BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ] 0 9 8 - ] ] 0 2
Vol. 180, No. 2, 1991 October 31, 1991
Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in v i t r o Yasufumi Sato ~, Tatsuo Shimada 2, and Ryosaburo Takaki ~ 'First Department of Medicine and 2Department of Anatomy, Oita Medica College Idaigaoka, Hasama-cho, Oita 879-55, Japan
Received
September
24,
1991
When bovine capillary endothelial (BCE) cells plated on type I collagen gel were covered with a second layer of collage gel, BCE cells reorganized into a network of capillary-like structures. In the presence of affinity purified anti-basic fibroblast growth factor (bFGF) antibody, this reorganization was inhibited. By using a computerized image analyzer, the formation of network structures and the effect of anti-bFGF antibody was quantitated. The inhibtory effect of anti-FGF antibody was dose dependent and maximal inhibition was observed at 2.0 pg/ml of antibody. Exogenously added bFGF potentiated network formation of BCE cells and coadministration of bFGF abrogated the inhibitory effect of anti-bFGF antibody. Platelet factor 4, which blocks the binding of bFGF to its receptor, inhibited network formation. These results indicate that bFGF produced by endothelial cells regulates angiogenesis as an autocrine factor. ® 1991 Academic Press, Inc.
Angiogenesis plays an important role in a wide range of physiological and pathological processes including wound healing, growth of solid tumor, diabetic retinoathy, and rheumatoid arthritits. At present, several growth factors have been identified as angiogenic factors. Among them, basic fibroblast growth factor(bFGF) is the best characterized factor (1). Angiogenesis is a complex phenomenon which includes proteolytic degradation of matrix, migration, proliferation, and tube formation by vascular endothelial cells (2). bFGF stimulates proteinase production which is required for matrix degradation, migration, and proliferation of endothelial cells (3) and induces angiogenesis in both IN VIVO (4) and IN VITRO systems (5). Many cell types including endothelial cells produce bFGF (6). Sato and Rifkin have reported that endogenous bFGF regulates plasminogen activator synthesis, migration, and DNA synthesis of endothelial cells (7); phenomena required for angiogenesis. Therefore it is reasonable to hypothesize that bFGF regulates angiogenesis as an autocrine factor. In the present study, we cultivated bovine capillary endothelial (BCE) cells in type 1 collagen gel and observed spontaneous formation of capillary-like structures.
The results indicate that
endogenously produced bFGF by endothelial cells regulated this phenomenon. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1098
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Materials: Bovine capillary endothelial (BCE) cells isolated from adrenal glands were grown in Dulbeco's modified essential medium (DME) containing 10% newborn calf serum (NCS). Affinity purified anti-bFGF antibody was prepared as descrived previously (7). Recombinant human bFGF was kindly provied by Synergen, Inc. (Boulder, CO). Platelet factor 4 (PF-4) was purchased from Sigma (St. Louis,MO). Tube Formation IN VITRO: Seven volumes of type 1 collagen gel solution (Nitta Gelatin, Osaka), 2 volumes of 10xDME, and 1 volume of 0.05N NaOH, 200mM HEPES, and 260mM NaHCO3, were mixed on ice. Half ml of collagen mixture was poured into a 35 mm plastic dish (Coming) and allowed to gel at 37" C. lxl05 of BCE cells were plated onto the collagen gel in DME containing 10% NCS and allowed to attach to collagen gel for 3 hr at 37" C in CO2 incubator. Then the medium was aspirated, half ml of collagen mixture was overlayed, 1 ml of DME containing 1% NCS was added. The indicated concentrations of affinity purified anti-bFGF antibody, recombinant bFGF or PF-4 were administered to both the collagen mixture and overlaying medium. After 24 hr incubation, BCE cells formed network structures. Quantitative Analysis of Network Structures: Phase contrast microscopic picture (200x magnification) was recorded in STILL VIDEO recordplayer (RS000H) (Fuji, Tokyo). Total length of the network structures composed of more than 2 cells recorded in VIDEO was mesured by using a Cosmozone 1S Image Analyzer (Nikon, Tokyo). Ten random fields were mesured and total length per singele field was calculated.
RESULTS AND DISCUSSION When BCE cells were plated onto the first layer of type I collagn gel and covered with a second layer of the gel, BCE cells contacted each other and formed network structures after 24 hr incubation (Fig. 1A). Cross-sectional analysis by electron microscopy revealed that there was a lumen inside of the network structures (Fig. 1C). Microvili could be observed on the luminal surface of the tube-like structure. To test the role of endogenous bFGF on the spontaneous network formation by BCE cells, affinity purified anti-bFGF antibody was applied.
As shown in Fig. 1B, there was a marked
inhibition of network formation by BCE cells. Image analyzer analysis by a Cosmozone 1A was introduced to quantitate the inhibitory effect of anti-bFGF antibody. The total length of network structures composed of at least 2 cells was calculated as described in the Methods. As shown in Fig.2, there was a dose-dependent inhibition of network formation by affinity purified anti-bFGF antibody. The inhibition reached a maximum at 2.0 !ag/ml of anti-bFGF antibody. Exogenously added recombinant bFGF potentiated network formation and also abrogated the inhibitory effect of anti-bFGF antibody (Fig.3).
These results strongly suggest that endogenous bFGF regulates
spontaneous tube formation of BCE cells. We have previously reported that platelet factor 4 (PF-4) blocked the binding of bFGF to its receptor and inhibited the migration of endothelial cells (8). Therefore we tesed whether PF-4 had any effect on nework formation by BCE cells. As shown in Fig.4, 5.0 ]ug/ml of PF-4 significantly inhibited network formation of BCE cells. 1099
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
250/Jm
Fig.1. Network formation of BCE cells in type 1 collagen gel. lx105 of BCE cells were plated onto type 1 collagen gel in 35 mm plastic dish and covered with another layer of collagen gel. A: BCE cells were incubated in DME cotaining 1% of NCS for 24 hr. B: BCE cells were incubated in the presence of affinity purified anti-bFGF antibody. C: Cross-sectional analysis by electron microscopy.
When endothelial cells were cultivated in three dimensional collagen gels, they spontaneously formed tube-like structures. This phenomenon indicated the exsistence of an endogenous regulator of angiogenesis.
Several growth factors have been identified as angiogenic factors (1,9,10). Among
them, bFGF is the factor that a endothelial cell makes by itself (11). bFGF produced by endothelial cells regulates migration, proteinase production, and proliferation of endothelial cells as an autocrine factor (7,12). Migrating endthelial cells express urokinase type plasminogen activator (uPA) (13), and uPA activity is reported to be required for spontaneous tube formation in type 1 collagen gels, 1100
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.~22s
2
v
1.75
w
15
i
ZE
i
"•
2 ~I7S 1.5
1,25
1.25
"~
S
.75
~ 75 o .5
25
.25
0
®
control
anti-bFGF 0.5
anti-bFGF I O
anti-bFGF 2.0
®
control
bFGF( IO)
anti-bFGF(2.0)
anti-bFGF÷bFGF
Fig.2. Dose responsive inhibition of anti-bFGF antibody on network formation. Network formation by BCE cells was quantitated by using Image Analyzer. BCE cells were incubated with 0, 0.5, 1.0, and 2.0 pg/ml of affinity purified anti-bFGF antbody for 24 hr and the length ofnetworkstructures composed of at least 2 cells was measured from 10 different fields. The results are presented as total length per field. Fie.3. Effect of exogenous bFGF and anti-bFGF on network formation by BCE cells. BCE cells were incubated with 10 ng/ml of recombinant bFGF and/or 2.0 pg/ml of affinity purified anti-bFGF antibody for 24 hr and the length of network structures was measured.
suggesting that uPA is a regulator of angiogenesis.(14).
However Odekon et al. have currently
shown that endgenous bFGF regulates the expession of uPA on migrating endothelial cells (15). Our present results further indicate that bFGF produced by endothelial cells plays a central role in the regulation of angiogenesis. Since bFGF lacks a classical signal sequence (11), bFGF must not be secreated and remain cell associated. However it was found that bFGF dose get out of cells and is deposited in extracellular matrix bound to heparan sulfate (16). More recently, a number of studies have shown that smoll amounts of bFGF, sufficient to induce biological antivities, are released from cells where it is postulated to act as an autocrine factor (17,18,19).
However the molecular mechanism for bFGF
release is currently obscure.
2
LO
W
control
PF4
Fig.4. Effect of PF-4 on network formation of BCE cells. BCE cells were incubated with 5.0 lag/ml of PF-4 for 24 hr and the length of network structures was measured. 1101
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Affinity purified anti-bFGF antibody blocked the spontaneous tube formation of endothelial cells in type 1 collagen gels. We could not detect any bFGF in type 1 collagen solution by immunoblot analysis (data not shown). These results indicated that endogenous bFGF regulates tube formation by endothelial cells as an autocrine factor. In this context, the regulation of production of bFGF in endothelial cells must be important for angiogenesis. Okamum et al. have currently observed that TNF induces production and release of bFGF from endothelial cells (20). Further study is required to elucidate the regulatory mechanism of production and release of bFGE
ACKNOWLEDGMENT We thank Dr. D. B. Rifkin at New York University Medical Center for the critical reading of this manuscript.
REFERENCES 1) Rifkin,D.B. and Moscatelli,D. (1989)J.Cell Biol. 109:1-6. 2) Folkman,J. (1986) Cancer Res. 46:467-473. 3) Moscatelli,D., Presta,M. and Rifkin,D.B. (1986) Proc.Natl.Acad.Sci.USA 83:2091-2095. 4) Gospodarowicz,D., Chen,J., Lui,G.M., Baird,A. and Bohlen,P. (1984) Proc.Natl.Acad.Sci. USA 81:6963-6967. 5) Montesano,R., Vasali,J.D., Baird,A., Guillemin,R. and Orci,L. (1986) Proc.Natl.Acad.Sci. USA 83:7297-7301. 6) Moscatelli,D., Presta,M., Joseph-Silverstein,J. and Rifkin,D.B. (1986) J.Cell.Physiol. 129: 273-276. 7) Sato,Y. and Rifkin,D.B. (1988) J.Cell Biol. 107:1199-1205. 8) Sato,Y., Abe M. and Takaki,R. (1990) Biochem.Biophys.Res.Commun. 172:595-600. 9) Ishikawa,E, Miyazono,K., Hellman,U., Drexler,H., Wemstedt,C., Hagiwara,K., Usuki,K., Takaku,E, Risau,W. and Heldin,C-H. (1989) Nature(Lond.) 338:557-562. 10) Leung,D.W., Cachianes,G., Kuang,W-J., Goeddel,D.V. and Ferrara,N. (1989) Science(Wash. DC) 246:1306-1309. 1I) Abraham,J.A., Whang,J.L., Tumolo,A., Mergia,A., Friedman,J., Gospodarowicz,D. and Fiddes,J.C. (1986) 233:545-548. 12) Tsuboi,R., Sato,Y. and Rifldn,D.B. (1990) 110:551-517. 13) Pepper,M.S., Vassalli,J-D., Montesano,R. and Orci,L. (1987) J.Cell Biol. 105:2535-2541. 14) Yasunaga,C., Nakashima,Y. and Sueishi,K. (1989) Lab.Invest. 61:698-704. 15) Odekon,L.E., Sato,Y. and Rifkin,D.B. (1991) J.Cell.Physiol. in press 16) Folkman,J., Klagsbrun,M., Sasse,J., Wadzinski,M., Ingber,D. and Voldavsky,I. (1988) Am.J. Pathol. 130:393-400. 17) Gajdusek,C.M. and Carbon,S. (1989)J.Cell.Physiol. 139:570-579. 18) Mcneil,P.L., Muthukrishnan,L., Warder,E. and D'Amore. (1989) J.Cell Biol. 109:811-822. 19) Maier,J.A.M., Rusnati,M., Ragnotti,G. and Presta,M. (1990) Exp.Cell Res. 186:354-361. 20) Okamura,K., Sato,Y., Matsuda,T., Hamanaka,R., Ono,M., Kohno,K. and Kuwano,M. (1991) J.Biol.Chem. in press.
1102
Vol. 180, No. 2, 1991 October 31, 1991
Autocrinological role of basic fibroblast growth factor on tube formation of vascular endothelial cells in v i t r o Yasufumi Sato ~, Tatsuo Shimada 2, and Ryosaburo Takaki ~ 'First Department of Medicine and 2Department of Anatomy, Oita Medica College Idaigaoka, Hasama-cho, Oita 879-55, Japan
Received
September
24,
1991
When bovine capillary endothelial (BCE) cells plated on type I collagen gel were covered with a second layer of collage gel, BCE cells reorganized into a network of capillary-like structures. In the presence of affinity purified anti-basic fibroblast growth factor (bFGF) antibody, this reorganization was inhibited. By using a computerized image analyzer, the formation of network structures and the effect of anti-bFGF antibody was quantitated. The inhibtory effect of anti-FGF antibody was dose dependent and maximal inhibition was observed at 2.0 pg/ml of antibody. Exogenously added bFGF potentiated network formation of BCE cells and coadministration of bFGF abrogated the inhibitory effect of anti-bFGF antibody. Platelet factor 4, which blocks the binding of bFGF to its receptor, inhibited network formation. These results indicate that bFGF produced by endothelial cells regulates angiogenesis as an autocrine factor. ® 1991 Academic Press, Inc.
Angiogenesis plays an important role in a wide range of physiological and pathological processes including wound healing, growth of solid tumor, diabetic retinoathy, and rheumatoid arthritits. At present, several growth factors have been identified as angiogenic factors. Among them, basic fibroblast growth factor(bFGF) is the best characterized factor (1). Angiogenesis is a complex phenomenon which includes proteolytic degradation of matrix, migration, proliferation, and tube formation by vascular endothelial cells (2). bFGF stimulates proteinase production which is required for matrix degradation, migration, and proliferation of endothelial cells (3) and induces angiogenesis in both IN VIVO (4) and IN VITRO systems (5). Many cell types including endothelial cells produce bFGF (6). Sato and Rifkin have reported that endogenous bFGF regulates plasminogen activator synthesis, migration, and DNA synthesis of endothelial cells (7); phenomena required for angiogenesis. Therefore it is reasonable to hypothesize that bFGF regulates angiogenesis as an autocrine factor. In the present study, we cultivated bovine capillary endothelial (BCE) cells in type 1 collagen gel and observed spontaneous formation of capillary-like structures.
The results indicate that
endogenously produced bFGF by endothelial cells regulated this phenomenon. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1098
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Materials: Bovine capillary endothelial (BCE) cells isolated from adrenal glands were grown in Dulbeco's modified essential medium (DME) containing 10% newborn calf serum (NCS). Affinity purified anti-bFGF antibody was prepared as descrived previously (7). Recombinant human bFGF was kindly provied by Synergen, Inc. (Boulder, CO). Platelet factor 4 (PF-4) was purchased from Sigma (St. Louis,MO). Tube Formation IN VITRO: Seven volumes of type 1 collagen gel solution (Nitta Gelatin, Osaka), 2 volumes of 10xDME, and 1 volume of 0.05N NaOH, 200mM HEPES, and 260mM NaHCO3, were mixed on ice. Half ml of collagen mixture was poured into a 35 mm plastic dish (Coming) and allowed to gel at 37" C. lxl05 of BCE cells were plated onto the collagen gel in DME containing 10% NCS and allowed to attach to collagen gel for 3 hr at 37" C in CO2 incubator. Then the medium was aspirated, half ml of collagen mixture was overlayed, 1 ml of DME containing 1% NCS was added. The indicated concentrations of affinity purified anti-bFGF antibody, recombinant bFGF or PF-4 were administered to both the collagen mixture and overlaying medium. After 24 hr incubation, BCE cells formed network structures. Quantitative Analysis of Network Structures: Phase contrast microscopic picture (200x magnification) was recorded in STILL VIDEO recordplayer (RS000H) (Fuji, Tokyo). Total length of the network structures composed of more than 2 cells recorded in VIDEO was mesured by using a Cosmozone 1S Image Analyzer (Nikon, Tokyo). Ten random fields were mesured and total length per singele field was calculated.
RESULTS AND DISCUSSION When BCE cells were plated onto the first layer of type I collagn gel and covered with a second layer of the gel, BCE cells contacted each other and formed network structures after 24 hr incubation (Fig. 1A). Cross-sectional analysis by electron microscopy revealed that there was a lumen inside of the network structures (Fig. 1C). Microvili could be observed on the luminal surface of the tube-like structure. To test the role of endogenous bFGF on the spontaneous network formation by BCE cells, affinity purified anti-bFGF antibody was applied.
As shown in Fig. 1B, there was a marked
inhibition of network formation by BCE cells. Image analyzer analysis by a Cosmozone 1A was introduced to quantitate the inhibitory effect of anti-bFGF antibody. The total length of network structures composed of at least 2 cells was calculated as described in the Methods. As shown in Fig.2, there was a dose-dependent inhibition of network formation by affinity purified anti-bFGF antibody. The inhibition reached a maximum at 2.0 !ag/ml of anti-bFGF antibody. Exogenously added recombinant bFGF potentiated network formation and also abrogated the inhibitory effect of anti-bFGF antibody (Fig.3).
These results strongly suggest that endogenous bFGF regulates
spontaneous tube formation of BCE cells. We have previously reported that platelet factor 4 (PF-4) blocked the binding of bFGF to its receptor and inhibited the migration of endothelial cells (8). Therefore we tesed whether PF-4 had any effect on nework formation by BCE cells. As shown in Fig.4, 5.0 ]ug/ml of PF-4 significantly inhibited network formation of BCE cells. 1099
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
250/Jm
Fig.1. Network formation of BCE cells in type 1 collagen gel. lx105 of BCE cells were plated onto type 1 collagen gel in 35 mm plastic dish and covered with another layer of collagen gel. A: BCE cells were incubated in DME cotaining 1% of NCS for 24 hr. B: BCE cells were incubated in the presence of affinity purified anti-bFGF antibody. C: Cross-sectional analysis by electron microscopy.
When endothelial cells were cultivated in three dimensional collagen gels, they spontaneously formed tube-like structures. This phenomenon indicated the exsistence of an endogenous regulator of angiogenesis.
Several growth factors have been identified as angiogenic factors (1,9,10). Among
them, bFGF is the factor that a endothelial cell makes by itself (11). bFGF produced by endothelial cells regulates migration, proteinase production, and proliferation of endothelial cells as an autocrine factor (7,12). Migrating endthelial cells express urokinase type plasminogen activator (uPA) (13), and uPA activity is reported to be required for spontaneous tube formation in type 1 collagen gels, 1100
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.~22s
2
v
1.75
w
15
i
ZE
i
"•
2 ~I7S 1.5
1,25
1.25
"~
S
.75
~ 75 o .5
25
.25
0
®
control
anti-bFGF 0.5
anti-bFGF I O
anti-bFGF 2.0
®
control
bFGF( IO)
anti-bFGF(2.0)
anti-bFGF÷bFGF
Fig.2. Dose responsive inhibition of anti-bFGF antibody on network formation. Network formation by BCE cells was quantitated by using Image Analyzer. BCE cells were incubated with 0, 0.5, 1.0, and 2.0 pg/ml of affinity purified anti-bFGF antbody for 24 hr and the length ofnetworkstructures composed of at least 2 cells was measured from 10 different fields. The results are presented as total length per field. Fie.3. Effect of exogenous bFGF and anti-bFGF on network formation by BCE cells. BCE cells were incubated with 10 ng/ml of recombinant bFGF and/or 2.0 pg/ml of affinity purified anti-bFGF antibody for 24 hr and the length of network structures was measured.
suggesting that uPA is a regulator of angiogenesis.(14).
However Odekon et al. have currently
shown that endgenous bFGF regulates the expession of uPA on migrating endothelial cells (15). Our present results further indicate that bFGF produced by endothelial cells plays a central role in the regulation of angiogenesis. Since bFGF lacks a classical signal sequence (11), bFGF must not be secreated and remain cell associated. However it was found that bFGF dose get out of cells and is deposited in extracellular matrix bound to heparan sulfate (16). More recently, a number of studies have shown that smoll amounts of bFGF, sufficient to induce biological antivities, are released from cells where it is postulated to act as an autocrine factor (17,18,19).
However the molecular mechanism for bFGF
release is currently obscure.
2
LO
W
control
PF4
Fig.4. Effect of PF-4 on network formation of BCE cells. BCE cells were incubated with 5.0 lag/ml of PF-4 for 24 hr and the length of network structures was measured. 1101
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Affinity purified anti-bFGF antibody blocked the spontaneous tube formation of endothelial cells in type 1 collagen gels. We could not detect any bFGF in type 1 collagen solution by immunoblot analysis (data not shown). These results indicated that endogenous bFGF regulates tube formation by endothelial cells as an autocrine factor. In this context, the regulation of production of bFGF in endothelial cells must be important for angiogenesis. Okamum et al. have currently observed that TNF induces production and release of bFGF from endothelial cells (20). Further study is required to elucidate the regulatory mechanism of production and release of bFGE
ACKNOWLEDGMENT We thank Dr. D. B. Rifkin at New York University Medical Center for the critical reading of this manuscript.
REFERENCES 1) Rifkin,D.B. and Moscatelli,D. (1989)J.Cell Biol. 109:1-6. 2) Folkman,J. (1986) Cancer Res. 46:467-473. 3) Moscatelli,D., Presta,M. and Rifkin,D.B. (1986) Proc.Natl.Acad.Sci.USA 83:2091-2095. 4) Gospodarowicz,D., Chen,J., Lui,G.M., Baird,A. and Bohlen,P. (1984) Proc.Natl.Acad.Sci. USA 81:6963-6967. 5) Montesano,R., Vasali,J.D., Baird,A., Guillemin,R. and Orci,L. (1986) Proc.Natl.Acad.Sci. USA 83:7297-7301. 6) Moscatelli,D., Presta,M., Joseph-Silverstein,J. and Rifkin,D.B. (1986) J.Cell.Physiol. 129: 273-276. 7) Sato,Y. and Rifkin,D.B. (1988) J.Cell Biol. 107:1199-1205. 8) Sato,Y., Abe M. and Takaki,R. (1990) Biochem.Biophys.Res.Commun. 172:595-600. 9) Ishikawa,E, Miyazono,K., Hellman,U., Drexler,H., Wemstedt,C., Hagiwara,K., Usuki,K., Takaku,E, Risau,W. and Heldin,C-H. (1989) Nature(Lond.) 338:557-562. 10) Leung,D.W., Cachianes,G., Kuang,W-J., Goeddel,D.V. and Ferrara,N. (1989) Science(Wash. DC) 246:1306-1309. 1I) Abraham,J.A., Whang,J.L., Tumolo,A., Mergia,A., Friedman,J., Gospodarowicz,D. and Fiddes,J.C. (1986) 233:545-548. 12) Tsuboi,R., Sato,Y. and Rifldn,D.B. (1990) 110:551-517. 13) Pepper,M.S., Vassalli,J-D., Montesano,R. and Orci,L. (1987) J.Cell Biol. 105:2535-2541. 14) Yasunaga,C., Nakashima,Y. and Sueishi,K. (1989) Lab.Invest. 61:698-704. 15) Odekon,L.E., Sato,Y. and Rifkin,D.B. (1991) J.Cell.Physiol. in press 16) Folkman,J., Klagsbrun,M., Sasse,J., Wadzinski,M., Ingber,D. and Voldavsky,I. (1988) Am.J. Pathol. 130:393-400. 17) Gajdusek,C.M. and Carbon,S. (1989)J.Cell.Physiol. 139:570-579. 18) Mcneil,P.L., Muthukrishnan,L., Warder,E. and D'Amore. (1989) J.Cell Biol. 109:811-822. 19) Maier,J.A.M., Rusnati,M., Ragnotti,G. and Presta,M. (1990) Exp.Cell Res. 186:354-361. 20) Okamura,K., Sato,Y., Matsuda,T., Hamanaka,R., Ono,M., Kohno,K. and Kuwano,M. (1991) J.Biol.Chem. in press.
1102
