JOURNAL OF CELLULAR PHYSIOLOGY 146:17&179 (1991)

Chondrocytes Inhibit Endothelial Sprout Formation In Vitro: Evidence for Involvement of a Transforming Growth Factor-Beta M.S. PEPPER,* R. MONTESANO, 1.-D. VASSALLI, AND 1. O R C l Institute of Histology and Embryology, University of Geneva Medical Center, 12 1 I Geneva 4, Switzerland Using a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti-TGF-P antibodies to the cocultures significantly reduces the inhibitory effect of chondrocytes on sprout formation. Chondrocyte-conditioned medium or exogenously added TGF-Pl have a similar albeit transient inhibitory effect. Depletion of TGF-P from chondrocyte conditioned medium with anti-TGF-P antibodies and solid-phase protein-A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte-derived TGF-P-like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF-P.

Angiogenesis, the formation of new capillary blood vessels, occurs in a wide range of developmental, physiological, and pathological settings. The process begins with localized breakdown of the basement membrane of the parent vessel (usually an existing capillary or a postcapillary venule), followed by the migration and outgrowth of endothelial cells into the surrounding extracellular matrix, resulting in the formation of a capillary sprout. A mature capillary, which results once a lumen and basement membrane have been formed, fuses with the tip of another maturing sprout or vessel to produce a functional capillary loop (reviewed in Furcht, 1986; Folkman and Klagsbrun, 1987). In an attempt to elucidate the mechanisms by which new blood vessels are formed, we and others (Montesano and Orci, 1985; Nicosia et al., 1986; Pepper et al., 1987; Kubota et al., 1988; Madri et al., 1988; Folkman et al., 1989;Mignatti et al., 1989)have developed a number of in vitro models that address different components of the sequence of events outlined above. Our present objective is to develop an in vitro model that can be used for the identification of physiological inhibitors of angiogenesis. To achieve this objective, we set out: (1)to devise a quantitative model of spontaneous capillary sprout formation, and (2) to develop a coculture system suitable for the study of interactions between microvascular endothelial cells and cell types that might produce antiangiogenic factors. Since hyaline cartilage is resitant to vascular invasion (reviewed in Kuettner and Pauli, 19831, and cartilage explants or extracts as well as isolated chondrocytes have been shown to inhibit angiogenesis in vivo (Brem and Folk0 1991 WILEY-LISS, INC.

man, 1975; Langer et al., 1976, 1980; Kaminski et al., 1977; Takigawa et al., 1987; Moses et al., 19901, cartilage was chosen as a potential source of antiangiogenic factors. We have adapted a previously described in vitro model (Nicosia et al., 1986) as a means of quantitating spontaneous capillary sprout formation to a coculture system with chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum, and have identified a chondrocyte-derived TGF-p-like molecule as an inhibitor of endothelial sprout formation, one of the earliest events that occur during angiogenesis.

MATERIALS AND METHODS Endothelial cell culture Bovine microvascular endothelial (BME) cells, a generous gift from Drs. M.B. Furie and S.C. Silverstein (Furie et al., 1984), were routinely subcultured in gelatin-coated tissue culture flasks (Falcon) in complete BME medium consisting of minimal essential medium, alpha-modification (a-MEM, Gibco), 15%heat inactivated donor calf serum (Flow Laboratories, Ayrshire, Scotland), penicillin (500 IU/ml), and streptomycin (100 pg/ml).

Received June 26, 1990; accepted October 4, 1990, *To whom reprint requestsicorrespondence should be addressed.

CHONDROCYTE TGF-p INHIBITS ENDOTHELIAL SPROUT FORMATION

Preparation of primary chick embryo chondrocytes Chondrocytes were prepared by a modification of the procedure described by Gibson et al. (1984). Sterna were removed from 18-20-day-old chick embryos and all adhering soft tissue dissected away. The sterna were then divided into cephalic and caudal halves, and the cephalic half discarded. The caudal half was then digested for 15 minutes at 37°C in digestion mixture consisting of 2 mg/ml collagenase (Cooper, CLSII) and 0.25% trypsin in calcium- and magnesium-free PBS. Remaining perichondrium was dissected away and the cartilage was cut into fine pieces. Fresh digestion mixture was added, and the digestion continued at 37°C for a further 60 minutes. At 10-15-minute intervals, the digest was vigorously pi etted to facilitate cell separation. Cells were then co lected by centrifugation and resuspended in complete CH medium consisting of Ham’s F12 medium (Gibco), 10% heat-inactivated fetal calf serum (Amimed, Muttenz, Switzerland), penicillin (500 IUiml), and streptomycin (100 kgiml). Cells were counted and seeded at 2 x lo5 cellsiml into 100 mm tissue culture dishes (Falcon) or tissue culture dishes coated with 0.5% agarose to prevent cell attachment (Nevo et al., 1972). Medium was changed every 2 3 days and attached cells on uncoated dishes passaged by trypsinization every 7 days.

P

Preparation of chondrocyte conditioned medium Chondrocytes were washed twice in PBS and seeded in serum-free F12 medium into freshly prepared 0.5% agarose-coated 60mm tissue culture dishes (Falcon). 24 hours later, cells were removed by centrifugation, cell number determined, and the conditioned medium (CM) dialyzed against distilled water. Dialyzed CM was lyophilized and stored at -20°C. Lyophilized CM was reconstituted by resuspending in a volume of complete BME medium calculated to give an equivalent of 5 x lo5 chondrocytes/ml. Sprout-formation assays BME cells were trypsinised and seeded onto 0.5% agarose-coated 35mm tissue culture dishes in complete BME medium. This procedure resulted after 24 hours in the formation of endothelial cell aggre ates in suspension above the agarose (Fig. 1)The en othelial cell aggregates were then decanted from single nonaggregated cells under gravitational force in conical plastic centrifuge tubes, by allowing the cells to stand for 20-30 minutes at room temperature. Endothelial cell aggregates were then suspended in 500 pl fibrin or collagen gels prepared essentially as previously described (Montesano et al., 1987).Briefly, for fibrin gels, bovine fibrinogen (Calbiochem, Lucerne, Switzerland) was dissolved immediately before use in calcium- and magnesium-free minimal essential medium (CMFMEM, Gibco), to obtain a final protein concentration of 2.5 mg/ml, unless stated otherwise. The endothelial cell aggregates were then resuspended in CMF-MEM containing fibrinogen, and clotting was initiated by adding 1/10 v/v of lox concentrated MEM containing 25U/ml thrombin (Sigma Chemical Co., St. Louis). The mixture was then immediately transferred into 15mm tissue-

%

171

Fig. 1. Formation of endothelial cell aggregates. Phase-contrastview of BME aggregates resulting from culture of BME cells in agarosecoated petri dishes (which provide a nonadhesive surface)for 24 hours. Bar = 200 Fm.

culture wells (Nunc) and allowed to gel for 2 minutes before adding 500 pl fresh complete medium to the wells. Collagen gels were prepared by resuspending endothelial cell aggregates in a solution of Type I collagen (obtained from rat tail tendons) in a sterile tube kept on ice. 8 volumes of this solution (approximately 1.5 mg/ml) were quickly mixed with 1volume of lox MEM and 1 volume of sodium bicarbonate (11.76 mg/ml), immediately dispensed into 15mm tissue culture wells (Nunc), and allowed to gel at 37°C for 10 minutes; 500 pl complete BME medium was added to each well. Where appropriate, Trasylol (Bayer) was added to the gel andlor culture medium at 200 KIUlml, and BME aggregates were treated with mitomycin C (10 pg/ml) (Sigma) for 4 hours prior to embedding; human platelet-derived TGF-P1 (5 ng/ml to 1 pg/ml) (R. and D. Systems Inc., Minneapolis) was added to the collagen or fibrin solutions prior to gel formation, and subsequently to the culture medium as well. For cocultures within fibrin or collagen gels, chondrocytes were resuspended with BME aggregates in a fibrinogen or collagen solution, and gel formation initiated as described above. For cocultures in which condrocytes were grown above the gels, chondrocytes were added in 500 ~1 culture medium to wells containing gels with BME aggregates. In all cocultures, medium consisted of a 1:l mixture of BME and CH complete medium. Protein-A purified rabbit antiporcine platelet TGFp l IgGs (R. & D. Systems Inc., Minneapolis) were resuspended in PBS and added to cocultures at a final concentration of 100 kg/ml. According to the manufacturer, the anti-TGF-pl antibody neutralizes the activity of porcine and human TGF-P1 and porcine TGF-P2, and shows no cross reactivity with acidic or basic fibroblast growth factors, platelet-derived growth factor, or epidermal growth factor. Dialysed and lyophilized chondrocyte conditioned medium reconstituted in complete BME medium was preincubated with nonimmune rabbit gamma-globulins (100 pg/ml, 50% ammonium sulfate cut) or anti-TGF-pl IgGs (100 pg/ml)

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for 4 hours before being added to BME aggregates in fibrin or collagen gels. For immunodepletion of TGF-P, reconstituted chondrocyte conditioned medium was incubated with nonimmune rabbit gamma-globulins (100 p,g/ml) or antiTGF-P antibodies (100 p,g/ml),at 4°Cfor 4 hours. Whole fixed S. Aureus Cowan I strain cells from 100 p1 of Pansorbin (Calbiochem) (washed twice with PBS) were then added to 500 pl aliquots of antibody-treated conditioned medium and tumbled at 4°C for a further 60 minutes. S. Aureus cells were removed by centrifugation. and the medium added to cultures of BME " aggregates in fibrin gels in the presence of Trasylol (200 KIU/ml). Quantification of sprout formation Cultures were fixed in situ with 2.5%glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and photographed under phase contrast using a Zeiss ICM 405 inverted photomicroscope. For each aggregate, average sprout length was determined by measuring the total length of all visible sprouts and dividing this number by the number of sprouts present. Where bifurcated, sprout length was determined along the longest segment. A roughly linear correlation was observed between aggregate size and sprout number, although sprout length was independent of these parameters (not shown). Processing for light microscopy Cultures that had been fixed in situ overnight were extensively washed in 0.1 M sodium cacodylate buffer (pH 7.4). Collagen or fibrin gels were then cut into 2mm x 2mm fragments, and the fragments postfixed in 1%osmium tetroxide in Verona1 acetate buffer for 45 minutes and further processed as previously described (Montesanoand Orci, 1985). Semithin sections were cut with an LKB ultramicrotome and photographed under transmitted light using an Axiophot photomicroscope (Zeiss, Germany).

RESULTS Endothelial aggregates embedded in fibrin or collagen gels give rise to capillary sprouts Bovine microvascular endothelial (BME) cells grown in tissue culture dishes coated with agarose (a nonadhesive substratum) aggregated in suspension to form, within 24 hours, free-floating spheroidal aggregates (Fig. 1).These aggregates were subsequently embedded into collagen or fibrin gels. Embedding the a gregates in collagen gels resulted after 3 days in the ormation of small hollow endothelial-lined saclike structures, with radially disposed capillarylike tubes (Fig. 2a,b). Similarly, in fibrin gels, this resulted after 3 days in the formation of endothelial-lined sacs, with tubelike structures and cell cords radiating out into the surrounding gel (not shown). Inhibition of fibrin lysis by addition of the serine protease inhibitor Trasylol (200KIU/ml),resulted in the formation of solid cords of endothelial cells radiating out from the compact aggregate into the surrounding gel (Fig. 3a,b). These findings demonstrate that the formation of hollow saclike structures and tubular but not solid sprouts, could be prevented by the addition of a serine protease inhibitor

f

Fig. 2. BME aggregates cultured within collagen gels for 3 days. Phase contrast view (a) and semithin action (b) of a small hollow endothelial-lined saclike structure with radially disposed capillarylike tubes. Bars = 150 km in (a) and 70 km in (b).

to fibrin gels. Mean sprout length per aggregate in fibrin gels in the presence of Trasylol was inversely proportional to fibrinogen concentration (Table 1). To determine whether cell division was necessary for sprout formation, BME aggregates in agarose-coated petri dishes were treated with mitomycin C (10 pg/ml) for 4 hours prior to embedding in fibrin gels. Mean sprout length per aggregate after 3 days culture in the presence of Trasylol was determined in control and mitomycin C-treated cultures. Sprout length was unaffected by treatment of aggregates with mitomycin C

CHONDROCYTE TGF-8 INHIBITS ENDOTHELIAL SPROUT FORMATION

173

TABLE 1. Mean sprout length per aggregate is inversely proportional to fibrinogen concentration'

Protein concentration 5.00 mg/ml 2.50 mg/ml 1.25 mg/ml

Mean sprout length 191 k 6pm

(n = 20) 237 rt 9pm (n = 20) 276 9pm (n = 20)

*

'

BME aggregates were embedded in fibrin gels prepared from fibrinogen solutions at the final protein concentrations indicated, and Trasylol(200 KIU/ml) was added to the medium above thegel. Mean sprout length per aggregate was determined after 72 hours as described in Materials and Methods. Values are mean SEM. n = total number of aggregates analysed

*

Fig. 3. BME aggregates cultured within fibrin gels in the presence of the fibrinolytic inhibitor, Trasylol. (a) Phase contrast view showing the formation of long radially disposed sprouts from the central endothelial cell aggregate after 3 days in culture. (b)Sernithin section reveals a solid aggregate with solid endothelial cords radiating out into the surrounding gel. Bars = 150 pm in (a) and 100 pm in (b).

prior to embedding (157 k 10 km and 158 f 7 pm in control and treated cultures respectively; n = 10 aggregates for each condition). Coculture with chondrocytes inhibits sprout formation Chondrocytes derived from the caudal half of the 18-20-day chick embryo sternum are a homogeneous population of cells destined to form the permanent cartilagenous region, whereas cells derived from the cephalic half are a heterogeneous population in the rocess of becoming hypertrophic (Gibson et al., 1984). y cell-size fractionation we observed that chondrocytes we had isolated from the caudal half of the sternum were a homogeneous population of relatively small cells (not shown). Furthermore, characteristic chondrocyte ultrastructural features were maintained

i

when the cells were grown within collagen or fibrin gels (not shown). Coculture of BME aggregates with chondrocytes from the permanent cartilagenous region (5 x lo5 cells/ml) resulted in a marked inhibition of sprout formation. This was observed whether chondrocytes were incorporated into the gel together with BME aggregates, or added to the medium above the gel containing the aggregates (Figs. 4,5). When mean sprout length per aggregate was used as a quantitative assessment of sprout formation, maximum inhibition was observed in fibrin gels in the presence of Trasylol (Fig. 5). Furthermore, coculture within fibrin gels in the absence of Trasylol also prevented the fibrinolysis and subsequent formation of saclike structures typically seen in control cultures after 3 days (not shown). Although chondrocyte phenotype is markedly affected by culture conditions (reviewed in von der Mark, 19861, we observed no difference in the inhibitory effect on sprout formation between chondrocytes utilized after culture on plastic (for up to 4 weeks, i.e., 4 passages), in suspension above agarose or used as primaries in cocultures immediately after isolation (not shown). The kinetics of chondrocyte inhibition of sprout formation were determined in cocultures in fibrin gels in the presence of Trasylol, since maximum inhibition was observed under these conditions (Fig. 5 ) . Inhibition was detectable after 24 hours and was constantly maintained until the end of the assay period (3 days) (Fig. 6). In controls to which chondrocytes were not added, a consistent and almost linear increase in sprout length was observed (Fig. 6).By decreasing the number of chondrocytes cocultured either within or above fibrin gels, a progressive reduction in the inhibitory effect on sprout formation was observed (Fig. 7). (Similar results were obtained with collagen gels; not shown). This experiment allowed us to determine the minimum concentration of chondrocytes required for a 50% decrease in sprout length, i.e., approximately 1 x lo4 cells/ml, and this concentration was used in initial experiments aimed at determining the factorb) mediating inhibition of sprout formation. Anti-TGF-P antibodies reduce the inhibitory effect of chondrocytes or chondrocyteconditioned medium on sprout formation In order to determine whether the inhibitory effect of coculture might be mediated by TGF-P, antiporcineTGF-P1 antibodies (100 pg/ml) were added to the

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Fig. 4. Inhibition of sprout formation by chondrocytes.Phase contrast views of BME aggregates in fibrin gels in the presence of Trasylol, 3 days aRer embedding in the absence (a) or the presence (b) of chondrocytes.(a) Long sprouts radiate out from the endothelial cell aggregate into the surrounding gel. (b)Sprout formation is inhibited when BME aggregates are coembedded with chondrocytes(arrowheads). Bars = 100 pm.

160

1

250

T

T

-

200

-5. -

rm

150

-

1 a,

0 a

100

v)

Chondrocytes

Control

Chondrocytes (Gel)

0

Chondrocytes (Medium)

Fig. 5. Quantitative assessment of inhibitory effect of chondrocytes. Mean sprout length per aggregate after 3 days was determined as described in Materials and Methods. Open bars represent cultures in fibrin gels, lightly shaded bars are cultures in fibrin gels in the presence of Trasylol, and heavily shaded bars are cultures in collagen gels. Control = cultures without chondrocytes; chondrocytes (Gel) = cocultures with chondrocytes coembedded in the gel; chondrocytes (medium) = cocultures with chondrocytes added to the medium above the gel. Values are mean sprout length per aggregate SEM. n = number of aggregates analysed, 5 aggregates per experiment.

24h

48h

72h

Fig. 6. Kinetics of sprout formation and inhibition in cocultures. Mean sprout length was determined (as described in Materials and Methods) after 24,48, and 72 hours in fibrin gels in the presence of Trasylol in control cultures and cultures in which chondrocytes(2.5 x 105/ml)were coembedded into the fibrin gel with BME aggregates. Values are mean sprout length per aggregate 2 SEM. Five aggregates were measured per condition in each of three separate experiments.

*

the 3-day assay period (Table 2). Addition of antibody alnno

medium of cocultures in fibrin gels in the presence of Trasylol, in which chondrocytes (1 x lo4 cellsiml) had been added above the gel. This resulted in approximately a 50% decrease in the inhibitory effect of chondrocytes when the antibody was added daily over

aid nnt cicmifirantlv affort cnrnnt lpnuth ITa-

tionea meaium was reconsciunea 111 cor~ipie~e mvin medium and added to cultures of BME aggregates embedded within fibrin gels in the presence of Trasylol. Chondrocyte conditioned medium alone resulted in an inhibition of sprout formation, although contrary to the

175

CHONDROCYTE TGF-p INHIBITS ENDOTHELIAL SPROUT FORMATION

Fig. 7. Quantitative assessment of reduction in sprout length in cocultures in fibrin (+ Trasylol) after 3 days with varying concentrations of chondrocytes. Control cultures (solid bar) and cocultures in which chondrocytes were either coembedded with BME aggregates (open bars) or added to the medium above the gel (shaded bars). Values are mean sprout length per aggregate 2 SEM. 5 aggregates were measured per condition in each of three separate experiments.

TABLE 2. Chondrocyte-mediated inhibition of sprout formation

Fig. 8. Effect of chondrocyte conditioned medium on sprout formation. Mean sprout length was determined (as described in Materials and Methods) after 24,48, and 72 hours in fibrin gels in the presence of Trasylol in control cultures ( C )and cultures to which reconstituted medium conditioned by 5 x lo5 chondrocyteshnl (CM) was added. Conditioned medium was preincubated with anti-TGF-pl antibodies (100 pgiml) for 4 hours at 4°C before addition to the cultures E M + anti-TGF); antiporcine platelet TGF-p1 antibodies (100pg/ml) were also added to the cultures alone. Values are mean sprout length per aggregate -t SEM. n = number of aggregates analysed, 5 aggregates per experiment.

is reduced bv anti-TGF-8 antibodies' ~

Mean sprout length2 Control (72 hours) Chondrocytes

(1 x 104/mi)

Chondrocytes + anti-TGFp daily dose (100 d m l ) Anti-TGFp daily dose (100 rg/ml) Chondrocytes + anti-TGFp single dose (100 pg/ml) Anti-TGFp single dose (100 d m l )

% inhibition

208 f 5 p m

(n = 25) 105 5 p m (n = 25) 152 12 pm' (n = 20)

+

* 200 * 11 pm (n = 15)

49.5

27.0 3.8

137 f 8 p m l (n = 20)

34.1

* 9 pm

6.1

196

(n = 15)

IChick embryo sternal chondrocytes (1 X 104/ml) were added to the medium of cultures of BME aggregatesin fibrin gels in the presenceof Trasylol(200 KIU/ml). Antiporcine platelet TGF-B1 antibodies (100 pg/ml) were added to the culture medium daily for 3 days or as a single dose at the start of the experiment, and the cultures fixed after 3 days. Mean sprout length per aggregate was determined as described in Materials and Methods. Values are mean & SEM. n =total number of aggregates analysed per condition (5 aggregates per experiment). 'Difference in mean sprout length between cocultures with and without antibodies: chondrocytes alone vs. chondrocytes plus antibody daily dose, p < 0.001; chondrocytes alone vs. chondrocytes plus antibody single dose, p < 0.005 (Student's T-test).

effect observed in co-culture, the inhibition was transient (Fig. 8). Preincubation of reconstituted conditioned medium with anti-TGF-p IgG prevented the inhibitory effect of conditioned medium on sprout formation (Fig. 8). When added alone in the absence of conditioned medium, the antibody had no effect (Fig. 8). (Similar results were obtained with collagen gels; not shown.) However, the effect of the anti-TGF-p antibodies might conceivably have been the result of an interaction with either a chondrocyte- or endothelial cell-derived TGF-p. To determine whether a chondro-

cyte-derived TGF-P might be involved, reconstituted chondrocyte conditioned medium was immunodepleted of TGF-P, and the medium added to cultures of BME aggregates embedded within fibrin gels in the presence of Trasylol. This resulted in a significant reduction of the inhibitory effect of conditioned medium on sprout formation (Table 31, demonstrating that a chondrocytederived TGF-P is responsible for the inhibitory effect. Exogenously added TGF-P1 has a transient inhibitory effect on sprout formation Exogenously added TGF-p1 (5 ng/ml) transiently inhibited sprout formation (not shown), thereby mimicking the transient inhibitory effect of reconstituted chondrocyte conditioned medium, which was maximal after 24 hours (Fig. 8). The inhibitory effect of TGF-P1 after 24 hours was observed with concentrations ranging from 100 pg/ml to 5 ng/ml; half maximal inhibition was observed with 250 pg/ml of TGF-P1 (Fig. 9). The inhibitory effect of TGF-Pl(5ng/ml) could be prevented by coaddition of anti-TGF-p antibodies (100 p.g/ml) to the cultures, thereby confirming the inhibitory nature of the antibodies (not shown). DISCUSSION In an attempt to identify hysiological inhibitors of angiogenesis, we have esta lished a quantitative in vitro assay of capillary-like sprout formation. Microvascular endothelial cell aggregates are embedded within fibrin or collagen gels, which resuits in the formation of long outgrowths of cells extending radially from the initial aggregate into the surrounding matrix. This phenomenon we interpret as mimicking one of the earliest events that occur during angiogenesis, namely,

1

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TABLE 3. Immunodepletion of reconstituted chondrocyte conditioned medium with anti-TGF-pantibodies reduces inhibition of sprout formation1

120 r

Mean sprout length 124 f 4 pm (n = 45) 80*3pm (n = 45) 76f3pm (n = 10) 120 f 5 pm (n = 10) 89 f 9 pm2 (n = 10) 120 f 5 pm2 (n = 10) 126 f 4 pm (n = 5) 110 f 7 pm (n = 5)

Control (24 hours) Conditioned medium (CM) CM NI y-globulins (100 pg/ml) CM a-TGF-0 IgG (100 pg/ml) CM NI y-globulins Pansorbin CM CY-TGF-P IgG Pansorbin NI y-globulins (100 pg/ml) a-TGF-/3IgG (100 pg/ml)

+

+

+ + + +

dialysed against distilled water, Iyophilized, and resuspended in complete BME medium to give an equivalent of 5 X lo5cells/ml. Antibodies and whole fixed S.Aureus Cowan Strain I cells were added and incubated as described in Materials and Methods. Medium was then added to cultures of BME aggregates in fibrin gels in the presence of Trasylol(200 KIU/ml). Mean sprout length per aggregate was determined as described in Materials and Methods. Values are mean f SEM.n = total number of aggregates analysed per condition (5 aggregates per experiment). *Difference in mean sprout length between control vs. anti-TGF-8 antibodies: p < 0.001 (Student’s T-test).

1

the formation of endothelial sprouts from existing vessels. When BME aggregates are cocultured with chick embryo sternal chondrocytes, sprout formation is inhibited by a chondrocyte-derived diffusible factor(s). We considered that TGF-P might be a potential mediator of this inhibition, since TGF-P is known to inhibit important components of the angiogenic process in vitro, namely endothelial cell proliferation (Baird and Durkin, 1986; Frhter-Schroder et al., 1986; Muller et al., 1987; Antonelli-Orlidge et al., 1989) migration (Heimark et al., 1986; Muller et al., 1987; Sato and Rifkin, 1989) and extracellular matrix invasion (Muller et al., 1987; Mignatti et al., 1989). This possibility was addressed by the addition of neutralizing anti-TGF-P antibodies to our coculture system. We found that the inhibitory effect of the chondrocytes was significantly reduced in the presence of the antibodies, indicating that TGF-P is at least partially responsible for the inhibition of sprout formation. Furthermore, the reduction in the inhibitory effect of chondrocyte conditioned medium following im-munodepletion with antiTGF-P antibodies demonstrates that TGF-p is chondrocyte derived. The transient nature of the effect of chondrocyte conditioned medium or exogenously added TGF-P1 on the inhibition of sprout formation is in contrast to the sustained inhibition observed in the presence of chondrocytes themselves, suggesting that a constant production of TGF-P is required for sustained inhibition. It is important to note that addition of anti-TGF-6 antibodies only reduced the chondrocyte-inhibitory effect in cocultures containing 1 x lo4 chondrocytes/ml, by approximately 50%. This finding may implicate the presence of an additional antiangio enic factor. A potential candidate is a recently descri ed collagenase inhibitor, which inhibits angiogenesis in vivo and capillary endothelial cell proliferation and migration

%

10

100

1000

10000

TGF-R1 (pgirnl)

I Serum-freechondrocyte-conditionedmedium was

Fig. 9. Dose-dependent reduction in mean sprout length by human platelet-derived TGF-pl. BME aggregates were embedded in fibrin gels, and human platelet-derived TGF-P1was added to the gels and to the culture medium above the gels. Trasylol (200 KIU/ml) was also added to the medium above the gel. Mean sprout length per aggregate for 10 aggre ates after 24 hours in the presence ofTGF-pl is expressed relative to t f e mean of 10 aggregates in control cultures in the same experiment, Values are mean 5 SEM of percent of controls. Number of aggregates analysed = 40 for 500 pgiml and 100 pgiml, 30 for 5 ngiml, 1 ngiml and 200 pgiml and 20 for the rest. P: values are significantly different from controls, p < 0,005; ’: values are highly significantly different from controls, p < 0,001; Student’s T-test).

in vitro (Moses et al., 1990). Since, however, the inhibitory effect of medium conditioned by 5 x lo5 chondrocytes/ml can be completely prevented by anti-TGF-P antibodies, this raises the possibility that the additional inhibitor, if present, is unstable in conditioned medium. The mechanisms by which TGF-P inhibits sprout formation in our assay are not known. Thus, whereas cartilage and chondrocyte-derived factors (Eisenstein et al., 1975; Sorgente and Dorey, 1980; Takigawa et al., 1985, 1987; Moses et al., 1990) and TGF-P (Baird and Durkin, 1986; Frater-Schroder et al., 1986; Muller et al., 1987; Antonelli-Orlidge et al., 1989) have been shown to inhibit endothelial cell replication, in our system mean sprout length is unaffected by treatment of BME aggregates prior to embedding with the DNA synthesis inhibitor mitomycin C, suggesting that the inhibitory effect is not related to effects on cell division. Previous re orts have likewise demonstrated that capillary endot elial cell migration and sprout formation are independent of cell division (Sholley et al., 1977, 1984). However, when BME aggregates and chondrocytes are cocultured within a fibrin gel in the absence of Trasylol, the formation of large endothelial-lined saclike structures is prevented, implicating the presence of an antifibrinolytic component in the chondrocyte-mediated effect. Similar results were obtained following addition of TGF-P1 to BME aggregates in fibrin gels in the absence of Trasylol (Pepper, unpublished observations). Since TGF-P induces the production of plasminogen activator inhibitor 1 (PAI-1) in microvascular endothelial cells (Saksela et al., 1987;

R

CHONDROCYTE TGF-P INHIBITS ENDOTHELIAL SPROUT FORMATION

Pepper et al., 1990), PAI-1 may play a role in the inhibition of sprout formation in our system. Additional mechanisms such as inhibition of cell migration (Heimark et al., 1986; Muller et al., 1987; Sat0 and Rifkin, 1989), modulation of the cytoskeleton (Kocher and Madri, 1989), the endothelial cell extracellular matrix (ECM) (Muller et al., 1987; Madri et al., 1988) or ECM adhesion receptors (Ignotz and Massague, 1987; Heino et al., 1989), might also affect sprout formation in our system. Most cultured cells examined secrete TGF-6 in an inactive (latent) form (Lawrence et al., 1984, 1985; Kryceve-Martinerie et al., 1985; Lyons et al., 1988; Antonelli-Orlidge et al., 1989; Sato and Rifkin, 1989). These and related observations raise a number of important questions about our findings. First, it has been observed that latent TGF-6 is activated in cocultures of endothelial cells and pericytes or smooth muscle cells and that this is dependent on close heterocellular proximity or cell-to-cell contact (AntonelliOrlidge et al., 1989; Sat0 and Rifkin, 1989). Our findings clearly differ from these observations since coculture with chondrocytes above the gel, which precludes heterocellular contact, is highly effective in preventing sprout formation. The reasons for these differences are not known. Second, the suggestion that activation of latent TGF-6 may be plasmin-dependent (Lyons et al., 1988; Sat0 and Rifkin, 1989) raises the important question as to why we see inhibition of sprout formation in the presence of a plasmin inhibitor. One possibility may be related to the recent demonstration that TGF-p2 can be secreted in an active form (Glick et al., 1989). The precise nature of the chick embryo chondrocyte-derived TGF-p-like molecule has not been determined and may differ significantly in its activation status or mechanism of activation from its mammalian pericyte or smooth muscle cell counterparts. Third, BME cells almost certainly produce TGF-b themselves. As demonstrated in this study, addition of anti-TGF-6 antibodies to cultures of BME aggregates embedded alone in fibrin or collagen gels does not significantly affect sprout formation. The inhibitory effect seen in coculture may therefore be related to quantitative differences in chondrocyte- versus BME-derived TGF-b. It has been suggested that factors that modulate the angiogenic process may exert their effect either directly on endothelial cells themselves, or indirectly via an intermediate cell type (reviewed in Folkman and Klagsbrun, 1987). In this context, TGF-P is an indirect angiogenic agent in vivo (Roberts et al., 1985), where the microvascular response is believed to be mediated by secretory products of TGF-6-recruited monocytes (Wahl et al., 1987; Wisemann et al., 1988).Results from in vitro assays that address different components of the angiogenic process demonstrate that the response to TGF-P varies depending on the assay used. Thus, as mentioned above, TGF-6 inhibits endothelial cell replication and migration in two dimensions. TGF-P1 also inhibits phorbol ester-induced invasion of a threedimensional collagen gel (Muller et al., 1987; Montesano, unpublished observation) and basic fibroblast growth factor (bFGF)-inducedinvasion of the explanted amnion (Mignatti et al., 1989). In addition, we have

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recently shown that TGF-b1 inhibits bFGF-induced capillary lumen formation within three-dimensional fibrin gels in vitro (Pepper et al., 1990). Our present results therefore support the observation that TGF-P is a direct-acting inhibitor of extracellular matrix invasion and tube formation. However, we have also observed a concentration-specific potentiating effect of TGF-61 on bFGF-induced invasion of a three-dimensional collagen or fibrin gel (Pepper et al., in preparation). In addition, it has been reported that culture of rat epididymal fat pad-derived endothelial cells within a three-dimensional collagen gel and subsequent treatment with TGF-61 (500 pg/ml), promotes gel contraction and organization of endothelial cells into tubelike structures with tight junctions and an abluminal basement membrane (Madri et al., 1988; Merwin et al., 1990). Taken together, these apparently conflicting results may be reconciled by considering that TGF-6 might have different functions on vessel formation at different stages of the angiogenic process. Thus it may modulate de novo bFGF-induced invasion, and in the absence of bFGF, inhibit vessel formation. Once sprout formation has occurred, TGF-P may be necessary for the inhibition of further endothelial cell replication and migration and might induce vessel organization and functional maturation. It is important to note, however, that when dealing with a complex phenomenon such as angiogenesis, the distinction between angiogenic factors as direct or indirect, positive or negative, although helpful in initially characterizing the many factors involved, is likely to be an oversimplification. It is likely that the temporally coordinated and concentration-dependent activity of a number of factors is necessary for the control of different elements of the angiogenic process in specific and appropriate settings in vivo. In conclusion, since TGF-P and related molecules have been observed immunohistochemically and by in situ hybridization in both developing and mature cartilage (Ellingsworth et al., 1986; Heine et al., 1987; Flanders et al., 1989; Lyons et al., 1989; Pelton et al., 1989; Thompson et al., 1989), and chick embryo chondrocytes have been shown to contain mRNA for chicken equivalents of previously described as well as new members of the TGF-6 family (Jakowlew et al., 1988a,b,c),our findings suggest that TGF-P may play a role in chondrocyte-mediated antiangiogenesis in developing and mature hyaline cartilage.

ACKNOWLEDGMENTS We are grateful to Drs. M.B. Furie and S.C. Silverstein for generously providing the BME cells. Excellent technical assistance was provided by M. Guisolan, D. Lacotte, and J. Rial, and photographic work was expertly done by G. Negro and P.-A. Ruttiman. This work was supported by grants from the Swiss National Science Foundation (no. 31-26625.89), the Juvenile Diabetes Foundation (no. 187.464), and the Sir Jules Thorn Charitable Trust. LITERATURE CITED Antonelli-Orlidge, A., Sanders, K.B., Smith, S.R.,and D’Amore, P.A. (1989)An activated form of transforming growth factor p is pro-

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