Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro.

Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro E . HELENE SAGE

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Department of Biological Structure, SM-20, University of Washington, Seattle, WA 98195, U.S.A. Received November 29, 1991 SAGE,E. H. 1992. Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro. Biochem. Cell Biol. 70: 579-592. Endothelioma cells expressing the polyoma virus middle T oncogene induced hemangiomas in mice by the recruitment of nonproliferating endothelial cells from host blood vessels (Williams et al. 1989). I now report that SPARC, a ca2+-bindingglycoprotein that perturbs cell-matrix interactions and inhibits the endothelial cell cycle, is produced by endothelioma cells and is in part responsible for the alterations in the morphology and growth that occur when nontransformed bovine aortic endothelial cells are cocultured with endothelioma cells. Normal endothelial cells cocultured with two different middle T-positive endothelial cell lines, termed End cells, exhibited changes in shape that were accompanied by the formation of cell clusters. Media conditioned by End cells repressed proliferation of normal endothelial cells, but enhanced that of an established line of murine capillary endothelium. Radiolabeling studies revealed no apparent differences in the profile of proteins secreted by aortic or capillary cells cultured in End cell conditioned media. Characterization of proteins produced by End cells led to the identification of type IV collagen, laminin, entactin, and SPARC as major secreted products. Although SPARC did not affect the morphology of End or capillary cells, it was associated with overt changes in the shape of aortic endothelial cells. Moreover, SPARC and a synthetic peptide from SPARC domain 11 inhibited the incorporation of [3~]thymidine by aortic cells, but had minimal to no effect on the capillary endothelial cell line. The inhibition of growth exhibited by aortic endothelial cells cultured in End cell conditioned media could be partially reversed by antibodies specific for SPARC and SPARC peptides. These studies indicate a potential role for SPARC in the generation of hemangiomas by End cells in vivo, a process that requires normal (host) endothelial cells to disengage from the extracellular matrix, withdraw from the cell cycle, migrate, and reassociate into the disorganized cellular networks that comprise cavernous and capillary hemangiomas. Key words: endothelial cells, hemangioma, cell proliferation, SPARC. SAGE,E. H. 1992. Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro. Biochem. Cell Biol. 70 : 579-592. Les cellules d'un endothtliome exprimant I'oncogtne moyen T du virus du polyome induisent des htmangiomes dans les souris par recrutement des cellules endothtliales non proliftrantes provenant des vaisseaux sanguins de I'hdte. Nous rapportons que la SPARC, une glycoprottine liant le c a 2 + qui perturbe les interactions de la matrice et des cellules et inhibe le cycle des cellules endothtliales, est produite par des cellules de l'endothtliome et est en partie responsable des alttrations dans la morphologie et la croissance quand les cellules endothtliales non transformtes de l'aorte bovine sont cultivtes avec les cellules de I'endothtliome. Les cellules endothtliales normales cultivtes en meme temps que deux difftrentes ligntes de cellules endothtliales sensibles ti I'angigtne T moyen, appeltes cellules terminales (End cells), exhibent des changements de forme accompagnts de la formation d'agrtgats cellulaires. Les milieux conditionnts par les cellules terminales rtpriment la proliferation des cellules endothtliales normales, mais stimulent celles d'une lignte ttablie de I'endothtlium capillaire murin. Les ttudes de radiomarquage ne rtvtlent aucune difftrence apparente dans le profil des prottines stcrkttes par les cellules de l'aorte ou les cellules capillaires cultivtes dans les milieux conditionnts par les cellules terminales. La caracttrisation des prottines produites par les cellules terminales permet I'identification du collagtne de type IV, de la laminine, de I'entactine et de la SPARC comme principaux produits stcrttes. Bien que la SPARC n'affecte pas la morphologie des cellules terminales ou des cellules capillaires, elle est associte avec des changements tvidents dans la forme des cellules endothtliales aortiques. D'ailleurs, la SPARC et un peptide synthttique issu du domaine I1 de la SPARC inhibent I'incorporation de la [3~]thymidine par les cellules aortiques, mais ils affectent peu ou pas la lignte des cellules endothtliales capillaires. L'inhibition de la croissance exhibte par les cellules endothtliales aortiques cultivtes dans les milieux conditionnts par les cellules terminales peut Etre partiellement renversk par les anticorps sptcifiques de la SPARC et des peptides de la SPARC. Ces ttudes montrent un r8le potentiel de la SPARC dans la gtntration des htmangiomes par les cellules terminales in vivo, processus qui requiert les cellules endothtliales (h8te) normales pour se dtgager de la matrice extracellulaire, se retirer du cycle cellulaire, migrer et se rtassocier dans les rtseaux cellulaires dtsorganists que renferment les htmangiomes caverneux et capillaires. Mots clbs : cellules endothtliales, htmangiome, proliftration cellulaire, SPARC. [Traduit par la rtdaction]

Introduction The middle T oncogene of polyoma virus has recently been implicated as a causative agent in the disruption of endothelial cell function, as well as normal development of the vasculature. Mice transgenic for middle T developed lethal hemangiomas (tumors of endothelial origin), a finding that identified endothelial cells as susceptible targets for this oncogene (Bautch ABBREVIATIONS: U- and t-PA, urokinase- and tissue-type plasminogen activators; PAI-1, type 1 plasminogen activator inhibitor; BAE, bovine aortic endothelial; IgG, immunoglobulin G; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphatebuffered saline (pH 7.4); TGF-0, transforming growth factor-p; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate - polyacrylamide gel electrophoresis; DTT, dithiothreitol; ED,,, mean effective dose; PDGF, platelet-derived growth factor; bFGF, basic fibroblast growth factor. Printed in Canada / Imprim6 au Canada


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et al. 1987). Moreover, injection of mice directly with a retrovirus bearing the middle T oncogene was associated with the rapid formation of cyst-like structures that were lined with endothelium (referred to as cavernous hemangiomas) (Williams et a/. 1988). Williams et al. (1988) also showed that embryonic stem cells, infected with middle T and subsequently introduced into normal host blastocysts, gave rise to chimeric mice which died at midgestation, ostensibly from the multiple foci of endothelial tumors that subverted vasculogenesis. Endothelial cells cultured from these tumors (End cells) continued to express middle T antigen, as well as the characteristic endothelial product von Wiebrand factor, and gave rise to hemangiomas in vivo upon injection into mice and several other species (Williams et al. 1988, 1989). Investigation into the mechanisms by which the middle T oncogene induced these tumors led to an unexpected finding. Histological and autoradiographic examination of the hemangiomas that arose subsequent to the injection of cultured, middle T-expressing endothelioma (End) cells revealed a preponderance of host-derived, nonproliferating endothelial cells (Williams et al. 1989). Since End cells did not stimulate the growth of normal (aortic) endothelial cells in vitro, it appeared that the recruitment of noncycling endothelium from the host vasculature was necessary for the development of the tumors (Williams et al. 1989). A second, related series of experiments focused on the behavior of End cells in the context of angiogenesis. Cultured End cells embedded in fibrin gels formed cystic structures that resembled the cavernous hemangiomas previously observed in vivo and secreted significant amounts of u-PA and t-PA (Montesano et al. 1990~).Diminution of this fibrinolytic activity by the addition of selected proteinase inhibitors was associated with the disappearance of the cystic structures and the formation of capillary-like networks reminiscent of the angiogenesis in vitro exhibited by strains of normal endothelial cells (Montesano et al. 1990~).This study emphasized the significant role of secreted products and the extracellular matrix, and particularly the ratio of proteolytic to antiproteolytic activity, in directing the behavior of endothelial cells, especially with respect to angiogenesis and vascular development (reviewed in Ingber and Folkman 1989a; Montesano et al. 1990b). It is important to consider, however, that an extensive temporal spectrum exists between the expression of the middle T oncogene by a transformed endothelial cell and the assembly of host endothelial cells into an hemangioma in vivo or a cystic structure in vitro, and that a complex series of biochemical events is responsible for each of the steps that has been isolated experimentally. It is our contention that an alteration of the balance between u-PA and t-PA, relative to their inhibitors (e.g., PAI-l), represents some of these events, and that the identification of agents which perturb this balance would increase our understanding of the process of hemangioma formation in this system. Recent studies have collectively identified a group of extracellular proteins (SPARC, tenascin, and thrombospondin) that modulate cell-matrix interactions (reviewed in Sage and Bornstein 1991). These proteins are expressed at high levels during embryonic morphogenesis and promote changes in cell shape and adhesion in vitro. We reasoned that End cells might secrete one or more of these antiadhesive proteins that could induce established host

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endothelial cells to reassociate into the disorganized structures typical of hemangiomas. Of particular interest was SPARC (also known as osteonectin (Termine et al. 1981) and BM-40 (Mann et al. 1987)), which has been shown in cultured endothelial cells to elicit a range of responses that are consistent with the recruitment of nonproliferating host cells by End cells. For example, SPARC changes cell shape and acts as an anti-spreading factor (Sage et al. 1989b; Everitt and Sage 1992), reduces focal contacts (MurphyUllrich et al. 1991), inhibits progression of the cell cycle (Funk and Sage 1991), induces the expression of PAI-1 (Hasselaar et al. 1991) and diminishes that of fibronectin (Lane et al. 1992), and is produced at high levels by sprouting endothelial cells undergoing angiogenesis in vitro (Iruela-Arispe et al. 1991b, 1991~).Confirmation of the production of SPARC by End cells was necessary, however, before the potential role of this or other anti-adhesive proteins in modulating normal cell behavior could be addressed. Initially, I established cocultures between BAE cells and two lines of End cells, and observed morphologic alterations in the BAE cells that included cell rounding and the formation of cell clusters. Culture media conditioned by End cells reduced the proliferation rate of BAE cells and was shown to contain significant quantities of SPARC. Since antiSPARC IgG partially reversed the inhibitory effect of End cell conditioned media on the growth of BAE cells, I propose that SPARC is an active factor in the signaling process between middle T-expressing endothelioma cells and the endogenous vasculature, which results in the disruption of normal endothelial function and interaction with the extracellular matrix. Materials and methods Cells and cell culture BAE cells were isolated from aortae of adult cows (Bos taurus) as previously described (Schwartz 1978; Sage et al. 19896). In this study, three different strains were used that ranged in passage number between 4 and 17. LE I1 cells, derived from murine lung capillary endothelium, were a gift from Dr. Tom Maciag (American Red Cross, Rockville, Md.). The cell line eEnd.2 (embryonic endothelioma) was established from hemangiomas in midgestation chimeric mouse embryos (Williams et al. 1989), which were initially derived from embryonic stem cells expressing polyoma middle T antigen (Williams et al. 1988). The bEnd.3 cell line was derived from primary cultures of normal mouse brain capillary endothelial cells that were infected with the middle T-expressing N-TKmT retrovirus (Montesano et al. 1990a). All cells were routinely cultured in DMEM (Sigma, St. Louis, Mo.) supplemented with antibiotics and 10% (by volume) FCS (HyClone, Logan, Utah). Coculture was performed according to two procedures. Initially, a small steel cloning ring was placed in the middle of each well of a 6-well tissue culture cluster dish (30 mm) (Corning, Corning, N.Y.) and was secured with sterile vacuum grease. To allow for the difference in area, cells were plated in the outer wells at a density of 8.5 times those plated in the inner wells. Typically, BAE cells were grown within the cloning rings and End cells were grown within the periphery, although all possible combinations were tried including those that were homotypic. Cell density, concentration of FCS, and duration of coculture were also varied systematically. Removal of the cloning ring and its isolated culture medium allowed the medium conditioned by cells in the periphery to cover the cells previously contained within the ring. The circle of vacuum grease acted as an effective barrier between the two cell types. The second method involved the use of Costar TranswellTM cell culture chamber inserts (Costar, Van Nuys, Calif.), which are removable wells with collagen-coated microporous membrane filters upon


which the cells are plated. Insertion of these chambers into the wells of cluster plates that contain one cell type allows independent access to both sides of a monolayer of another cell type. The filter did not permit transcellular migration and did not alter the attachment, spreading or proliferation rate of BAE or End cells in comparison to a plastic substrate. Measurement of cell growth A total of 16 separate experiments were performed to assess the effect of End cell conditioned media on the growth of BAE and LE I1 cells. Although the successive experiments produced consistent results, they accommodated a range of variables that were tested systematically for their potential contribution to observed effects on cell growth. For the production of conditioned media, BAE (two strains), LE 11, eEnd.2, and bEnd.3 cells were passaged into 175-cm2Falcon flasks (Becton Dickinson, Oxnard, Calif.) at subconfluent densities. Twenty-four hours later, the growth medium was replaced with 12 mL serum-free DMEM, DMEM containing 1, 2, 4, or 10% FCS, or DMEM containing 1, 2, or 4% of the same lot of heat-inactivated FCS. Conditioned media were collected 24,48, or 72 + 4 h later (cells became confluent between 48 and 72 h) and were clarified by centrifugation in a clinical centrifuge for 10 min. For some experiments, FCS or heat-inactivated FCS was added to serum-free conditioned DMEM as an alternative to the provision of serum that had itself been previously exposed to cells. Likewise, conditioned media were in some instances sterilefiltered (0.22-pn filter), before or after supplementation with sera. BAE and LE I1 cells were dissociated in trypsin-EDTA and replated in conditioned media at a density of 20 000 cells/4cm2 (i.e., one well of a 12-well plate) (Costar). Alternatively, the cells were cocultured in 1-cm2insert ~ r a n s w e l l over s ~ ~ monolayers of eEnd.2 or bEnd.3 cells grown in 4-cm2 wells. Cell number was determined 1, 2, and (or) 3 days later by Coulter counter or hemocytometer. Measurements of the incorporation of ['~lthymidinewere performed as previously described (Funk and Sage 1991). Briefly, cells were pulsed with 1.0 pCi [methyl-'~]thymidine/m~for 24 h and subsequently rinsed with cold PBS (pH 7.4), fixed 20 min at 4°C in 10% trichloroacetic acid, rinsed with cold ethanol, and air-dried. Material insoluble in trichloroacetic acid was hydrolyzed in 0.4 M NaOH at 60" for 25 min, neutralized with glacial acetic acid, and solubilized in Ecolume (ICN Biochemicals, Irvine, CA) for scintillation counting. Assessment of cell number or incorporated [3~]thymidine was performed on individual wells of cells plated in multiples of 2, 3, 4, or 6, and data were analyzed as means k SEM.

20% ammonium sulfate was used as a control for the addition of immune IgG, in equivalent or greater amounts (i.e., in pg/mL). Analysis and characterization of secreted proteins The levels of SPARC secreted by eEnd.2, bEnd.3, and LE I1 cells were measured quantitatively by a radioimmune assay developed in our laboratory for murine SPARC (Everitt and Sage 1992). Cocultures (usually cells in the inserts or inner wells) were incubated approximately 24 h (individual experiments ranged from 18 to 26 h) in serum-free conditioned DMEM supplemented with 50 pg sodium ascorbate/mL, 64 pg P-aminopropionitrile fumarate/mL, and 20-25 pCi [ 2 , 3 , 4 , 5 - 3 ~ ] p r o l i n e / m ~ (5 mCi/mL; ICN Biochemicals, Irvine, Calif.). Culture media were clarified in the presence of 0.2 mM PMSF, 25 mM EDTA (pH 7.5). and 10 mM N-ethylmaleimide (final concentrations), dialyzed at 4°C against 0.1 M acetic acid, and lyophilized. For preparative-scale analysis of proteins secreted by End cells, eEnd.2 and bEnd.3 cells (approximately nine 175-cm2 flasks of each per experiment)were radiolabeled as described in the preceding paragraph (typically 12 mL supplemented DMEM/flask). Culture media were collected as noted above, and radiolabeled proteins were precipitated overnight at 4OC by the slow addition of ammonium sulfate to the media (final concentration was 50%, weight to original volume ratio) (Sage and Bornstein 1992). Precipitated protein was concentrated by centrifugation, dissolved in and dialyzed against a 4 M urea - 50 mM Tris-HCI buffer (pH 8.0), and chromatographed over DEAE-cellulose equilibrated in the same buffer (Sage and Bornstein 1992). Gradient elution was performed with 0-200 mM NaCl in 200 mL column buffer. Elution profiles were determined by scintillation counting, and pooled fractions were subsequently dialyzed against 0.1 M acetic acid and lyophilized.

Other procedures SDS-PAGE and immunoblotting, with and without prior reduction of the samples in 50 mM DTT, were performed as previously described (Sage et al. 1989b; Sage and Bornstein 1992). Antibodies against murine entactin were a gift from Dr. A. Chung (University of Pittsburgh, Pittsburgh, Pa.). Anti-murine laminin antibodies were purchased from Telios (San Diego, Calif.), and affinitypurified anti-human thrombospondin antibodies (cross-reactive with murine thrombospondin) were a gift from Dr. P. Bornstein (University of Washington, Seattle, Wash.) (Iruela-Arispe et al. 1991~). Fractions from DEAE-cellulose chromatography of End cell conditioned medium were dissolved in 100 pL Tris-saline (0.15 M NaCl, Addition of SPARC, SPARC peptide, and antibodies to cells 50 mM Tris-HC1, pH 7.5) containing 1.0 mM PMSF, and aliquots SPARC was purified from the murine PYS cell line as previwere incubated with 0.256 U (1.0 pL) bacterial collagenase ously described (Sage et al. 1989a; Sage and Bornstein 1992). (Form 111; Advance Biofactures, Lynbrook, N.Y.) at 37°C for SPARC peptide 2.1 is a synthetic 20-mer comprising amino acids 45 min. The samples, including a control incubated at 37°C in the 54-73 located in domain I1 of SPARC (Lane and Sage 1990); pepabsence of enzyme, were subsequently analyzed directly by SDStide 2. l a represents the first 10 amino acids (54-63) of peptide 2.1. PAGE (Sage et al. 1984). SPARC and peptide 2. l a were dissolved in PBS and used on cells Similar or identical samples were also chromatographed on a at concentrations of approximately 10-50 pg/mL (0.3-1.5 @ andI) column of heparin-Sepharose (Pharmacia, Piscataway, N.J.) that 0.8 mM, respectively (Lane and Sage 1990; Funk and Sage 1991). was equilibrated in sterile Tris-saline containing 25 pM EDTA. Antibodies were added to cells cultured in conditioned media Proteins eluted in PBS (unbound material), 250 mM NaCl, in the presence of heat-inactivated FCS. Affinity-purified anti500 mM NaCI, and 2 M NaCl were dialyzed at 4°C against H 2 0 murine SPARC IgG (Sage et al. 1989a, 1989b) was used at confollowed by 0.1 N acetic acid, lyophilized, and analyzed by centrations ranging from 450 ng/(mL- 10 000 cells) t o SDS-PAGE. 1.2 pg/(mL. 10 000 cells). For some experiments, a fraction of the same anti-SPARC antiserum was precipitated in 20% ammonium Results sulfate and used at a concentration of 84 pg/(mL-5000 cells). Endothelial cell lines expressing polyoma middle T antigen Antibodies against SPARC peptide 1.1 (Lane and Sage 1990) were modulate the morphology and growth of normal aortic used at 80 ng/(mL. 10 000 cells). Antibodies specific for SPARC endothelial cells peptide 2.1 (Lane and Sage 1990) were affinity purified and used Noncontiguous cocultures were initially established at concentrations of 495-990 ng/(0.5 mL-10 000 cells). Anti-TGF-P between several strains of BAE cells and the transformed IgG, an affinity-purified neutralizing antibody preparation (R & D endothelial cell lines eEnd.2 (derived from hemangiomas of Systems. Minneapolis, Minn.), was added to 10 000 cells at conchimeric embryos) and bEnd.3 (capillary endothelial cells centrations of 0.5 and 1.0 pgI0.5 mL conditioned media. An IgG fraction of preimmune rabbit serum that had been precipitated in infected directly with a middle T-expressing retrovirus). As

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F I G . 1 . Conditioned media from murine endothelioma cell lines (eEnd.2 and bEnd.3) alter the morphology of bovine aortic endothelial (BAE) cells. Cocultures were established between BAE cells and eEnd.2 or bEnd.3 cells; all cell types were grown to greater than 90% confluence in DMEM containing 10% FCS. Twenty-four hours after the initial plating, cloning rings and media were removed from the inner island of cells, which were subsequently exposed to media conditioned by the cells in the periphery of the dish. Photographs were taken of cells in the inner wells 7.5 h after the cloning rings were removed. (A-C) Control cocultures showing BAE cells in BAE cell medium (A), eEnd.2 cells in eEnd.2 medium (B), and bEnd.3 cells in bEnd.3 medium (C). (D-F) BAE cells exposed to eEnd.2 conditioned medium; three independent fields of increasing cell density are shown. (G-I) BAE cells exposed to bEnd.3 conditioned medium; three independent fields of increasing cell density are shown. In the presence of End cell conditioned media. BAE cells exhib-


shown in Fig. 1, exposure of BAE cells to medium conditioned 24 h by eEnd.2 cells (panels D-F) or by bEnd.3 cells (panels G-I) was associated with marked alterations in both the shape and intercellular contacts of the BAE cells. Despite the initial plating of equal numbers of cells that exhibited a uniform adhesion over the entire surface of the dishes (e.g., panel A), the BAE cells subsequently exposed to End cell conditioned media displayed areas of irregular density. Stellate or elongate cells were prevalent in areas of sparse density (panels D, G, and H, thin arrows), whereas foci of rounded cells were associated with higher cell densities (panels E, F, and H, open arrows). Coalescence of the foci into aggregates (panel I, thick arrows) occurred at a frequency that increased with time in coculture. The cocultures shown in Fig. 1 were photographed 7.5 h after exposure of BAE cells to End cell conditioned media; the changes seen after an additional 20.5 h were similar, with more areas of clearing (e.g., as seen in panel D) and increased numbers of foci (data not shown). More extreme changes in morphology were observed in coculture experiments in which Transwell inserts were used for the BAE cells; in this case, BAE cells, from the time of plating, were exposed constantly to End cell conditioned media (data not shown). Homotypic cocultures of BAE, eEnd.2, and bEnd.3 cells showed no alterations in morphology (panels A-C), even after extended times in culture. BAE cells are known to exhibit contact inhibition at confluence and do not increase in density as a function of time in culture (Schwartz 1978). Although these initial experiments indicated that BAE cells might react to End cells via changes in cell motility or growth as a consequence of altered cell-cell or cell-matrix interactions, a quantitative assessment of cell proliferation in this system was clearly necessary. As the effects of conditioned medium on cells can be primary or derivative, and cellular responses complex and interrelated, I chose to restrict investigation of the phenomenon shown in Fig. 1 to two questions: (i) do End cells affect the rate of proliferation of BAE cells, and (ii) do End cells secrete a protein(s) that can be shown to modulate growth or cell shape? It was reasonable to include both End lines in subsequent experiments, as they were derived from different sources of middle T-expressing murine endothelial cells. In addition, an established line of murine lung capillary endothelial cells, LE 11, was used to control for differences in both the species and vascular bed of origin. A series of experiments was performed to assess the potential effects of End cell conditioned media on the proliferation of BAE and LE I1 cells. The results are represented by the experiment shown in Fig. 2. Compared to growth in homologous conditioned medium over a period of 72 h, eEnd.2 and bEnd.3 conditioned media repressed the growth of BAE cells by 35 and 40%, respectively. In contrast, a positive response was observed with LE I1 cells. which showed respective increases of 28 and 40% in the presence of eEnd.2 and bEnd.3 conditioned media. In 16 independent experiments, I deliberately varied the culture conditions, concentration of FCS, plating density, time of

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FIG. 2. Conditioned media from eEnd.2 and bEnd.3 cells diminish the proliferation of normal aortic endothelial cells but increase that of an established line of capillary endothelial cells. BAE and LE I1 cells were plated at a density of 20 000 cells/ (mL well) and grown in media (DMEM containing 2% FCS) conditioned for 52 h by BAE, LE 11, eEnd.2, or bEnd.3 cells. Media were changed daily; 72 h after the initial plating, cells were released by trypsin and counted by hemocytometer. Initial losses of cells due to differences in adhesion in the various conditioned media were negligible. The bars show total cell number at 72 h and represent the mean of triplicate wells + SEM. CM, conditioned medium. Compared with growth in their respective homologous CM, BAE cells exhibited decreases in cell number, whereas LE I1 cells showed increases in cell number, when cultured in CM from End cells. Differences between End cells cultured in the same CM were not significant.

exposure to conditioned media, and method of measurement of cell proliferation, and obtained results as shown in Fig. 2. Effects observed with bEnd.3 conditioned media were generally more extreme than those seen with eEnd.2 media. The degree of inhibition or stimulation of growth, however, was clearly influenced by the procedure used for the production of conditioned media. Although the duration of conditioning (between 24 and 72 h) was itself not significant, the degree of cell confluence was critical; more inhibitory activity with respect to BAE cells was recovered when the conditioned medium was removed from subconfluent cultures of End cells. The presence of FCS during the conditioning period, or added to DMEM conditioned in the absence of FCS, did not alter the growth response appreciably. However, it was important to use an homologous conditioned medium, rather than fresh DMEM, to achieve a control level of proliferation for BAE and LE I1 cells, as depletion of nutrients invariably occurs during the process of conditioning. Despite the diminution in growth, BAE cells did not appear necrotic in End cell conditioned media.

End cells do not alter the secretory phenotype of BAE or LE II cells Although changes in BAE cell morphology were apparent within 12 h after their exposure to End cell conditioned

ited nonuniform densities and a range of altered cell shapes (from stellate to rounded, as indicated by the thin black arrows in panels D. G, and H). In panel D, the edge of the cloning ring is on the right and can be seen as an opaque image at the top of panels G and H. In areas of higher cell density, BAE cells formed apparent clusters consisting of both round and polygonal cells (open arrows). Spherical clumps of BAE cells were observed in those fields containing large numbers of closely apposed cells (panel I, thick black arrows). Bar = 100 pm.


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FIG. 3. Biosynthesis of secreted proteins by aortic and capillary endothelial cells cocultured with eEnd.2 and bEnd.3 cells. (A) BAE cells were cocultured with eEnd.2 or bEnd.3 cells for 72 h and were subsequently reisolated with cloning rings and incubated with [3~lproline in DMEM for an additional 23 h. Radiolabeled culture media proteins from equal numbers of cells were resolved by SDSPAGE on 4-8% composite gels in the presence of 50 mM DTT and were visualized by autoradiography. Protein molecular mass standards are indicated on the left of each gel. Proteins secreted by BAE cells cocultured with BAE cells (control, lane I), eEnd.2 cells (lane 4), or bEnd.3 cells (lane 5) are shown. Proteins secreted by eEnd.2 cells cocultured with eEnd.2 cells (lane 2) and by bEnd.3 cells cocultured with bEnd.3 cells (lane 3) are included as controls. Radiolabeled components synthesized by BAE cells include fibronectin (FN), type I11 procollagen (pro a1 (111)), thrombospondin (TSP), and SPARC (SP). Products of End cells include laminin (LM), type IV procollagen (pro a1 (IV) and pro a2 (IV)), and a component of M, 165 000 (indicated by the open arrow). Identical results were obtained when BAE cells were radiolabeled in DMEM that had been previously conditioned by End cells. (B) Proteins secreted by LE I1 cells radiolabeled in DMEM (lane 1, control), cultured in serum-free DMEM conditioned 24 h by either eEnd.2 cells (lane 2) or bEnd.3 cells (lane 3). The products shown, which include fibronectin (FN)and type I procollagen (pro or1 (I) and pro or2 (I)), were resolved as described in A and represent equal numbers of cells. There were no apparent changes in the biosynthetic profile of BAE cells or LE I1 cells as a result of coculture in eEnd.2 or bEnd.3 cell conditioned media.

media, it was nevertheless formally possible that changes in the synthesis of secreted protein by BAE cells could be responsible in part for the observed modulation. BAE cells that had been cocultured with eEnd.2 or bEnd.3 cells were, therefore, reisolated and incubated with [3~]prolinein fresh DMEM or in DMEM previously conditioned by End cells. Analysis of the proteins secreted into the culture media is shown in Fig. 3A. The major secretory products of BAE cells (fibronectin, type 111 procollagen, thrombospondin, and SPARC) were present in similar proportions in media from cells cocultured with BAE, eEnd.2, or bEnd.3 cells (cf. lanes 1, 4, and 5). Among four independent experiments, there were minor increases in the levels of SPARC (e.g., lane 1 versus lane 5), despite the equal number of BAE cells plated prior to coculture. The induction of foci in BAE monolayers by End cell conditioned medium could be associated with increases in SPARC, based on results obtained with BAE cells undergoing angiogenesis in vitro (Iruela-Arispe et al. 1991b, 1991~).However, changes in the specific activities of secreted proteins cannot be excluded in these experiments. Microvascular LE I1 cells displayed a profile different from that of the aortic endothelial cells. As shown in Fig. 3B, type I procollagen and fibronectin were the prin-

cipal secreted products (lane 1). Digestion with pepsin, followed by differential fractionation in NaCl and SDS-PAGE with or without DTT, confirmed the identity of this collagen type (data not shown). However, in the presence of eEnd.2 or bEnd.3 conditioned medium, there also appeared to be no change in the secretory profile of LE I1 cells (lanes 2 and 3). Also shown in Fig. 3A is the secretory phenotype of eEnd.2 and bEnd.3 cells (lanes 2 and 3). Based on data from immunoblots (not shown), the identities of a B chain of laminin and of SPARC were confirmed. Type IV procollagen was identified by its position of migration on SDS-PAGE with or without DTT, prior and subsequent to treatment with pepsin (data not shown). Since differences in the biosynthesis of secreted proteins by BAE or LE 11 cells cultured in the presence of End cell conditioned medium were not observed, I focused on the products elaborated by End cells as potential candidates that might participate in the modulation of normal endothelial cell cycle and morphology. The comparatively high level of SPARC protein associated with the cells was particularly intriguing, as well as the presence of a component of M, 165 000, the identity of which was not immediately obvious (shown in Fig. 3A as a light band of a slightly greater mobility than thrombospondin and indicated by the open arrow).

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, pro a1 (IV) pro a2 (IV)

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FIG. 4. Resolution of End cell proteins by chromatography on DEAE-cellulose and by SDS-PAGE. eEnd.2 and bEnd.3 cells were incubated 24 h in serum-free DMEM containing ascorbate, 8-aminopropionitrile fumarate, and [3~]proline as described in Materials and methods. Radiolabeled culture media proteins were fractionated by precipitation in 50% ammonium sulfate, followed by chromatography on DEAE-cellulose equilibrated in a 4 M urea - 50 mM Tris-HCI buffer (pH 8.0). Unbound material is indicated as peak I; protein peaks eluted within a gradient of 0-200 mM NaCl are designated as 11-V. (A) A representative chromatogram is shown for eEnd.2 culture media. (B) SDS-PAGE of peak fractions I-V from DEAE-cellulose chromatography of eEnd.2 and bEnd.3 radiolabeled culture medium protein. Samples were both reduced and unreduced (with or without DTT) (not all combinations are shown), and proteins were subsequently visualized by fluorescent autoradiography. Laminin (LM), type IV procollagen (pro a 1 (IV) and pro a 2 (IV)), and SPARC (SP) are identified; the open arrow designates a protein biochemically similar to entactin (M, 165 000) (see Fig. 5). Protein molecular mass standards are shown on the left. df, dye front. Two separate gels are shown.

Identification of proteins secreted by eEnd.2 and bEnd.3 cells Radiolabeled proteins, secreted by End cells into the culture medium over an interval of 24 h, were separated by chromatography on DEAE-cellulose and further analyzed

by SDS-PAGE. Figure 4A is a chromatographic profile of eEnd.2 protein; a similar elution profile was obtained for culture medium protein from bEnd.3 cells (data not shown). Proteins in the peaks identified by Roman numerals in Fig. 4A are shown on SDS-polyacrylamide gels, after


586


resolution in the presence or absence of DTT (Fig. 4B). The mobilities of relevant proteins (e.g., fractions IV and V) have been shown before and after reduction for both cell lines. The other fractions are shown either reduced or unreduced (Fig. 4B). Type IV procollagen characteristically did not bind to the DEAE-cellulose (End.2 and End.3, lanes I). Laminin (as confirmed by immunoblot) was eluted more or less throughout the gradient (lanes 11-V, some chains also present in the corresponding unreduced lanes). SPARC was eluted at the characteristic conductivity (150-175 mM NaCl at pH 8.0) and displayed a shift in mobility in the presence of DTT (from Mr 39 000 to M, 43 000) (lanes V). A component of reduced apparent Mr 165 000, which displayed a slightly lower mobility prior to reduction, was purified essentially to radiochemical homogeneity (lanes IV and V with or without DTT, designated by open arrows). Initially, this component was thought to be a chain of thrombospondin, possibly a truncated form as described by Good et al. (1990). Since thrombospondin has recently been shown to be an inhibitor of angiogenesis in vivo (Rastinejad et al. 1989) and in vitro (Iruela-Aripse et al. 1991a),it was important to confirm the identity of the M, 165 000 protein produced by the End cells. A summary of several of these experiments is shown in Fig. 5. Digestion of a fraction containing the M, 165 000 component and SPARC (which served as an internal control) with bacterial collagenase confirmed the absence of collagenous sequences (Fig. 5, lanes 1 and 2). Although both chymotryptic digests and immunoblots with antithrombospondin and anti-laminin IgGs did not support an identity of the M, 165 000 protein with thrombospondin or laminin, chromatography on heparin-Sepharose was also performed as a further attempt to distinguish among these proteins. As shown in lanes 3 and 4, both the Mr 165 000 protein and SPARC failed to bind to heparin, although laminin was retained. The identity of the M, 165 000 protein was subsequently made apparent by the positive reactivity of three out of four different preparations of antisera against murine entactin, a basement membrane glycoprotein secreted by both transformed and normal cells (shown with one anti-entactin antiserum in lane 5) (Chung and Durkin 1990). Since laminin, type IV collagen, and entactin are wellestablished components of basement membranes that generally facilitate the attachment and spreading of epithelial cells, their role as End cell products in the modulation of BAE cell growth and promotion of a rounded morphology seemed questionable. Alternatively, SPARC has been shown to interfere with cell-matrix interactions, change cell shape, and retard cell cycle progression in BAE cells (Sage and Bornstein 1991). I therefore performed three separate experiments to probe the potential role of SPARC in mediating interactions between End cells and BAE or LE I1 cells.

End cell SPARC is responsible in part for the changes in morphology and the inhibition of growth of BAE cells I first asked whether SPARC would act as an antispreading factor for End cells. Previously it had not been our experience that exogenous SPARC influenced the shape of transformed cells, a result that was unrelated to the levels of endogenous SPARC secreted by these cells (Sage et al. 1989b). In several experiments, I was unable to show rounding of eEnd.2 or bEnd.3 cells cultured in the presence of exogenous murine SPARC. In Fig. 6, high levels of SPARC

I r e

FIG. 5. Characterization of the M, 165 000 protein secreted by eEnd.2 and bEnd.3 cells. Protein from eEnd.2 culture medium was fractionated on DEAE-cellulose (e.g., peak V in Fig. 4B) and subjected to bacterial collagenase (lanes 1 and 2). chromatography on heparin-Sepharose (lanes 3 and 4), and immunoblotting with anti-entactin antibodies (lane 5). Fractions containing a small amount of laminin (LM), the M, 165 000 protein (open arrow), and SPARC (SP) were resolved on an 8% SDS-polyacrylamide gel in the presence of 50 mM DTT, and proteins were visualized by fluorescent autoradiography. After incubation 1 h with collagenase (lane 2) or buffer alone (lane l), there was no apparent change in either the Mr 165 000 protein or SPARC. Neither protein bound to heparin-Sepharose (lane 3), but laminin was subsequently eluted with 250 mM NaCl (lane 4). Immunoblotting with anti-entactin antibodies revealed a positive reaction with the Mr 165 000 protein (lane 5).

were used (the EDso for SPARC to inhibit spreading of BAE cells is 20 pg/mL (Sage et al. 19896)) that elicited changes in shape of nearly confluent BAE cells within 4 h (cf. panel A with B). In contrast, neither eEnd.2 cells (panels C and D) nor bEnd.3 cells (panels E and F) demonstrated obvious changes in morphology, even after 26 h (not shown). These results were confirmed with at least three different preparations of SPARC. In similar experiments, LE I1 cells were also found to be unresponsive to SPARC (data not shown). As shown in Fig. 2, End conditioned media had an inhibitory effect on the proliferation of BAE cells, but a stimulatory one on that of LE I1 cells. If SPARC in conditioned media were an active participant in the modulation of LE I1 cell growth, it follows that exogenous SPARC, tested directly on LE I1 cells in a proliferation assay, should also stimulate their growth. The data shown in Fig. 7 do not support this prediction. Whereas SPARC (10 or 20 pg/mL) clearly inhibited the incorporation of [3~]thymidineby BAE cells, there was a minimal effect observed with LE I1 cells. We have shown that SPARC pep-


SAGE

587

FIG.6. SPARC does not function as an anti-spreading factor for End cells. BAE cells (A,B), eEnd.2 cells (C,D), and bEnd.3 cells E.F) were pIated in DMEM containing 2.5% FCS and 46.6 ~ g / r n LSPARC (B,D,F) or an equivalent volume of PBS (A,C,E). Cultures were photographed 4 h later. eEnd.2 (D)and bEnd.3 cells ( f ) exhibited no apparent changes in shape, attachment, or spreading, compared with control cultures (C and E, respectiveIy), whereas B h E cells were retracted and displayed morphologies (B) that were altered from those of controI cultures (A). Bar = 100 pm.

tide 2.1 (EDSo of 0.4 mM) is a potent inhibitor of [3~]thymidine incorporation in BAE cells (Funk and Sage 1991) and that a shorter form of this peptide, 2. la, is at least equally effective (Funk and Sage 1992). When peptide 2. l a was added to cells at a concentration of 0.8 mM, there was no significant change in the level of [3~]thymidine incorporated by LE I1 cells, although that of BAE cells was markedly suppressed. It therefore seemed unlikely that

SPARC was responsible for the stimulatory effect of End cell conditioned media on the proliferation of LE I1 cells. This result was not surprising, given the high rate of cell division and apparent immortality of this cell line. In contrast, the repression of [3~]thymidineincorporation by BAE cells cultured in the presence of SPARC was consistent with a functional role for this protein, as an End cell product, in mediating the growth of normal endothelial cells.

BIOCHEM. CELL

588

BIOL. VOL. 70,

BAE


LE l l

PBS

0.8mM 2.la

SPARC (1)

SPARC (2)

FIG. 7. Effect of SPARC and SPARC peptide 2.la on incorporation of [3~]thymidine by aortic (BAE) and capillary (LE 11) endothelial cells. BAE and LE I1 cells were released with trypsin and plated in DMEM containing 2.5070 FCS and [3~]thymidine. Media were further supplemented with SPARC (approximately 10 or 20 pg/mL) (SPARC 1 and 2, respectively), SPARC peptide 2.la (final concentration of 0.8 mM), or equivalent volumes of PBS. After 24 h, cells were washed with cold PBS, fixed in 10% trichloroacetic acid, and solubilized in 0.4 NaOH. Bars represent mean values derived from triplicate wells (+ SD). Whereas BAE cells (lightly hatched bars) exhibited a decrease in the incorporation of [3~]thymidine in the presence of SPARC and peptide 2.la, LE I1 cells (darkly hatched bars) were unaffected by peptide 2.la and showed minimal diminishment in [3~]thymidine incorporation in the presence of SPARC.

This point was addressed by attempting to correct the growth-inhibitory properties of End cell conditioned media on BAE cells by the inclusion of anti-SPARC antibodies in the proliferation assays. Initially the quantities of antibodies to be used were estimated from the values of SPARC in eEnd.2 and bEnd.3 conditioned media, as determined by radioimmune assay. These values were nearly identical between eEnd.2 and bEnd.3 cells and ranged from 0.61 to 1.09 pg/(106 cells. mL serum-free conditioned medium) (we have shown that the inhibitory effects of SPARC on spreading and the cell cycle require a minimum of 1-2 pg SPARC/mL (Funk and Sage 1991)). Growth assays were generally performed in 0.25-0.5 mL conditioned medium, with the addition of fresh, heat-inactivated FCS immediately prior to the experiment. Under these conditions, cell lysis was not observed. Data from these experiments are shown in Fig. 8. Antibodies against SPARC or SPARC peptide 1.1 were associated with elevated levels of [3~]thymidine incorporated by BAE cells cocultured on insert wells with eEnd.2 cells (Fig. 8A). The level of incorporation achieved in eEnd.2 conditioned medium alone has been plotted as 100%. An increase in cell number of 74 and 43% was found in the presence of anti-peptide 1.1 and anti-SPARC IgG, respectively. Similar results were obtained with these antibodies on BAE cells cultured in eEnd.3 conditioned medium (data not shown), although the anti-SPARC peptide 2.1 IgG produced a correction only 20% greater than the control value (Fig. 8B). However, it is known that this antibody has a relatively low affinity for native SPARC (Lane and Sage 1990). In a parallel experiment, the percent inhibition of BAE cell proliferation in medium conditioned by eEnd.2 or bEnd.3 cells was 58 or 60070, respectively (means of six

1992

wells per sample after 3 days) (data not shown). The extent of reversal of the growth-inhibitory effect of eEnd.2 cell conditioned medium on BAE cells was nearly complete (within error), but the correction of bEnd.3 conditioned medium was only partial, even when other anti-SPARC antibodies were used (data not shown). The anti-SPARC and antiSPARC peptide antibodies in DMEM were associated with negligible increases in BAE cell proliferation or [3~]thymidineincorporation. In contrast, the anti-peptide 2.1 antibody was associated with a decrease in the levels of [3~]thymidineincorporated by LE I1 cells (Fig. 8C). Controls included the addition of preimmune rabbit IgG and a preparation of neutralizing antibodies against TGF-0. With the latter reagent, there was a slight inhibition of [3~]thymidine incorporated by both cell types (Figs. 8B and 8C).

Discussion It had originally been assumed that the hemangiomas induced in transgenic and chimeric mice by the polyoma virus middle T oncogene had arisen by proliferation of endothelial cells (Bautch et al. 1987; Williams et al. 1988). However, Williams et al. (1989) recently demonstrated that significant proportions of these tumors induced in young mice consisted of nonproliferating endothelial cells that were derived from the host, and consequently proposed that endothelioma cells bearing middle T induced vascular tumors in a paracrine manner by the recruitment of normal endothelium. Since the inducing activity was not diffusible and appeared to act over a limited distance, these investigators also suggested that the putative factor was associated with cell membranes or with components of the extracellular matrix. It was in part this suggestion that prompted investigation of the properties of SPARC on cocultures of normal and middle T-expressing endothelial cells, as SPARC is a secreted glycoprotein that binds to collagen types produced by endothelial cells, but does not itself become incorporated into their matrix (Sage 1984; Sage et al. 1989b; Lane and Sage 1990). As part of this study, the secretory phenotype in vitro of two independently derived lines of End cells was defined. Hemangioendothelioma cells in culture (i) form tubular or cystic structures when plated in plasma clots (Mulliken et al. 1981), in fibrin gels (Montesano et al. 1990a), or on a reconstituted gel of basement membrane components (Obeso et al. 1990); (ii) produce the endothelial products such as von Willebrand protein (Williams et al. 1989; Montesano et al. 1990a; Obeso et al. 1990), angiotensin-converting enzyme (Obeso et al. 1990), and surface receptors for acetylated low density lipoprotein (Obeso et al. 1990); (iii) secrete low to undetectable levels of known endothelial mitogens and (or) angiogenesis factors (Williams et al. 1989); (iv) are thrombogenic and produce low levels of prostacyclin (Hoak et al. 1971; Fry et al. 1980); and (v) generate tumors upon reintroduction into host animals (Hoak et al. 1971; Williams et al. 1989). In agreement with a previous study on another hemangioendotheliomacell line (Sage and Bornstein 1982), both End cell lines synthesized the basement membrane collagen type IV and did not secrete either fibronectin or thrombospondin at levels detectable in these experiments. Other major products produced by End cells included laminin and entactin (also known as nidogen).


SAGE

FIG. 8. Anti-SPARC IgG partially reverses the End cell mediated growth inhibition of BAE but not LE I1 cells. (A) BAE cells were cocultured on insert wells above eEnd.2 cells in DMEM containig 2% heat-inactivated FCS. Anti-SPARC IgG or antiSPARC peptide 1.1 IgG (anti-1.1) was added to the insert wells; 48 h later, BAE cells were released by trypsin and counted by hemocytometer. Bars represent the average of duplicate wells, and values are plotted as percent of the control value obtained for BAE cells in DMEM containing 2% heat-inactivated FCS in the absence of antibody (63).(B) BAE cells were plated in DMEM that was conditioned 48 h by bEnd.3 cells and were subsequently supplemented with 4% heat-inactivated FCS and normal rabbit IgG (IgG), anti-TGF-/3IgG, or anti-SPARC peptide 2.1 IgG (anti-2.1). Forty-eight hours later the cells were pulsed 2 h with [3~]thymidine and subsequently fixed in 10% trichloroacetic acid prior to scintillation counting. Bars represent the mean of quadruplicate wells and values are plotted as percent of control O ([3~]thymidine incorporated by cells in the absence of antibody). (C) LE I1 cells were treated identically to BAE cells as described in B; bars represent means of quadruplicate wells. In the presence of antibodies specific for SPARC or peptides derived from SPARC, BAE cells demonstrated a positive growth response in eEnd.2 and bEnd.3 conditioned media, whereas LE I1 cells exhibited a negative response. Antibodies against TGF-/3 were associated with a slight decrease in growth for both BAE and LE I1 cells.

These noncollagenous glycoproteins are ubiquitous components of basement membranes that contribute to the structural integrity of these matrices (reviewed in Beck et al. 1990; Chung and Durkin 1990) and serve as attachment and (or) adhesion molecules for a number of cell types (Chakravarti et al. 1990). Moreover, the extreme sensitivity of entactin to proteases is thought to facilitate the degradation of basement membranes that accompanies remodeling during metastasis and embryogenesis (Chung and Durkin 1990). Although it is not clear why End cells secrete rather significant levels of entactin, a recent study by Aumailley et al. (1991) has shown that these cells display enhanced adhesion on fragments of laminin and on laminidentactin, in comparison to untransformed endothelial cells that failed to spread on laminin/entactin. These studies are yet another example of the specific controls that can be exerted by the extracellular matrix on cells, and they emphasize the importance of cell-matrix interactions in the development of a given cellular phenotype (Kubota et al. 1988; Ingber and Folkman 1989a; Ingber 1990).

589

A fourth major biosynthetic product identified in End cells was SPARC. Although not an integral component of basement membranes per se (Mason et al. 1986b; Sage et al. 1989a), SPARC is associated in vivo with cells of tissues actively undergoing morphogenesis (somites), repair and remodeling (bone), steroidogenesis (testicular Leydig cells), and proliferation (Termine et al. 1981;Mason et al. 1986a, 1986b; Holland et al. 1987; Wewer et al. 1988; Sage et al. 1989a) (reviewed in Sage and Bornstein 1991). In vitro, the protein is expressed by a rather wide spectrum of cells that generally have been plated at low densities on plastic substrates (Sage and Bornstein 1991). Although there is a limited number of studies on the production of SPARC by virally transformed cells, the results with End cells are consistent with earlier reports in which SPARC was shown to be a constitutive product of several transformed cell lines (Sage et al. 1984; Mason et al. 1986a; Young et al. 1986; Howe et al. 1990). Mason et al. (1986a) demonstrated a reduction of SPARC mRNA in NIH-3T3 cells transformed by v-abl and v-src compared with steady-state levels in the parental strains, and similar results were obtained with fibroblasts after transformation in vitro by Kirsten-MSV and SV-40 (Colombo et al. 1991). Although a systematic study of SPARC production by endothelial cells before and after transformation by middle T oncogene was not performed, it was nevertheless interesting that SPARC was one of the principal proteins secreted by End cells in vitro. At the present time these findings cannot be extrapolated to the development of hemangiomas in vivo. However, examination of human carcinoma in situ revealed expression of SPARC in the cytoplasm and (or) associated with the basement membrane in 13 of 38 cases (Wewer et al. 1988), and a preliminary report has suggested that v-ki-ras-transformed fibroblasts producing elevated levels of SPARC are associated with increased tumorigenicity (Colombo et al. 1989). The ability of SPARC to modulate cell shape and to inhibit cell spreading is consistent with current data that implicate components of the extracellular matrix as direct coordinators of endothelial cell growth and differentiation (Jaye et al. 1985; Kubota et al. 1988; Risau and Lemmon 1988; Ingber and Folkman 1989a, 19896). It is in the context of SPARC as a protein that perturbs cell-matrix interactions that we envision its role in the vascular system, particularly with respect to angiogenesis (Sage and Bornstein 1991). Modifications of endothelial cell behavior that are pertinent to angiogenesis include the induction of migration or invasion, altered rates of proliferation, and changes in the expression of proteolytic activity and receptors for growth (morphogenic) factors (Madri et al. 1988; Pepper et al. 1990; Tsuboi et al. 1990). Since SPARC diminishes focal contacts in cultures of BAE cells (Murphy-Ullrich et al. 1991), induces the expression of PAI-1 (Hasselaar et al. 1991) and reduces that of fibronectin (Lane et al. 1992), and binds to several proteins of vascular extracellular matrices (Sage et al. 1989b), its secretion by a population of cells (e.g., eEnd.2 cells) could directly affect the morphology and subsequent behavior of neighboring, but different, cells (e.g., endothelial cells lining a vascular channel). Since confluent BAE cells, such as those that line vessel lumina, produce very low levels of SPARC, minimal effects on endothelial cell adhesion are expected to occur under normal conditions in vivo. From other studies (Everitt and Sage 1992), we have proposed that


590


a balance of opposing forces maintains cell spreading and that excessive levels of anti-adhesive proteins disrupt this equilibrium toward cell detachment (Sage and Bornstein 1991). The secretion of SPARC by End cells in the vicinity of confluent, quiescent normal endothelial cells would presumably effect a disruption of adhesive forces between cells and their extracellular matrix, which is similar to that seen when exogenous SPARC is added to normal endothelial cells in vitro. The identification of structural domains in SPARC by several groups (Engel et al. 1987; Bolander et al. 1988; Domenicucci et al. 1988; Villarreal et al. 1989) has facilitated functional studies in vitro with synthetic peptides (Lane and Sage 1990). SPARC and the cationic peptide 2.1 inhibited the uptake of [3~]thymidineby synchronized cultures of BAE cells and retarded the progression of the cell cycle within the first 12 h of GI (Funk and Sage 1991). The recently described sensitivity of capillary endothelial cells to PDGF (Smits et al. 1989) is relevant to the effect of SPARC on endothelial proliferation, as SPARC binds specifically to the B chain of PDGF and inhibits the binding of this mitogen to its cognate receptor on fibroblasts (Raines et al. 1992). During angiogenesis, SPARC could potentially function as a mediator of endothelial cell growth that facilitates the temporary withdrawal from the cell cycle necessary for cell migration or the acquisition of a differentiated phenotype. A second mitogen, bFGF, stimulates both the migration and proliferation of a wide spectrum of endothelial cells (Gospodarowicz et al. 1987). We have recently shown that its effects on BAE cells can be modulated by SPARC, although SPARC neither bound to bFGF nor interfered with the binding of bFGF to its high affinity receptor (Hasselaar and Sage 1992). SPARC inhibited both the migration and the incorporation of [3~]thymidinethat are induced by bFGF in these cells. However, the latter activity required a factor(s) present in serum. This result is consistent with our proposal that SPARC acts indirectly as a mediator of endothelial cell migration and proliferation, in the context of other extracellular components (Sage and Bornstein 1991; Raines et al. 1992; Hasselaar and Sage 1992). During wound repair, tumor growth, or scheduled remodeling (events in which serum components as well as bFGF are often present), SPARC might activate migration and concomitantly inhibit growth of endothelial cells. This mechanism is also consistent with the data presented in this communication: SPARC secreted by End cells inhibits [3~]thymidine incorporation by BAE cells and might possibly contribute to the recruitment of normal cells in the formation of hemangiomas as proposed by Williams et al. (1989). In this study I have shown that SPARC is at least partially responsible for the inhibitory effect of End cells on BAE cell proliferation. Some of the activities of End cell conditioned medium on endothelial cells described by Williams et a/. (1989) are in agreement with these data: SPARC does not act as a mitogen or an angiogenic factor and is at least equally effective in its ability to round fetal cells compared to normal cells. However, Williams et al. (1989) found essentially insignificant levels of growth inhibition when BAE cells were cultured in eEnd.2 cell conditioned medium and observed no morphological changes upon coculture of the two cell types. Furthermore, End cell conditioned medium did not stimulate growth of LE I1 cells,

a result different from that of the present study. Our explanation lies principally in the procedure used for collection of conditioned medium and the recovery of sufficient amounts of SPARC; SPARC is not produced by confluent cells in vitro and is diminished when cells are cultured in medium supplemented with serum (Sage et a/. 1984, 1989b). Concentration of medium also contributes to losses of SPARC, as the protein has a high affinity for most ultrafiltration membranes (E.H. Sage, unpublished observations). Our previously published studies have clearly shown that the activities of SPARC as an anti-spreading factor (Sage et al. 19896) or as a cell-cycle inhibitor (Funk and Sage 1991) are dose dependent with respect to target cells. The formation of cavernous hemangiomas by normal, nonproliferating endothelial cells is ostensibly different from angiogenesis. However, several of the cellular events required for these processes appear to be similar or perhaps identical. Montesano et al. (1990a) described the formation of cystic structures by End cells embedded in fibrin gels and the reversion of End cells to the formation of capillary-like tubes in the presence of protease inhibitors. A cyst might in fact be a tube that is poorly constrained by its extracellular matrix, and the excessive levels of PA produced by End cells could account for its compromised integrity. The production of PAI-1 therefore represents a critical level of control of both hemangioma and tube formation. End cell SPARC could induce PAI-1 in BAE (or host) cells, modulate the ratio between protease and antiproteases, and thereby affect host cell migration. The significance of certain extracellular matrix components to the process of hemangioma formation will not be fully answered until the action of the middle T oncogene on endothelial cell function is understood. Although a cellular homolog of middle T has not been described, the antigen forms a complex with pp60C'SrCor a src-related protein and is thought to modulate the activity of src-tyrosine kinases (Courtneidge and Smith 1983). This signaling pathway might be of major relevance in mediating the morphogenetic changes that precede or facilitate subversion of normal endothelial cell function. Acknowledgments I thank Dr. Werner Risau for providing End cell lines, stimulating discussions, and his helpful comments on the manuscript. Appreciation is due to Dr. R. Hallmann for his advice on End cells, Dr. A. Chung for anti-entactin antibodies, Dr. M. Kinsella for a strain of BAE cells, S. Funk for experiments with SPARC and SPARC peptides, and T. Lane for anti-SPARC antibodies. Special appreciation is due to Dr. L. Iruela-Arispe and T. Lane for their help in the identification of entactin, and Dr. J. Yost and E. Everitt for the SPARC radioimmune assays. I also thank B. Wood for her skillful word processing and her assistance with the manuscript. These studies were supported in part by National Institutes of Health grants HL03174 and GM40711. Aumailley, M., Timpl, R., and Risau, W. 1991. Differences in laminin fragment interactions of normal and transformed endothelial cells. Exp. Cell Res. 196: 177-183. Bautch, V.L., Toda, S., Hassell, J.A., and Hanahan, D. 1987. Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle T oncogene. Cell, 51: 529-538.


SAGE

Beck, K., Hunter, I., and Engel, J. 1990. Structure and function of laminin: anatomy of a multidomain glycoprotein. FASEB J. 4: 148-160. Bolander, M.E., Young, M.F., Fisher, L.W., et al. 1988. Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc. Natl. Acad. Sci. U.S.A. 85: 2919-2923. Chakravarti, S., Tam, M.F., and Chung, A.E. 1990. The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions. J. Biol. Chem. 265: 10 597 - 10 603. Chung, A.E., and Durkin, M.E. 1990. Entactin: structure and function. Am. J. Respir. Cell Mol. Biol. 3: 275-282. Colombo, M.P., Biondi, G., Galasso, D., et al. 1989. Osteonectin transcript and metastatic behavior of v-ki-ras transformed fibroblasts. Int. J. Cancer, (Suppl. 4): 76-77. Colombo, M.P., Ferrari, G., Biondi, G., et al. 1991. Downregulation of SPARC/osteonectin/BM-40 expression in methylcholanthrene-induced fibrosarcomas and in Kirsten-MSV transformed fibroblasts. Eur. J. Cancer, 27: 58-62. Courtneidge, S.A., and Smith, A.E. 1983. Polymoma virus transforming protein associates with the product of the c-src cellular gene. Nature (London), 303: 435-439. Domenicucci, C., Goldberg, H.A., Hofmann, T., et al. 1988. Characterization of porcine osteonectin extracted from foetal calvariae. Biochem. J. 253: 139- 151. Engel, J., Taylor, W., Paulsson, M., et al. 1987. Calcium-binding domains and calcium-inducedtransition in SPARC (osteonectin, BM-40), an extracellular glycoprotein expressed in mineralized and nonmineralized tissues. Biochemistry, 26: 6958-6965. Everitt, E.A., and Sage, E.H. 1992. Expression of SPARC is correlated with distinct morphologies in F9 murine embryonal carcinoma cells. Exp. Cell Res. 199: 134-146. Fry, G.C., Czervionke, R.L., Hoak, J.C., et al. 1980. Platelet adherence to cultured vascular cells: influence of prostacyclin (PGI,). Blood, 55: 271-275. Funk, S.E., and Sage, E.H. 1991. SPARC modulates cell cycle progression in bovine aortic endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 88: 2648-2652. Funk, S.E., and Sage, E.H. 1992. SPARC and cationic SPARC peptides differentially regulate DNA synthesis by fibroblasts and endothelial cells. J. Cell. Physiol. In press. Good, D.J., Polverini, P., Rastinejad, F., et al. 1990. A tumor suppressordependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. U.S. A. 87: 6624-6628. Gospodarowicz, D., Ferrara, N., Schweigerer. L.. and Neufeld, G. 1987. Structural characterization and biological functions of fibroblast growth factor. Endocr. Rev. 8: 95-114. Hasselaar, P., and Sage, E.H. 1992. SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells. J. Cell Biochem. 49: 272-283. Hasselaar, P., Loskutoff, D.J., Sawdey, M., and Sage, E.H. 1991. SPARC induces the expression of type I plasminogen activator inhibitor in cultured bovine aortic endothelial cells. J. Biol. Chem. 266: 13 178 - 13 184. Hoak, J.C., Warner, E.D., Cheng, H.P., et al. 1971. Hemangioma with thrombocytopenia and microangiopathic anemia (KasabachMerritt syndrome): an animal model. J. Lab. Clin. Med. 77: 941-950. Holland, P., Harper, S., McVey, J., and Hogan, B.L.M. 1987. In vivo expression of the mRNA for the Ca binding protein SPARC (osteonectin) revealed by in situ hybridization. J. Cell Biol. 105: 473-482. Howe, C.C., Kath, R., Mancianti, M.L., et al. 1990. Expression and structure of human SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some of these cells show an unusual expression pattern. Exp. Cell Res. 188: 185-191. +

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Thrombospondin-1 is produced by retinal glial cells and inhibits the growth of vascular endothelial cells.

Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T antigen.

Endothelial cells transformed by SV40 T antigen cause Kaposi's sarcomalike tumors in nude mice.

MiR-302c inhibits tumor growth of hepatocellular carcinoma by suppressing the endothelial-mesenchymal transition of endothelial cells.

Characterization of endothelin secretion by vascular endothelial cells.

Purification of polyoma T antigen from transformed cells.

Astrocyte growth stimulation by a soluble factor produced by cerebral endothelial cells in vitro.

Regulation of the migration of endothelial cells by a gradient density of vascular endothelial growth factor.

Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.

Shaping of Peripheral T Cell Responses by Lymphatic Endothelial Cells.

Inhibition of in vitro T cell activation by corneal endothelial cells.

Delphinidin Inhibits Tumor Growth by Acting on VEGF Signalling in Endothelial Cells.

SPARC expression by cerebral microvascular endothelial cells in vitro and its influence on blood-brain barrier properties.

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation.

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro.

Oncogene alterations in in vitro transformed rat tracheal epithelial cells.

Stimulus-secretion coupling in vascular endothelial cells.

Induction of transformational changes in normal endothelial cells by cultured human astrocytoma cells.

Photodynamic treatment of normal endothelial cells or glioma cells in vitro.

Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyoma virus.

Differentiation of human hair follicle stem cells into endothelial cells induced by vascular endothelial and basic fibroblast growth factors.

Virus-secific transcription in 3T3 cells transformed by the ts-a mutant of polyoma virus.

Production of prostacyclin by vascular endothelial cells.