JOURNAL OF CELLULAR PHYSIOLOGY 151549-554 (1992)

Stimulation of Endothelial Cell Proliferation by Vanadate I s Specific for Microvascular Endothelial Cells PAMELA A. MAHER Department of Molecular and Cellular Growth Biology, Whittier lnstitute for Diabetps and Endocrinology, La jolia, California 92037 Micromolar conccntrations of sodium orthovanadate 5timulated the proliferation of bovine capillary endothelial cclls, but no1 bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadatc treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadatc was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF-p, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect a1 higher vanadate concentrations. B 1992 Wiley-Liss, Inc

Although the proliferation of blood vessels is a n essential part of embryonic development, angiogenesis occurs infrequently in most adult tissues. In the adult, controlled angiogenesis is a n essential part of the wound healing process, whereas uncontrolled angiogenesis occurs in growing tumors and other pathological conditions. Since angiogenesis requires proliferation of endothelial cells, much effort has been directed towards identifying and characterizing endothelial cell mitogens. Endothelial cells can be induced to proliferate in uitro by a number of different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin, and insulin-like growth factors (for reviews see Bar et al., 1988; Klagsbrun and D’Amore, 1991). The receptors for all of these growth factors have been characterized and they all contain tyrosine kinase domains (for review see Ullrich and Schlessinger, 1990). Thus, addition of any of these ligands to endothelial cells will stimulate tyrosine kinase activity and lead to the tyrosine phosphorylation of specific proteins which eventually can give rise to endothelial cell proliferation. Although all endothelial cells perform similar structural functions in the animal, there appears to be significant physiological differences between microvascular or capillary endothelial cells and the macrovascular endothelial cells which line the arteries and veins (Zetter, 1984). For instance, several of the growth factors described above, including EGF, PDGF (Boes et al., 1991; Beitz et al., 1991) and insulin (Bar e t al., 1988), are specific for one or the other type of endothelial cell. Thrombin has been shown to stimulate protein phosphorylation and proliferation in endothelial cells derived from large vessels, but not those derived from capillaries (Dupuy et al., 1989). The protein kinase C (L)

1992 WILEY-LISS, INC

activator, p-phorbol12,13-dibutyrate,can suppress the proliferation of capillary endothelial cells induced by bFGF, but has no effect on aortic endothelial cells (Doctrow and Folkman, 1987). Thus, the two types of endothelial cells differ not only in the array of growth factor receptors present on their surfaces, but also in their intracellular regulatory mechanisms. The level of protein tyrosine phosphorylation in cells can be regulated by the activity of both tyrosine kinases (for review see Hunter, 1989) and protein tyrosine phosphatases (for reviews see Alexander, 1990; Tonks and Charbonneau, 1989). Thus, even if tyrosine kinase activity is low, inhibition of tyrosine phosphatase activity can lead to increased protein tyrosine phosphorylation (Klarlund, 1985; Marchisio et al., 1988; Owada et al., 1989) and, in some cases, increased cell proliferation (Klarlund, 1985). When used a t micromolar concentrations sodium orthovanadate can act as a specific inhibitor of tyrosine phosphatases (Swarup et al., 1982). Based on these observations, we asked whether or not sodium orthovanadate could promote the proliferation of aortic or capillary endothelial cells and whether or not it could potentiate the action of bFGF, a n agent whose receptor is a protein tyrosine kinase. The results of these studies show a differential effect of sodium orthovanadate on the proliferation of the two types of endothelial cells. MATERIALS AND METHODS Cell culture and mitogenic agents Bovine aortic endothelial (ABAE) cells were prepared from the aortic arch and cultured as described Received July 8,1991; accepted January 22,1992.

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(Baird and Durkin, 1986).Bovine capillary endothelial (ACE) cells were derived from the adrenal cortex and cultured a s described (Gospodarowicz et al., 1986).Human and recombinant bFGF were obtained from Dr. Andrew Baird. TGF-P was from R&D Systems. Sodium orthovanadate was purchased from Sigma and prepared as a 1 mM stock in water.

Cell proliferation assays This assay was a modification of a previously described method (Baird and Durkin, 1986).Aortic and capillary endothelial cells were trypsinized and plated in 24-well dishes a t 5 x l o 3 cellsiwell in Hepes buffered Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum. After 24 h r sodium orthovanadate andlor growth factors were added. After 5 days of incubation the cells were harvested by trypsinization and counted using a Coulter particle counter. The results are expressed as the fold increase in cell number relative to control cultures to which the appropriate solvent was added. A fold increase of 1 indicates no change relative to control cultures. Control cultures showed a two-fold increase in cell number over the course of the experiment. The data are the mean of triplicate determinations. All experiments were done 3-7 times with similar results. Protein tyrosine phosphorylation To assay for the presence of phosphotyrosine in cellular proteins, 5 x lo4 aortic or capillary endothelial cells were plated on 6 cm dishes to give the same number of cells/cm2 a s was used in the cell proliferation assays. After 24 hr sodium orthovanadate was added. After 1-5 days the cell were harvested by scraping and then solubilized in sodium dodecyl sulfate sample buffer. Equal amounts of cellular proteins were separated by SDSPAGE on 7.5% gels, transferred to nitrocellulose, and immunoblotted with rabbit anti-phosphotyrosine antibodies and lZ5I-proteinA a s described previously (Maher and Pasquale, 1988). Immunofluorescence microscopy Aortic or capillary endothelial cells were plated onto glass coverslips in 35 mm dishes a t 1 x lo4 cellsidish to give the same number of cells/cm2 as was used in the cell proliferation assays. After 24 h r sodium orthovanadate was added and 5 days later the cells were fixed, permeabilized, and immunolabeled with anti-phosphotyrosine antibodies and NBD-phallacidin as described previously (Maher and Pasquale, 1988).Labeled cells were examined with a Zeiss microscope using a x63 numerical aperture 1.4 planapochromat oil objective and photographed on Tri-X film. Protein tyrosine kinase assay Aortic or capillary endothelial cells were plated on 6 cm dishes and treated with sodium orthovanadate for 1-5 days as described above. The cells were rinsed with PBS? scraped into homogenization buffer, and sonicated, and equal amounts of protein were assayed for tyrosine kinase activity as described previously (Maher, 1991)using poly g1u:tyr (4:l)a s a substrate. The

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Fig. 1. Effect of (A) sodium orthovanadate and (B) bFGF on ACE and ARAE cell proliferation. 5 x 10’ cells were plated per well of a 24-wcll dish. After 24 hr the indicated concentrations of sodium orthovanadate or hFGF were added directly to the culture medium. After 5 days the cells were trypsinized and counted in a Coulter counter. The results are expressed as the fold increase in cell number relative to control cultures to which the appropriate solvent was added. A fold increase of 1 indicates no change relative to control cultures. Control cultures showed a doubling in cell number over the course of the experiment. The data are the mean -t SD of triplicate determinations. All experiments were done at least 3 times with similar results. .-H ACE cells: e--. ABAE cells.

amount of phosphate incorporated was determined by Ceren kov counting.

Protein assay Protein was determined using the BCA method (Pierce). RESULTS When bovine capillary endothelial cells (ACE cells) were plated a t low density in serum-containing me-

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VANADATE STIMULATES ENDOTHELIAL CELL GROWTH

dium for 24 h r and then treated with sodium orthovanadate for 5 days, a marked increase in cell proliferation was observed (Fig. la). The most effective concentration was 20 pM vanadate, although concentrations a s low a s 1 p M caused a small, but reproducible, increase in cell number above untreated cells. Concentrations above 20 pM brought about a progresssive decrease in cell number. ACE cells treated in parallel with bFGF, a well-characterized endothelial cell mitogen, showed a similar increase in cell proliferation with only the highest bFGF concentrations more effective than 20 FM vanadate (Fig. lb). To determine if vanadate could enhance the effect of bFGF on ACE cells, the cells were treated simultaneously with various doses of both bFGF and vanadate. The results are shown in Figure 2a. At bFGF concentrations below 0.5 ngiml, the effects of vanadate and bFGF were additive below 20 pM vanadate. At the highest concentration of bFGF used (5.0 ng/ml), the effects of bFGF and vanadate on cell proliferation were somewhat more than additive a t vanadate concentrations below 20 pM. In contrast to the above finding, when bovine aortic endothelial cells (ABAE cells) were plated a t low density and treated with vanadate as described above for capillary endothelial cells, no increase in cell number was observed (Fig. l a ) . Cells treated in parallel with bFGF showed a 2-3-fold increase in cell number (Fig. lb). There was also no interaction between bFGF and vanadate in ABAE cells (not shown). TGF-P is a potent inhibitor of bFGF stimulation of endothelial cell proliferation (Baird and Durkin, 1986). The mechanism for this inhibition is not known. If vanadate was stimulating ACE proliferation by the same pathway as bFGF, then TGF-P might also block vanadate-induced cell proliferation. When ACE cells were treated with varying amounts of vanadate in the presence of a saturating concentration of TGF-P, a slight stimulation of the effects of vanadate was observed a t the lower vanadate concentrations. However, in the presence of TGF-P, 20 pM vanadate was no longer effective a t stimulating cell proliferation (Fig. 2b). To determine if a n increase in protein tyrosine phosphorylation correlated with the mitogenic effects of vanadate, cells were treated with vanadate for 1-5 days and then equal amounts of cellular proteins were analyzed for the presence of phosphotyrosine by SDSPAGE and immunoblotting with anti-phosphotyrosine antibodies. Untreated ACE cells showed phosphotyrosine in proteins of 116-120,70,60 and 55 kD (Fig. 3 ) . Treatment with 10 pM vanadate produced a n increase in protein tyrosine phosphorylation in a number of proteins ranging in molecular weight from 43-200 kD which was first apparent after 2 days and reached a maximum after 4 days (Fig. 3). Vanadate also increased the level of phosphotyrosine in proteins in ABAE cells with a time course similar to that seen with ACE cells (Fig. 3 ) . To further investigate the increases in protein tyrosine phosphorylation induced by vanadate, control and vanadate-treated ACE cells were stained with anti-phosphotyrosine antibodies and NBD-phallacidin to label F-actin and examined by immunofluorescence microscopy. As shown in Figure 4, control cells were

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Fig. 2. A Etfect ofdifferent bFGF conccntrations on the stiniulation of ACE proliferation by sodium orthovanadate; 6 X 10' cells were plated per well of a 24-well dish. After 24 hr the indicated concentrations of Podium orthovanadate and bFGF were added directly to the culture medium. After 5 days the cells were trypsinized and counted in u Coulter counter. The results are expressed a s the fold increase in cell number relative to control cultures to which the appropriate solvent was added. A fold increase of 1indicates no change relative to control cultures. Control culturcs showed a doubling in cell number over the course of the experiment. The data are the mean of triplicate determinations. All experiments were done 3 times with similar results. The variation in the results was less than 10% and the error is not plotted. H-m no bFGF; .-•0.05 n g h l bFGF; c-u 0.5 ng/ml bFGF; A-A 5.0 n g h l bFGF. B: Effect of TGF-P on the stimulation of ACE proliferation by sodium orthovanadate; 5 x 10" cells were plated per well of a 24-well dish. After 24 hr the indicated concentrations of sodium orthovanadate and TGV-P (1 ng!mli wcrc added directly to the culture medium. After 5 days the cells were trypsinized and counted in a Coulter counter. The results are expressed as the fold increase in cell number relative to control cultures l o which the appropriate solvent was added. A fold increase of 1 indicates no change relative to control cultures. Control cultures showed a doubling in cell numher over the course of the experiment. The data are the mean ? SD of triplicate determinations. All experiments were done 3 times with similar results. no TGF-B; 1 ngiml TGP-p.

.-= .--.

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Fig. 3. Stimulation of protein tyrosine phosphorylation hy sodium orthovanadate. Immunoblot, with rabbit anti-phosphotyrosine antibodies and 1Z51-proteinA, of extracts from ACE (100 pg total cellular proteidlane) and ABAE cells (50 pg total cellular proteinllane) either untreated ( -) for 1-5 days or treated (+) with 10 p M sodium orthovanadate for 1-5 days. Molecular weight markers, in kD, are at right.

generally well-spread and had prominent actin-containing stress fibers. The low levels of phosphotyrosine were present at focal contact-like regions. These regions also stained with a n antibody to vinculin, a characterized focal contact protein (Geiger, 1979). With vanadate concentrations a s low as 5 FM some cells showed a n increase in labeling of their focal contacts. Higher concentrations of vanadate led to progressively more cells showing a n increased labeling in focal contacts. In addition, more and more cells appeared which had extremely high levels of phosphotyrosine present throughout their cytoplasms (Fig. 4).Although these cells frequently had a n abnormal, elongated morphology, there were also a number of cells whose morphology appeared normal, despite the presence of high amounts of phosphotyrosine. Similar results were obtained with the ABAE cells (not shown). Since vanadate appeared equally effective in increasing protein tyrosine phosphorylation in both aortic and capillary endothelial cells, it was possible that the lack

of effect on aortic endothelial cell proliferation was due to the inhibition of a tyrosine kinase by phosphorylation. Some intracellular tyrosine kinases are known to be inhibited by tyrosine phosphorylation (Cooper and King, 1986; Mustelin and Altman, 1989). Thus, high levels of tyrosine phosphorylation could lead to the inhibition of one or more tyrosine kinases involved in cell proliferation. To test this hypothesis, aortic and capillary endothelial cells were plated at low density and treated with 10 J.LMvanadate as in the previous experiments. After 1-5 days, cell lysates were prepared and assayed for total tyrosine kinase activity using a synthetic substrate. The results are shown in Table 1. AIthough vanadate treatment slightly stimulated tyrosine kinase activity in the ACE cells, it led to a decrease in tyrosine kinase activity in the ABAE cells.

DISCUSSION These results shour that the proliferation of capillary endothelial cells can be stimulated by vanadate in a dose-dependent fashion. The finding that vanadate stimulated the proliferation of capillary endothelial cells but not aortic endothelial cells suggests that the two types of endothelial cells have different mechanisms of growth regulation. However, in both types of endothelial cells, vanadate increased the level of phosphotyrosine in cellular proteins, indicating that the lack of a proliferative effect on aortic endothelial cells was not due to a failure to inhibit protein tyrosine phosphatases. In addition, vanadate greatly altered the morphology of both types of endothelial cells. Therefore, the failure of aortic endothelial cells to respond to vanadate with increased cell proliferation suggests that a different mechanism exists in these cells to control their growth. These results are consistent with thc finding that activators of protein kinase C can inhibit the proliferation of capillary endothelial cells induced by bFGF, but have no effect on aortic endothelial cells (Doctrow and Folkman, 1987). Vanadate could be inducing an increase in the proliferation of the capillary endothelial cells, either by stimulating more cells to divide or by decreasing the length of the cell cycle of cells which are already dividing. Preliminary studies using bromodeoxyuridine treatment of cells followed by labeling with a n anti-bromodeoxyuridine antibody indicate that vanadate increases the number of labeled nuclei above that seen in control cells (not shown). Vanadate, a t the concentrations used in this study, is a specific inhibitor of protein tyrosine phosphatases (Swarup, 1982; Klarlund, 1985). However, the lack of response of the aortic endothelial cells to vanadate may be due to the inhibition of tyrosine kinase activity. This result appears inconsistent with the similar increases in protein tyrosine phosphorylation seen in the two types of cells. However, these increases in tyrosine phosphorylation are seen over several days and tyrosine kinase activity is only partially reduced, so that even low levels of tyrosine kinase activity would be able to give rise to increased phosphotyrosine levels in the absence of phosphatase activity. Furthermore, some tyrosine kinases, most notably those of the src family, are inactivated by tyrosine phosphorylation (King and Cooper, 1986; Mustelin et al., 1989).Thus, the data suggest

VANADATE STIMULATES ENDOTHELIAL CELL GROWTH

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Fig. 4. Indirect immunofluorescent labeling of cultures of ACE cells with rabbit antiphnsphotyrosine antibodies, followed by rhodamine-conjugated goat anti-rabbit (B, D) and NED-phallacidin to label F-actin (A, C). Cells were grown for 5 days in the absence (A. B) or presence (C, DJ of 10 FM sodium orthovanadate

TABLE 1. Protein tyrosine kinase activity in endothelial cells1 Time 1 day 2 days 3 days 4 days 5 days

ACE

ABAE

1.10 i .10 1.36 f .10 1.20 .01 1.21 i .14 1.08 f .06

0.97 f .10 0.67 f .10 0.77 .07 0.72 .04 0.85 f .08

+

**

'Protein tyrosine kinase activity was assayed in homogenates of endothelial cells as described in Materials and Methods. The results are the average of triplicate determinations performed on each of two separate experiments.

that one or more of these tyrosine kinases play a critical role in aortic endothelial cell proliferation, but not capillary endothelial cell proliferation. Since bFGF and a number of the other growth factors for endothelial cells stimulate tyrosine kinase activity, i t was possible that by blocking phosphatase activity vanadate could significantly increase the effectiveness of any such growth factors which were present in the culture medium. In other words, the effect of those growth factors whose receptors possess tyrosine kinase activity would be potentiated by vanadate, since the phosphorylation of the substrates would persist. How-

ever, the results with bFGF and vanadate suggest that this does not occur. Although cell growth at 5 ngiml bFGF and 5 or 10 pM vanadate was slightly more than additive, cell growth in the presence of lower concentrations of bFGF (50-500 pgiml) and 1-20 p M vanadate was never more than additive, indicating that vanadate does not potentiate the mitogenic effect of low doses of bFGF on capillary endothelial cells. Further evidence that vanadate and bFGF are not acting through the same pathways to stimulate cell proliferation is provided by the experiments with TGF-P. TGF-P can inhibit bFGF-stimulated endothelial cell proliferation over a wide range of bFGF concentrations (Baird and Durkin). However, with vanadate, TGF-P appears to shift the dose response curve such that lower concentrations of vanadate are slightly more effective a t stimulating cell proliferation and 20 pM vanadate is ineffective or slightly toxic. Vanadate induces the transformation of fibroblasts (Klarlund, 1985; Marchisio et. al., 1988) and chondrocytes (Owada et al., 1989). Among the criteria used to characterize the vanadate-induced transformation of fibroblasts and chondrocytes were a change in cellular

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morphology and a decrease in density-dependent growth inhibition. These two criteria are similar to the changes induced by vanadate on the capillary endothelial cells. These cells exhibited a marked change in morphology, similar to that of transformed cells, and a n increase in cell proliferation. Therefore, vanadate may be acting on the capillary endothelial cells as a transforming agent. Although vanadate does induce morphological changes in aortic endothelial cells, it does not induce a n increase in cell proliferation. Thus, these two events can be uncoupled. Vanadate induces angiogenesis in a n in uitro model system using capillary endothelial cells (Montesano et al., 1988).When confluent capillary endothelial cells were grown to confluence on a collagen gel matrix and then were treated with 5-10 pM vanadate, the cells were found to invade the underlying matrix. Changes in cellular morphology and invasion of normal tissue are two properties that are also associated with cellular transformation. Vanadate also induces increased synthesis and secretion of plasminogen activator in low density capillary endothelial cell cultures (Montesano et al., 1988). An increased synthesis and secretion of proteolytic enzymes is also characteristic of transformed cells. Thus, in this system vanadate may be acting to promote transformation of the capillary endothelial cells rather than angiogenesis. Vanadate is able to induce changes in capillary endothelial cells similar to some of those observed in transformed cells. Since transformation can be induced by hyperactive protein tyrosine kinases, this effect of vanadate is not unexpected. What is unexpected is that despite high levels of protein tyrosine phosphorylation and dramatic changes in cellular morphology, the aortic endothelial cells do not respond with a n increase in cell proliferation. The data suggest that this lack of responsiveness is due to a n inhibition of tyrosine kinase activity. Apparently the tyrosine kinases which are important in cell proliferation in aortic endothelial cells are distinct from those in capillary endothelial cells. Further investigation into the nature of the tyrosine kinases in the two types of endothelial cells should help answer this question.

ACKNOWLEDGMENTS The author wishes to thank Drs. D. Schubert and A. Baird for critical reading of the manuscript. This work was supported by grants from the U S . Public Health Service (HD-25005) and Erbamont N. V. LITERATURE CITED Alexander, D.R. (1990) The role of phosphatases in signal transduction. The New Biologist, 2r1049-1062. Baird, A , and Durkin, T. (1986) Inhibition of endothelial cell proliferation by type p-transforming growth factor: Interactions with acidic and basic fibroblast growth factor. Biochem. Biophys. Res. Commun., 138:47&482.

Bar, R.S., Boes, M., Dake, B.L., Booth, B.A., Henley, S.A., and Sandra, A. (1988) Insulin, insulin-like growth factors, and vascular endothelium. Am. J. Med., 85:59-70. Beitz, J.G., Kim, 1.3, Calabresi, P., and Frackelton, A.R. (1991) Human microvascular endothelial cells express receptors for plateletderived growth factor. Proc. Natl. Acad. Sci. U.S.A.,88:2021-2025. Boes, M., Dake, R.L., and Bar, R.S. (1991) Interactions of cultured endothelial cells with TGF-P, bFGF, PUGF, and IGF-I. Life Sci., 48:811-821. Cooper, J.A., and King, C.S. (1986) Dephosphorylation or antibody binding to the carboxyl terminus stimulates pp6OC-"". Mol. Cell. Biol., 6:44674477. Doctrow, S.R., and Folkman, J. (1987) Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens. J. Cell Biol., 104.579487. Dupuy, E., Bikfalvi, A., Rendu, F., Toledano, S.L., and Tobelem, G. (1991) Thrombin mitogenic responses and protein phosphorylation are different in cultured human endothelial cells derived from large and microvessels. Exp. Cell Res., 185:363-372. Folkman, J., and Klagsbrun, M. (1987) Angiogcnic factors. Science, 235:442-447. Geiger, B. (1979)A 130K protein from chicken gizzard: Its localization at the termini of microfilament bundles in cultured cells. Cell, 18t193-205. Gospodarowicz, D., Massoglia, S., Cheng, J., and Fujii, D.K. (1986) Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries. J. Cell. Physiol., 127r121136. Hunter, T. (1987) A thousand and one protein kinases. Cell, 50t823829. Klagsbrun, M., and D'Amore, P.A. (1991) Regulators of angiogcnesis. Annu. Rev. Physiol. 53.217-239. Klarlund, J.K. (198.5)Transformation of cells by an inhibitor of phosphatases acting on phosphotyrosine in proteins. Cell, 41 :707-717. Maher, P.A. (1991) Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development. J. Cell Biol., 112r955-963. Maher, P.A., and Pasquale, E.B. (1988)Tyrosine phosphorylatcd proteins in different tissues during chick embryonic development. J. Cell Biol., 106:1747-1755. Marchisio, P.C., DUrso, N., Comoglio, P.M., Giancotti, F.G., and Tarone, G . (1988) Vanadate-treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus-transformed counterparts. J. Cell. Biochem., 37:151-159. Montesano, R., Pepper, M.S., Bclin, D., Vassalli, J.-D., and Orci, L. (1988) Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases. J . Cell. Physiol., 134t460-466. Mustelin, T., Coggeshall, K.M., and Altman, A. (1989) Rapid activation of the T-cell tyrosine protein kinase ~ ~ 5 by6 the " ~ CD45 phosphotyrosine phosphatase. Proc. Natl. Acad. Sci. U.S.A., 86:63026306. Owada, M.K., Iwamoto, M., Koike, T., and Kato, Y. (1989) Effects of vanadate on tyrosine phosphorylastion and the pattern of glycosaminoelvcan svnthesis in rabbit chondrocvtes in culture. J. Cell. Physiol. fi8:484292. Presta, M., and Rifkin, D.B. (1988)New aspects ofblood vcssel mowth: Tumor and tissue-derived angiogenesis fictors. Haemostasis, 18.617. Swarup, G., Cohen, S., and Garbers, D.L. (1982) Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem. Biophys. Res. Commun., 107rllOP1109. Tonks, N.K., and Charbonneau, H. (1989) Protein tyrosine dephosphorylation and signal transduction. Trends Biochem. Sci., 14:497-500. Ullrich, A., and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell, 61r203-212. Zetter, B.R. i1984) Culture of capillary endothelial cells. In: Biology of Endothelial Cells. E.A. Jaffe, ed. Martinus Nijhoff, Boston, pp. 14-26. ~

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Stimulation of endothelial cell proliferation by vanadate is specific for microvascular endothelial cells.

Micromolar concentrations of sodium orthovanadate stimulated the proliferation of bovine capillary endothelial cells, but not bovine aortic endothelia...
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