Effects of cocultivation of Human Umbilical Vein Endothelial Cells with Bowes Melanoma Cells K. ADDO-BOADU, G. CHRIST, AND B. R. BINDER Clinical and E a p i m m l Ph@& U n M t y of Vienna Schmpanimnassc 17 A-1090 V ~ WAw2ria , Fibrinolytic activity is thought to be important not only for intravascular thrombolysis but also for extravascular proteolysis occurring in processes like tumor invasion and metastasis. In the latter situation endothelid and smooth muscle cells are thought to contribute to the fibrinolytic status by providing plasminogen activator inhibitor (PAI) capacity whereas tumor cells are thought to generate plasminogen activating activity. Most tumor cells regulate local proteolytic activity by expression of urokinase and its specific receptor,*V2while only a few tumor cells are known to generate fibrinolytic activity by elaborating tissue-type plasminogen activator (tPA). The most common example of a tumor cell line with high tPA activity is the Bowes melanoma cell line. It was the aim of this study to cultivate human umbilical vein endothelial cells (HUVEC) together with Bowes melanoma cells in order to analyze whether in such a system the cultured cells can influence each other's fibrinolytic potential.
MATERIALS AND METHODS HUVEC were isolated by mild collagenase treatment3 and seeded on amniotic membranes4 stretched across the bottom of hollow teflon cylinders. Bowes cells were seeded in six well plastic plates (COSTAR). Both cell types were cultured to confluency in M199 (Sigma) containing 20% sutplemented calfserum (SCS, Hyclone), 50 &ml endothelial cell growth supplement (ECGS),5 and 5 U/ml heparin (Liquemin, Hoffmann-LaRoche). The cocultivation system was established by placing the teflon cylinders containing the HUVEC cultures into the six well plates containing the Bowes cells in such a way that the supernatants in the resulting two chambers were in contact only across the amniotic membrane. Experiments were perbrmed by cocultivating the confluent cells for 24 h in serum-free media containing 1%bovine serum albumin (BSA). For control purposes, conditioned media of Bowes cells were used instead of coculturing. tPA and PAI-1 antigen were determined in conditioned media by specific ELISAS (Technoclone) using monoclonal catching and detecting antibodies for the PAI-1 ELISA and polyclonal catching and monoclonal detecting antibodies fbr the tPA 186
ADDO-BOADU ct aL.: EPPECTS OP COCUL'IWATION
vI L 3
2 FIGURE 1. PAL1 antigen in conditioned media of HUVEC (A), Bowes cells (B), coculture of HUVEC and Bowes (C), and HUVEC grown in F media conditioned for 24 h by Bowes cells (D). 7
187
1250 1000
750
500
Y
4
250 n "
A
B
C
D
ELISA. The PAI-1 ELI% recognizes all three forms of PAI-1 (active, latent, and complexed with tPA) while the tPA ELISA recognizes active and complexed tPA.
RESULTS AND DISCUSSION The results are shown in FIGURES1 and 2. In the cocultivation system a reduction in PAI-1 antigen by 36%and tPA antigen by 25% was observed. These changes in PAI-1 or tPA secretion cannot be accounted for by the amount of tPA or PAI-1 contained in the conditioned media of the respective cells because when only conditioned media of Bowes were added to HUVEC no significant change in secreted PA1 could be observed, indicating that simple complex formation of tPA and PAI-1 did not change the amount of PAI-1 released by the endothelid cells. On the other hand the amount of tPA detected in the supernatant of HUVEC exposed to Bowes conditioned media was reduced by 72%,indicating that free tPA contained in the conditioned media might become complexed with PAI-1 and/or a tPA receptor both present on the surfice of HUVEC. However, this explanation is in contrast to the insignificant reduction of tPA in a coculturing system. This difference might be explained by a steady state of HUVEC taking up or degrading tPA and Bowes cells secreting tPA at a possibly stimulated rate. Otherwise the differences in the tPA content of the conditioned media be-
__
7,1313C
,,,L
T
600
2 500 ;= 4oo L
FIGURE 2. tPA antigen in conditioned media of HUVEC (A), Bowes cells (B), coculture of HUVEC and Bowes (C), and HUVEC grown in media conditioned for 24 h by Bowes cells (D).
\
5" 300 4+ 200 100
n-
A
B
C
D
188
ANNALS NEW YORK ACADEMY OF SCIENCES
tween a coculture system and HUVEC grown in conditioned media of Bowes cells remain unexplained. The data presented indicate that in a system of cocultivation between HUVEC, providing essentially an antiiibrinolytic environment, together with tPA-producing Bowes melanoma cells, the resulting overall fibrinolytic inhibiting capacity is significantly reduced by approximately one-third owing to a decrease in PAI-1 production by endothelial cells while the overall amount of t-PA in the conditioned media is only slightly reduced as compared to a system where no Bowes cells are present. Therefore, fictors other than PAI-1 and tPA m&t influence the PAI-1 production in human umbilical vein endothelial cells leading to a down replation of the total fibrinolytic inhibitory potential and thereby ficilitating invasion and metastasis by Bowes melanoma cells. REFERENCES 1. 2. 3. 4. 5.
HOLLAS, W. & D. BOYD. 1991. Semin. Thromb. Haemostasis 17: 225-230. KWMN,H. C., ctal. 1991. Semin.Thromb. Haemostasis 17: 175-182. GIMBRONE, M.A., R CoTRMl & S. S. FOLKMAN.1974. J. Cell Biol. 60:673684. FURIE,M. B., B. C. NAPRSTEK & C. SILVERSTEIN. 1987. J. Cell Sci. 88: 161-175. GOSIJODAROWICZ, J. 1975. J. Biol. Chem. 250: 2515-2520.
MATERIALS AND METHODS HUVEC were isolated by mild collagenase treatment3 and seeded on amniotic membranes4 stretched across the bottom of hollow teflon cylinders. Bowes cells were seeded in six well plastic plates (COSTAR). Both cell types were cultured to confluency in M199 (Sigma) containing 20% sutplemented calfserum (SCS, Hyclone), 50 &ml endothelial cell growth supplement (ECGS),5 and 5 U/ml heparin (Liquemin, Hoffmann-LaRoche). The cocultivation system was established by placing the teflon cylinders containing the HUVEC cultures into the six well plates containing the Bowes cells in such a way that the supernatants in the resulting two chambers were in contact only across the amniotic membrane. Experiments were perbrmed by cocultivating the confluent cells for 24 h in serum-free media containing 1%bovine serum albumin (BSA). For control purposes, conditioned media of Bowes cells were used instead of coculturing. tPA and PAI-1 antigen were determined in conditioned media by specific ELISAS (Technoclone) using monoclonal catching and detecting antibodies for the PAI-1 ELISA and polyclonal catching and monoclonal detecting antibodies fbr the tPA 186
ADDO-BOADU ct aL.: EPPECTS OP COCUL'IWATION
vI L 3
2 FIGURE 1. PAL1 antigen in conditioned media of HUVEC (A), Bowes cells (B), coculture of HUVEC and Bowes (C), and HUVEC grown in F media conditioned for 24 h by Bowes cells (D). 7
187
1250 1000
750
500
Y
4
250 n "
A
B
C
D
ELISA. The PAI-1 ELI% recognizes all three forms of PAI-1 (active, latent, and complexed with tPA) while the tPA ELISA recognizes active and complexed tPA.
RESULTS AND DISCUSSION The results are shown in FIGURES1 and 2. In the cocultivation system a reduction in PAI-1 antigen by 36%and tPA antigen by 25% was observed. These changes in PAI-1 or tPA secretion cannot be accounted for by the amount of tPA or PAI-1 contained in the conditioned media of the respective cells because when only conditioned media of Bowes were added to HUVEC no significant change in secreted PA1 could be observed, indicating that simple complex formation of tPA and PAI-1 did not change the amount of PAI-1 released by the endothelid cells. On the other hand the amount of tPA detected in the supernatant of HUVEC exposed to Bowes conditioned media was reduced by 72%,indicating that free tPA contained in the conditioned media might become complexed with PAI-1 and/or a tPA receptor both present on the surfice of HUVEC. However, this explanation is in contrast to the insignificant reduction of tPA in a coculturing system. This difference might be explained by a steady state of HUVEC taking up or degrading tPA and Bowes cells secreting tPA at a possibly stimulated rate. Otherwise the differences in the tPA content of the conditioned media be-
__
7,1313C
,,,L
T
600
2 500 ;= 4oo L
FIGURE 2. tPA antigen in conditioned media of HUVEC (A), Bowes cells (B), coculture of HUVEC and Bowes (C), and HUVEC grown in media conditioned for 24 h by Bowes cells (D).
\
5" 300 4+ 200 100
n-
A
B
C
D
188
ANNALS NEW YORK ACADEMY OF SCIENCES
tween a coculture system and HUVEC grown in conditioned media of Bowes cells remain unexplained. The data presented indicate that in a system of cocultivation between HUVEC, providing essentially an antiiibrinolytic environment, together with tPA-producing Bowes melanoma cells, the resulting overall fibrinolytic inhibiting capacity is significantly reduced by approximately one-third owing to a decrease in PAI-1 production by endothelial cells while the overall amount of t-PA in the conditioned media is only slightly reduced as compared to a system where no Bowes cells are present. Therefore, fictors other than PAI-1 and tPA m&t influence the PAI-1 production in human umbilical vein endothelial cells leading to a down replation of the total fibrinolytic inhibitory potential and thereby ficilitating invasion and metastasis by Bowes melanoma cells. REFERENCES 1. 2. 3. 4. 5.
HOLLAS, W. & D. BOYD. 1991. Semin. Thromb. Haemostasis 17: 225-230. KWMN,H. C., ctal. 1991. Semin.Thromb. Haemostasis 17: 175-182. GIMBRONE, M.A., R CoTRMl & S. S. FOLKMAN.1974. J. Cell Biol. 60:673684. FURIE,M. B., B. C. NAPRSTEK & C. SILVERSTEIN. 1987. J. Cell Sci. 88: 161-175. GOSIJODAROWICZ, J. 1975. J. Biol. Chem. 250: 2515-2520.
