Vol.
170,
July
31,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
SPECIFIC
INHIBITION
PROLIFERATION
OF ENDOTHELIAL
867-872
CELL
BY THROMBOSPONDIN
P. Bagavandossand J.W. Wilks Cancer Research The Upjohn Laboratories Kalamazoo. MI 49001 Received
June
20,
1990
SUMMARY: Angiogenesis is a multi-step event involving endothelial cell migration, attachment, and proliferation. A thrombospondin (TSP)-like protein has recently been described as a naturally-occurring inhibitor of angiogenesis (1). We now report that human platelet TSP inhibits the in vitro proliferation of endothelial cells from the rabbit corpus luteum, bovine adrenal cortex and pulmonary artery, and human umbilical vein. The antiproliferative effect of TSP was neutralized by monoclonal antibodies against TSP. The growth arrest seen with TSP was specific for endothelial cells since TSP actually stimulated the growth of vascular smooth muscle cells and human foreskin fibroblasts. These results imply that the angiogenesis-inhibiting effect of TSP is mediated through an inhibition of endothelial cell proliferation. Elucidation of the mechanism of action of TSP on endothelial cell proliferation may lead to potential therapeutic approaches for the control of neovascular diseases. 0199DAcademic Press.Inc. TSP, a trimeric multi-functional glycoprotein, is widely distributed in tissues during development (2), wound healing (3), and malignancy (4). Through its multiple binding domains, TSP interacts with extracellular molecules (5) and cell surface receptors (6-9). Through these receptors, TSP may facilitate cell attachment (10, 1l), tumor cell migration (12), and proliferation of smooth muscle cells (13) and fibroblasts (14). Endothelial cells secrete TSP (15, 16) and possess cell surface receptors for TSP (17). However, the functional significance of TSP for endothelial cells is unknown. Recently, the presence of a tumor suppressor gene in hamster somatic cell hybrids was correlated with the cellular production of a protein inhibitor of angiogenesis (18); this protein is similar to human TSP (1). Themfore, we examined the effect of TSP on endothelial cell proliferation in culture. Materials
and Methods
Materials Microvascular endothelial cells from the rabbit corpus luteum undergoing angiogenesis were isolated as before (19). Bovine adrenal cortex endothelial cells (ACE) were kindly provided. by Dr. D. Gospodarowicz. Rat aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) were kindly provided by Ms. L. Goodman and Ms. N. Oien, respectively. Human foreskin fibroblasts (FF) were kindly provided by Mr. R. Ulrich . Pulmonary artery endothelial cells (PAE) were obtained from ATCC, MD.
867
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tissue culture reagents were obtained from the following sources: endothelial basal medium (EBM), trypsin, and trypsin inhibitor from Clonetics, CA; Hanks balanced salt solution (HBSS) and EDTA (0.02%) from Irvine Scientific, CA; GRGDSP and GRGESP peptides from Peninsula Labs., CA; heparin from Hepar, OH; mouse IgG, enzyme-free dissociation buffer, and epidermal growth factor (EGF) from Sigma, MO; basic fibroblast growth factor (FGF) and rat IgG from Cappel, PA; fetal bovine serum (FBS) from Hyclone, UT, monoclonal antibodies to TSP (20,21) from AMAC, ME. TSP was purified from fresh human platelets as described previously (22). Methods 2500 bovine ACE (passages 9-14) and PAE cells (passages 20-22), rabbit CLE cells (passages 7-14), primary HUVEC, and human FF (passages 20-22) were plated in 24 well plates in EBM containing 1% FBS and incubated at 37 C in an atmosphere of 95% air-Y% CO2. Sixteen hours later, four hundred microliters of fresh EBM-1% FBS, or medium containing of FGF (10 rig/ml), TSP (30 or 60 pg/ml), or FGF+TSP were added to the wells. The medium was changed every other day and the cells were trypsinized and counted in a Coulter counter at selected intervals. Rat SMC at passage 7-8 were plated at 5000 cells/cm2 in EBM-10% FBS. The following day the medium was changed to serumfree medium. Two days later, TSP (30 p&ml), EGF (5 @ml), or EGF+TSP was added in serum-free medium and the cells were incubated, trypsinized, and counted as above. The results shown are from a representative experiment. Each experiment was repeated at least twice and the results were confirmed. Statistical analyses were performed by one way analysis of variance (ANOVA) and Scheffe’s F-test.
Results
and Discussion
When microvascular bovine adrenal cortex endothelial cells (ACE) were exposed to TSP in culture, their basal and basic fibroblast growth factor (FGF)-stimulated proliferation was completely inhibited by TSP (Fig. 1). The inhibition was observed as early as two days after exposure to TSP. However, this inhibition is reversible since the removal of TSP and subsequent addition of growth factor alone leads to a resumption of endothelial cell proliferation (data not shown). As shown in Fig. 2, monoclonal antibodies to TSP not only neutralized the growth arrest seen with TSP but also stimulated the bovine ACE cell growth above that seen with FGF alone. The stimulatory effect of TSP antibodies could be due to their ability to neutralize the endogenous TSP produced by these cells. Heparin at as low as 10 yg/ml inhibits the binding of TSP to bovine aortic endothelial cells (17). Therefore, we examined the effect of heparin on TSP-mediated inhibition of endothelial cell growth. Heparin, even at 50 pg/ml, did not neutralize the inhibitory effect of TSP on bovine ACE cells (FGF, 216,000?15716 cells; FGF+TSP+heparin 591of995 cells). Recently, platelet factor-4 has been shown to be an inhibitor of angiogenesis and human umbilical vein endothelial cell (HUVEC) growth (23). Unlike platelet factor-4, TSP mediated inhibition of cell proliferation is insensitive to heparin. These results argue against the possibility that platelet factor-4 is a potential contaminant of our TSP preparation. Further, the effect of TSP on the endothelial cell growth appears to be mediated by non-heparin binding domain of TSP. TO determine the species specificity of TSP inhibition, we examined its effect on the growth of endothelial cells derived from the 5 day old corpora lutea undergoing angiogenesis in the rabbit (CLE). TSP also inhibited rabbit microvascular endothelial cell 868
Vol.
170,
No.
2,
BIOCHEMICAL
1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-r L
-
0
01
0
4
2
6
Days in culture
02
u
3 F c L
Figure 1. Inhibition of bovine ACE cell proliferation by TSP. The ACE cells were plated and grown as described in “Methods”. The medium was replaced every other day and the cells were trypsinized and counted on days 2, 4, and 6. FGF stimulated the proliferation of ACE cells (P
170,
July
31,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1990
SPECIFIC
INHIBITION
PROLIFERATION
OF ENDOTHELIAL
867-872
CELL
BY THROMBOSPONDIN
P. Bagavandossand J.W. Wilks Cancer Research The Upjohn Laboratories Kalamazoo. MI 49001 Received
June
20,
1990
SUMMARY: Angiogenesis is a multi-step event involving endothelial cell migration, attachment, and proliferation. A thrombospondin (TSP)-like protein has recently been described as a naturally-occurring inhibitor of angiogenesis (1). We now report that human platelet TSP inhibits the in vitro proliferation of endothelial cells from the rabbit corpus luteum, bovine adrenal cortex and pulmonary artery, and human umbilical vein. The antiproliferative effect of TSP was neutralized by monoclonal antibodies against TSP. The growth arrest seen with TSP was specific for endothelial cells since TSP actually stimulated the growth of vascular smooth muscle cells and human foreskin fibroblasts. These results imply that the angiogenesis-inhibiting effect of TSP is mediated through an inhibition of endothelial cell proliferation. Elucidation of the mechanism of action of TSP on endothelial cell proliferation may lead to potential therapeutic approaches for the control of neovascular diseases. 0199DAcademic Press.Inc. TSP, a trimeric multi-functional glycoprotein, is widely distributed in tissues during development (2), wound healing (3), and malignancy (4). Through its multiple binding domains, TSP interacts with extracellular molecules (5) and cell surface receptors (6-9). Through these receptors, TSP may facilitate cell attachment (10, 1l), tumor cell migration (12), and proliferation of smooth muscle cells (13) and fibroblasts (14). Endothelial cells secrete TSP (15, 16) and possess cell surface receptors for TSP (17). However, the functional significance of TSP for endothelial cells is unknown. Recently, the presence of a tumor suppressor gene in hamster somatic cell hybrids was correlated with the cellular production of a protein inhibitor of angiogenesis (18); this protein is similar to human TSP (1). Themfore, we examined the effect of TSP on endothelial cell proliferation in culture. Materials
and Methods
Materials Microvascular endothelial cells from the rabbit corpus luteum undergoing angiogenesis were isolated as before (19). Bovine adrenal cortex endothelial cells (ACE) were kindly provided. by Dr. D. Gospodarowicz. Rat aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) were kindly provided by Ms. L. Goodman and Ms. N. Oien, respectively. Human foreskin fibroblasts (FF) were kindly provided by Mr. R. Ulrich . Pulmonary artery endothelial cells (PAE) were obtained from ATCC, MD.
867
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tissue culture reagents were obtained from the following sources: endothelial basal medium (EBM), trypsin, and trypsin inhibitor from Clonetics, CA; Hanks balanced salt solution (HBSS) and EDTA (0.02%) from Irvine Scientific, CA; GRGDSP and GRGESP peptides from Peninsula Labs., CA; heparin from Hepar, OH; mouse IgG, enzyme-free dissociation buffer, and epidermal growth factor (EGF) from Sigma, MO; basic fibroblast growth factor (FGF) and rat IgG from Cappel, PA; fetal bovine serum (FBS) from Hyclone, UT, monoclonal antibodies to TSP (20,21) from AMAC, ME. TSP was purified from fresh human platelets as described previously (22). Methods 2500 bovine ACE (passages 9-14) and PAE cells (passages 20-22), rabbit CLE cells (passages 7-14), primary HUVEC, and human FF (passages 20-22) were plated in 24 well plates in EBM containing 1% FBS and incubated at 37 C in an atmosphere of 95% air-Y% CO2. Sixteen hours later, four hundred microliters of fresh EBM-1% FBS, or medium containing of FGF (10 rig/ml), TSP (30 or 60 pg/ml), or FGF+TSP were added to the wells. The medium was changed every other day and the cells were trypsinized and counted in a Coulter counter at selected intervals. Rat SMC at passage 7-8 were plated at 5000 cells/cm2 in EBM-10% FBS. The following day the medium was changed to serumfree medium. Two days later, TSP (30 p&ml), EGF (5 @ml), or EGF+TSP was added in serum-free medium and the cells were incubated, trypsinized, and counted as above. The results shown are from a representative experiment. Each experiment was repeated at least twice and the results were confirmed. Statistical analyses were performed by one way analysis of variance (ANOVA) and Scheffe’s F-test.
Results
and Discussion
When microvascular bovine adrenal cortex endothelial cells (ACE) were exposed to TSP in culture, their basal and basic fibroblast growth factor (FGF)-stimulated proliferation was completely inhibited by TSP (Fig. 1). The inhibition was observed as early as two days after exposure to TSP. However, this inhibition is reversible since the removal of TSP and subsequent addition of growth factor alone leads to a resumption of endothelial cell proliferation (data not shown). As shown in Fig. 2, monoclonal antibodies to TSP not only neutralized the growth arrest seen with TSP but also stimulated the bovine ACE cell growth above that seen with FGF alone. The stimulatory effect of TSP antibodies could be due to their ability to neutralize the endogenous TSP produced by these cells. Heparin at as low as 10 yg/ml inhibits the binding of TSP to bovine aortic endothelial cells (17). Therefore, we examined the effect of heparin on TSP-mediated inhibition of endothelial cell growth. Heparin, even at 50 pg/ml, did not neutralize the inhibitory effect of TSP on bovine ACE cells (FGF, 216,000?15716 cells; FGF+TSP+heparin 591of995 cells). Recently, platelet factor-4 has been shown to be an inhibitor of angiogenesis and human umbilical vein endothelial cell (HUVEC) growth (23). Unlike platelet factor-4, TSP mediated inhibition of cell proliferation is insensitive to heparin. These results argue against the possibility that platelet factor-4 is a potential contaminant of our TSP preparation. Further, the effect of TSP on the endothelial cell growth appears to be mediated by non-heparin binding domain of TSP. TO determine the species specificity of TSP inhibition, we examined its effect on the growth of endothelial cells derived from the 5 day old corpora lutea undergoing angiogenesis in the rabbit (CLE). TSP also inhibited rabbit microvascular endothelial cell 868
Vol.
170,
No.
2,
BIOCHEMICAL
1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-r L
-
0
01
0
4
2
6
Days in culture
02
u
3 F c L
Figure 1. Inhibition of bovine ACE cell proliferation by TSP. The ACE cells were plated and grown as described in “Methods”. The medium was replaced every other day and the cells were trypsinized and counted on days 2, 4, and 6. FGF stimulated the proliferation of ACE cells (P
