JOURNAL OF CELLULAR PHYSIOLOGY 142117-128 (19901

Transforming Growth Factor Beta, Modulates Extracellular Matrix Organization and Cell-Cell Junctional Complex Formation During In Vitro Angiogenesis JUNERAE MERWIN*, JAMESM. ANDERSON, OLlVlER KOCHER, CHRISTINA M. VAN ITALLIE, AND JOSEPHA. MADRI Departments of Palhology i) R M I 0 K , 1 A M i and Internal Medicine (1 M A , C M V Ij, Yale School of Medicine, New Haven, Connectirut 065 10 Transforming growth factor-beta, (TGF-P,) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-Pi; however, in three-dimensional (3-1)) collagen gels TGF-P, is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-P, treated cultures. In 2-D cultures TGF-Pi i s known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-P, treated cells as established by ELSA te5ting for type IV collagen, fibroneclin, and laminin. In 3-D cultures there i s increased 5ynthesis and secretion of type V collagen in both control and TGF-P, treated cultures over 2-D cultures. Even though an equal amount of type V collagen i s seen in both 3-D conditions, there i s a reorganization of the protein with concentration along an organizing basal laniina in TGF-P, treated cultures. EM morphological analyses on 3-D cullures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-P, treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-P, treated cells also appear to form junctional complexes between adjoining cells. lmmunofluorescence using specific antibodies to the tight junction protein LO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the LO-1 tight junction protein, reveal the presence of the 7.8 kb mKNA. Western blots of rat epididymal fat pad endothelial cells (KFC) monolayer lysates probed with anti-LO-1 label a 220 kd band which co-migrates with the bonafide ZO-1 protein. These data confirm and support the hypothesis that TGF-b, is angiogenic in vitro, eliciting microvascular endothelial cells to form tube-like structures with apparent tight junctions and abluminal basal lamina deposition in three-dimensional cultures.

Angiogenesis (the formation of new blood vessels) is a complex process involving endothelial cell activation, migration, and proliferation as well as matrix synthesis and differentiation, multicellular organization, sta-

bilization of newly formed vessels, and down-regulation of local endothelial cell populations (Folkman, 1982; Madri and Pratt, 1987). The association between angiogenesis, tumor growth, inflammation, and tissue repair is well documented (Madri and Pratt, 1987). Transforming growth factor-betas (TGF-ps) are polypeptides that act hormonally to regulate differentiation and proliferation of a variety of cell types depending on the environment (see reviews Sporn et al., 1987; Roberts and Sporn, 1988). Presently, there are six known TGF-f.3 homologs: TGF-P,, TGF-P,, TGF-

Cj 1990 WILEY-LISS, INC.

P1 1 , TGF-P,, TGF-P,, and TGF-P,. TGF-8, and TGFP5, the most recently discovered isoforms of the TGF-(3 family, have been cloned (Roberts and Sporn, 1989) and preliminary investigations are beginning. TGF-P, is of mesoderm origin and has been identified by cDNA characterization (Derynck et al., 1988). TGF-P, and TGF-P, are both platelet-derived homodimers, while TGF-PI, (Massague, 1987) results from a heterodimeric combination of a subunit of TGF-6, and TGF-P,. TGF-P, (Assoian et al., 1983), the polypeptide originally described a s TGF-P, exhibits a 70% amino Received May 31, 1989; accepted September 5, 1989.

*To whom reprint requestsicorrespondence should be addressed.

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acid N-terminal identity with TGF-P, (Marquardt et al., 1987). Current work in our lab is focused on differential vascular cell responses t o TGF-P, and TGF-P, (Merwin et al., in preparation). Transforming growth factor-beta, (TGF-P,), a 25 kd disulfide-linked homodimer originally characterized based on its ability to promote the anchorage independent growth of mesenchymal cells, has been shown to be angiogenic in vivo and in vitro (Roberts et al., 1986; Madri et al., 1988a). In vitro angiogenesis is defined in this manuscript as the organization of microvascular endothelial cells into tubular structures. However, it is now known that TGF-P, is multifunctional (Sporn and Roberts, 1986) and has diverse effects on cells depending on the environment; cell type, extracellular matrix (ECM), and other growth factors present. TGF-P, alters proliferation of many cell types, acting either a s a n inhibitor (Heimark et al., 1986; Ristow, 1986) or enhancer (Hill et al., 1986) of cell growth rate. In addition TGF-P, influences processes such as osteogenesis (Centralla et al., 1986), chondrogenesis (Seyedin et al., 19851, adipogenesis (Ignotz and Massague, 19851, myogenesis (Florini et al., 1986; Olsen et al., 19861, and epithelial cell differentiation (Masui et al., 1986). It also stimulates increased expression of fibronectin (Fn) and type I collagen (Ignotz and Massague, 1986; Varga and Jimenez, 1986; Sporn et al., 1983). TGF-P, is a member of a family of homologous polypeptides including various inhibins (Mason et al., 1985), activins (Vale et al., 1986), Mullerian inhibiting substance (Cate et al., 1986), and decapentaplegic gene complex of the Drosophila (Padgett et al., 1987). There is 25-35% amino acid sequence shared within the bioactive domains of these peptides. Endothelial cells of the various vascular beds exhibit a broad range of diverse functions and appearances along with their shared features of nonthrombogenicity, polarity, and metabolic and transport functions (Madri, 1987). Neovascularization is a common response t o injury, tumors, and a variety of growth factors, yet it varies depending on whether the endothelium is derived from large vessels or microvasculature. Large vessel endothelial cells form a flat sheet lining the vessel and responding to injury by sheet migration and proliferation, whereas microvascular endothelial cells express a significant arc of curvature necessary for the lumen formation by a single endothelial cell and respond to injury by migration into the three-dimensional surroundings followed by tube formation (Madri et al., 198813). In this study, microvascular endothelial cells were cultured in both two- and three-dimensional environments to examine the effects of TGF-PI treatment. Changes in ECM organization (but not ECM accumulation) and formation of cell-cell junctional complexes were documented during TGF-P1 induced in vitro angiogenesis. “Activation,” as seen in our system, is defined as increased activity assessed by enlarged endoplasmic reticulum, increased numbers of ribosomes and vacuoles, extensive pseudopods, multicellular aggregations with apparent junctional complexes, increased contraction of surrounding collagen, and possible polarity shown by slit lumen-like formation apically and deposition of apparent ECM proteins basal-laterally. Our results support the concept that

responses to TGF-P, are modulated, in part, by the cell type studied and the culture conditions,

METHODS AND MATERIALS Cells Microvascular endothelial cells (RFCs) were isolated and cultured from the epididymal fat pads of SpragueDawley rats as described (Madri and Williams, 1983). Culture Two-dimensional cultures of RFCs were grown on 1.5%gelatin-coated tissue culture vessels in Dulbecco’s modified eagle’s medium (DMEM) mixed 4:l with sterile-filtered conditioned bovine aortic endothelial cell media as described (Madri and Williams, 1983) containing 10% heat inactivated fetal calf serum (FCS). Three-dimensional RFC cultures were made a s described (Madri et al., 1988a) from gels composed of acid-soluble calf dermis type I collagen (collagen I). The purity of the collagen was analyzed by ELISA, RIA, rotary shadowing, and SDS-PAGE (Roll et al., 1979; Madri and Furthmayr, 1979; Furthmayr and Madri, 1982). Briefly, the purified collagen was solubilized in 10 mM acetic acid a t a concentration of 5.0 mgiml and stored a t 4°C. A measured amount of the collagen with 1/10 volume of 10 x DMEM was neutralized with sterile 1 M NaOH and the solution kept on ice. Cultured RFCs were added to the collagen preparation to achieve a concentration of 106/ml; 0.5 ml of the cell/ collagen suspension was added to 12 mm Millicell-HA (Millipore Products Division, Bedford, MA) filter wells set in a Costar 24-well cluster tissue culture dish (Costar Corp., Cambridge, MA) and placed in a 37°C humidified 5% CO, incubator for 10 min to permit polymerization of the collagen. Following gel formation, 1.5 ml of medium (20.5 ngiml TGF-(3,) were added. Growth factors TGF-PI, prepared a s described (Assoian et al., 19831, was a generous gift of Drs. Anita Roberts and Michael Sporn, Laboratory of Chemoprevention, National Cancer Institute, NIH, Bethesda, Maryland. EGF was purchased from Collaborative Research (Lexington, MA) and used without further purification. Recombinant bFGF was a generous gift of Drs. Michael Klagsbrun and Judah Folkman, Harvard University, Boston, Massachusetts.

Antibodies Antibodies (Abs) to ZO-1 (a specific, internal peripheral tight junction protein) were prepared and characterized as described (Stevenson et al., 1986). Affinitypurified Abs to human plasma fibronectin (Fn), human placental membrane collagen V, murine EngelbrethHolm-Swarm laminin (Ln), and collagen IV were produced a s described along with determination of crossreactivity profiles (Roll et al., 1980; Madri and Furthmayr, 1980). Anti-Factor VIII was purchased from Calbiochem-Behring (LaJolla, CA). Proliferation assay Cell number. Collagen I coated bacteriologic culture dishes were washed in PBS before the addition of cell suspensions (1.4 x lo5 cellddish). After 6 h , samples

TGF-PI MODULATES CELL-CELL AND ECM ORGANIZATION

were counted to determine starting cell numbers. At this time fresh medium kTGF-P, was added to the cultures and replaced again on the third day. Cell numbers were determined after trypsinization of quadruplicate samples using a Coulter Counter (Coulter Electronics, Inc., Hialeah, FL). The mean number of cells per condition was then calculated. DNA. DNA was quantitated using DAPI (4',6"-diamidino-2-phenylindole: Hoechst No. 33258) in a fluorescence assay as described by Labarca and Paigen (1980). Amounts of DNA were related t o cell number by correlating p,g of DNA with cell numbers using serial dilutions of cells.

Elisa Quantitation of ECM proteins were analyzed using ELISAs a s described by Madri and Williams 11983). Immunofluorescence (IF) The procedure according to Let0 e t al. (1986) was modified a s follows: Monolayer cultures were fixed with MeOH, 10% normal buffered formalin, or the periodate-lysine-paraformaldehyde (PLP) method of McLean and Nakane (1974). The samples were rinsed 3 times with 1 x PBS (PBS), treated with 0.2% TritonXlOO in PBS for 20 min, and washed exhaustively. They were incubated overnight in 4°C with PBS containing 3%)BSA (3%PBSA). After rinsing 3 times with 1% PBSA, the primary Ab was applied to the sample for 40 min at 22"C, washed 3 times with 1%PBSA, and a fluorescein-conjugated goat-anti-rabbit secondary Ab (Cappel Laboratories, Malvern, PA) was incubated under the same conditions. For dual-label experiments actin stress fibers were detected with a philloidinrhodamine conjugate (Molecular Probes, Inc., Junction City, OR). The samples were examined under a Zeiss 14 binocular microscope (Carl Zeiss Inc., Oberkochen, West Germany) and photographs were taken on Ektachrome ASA 400 film (Eastman Kodak Co., Rochester, NY). Immunofluorescence (IF) of 3-D cell cultures was also performed. The collagen gels were rinsed three times with PBS, removed from the millicells, and quickly frozen in O.C.T. embedding compound (Miles Scientific Co., Kankakee, IL). Eight micron cryostat sections were placed on albumin-coated glass slides, acetone fixed for 1 min a t -2o"C, and air dried. The procedure continued as above, beginning with rinsing 3 times with 1%PBSA followed by primary Ab incubation. Electron microscopy Collagen gels were rinsed 3 times with PBS, treated for 2 h with formaldehyde-glutaraldehyde fix at 22"C, and placed in a 10% sodium cacodylate holding buffer until used (Karnovsky, 1965). The samples were then postfixed in 1%osmium tetroxide buffered with 0.2 M s-collidine for 1h a t 4°C. After rinsing 3 times with 0.1 M s-collidine, the samples were stained with uranyl acetateioxalic acid for 1 h at 4°C. Dehydration steps were 10-30 min, going from '70-100% ETOH, ending with 100% propylene oxide before embedding in Epon 812. Ultrathin sectioning was done on a LKB I11 8800 Ultramicrotome (LKB Produkter AB, Bromma, Sweden) after which 2% uranyl acetatellead citrate stain-

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ing was performed. The sections were viewed on a Zeiss EM 1OB electron microscope. Photographs were taken on Kodak film.

Immunoelectron microscopy The procedure was done a s described by Kashgarian et al. (1985). Briefly, collagen gels were rinsed 3 times with PBS and fixed with PLP for 30 min a t 4°C. They were then rinsed as above, removed from the millicell, cut in 1 mm blocks, treated with 10% DMSO for 10 min, fast frozen in freon 22 slurry, and stored in liquid nitrogen until used. Semi-thin sections were cut on a cryostat and incubated overnight at 4°C with the primary Ab in 1% PBSA. After 3 washes with 0.1% PBSA, 10 min a t 22"C, the secondary Ab (sheep anti-rabbit conjugated t o horseradish peroxidase, Biosys, Compiegne, France) was incubated with the samples for 2 h a t 22°C. Glutaraldehyde fixation, with 3 washes before and after, was done followed by diaminobenzidine reaction using H,O, a s the substrate. The reaction was stopped by four rinses with 50 mM Tris buffer containing 7.5% sucrose. The remaining steps followed the above EM procedures from postfixation with osmium tetroxide on. RNA extraction Three-dimensional cultures were homogenized in a solution containing 4.5 M guanidium thiocyanate, 50 mM EDTA, 25 mM sodium citrate, 0.1 M 2-p-mercaptoethanol, and 2% sodium-N-lauroylsarcosine using a polytron (Tissumizer, Tekmar, Cincinnati, OH), and further homogenized using a syringe with a 25-gauge needle. RNA was purified by ultracentrifugation through a cushion of 5.7 M CsC1, a s previously described by Chirgwin et al. (19791, and the RNA pellet was recovered as previously described by Kocher and Gabbiani (1987). Northern blot hybridization RNAs (5 kg per lane) were denatured with formaldehyde and electrophoresed in 1%agarose formaldehyde gels containing 0.5 p,g/ml of ethidium bromide, examined under UV light, and transferred to Biodyne filters (Pall Filter Co., Glen Cove, NY). After blotting, filters were baked for 2 h a t 80°C under vacuum. Northern blots were hybridized a s previously described by Kocher and Gabbiani (1987), using SP6-RNA polymerase transcribed ZO-1 cRNA probe according to the procedure described by Melton et al. (1984). The Northern blots were washed 2 times 20 min a t 58°C in 3 x SSC (SSC is 0.15 M NaC1, 0.015 M Na citrate, pH 7.0) and 2 x Denhardt's solution, and subsequently washed 3 times a t 75°C in 0.2 x SSC, 0.1% SDS, and 0.1% Na pyrophosphate. The filters were exposed to Kodak XOmat AR films at 70°C between intensifying screens. The films were analyzed using computerized densitometric scanning a s described by Kocher and Gabbiani (1987). ~

Western blots The procedure according to Towbin et al. (1979) was modified. Due to the nature of the three-dimensional collagen gels, several extraction methods had to be used in order to solubilize the collagen I. Various procedures included hot SDS, 4.6 M acetic acid, 6 M urea,

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RFC Proliferation

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Fig. 1. Proliferation assays. RFC proliferation (A). Two-dimensional cultures of microvascular endothelial cells cultured on collagen I were counted at 6 h, 3 days, and 5 days. Proliferative inhibition by TGF-(3, was dose dependent. There were no differences in proliferation from control when using the various concentrations of EGF and bFGF (0.05, 0.5 and 5.0 ngiml). RFC DNA analysis (B). Three-dimensional cultures of microvascular endothelial cells in collagen I gels were assayed for total DNA content, correlating kg of DNA with cell nuniber. There were no significant differences between control, bFGF, EGF, or TGF-p, treated cell cultures.

collagenase, homogenization and sonication. After centrifugation, the cell pellet was lysed by boiling in 1 x SDS loading buffer (Laemmli, 1970). The proteins were then subjected to SDS-PAGE electrophoresis and transferred to a nitrocellulose sheet. After overnight incubation with a blocking buffer consisting of 1 x TBS, 0.05% sodium azide, 0.2% Tween 20, and 3% nonfat dry milk, the nitrocellulose was rinsed with 10 mM Tris-HC1 buffer containing 0.210 Tween 20, incubated with the specific primary Ab diluted in 1 x TBS 1h a t 22"C, and rinsed. I t was then incubated with the secondary Ab, anti-rabbit IgG Fc conjugated to alkaline phosphate (Promega Co., Madison, WI) for 1h at 22"C, rinsed three times as above and one time with AP buffer (10 mM Tris-HC1 [pH 9.51, 100 mM NaC1, 5 mM MgC1,). The substrate (nitro blue tetrazolium and 5bromo-4-chloro-3 indolyl phosphate; Sigma Chemical Co., St. Louis, MO) was added as per directions by manufacturer, the samples placed on a shaker until bands appeared, and the reaction terminated with alkaline phosphate buffer (20 mM Tris-HCI, 5 mM EIITA, pH 6.8). RESULTS TGF-(3, effects on RFC proliferation The ability of TGF-P1 to elicit a n in vitro mitogenic response by microvascular endothelial cells was first

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Fig. 2. Immunofluorescence (IF).Top: One-week, three-dimensional RFC cultures with ( i ) and without (-1 TGF-p, treatment showing marked contraction of the gels treated with TGF-PI. Bottom: IF of 5-day, 3-D RFC cultures with and without TGF-(3, treatment. Unfixed frozen, acetone treated, 8 K r n sections probed with anti-rollagen type V showing equal intensities of staining but dramatic organization of the staining outlining tube-like structures in the TGF-p, treated cultures ( + ) compared to the single cell-associated staining in nontreated cultures ( - 1.

investigated using low passage (%fold increase of F n over control cultures (Madri et al., 1988a) with no increased accumulation of Ln, collagen IV or V. Furthermore, when RFCs were grown in a 3-D environment, there was no increase in any of the ECM proteins tested as determined by competitive ELISAs in 1-21day cell lysates and media fractions, but there was a n overall increase in collagen V in both control and TGFP1 treated cells in the 3-D system compared to 2-D cultures (Madri et al., 1988a). In this study we have investigated the matrix components’ localization and the formation of junctional complexes by RFCs in response to TGF-PI. Inhibition of proliferation by TGF-PI in many monolayer cell culture systems has been well documented (Heimark et al., 1986; Ristow, 1986). Since neovascu-

DISCUSSION Angiogenesis (the formation of new blood vessels) has a central role in development, repair, and tumor growth (Madri and Pratt, 1987). Although a great deal of research has been done in 2-D culture systems investigating microvascular endothelial cell behavior (Lobb et al., 19861, 3-Dcultures are felt to allow for in vitro conditions that more closely mimic the in vivo environment. We observed major differences in the proliferative response to TGF-P, depending on the composition of the ECM the cells are cultured on and the dimensionality of the culture (Madri et al., 1988a) and have in-

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Fig. 6. Structural analysis. Four-day, three-dimensional RFC, TGFPI treated cultures, TEM micrographs. a: Slit lumen formation sealed off by junctional complex (large arrows); engorged ER; increased vacuoles and ribosomes; deposition of ECM proteins (small arrows) expressed abluminally indicating cellular polarity. b: Abluminal aspect of the cell revealing organizing basal lamina (small arrows) and a cross-section of organized cytoskeletal fibers (arrowheads). Bar = 1 pm.

larization (as observed in vivo) requires proliferation, it was unclear if TGF-P, had a direct angiogenic effect on endothelial cells. The mechanism(s) of action for TGF-P, is not known and may, therefore, work either directly, where the presence of TGF-P, is all that is necessary to initiate a n endothelial angiogenic response or it could work through indirect mechanisms. These pathways may include stimulation of another local cell population which, in turn, activates endothelial cells or stimulation of endothelial cells in the presence of other factors necessary for the activity of TGFPI. Our results show that RFCs cultured in the presence of TGF-p, in a 3-D environment proliferate at a level equal to cultures treated with other known angiogenic factors and to non-treated cultures (Fig. 1). These findings suggest that the markedly different responses by the microvascular endothelium to TGF-p, noted in 2-D cultures may be the results of cell-cell and/or cell-matrix interactions, which are not present or are different in the 3-D cultures. Since TGF-P, is known to be angiogenic in vivo, we feel the lack of inhibition of proliferation and the rapid and extensive tube formation (Fig. 2) may be more physiologic and may be directly related to the 3-D environment of the cells. The second major difference between the 2-D and 3-D cultures is the matrix synthetic profiles in the absence and presence of TGF-6,. Our laboratory has previously shown that TGF-6, treatment of 2-D RFC cultures

Fig. 7. Immunohistochemistry. Four-day, three-dimensional RFC, TGF-P, treated cultures, incubated with anti-collagen V illustrating positive staining of the organizing basal lamina closely apposed to the putative abluminal aspect of the cell. a: Electron micrograph showing deposition of organizing basal lamina. b,c: Electron micrographs of cells expressing positive staining for anti-collagen V. Bars = 1 pm.

could elicit the increased deposition and accumulation of Fn and that soluble Fn added to 2-D RFC cultures could, in part, mimic the TGF-P, proliferative inhibition effect (Madri et al., 1988a). This suggested t h a t perhaps TGF-PI was stimulating F n accumulation, which in t u r n elicited the cellular response. In light of these findings, we investigated whether alteration of RFC matrix synthesis, secretion, and deposition might be driving the angiogenic response, However, in our 3-D cultures there was no increased accumulation in any ECM protein tested in response to TGF-P,, and soluble Fn added to the cultures had no apparent effect (data not shown), and yet there was a activation of the TGF-IJ, treated cultures in the form of rapid and extensive tube formation (Fig. 2). Therefore, the increased F n accumulation noted in monolayer cultures

TGF-f3, MODULATES CELL-CELL AND ECM ORGANIZATION

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Fig. 10. EDTA trcatment. Phase photographs taken of unfixed frozen, acetone treated, 8 km sections. Left: Four-day, threedimensional RFC, TGF-Pi treated cultures showing complex branching, tube-like formations. Right: Additional treatment of cultures with 5 mM EDTA for 24 h caused dissolution of the tuhe-like structures with cells appearing as individual entities as noted in control cultures where tube formation is rarely seen at this time. Inset: Fourday, TGF-6, treated collagen I gels were removed from the millicell and measured to observe contraction of gel by TGF-PI treatment and reversal of this response by the addition of EDTA. Gel measurements: TGF-P, treated - EDTA = 4 mm; TGF-P, treated + EDTA = 1 cm. Bar = 15 p.m.

Fig. 8. Immunofluorescence. Freshly isolated epididymal fat pads, unfixed frozen, acetone treated, 8 pm sections were used for duallabeling experiments. a,c: Sections incubated with a phalloidinrhodamine conjugate stained positive for actin stress fibers in a punctate and short linear pattern associated with the capillary network (arrows).b,d: Sections probed with anti-ZO-1 revealed a delicate, less intense staining associated with cell membrane-cell membrane contact points (arrowheads). Bar = 5 pm.

Fig 9 Immunofluorescence Four-day, three-dimensional RFC, TGF- PI treated Lulture, unfixed frozen, acetone treated, 8 p,m sections probed with anti-ZO-1 a: Phase contrast b IF showing tuhelike formation positive for ZO-1 Bar 50 pm ~

and the tube formation in 3-D cultures appear to be two independent responses by the cells to TGF-PI. While cells in untreated 3-D cultures remained isolated and quiescent, TGF-P, treated cells showed pseudopod formation along aggregated multicellular complexes within a 24 hour period (Fig. 3). The TGF-PI treated cells also expressed increased numbers of vacuoles and ribosomes as well as markedly enlarged endoplasmic reticulum suggesting metabolic activation and increased protein synthesis. The TGF-f3, stimulated cells were polarized as noted by the formation of lumen sealed off by junctional complexes and deposition of an organized abluminal basal lamina (Figs. 5,6). The establishment of basement membranes is a major requirement for the formation of new blood vessels in vivo (Madri and Pratt, 1987). TGF-P, treated cultures appear t o mimic this natural activity by the deposition of basal lamina along the newly forming tubular structures (Fig. 7). Also seen during the activation of the treated cells is the condensation of the surrounding collagen. With the aggregation of cells, collagen appears to be pulled in toward the activated cellular aggregates. This is not seen in the control cultures where there is little tube formation and collagen fibrils remain randomly organized (Fig. 4).Therefore, along with the metabolic stimulation of the cells, TGF-P, treatment causes the cell to alter the surrounding environment through redistribution and reorganization of the existing and newly synthesized

ECM. The lumina of blood vessels need to be sealed off from their surroundings. In capillary beds tight junctions are present in vivo. Capillaries of epididymal fat pads, upon removal from the rat, show tight junctions at the point of lumen closure. The junctional complexes ob-

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a 20-1 RFC

A

B

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C

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7.8KbI)

I)

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28S+ Fig. 11. Northern blot. Autoradiographs of total RNA transfers hybridized with SP6-RNA polymerase transcribed ZO-1 cRNA probe revealed the presence of a 7.8 kb mRNA i n A) freshly isolated RFCs; B) 4-day, 2-D RFC monolayer cultures + TGF-P,; C) 4-day, 3-D RFC cultures i TGF-P,. TGF-pl did not appear to induce 20-1 mRNA expression i n any condition.

467

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served during our EM structural analysis led us to question whether ZO-1, a n internal, membrane-associated protein specific for tight junctions, was present in our newly formed tubular structures. Epididymal fat pad capillaries exhibit tight junctions a t cell-cell processes and stain positively with anti-ZO-1 in patterns consistent with tight junction complexes (Fig. 8). In addition, long-term 2-D cultures exhibiting occasional tube formation and TGF-P, treated 3-D cultures in which there are extensive tube-like structures also stain positively for ZO-1 in areas of tube formation (Fig. 9). Our 3-D culture system is one of random orientation where tubular growth has a n unpredictable pattern. The ZO-1 signal is positive wherever junctions form: at pseudopods folding back on the cell, a t cell-cell contact points, and following longitudinal “seams” of the tubes. Because of the absence of ZO-1 membrane staining in early 2-D cultures and 3-D cultures not exposed to TGF-P,, we performed Northern analysis (Fig. 11)to determine whether ZO-1 mRNA was being expressed under these conditions. We found ZO-1 mRNA expressed in both culture systems, and the levels did not change in response to TGF-PI. In addition, immunoblots (Fig. 12) revealed the presence of ZO-1 protein in 2-D cultures even though it was not assembled into tight junctions. Thus, while ZO-1 mRNA and protein are expressed in 2-D cultures, tight junctions do not assemble in early cultures, and assembly is not responsive to TGF-P,. In contrast, growth in 3-D cultures confers the ability of cells to assemble ZO-1 into tight junctions in reponse to TGF-p,. The data presented above indicate the importance of mimicking the in vivo environment when doing in vitro experimentation. Culturing microvascular endothelial cells in monolayers causes them to flatten and spread, eliciting a particular set of responses when the cells are exposed to TGF-P1 (Heimark et al., 1986; Saksela et al., 1987). In contrast, when the same cells are cultured in a 3-D environment, the response to a particular factor (TGF-PI) is one of rapid, extensive tube formtion and maintenance of a differentiated pheno-

Fig. 12. Western blot. Four-day, two-dimensional RFC cultures were extracted with hot SDS, run on 6% reduced SDS-PAGE, transferred unto nitrocellulose, and incubated with pAb against ZO-1 tight junction protein, labeling a protein with a mw of -220 kd. Molecular weight markers: 220 kd = fibronectin; 95 kd = phosphorylase b; 67 kd = bovine serum albumin.

type which mimics, to some degree, small vessel endothelium undergoing neovascularization in vivo. Thus, TGF-p, appears to act on microvascular endothelial cells directly, eliciting the assembly of junctional complexes between cells and cell processes and the organization of abluminal basal lamina. Further studies directed a t determining what specific genes are up- and down-regulated following TGF-P,-induced angiogenesis are in progress and will ultimately lead to a better understanding of angiogenesis and its control.

ACKNOWLEDGMENTS We thank Thomas Ardito for his electron microscopy assistance, Adeline Tucker for her technical assistance, and Robert Specht for his photographic assistance. This work was supported by a Joshua Macy Predoctoral Fellowship to J.R.M.; Lucille P. Markey Scholarship in Biomedical Sciences t o J.M.A.; Swiss National Science Foundation Postdoctoral Fellowship to O.K.; Yale Liver Center Pilot Project grant DK 34989 to C.M.V.I., and USPHS grants R01-HL-28373 and PO1 DK 38979 to J.A.M.. LITERATURE CITED Alberts, B., Bray, D., Lewis, J.,Raff, M., Roberts, K., and Watson, J.D. (1983) Molecular Biology of the Cell. Garland Publishing Company, New York. Assoian, R.K., Komoriya, A,, Meyers, C.A., Miller, D.M., and Sporn, M.B. (1983) Transforming growth factor-beta in human platelets: Identification of a major storage site, purification and characterization. J. Biol. Chem., 258fl1):7155-7160. Cate, R.L., Mattaliano, R.J., Hession, C., Tizard, R., Farber, N.M., Cheung, A,, Ninfa, E.G., Frey, A.Z., Gash, D.J., Chow, E.P., Fisher, R.A., Bertonis, J.M., Torre, G., Wallner, B.P., Ramachandran, K.L., Ragin, R.C., Manganaro, T.F., MacLaughlin, D.T., and Donahoe, P.K. (1986) Isolation of t h e bovine and human genes for Mullerian

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