matrices. et al., 1991). How the composition of the basement membrane Rather, our results show that permissive and inhibitory may affect angiogenesis is not known. In vitro, endothelial matrices differ in the relative levels of tenascin and throm- cells synthesise and deposit an extracellular matrix, the com- bospondin-1 they contained. Namely, matrices which were 806 A. E. Canfield and A. M. Schor

Fig. 6. Immunolocalisation of tenascin and thrombospondin-1 in endothelial cells. Endothelial cells plated on gelatin-coated dishes were grown in control medium until the cells were confluent (A,B) and post-confluent (C-H). At this time, cultures were stained either with polyclonal antiserum to tenascin (Tn-2; A-D) or with monoclonal antibodies to thrombospondin-1 (MA-IV, E and F; TSP-B7, G and H) as described in Materials and Methods. (A,B) Confluent culture of endothelial cells. Phase (A) and immunofluorescence (B) micrographs of the same field showing a weak cytoplasmic staining of cobblestone cells with anti-tenascin serum. (C,D) Post-confluent culture of endothelial cells. Phase (C) and immunofluorescence (D) micrographs of the same field showing that sprouting cells stain preferentially with antibodies to tenascin. A diffuse, extracellular staining is also seen. (E,F) Post-confluent culture of endothelial cells. Phase (E) and immunofluorescence (F) micrographs of the same field showing that cobblestone and sprouting cells stain with monoclonal antibody MA-IV. (G,H) Post-confluent culture of endothelial cells. Phase (G) and immunofluorescence (H) micrographs of the same field showing that only the cobblestone cells, and not the sprouting cells, stain with monoclonal antibody TSP-B7. Arrowheads are used to identify the same point of reference in both micrographs. Bar, 150 µm. inhibitory for sprouting were found to contain relatively less tenascin (not shown). It is possible that xylosides may also tenascin and more thrombospondin-1 than matrices which have modified the synthesis of other matrix components that were permissive (Figs 2 and 3). Neither tenascin nor throm- we have not investigated. Similarly, we have not examined bospondin could be identified in the total protein gels, sug- whether post-translational modifications of these proteins (for gesting that these proteins were only minor components of the example, glycosylation) may have been altered by the matrices. The relative levels of fibronectin were also decreased xylosides. in the inhibitory matrices, although to a lesser extent than Our results clearly show that both tenascin and throm- Endothelial sprouting 807 bospondin-1 are involved in endothelial sprouting. Immunolo- al., 1991). However, we cannot exclude the possibility that calisation studies demonstrated that tenascin was preferentially tenascin-X is also synthesised by endothelial cells and that this expressed by cells displaying the sprouting phenotype (Fig. 6). molecule may also be recognised by the antibodies used in this Thrombospondin-1 was also localised in sprouting cells (Fig. study. At present, therefore, we cannot say definitively which 6, this study; Iruela-Arispe et al., 1991), although our results tenascin isotype is involved in angiogenesis. This question using two different monoclonal antibodies suggested that the should be resolved in future studies using monoclonal anti- thrombospondin-1 expressed by these cells may differ in some bodies specific for the different proteins. way from that expressed by cobblestone cells (Fig. 6). That is, Our results showing that matrices containing increased either cobblestone and sprouting cells synthesise different levels of thrombospondin-1 are inhibitory for endothelial isoforms of thrombospondin-1, or the epitope corresponding to sprouting support and extend previous in vitro and in vivo the 70 kDa core fragment of thrombospondin-1 (which is studies. We have previously shown that thrombospondin-1 is recognised by one of the antibodies) is masked in sprouting a major secreted product of endothelial cells displaying the cells. resting phenotype and both mRNA and protein levels of this Antibodies to tenascin inhibited sprouting on permissive protein are greatly reduced when cells assume the sprouting matrices and on gelatin-coated dishes in the absence of any phenotype (Canfield et al., 1990a). Furthermore, Iruela-Arispe effects on cell growth (Figs 4 and 5; Tables 2 and 3). Anti- et al. (1991) have shown that polyclonal antibodies to throm- bodies to thrombospondin-1 promoted sprouting on inhibitory bospondin-1 promote the formation of sprouting cells in endo- matrices (Fig. 4 and Table 2). It is important to note that in thelial cells cultured on plastic tissue culture dishes. Interest- each case, antibodies against one of the proteins alone was suf- ingly, exogenous thrombospondin-1 has been shown to inhibit ficient to either promote or inhibit endothelial sprouting. That bFGF-stimulated angiogenesis in the rat cornea, and this is, antibodies to tenascin inhibited sprout formation even inhibitory activity has been attributed to the domain showing though the relative levels of thrombospondin-1 in the permis- homology to procollagen and the properdin-like type I repeats sive matrices were low, and antibodies against throm- (Good et al., 1990; Tolsma et al., 1993). Taken together, these bospondin-1 promoted sprouting even though the relative studies therefore suggest that thrombospondin-1 may inhibit levels of tenascin in the inhibitory matrices were low. Anti- angiogenesis by preventing the formation of sprouting cells bodies to thrombospondin-1 have previously been shown to and the subsequent morphogenetic interactions between these promote sprouting in endothelial cells cultured on plastic tissue cells. It has also been suggested that the localisation of throm- culture dishes (Iruela-Arispe et al., 1991). Antibodies to bospondin-1 in filamentous arrays around sprouting endo- tenascin have not been used previously in angiogenesis-related thelial cells may serve to stabilise these structures and inhibit studies. It has, however, been shown that anti-tenascin serum their progression (Iruela-Arispe et al., 1991). It is important to can inhibit the migration of neural crest cells and ganglion cells note, however, that the precise cellular and macromolecular (Bronner-Fraser, 1988; Choung et al., 1987). Preliminary environment surrounding the endothelial cells may also play a experiments attempting to either (a) restore sprouting on crucial role in determining the response of these cells to throm- inhibitory matrices or (b) inhibit sprouting on permissive bospondin-1. Thus, we have shown that antibodies to tenascin matrices by the simple addition of tenascin or thrombospondin- will inhibit sprouting on a matrix in which the levels of matrix- 1 (respectively) to these matrices did not produce clear-cut bound thrombospondin-1 are low and apparently ‘permissive’ results (A. E. Canfield and A. M. Schor, unpublished observa- for sprouting (this study). Furthermore, Nicosia and Tuszynski tions). Our results suggest that the importance of individual (1994) have recently shown that matrix-bound throm- components for the regulation of endothelial sprouting may be bospondin-1 can promote angiogenesis in vitro through a influenced by the presence and relative levels of other proteins primary effect on myofibroblasts. Ben Ezra et al. (1993) have as well as by their organisation within the matrix. similarly observed that TSP-1 enhanced angiogenesis in the The expression of tenascin is known to be enhanced in the rabbit cornea assay. stroma of tissues in which angiogenesis is occurring (e.g. The relative levels of laminin were similar in the permissive tumours and healing wounds; Chiquet-Ehrismann et al., 1986; and inhibitory matrices isolated in this study (Fig. 3). These Mackie et al., 1988; Whitby et al., 1991). In spite of this obser- results suggest that this molecule may not play a major role in vation, the possible involvement of tenascin in the formation the early stages of angiogenesis. It is likely, however, that of new blood vessels has, until now, received little attention. laminin may be important in the later stages of angiogenesis. The data presented in this paper show that: (i) tenascin is In this regard, it has been shown that laminin expression is expressed preferentially in sprouting cells both in terms of its increased during the maturation of newly-formed vessels in immunolocalisation (Fig. 6), and protein levels (Figs 2 and 3), vivo (Risau and Lemmon, 1988; Wakui et al., 1990). Further- and (ii) tenascin may play a direct role in the initial stages of more, laminin and specific peptides derived from laminin angiogenesis by promoting the formation of sprouting endo- chains have been shown to promote ‘tubule’ formation by thelial cells. Three members of the family of have endothelial cells in vitro (Grant et al., 1989, 1992). Several now been described (tenascin-C, tenascin-R and tenascin-X; different laminin chains are now known to exist (Tryggvason, Erickson, 1993; Bristow et al., 1993). The precise role(s) 1993). These isoforms appear to be expressed in a tissue- played by these proteins in development is not clear. Recent specific manner (Engvall et al., 1990). The majority of studies evidence suggests that tenascin-X, and not tenascin-C, may be to date (including our own) have used laminin isolated from essential for normal development (Saga et al., 1992; Bristow the EHS tumour or antibodies directed against this protein. The et al., 1993). The molecular mass of the molecule (220,000) possibility that other laminin isoforms may also be involved in detected by western blotting in this study corresponds to one angiogenesis cannot, therefore, be excluded. of the alternatively spliced isoforms of tenascin-C (Chiquet et The modified matrices isolated in this study may have 808 A. E. Canfield and A. M. Schor inhibited endothelial sprouting in a number of ways. For Bigner, D. D. (1983). Human glioma-mesenchymal extracellular matrix example, the attachment of endothelial cells to a modified antigen defined by monoclonal antibody. Cancer Res. 43, 2796-2805. matrix may be such that these cells are physically ‘constrained’ Bristow, J., Tee, M. K., Gitelman, S. E., Mellon, S. H. and Willer, W. L. (1993). Tenascin-X: A novel extracellular matrix protein encoded by the in a cobblestone phenotype. This ‘constraint’ may occur via human XB overlapping p450c21B. J. Cell Biol. 122, 265-278. extracellular matrix-cytoskeletal interactions which are Bronner-Fraser, M. (1988). Distribution and function of tenascin during mediated by cell surface receptors. These interactions may then cranial neural crest development in the chick. J. Neurosci. Res. 21, 135-147. prevent the cells from undergoing the changes in shape Canfield, A. E., Schor, A. M., Schor, S. L. and Grant, M. E. (1986). The biosynthesis of extracellular matrix components by bovine retinal necessary for sprout formation to occur (see reviews by Ingber endothelial cells displaying distinctive morphological phenotypes. Biochem. and Folkman, 1989b; Adams and Watt, 1993). An alternative J. 235, 375-383. explanation is that the attachment of cells to an inhibitory Canfield, A. E., Boot-Handford, R. P. and Schor, A. M. (1990a). matrix via cell surface receptors may modulate gene expression Thrombospondin gene expression by endothelial cells in culture is by the cells such that sprouting is prevented (Bissell and modulated by cell proliferation, cell shape and the substratum. Biochem. J. 268, 225-230. Barcellos-Hoff, 1987; Adams and Watt, 1993). In support of Canfield, A. E., Allen, T. D., Grant, M. E., Schor, S. L. and Schor, A. M. this theory, we have preliminary evidence showing that the (1990b). Modulation of extracellular matrix biosynthesis by bovine retinal mRNA levels of thrombospondin-1 appear to be increased pericytes in vitro: effects of substratum and cell density. J. Cell. Sci. 96, 159- when endothelial cells are grown on inhibitory matrices in 169. comparison with permissive matrices (A. E. Canfield and A. Canfield, A. E., Sutton, A. B., Hiscock, D. R. R., Gallagher, J. T. and Schor, A. M. (1994). Alpha- and beta-xylosides modulate the synthesis of M. Schor, unpublished observations). The modulation of endo- fibronectin and thrombospondin by endothelial cells. Biochim. Biophys. thelial sprouting by antibodies to tenascin and throm- Acta. 1200, 249-258. bospondin-1 seen in this study may have interfered with either Chiquet, M., Vrucinic-Filipi, N., Schenk, S., Beck, K. and Chiquet- mechanism. Ehrismann, R. (1991). Isolation of chick tenascin variants and fragments. Eur. J. Biochem. 199, 379-388. The composition of the extracellular matrix may be modified Chiquet, M. (1992). Tenascin: an extracellular matrix protein involved in in vivo in several ways. For example, angiogenic factors are morphogenesis of epithelial organs. Kidney Int. 41, 629-631. known to modulate both the synthesis of matrix proteins Chiquet-Ehrismann, R., Mackie, E. J., Pearson, C. A. and Sakakura, T. (Madri et al., 1988; Sutton et al., 1991) as well as the synthesis (1986). Tenascin: an extracellular matrix protein involved in tissue of certain proteases and their inhibitors (Mignatti et al., 1989; interactions during fetal development and oncogenesis. Cell 47, 131-139. Choung, C. M., Crossin, K. L. and Edleman, G. M. (1987). Sequential Pepper et al., 1990; Sutton et al., 1991). Furthermore, other expression and differential function of multiple adhesion molecules during peri-endothelial cells (e.g. pericytes) may also play a role in the formation of cerebrellar cortical layers. J. Cell Biol. 104, 331-342. altering the composition of the extracellular matrix (Canfield Church, G. M. and Gilbert, W. (1984). Genomic Sequencing. Proc. Nat. et al., 1990b). We suggest that the composition of the extra- Acad. Sci. USA 81, 1991-1995. Dardik, R. and Lahav, J. (1991). Cell-binding domain of endothelial cell cellular matrix in blood vessels is tightly regulated. 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