American Journal of Pathology, Vol. 152, No. 6, June 1998 Copynight ©) American Societyfor Investigative Patbology
Effects of Platelet-Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase, Substrate, and Products in a Three-Dimensional Model of Angiogenesis
Darren Paul Stevenson,* Stuart Richard Milligan,t and William Patrick Collins* From the Diagnostics Research Unit,* Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, London, United Kingdom and the Physiology Group,t Biomedical Sciences Division, King's College, London, United Kingdom
Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is associated with angiogenesis and the progression of human breast and ovarian cancers. The aim of this study was to obtain information about the possible mechanisms of PD-ECGF/TP activity in an established three-dimensional model of angiogenesis. The plan was to study the effects of the enzyme, substrate, products, and further metabolites on the formation and rate of microvessel growth from cultured segments of rat aorta in serum-free media. The end-points were the number and length of microvessels compared with controls after 4, 7, 11, and 14 days in culture. Thymidine (10 to 1000 ,umoVL), thymidine-5'-monophosphate (1000 ,imolL), and 2'-deoxy-D-ribose-l-phosphate (1000 ,umoVL) inhibited the number of microvessels produced. Conversely PD-ECGF/TP (50 to 100 ng/ml) and (8-amino-iso-butyric acid (1000 ,umol/L a metabolite of thymine) had a significant stimulatory effect (P < 0.05, P < 0.01, P < 0.001 respectively on culture day 11). PD-ECGF (10 ng/ml), ,B-amino-iso-butyric acid (1000 ,umoVL), and 2-deoxy-D-ribose (100 to 1000 ,umo/L) significantly (P < 0.001, P < 0.01, P < 0.01, respectively) stimulated microvessel elongation by day 11. We conclude that PD-ECGF/TP may affect anglogenesis by changing the relative concentrations of pyrimidine-based compounds and their metabolites in interstitial fluid surrounding endothelial cells. Drugs that inhibit PD-ECGF/TP activity may therefore delay abnormal angiogenesis and the progression of various cancers. (Amj Patbol 1998, 152:1641-1646) -
Angiogenesis plays a vital role in several physiological processes (eg, follicular development and wound repair), and the process is sometimes reversible (eg, during the
regression of a corpus luteum).' Persistent, up-regulated angiogenesis is a feature of many diseases (eg, rheumatoid arthritis and atherosclerosis),2 and the process sometimes appears to be distinctly disorganized (eg, in ovarian cancer).3 Angiogenesis involves the migration, proliferation, proteolytic activity, and organizational behavior of endothelial cells.4 These events are stimulated by a variety of growth factors, some of which may be relatively specific for certain tissues or diseases. In particular, it has been shown that platelet-derived endothelial cell growth factor (PD-ECGF) is important in the progression of breast and ovarian cancers.56 Moreover, the factor stimulates the migration of cultured endothelial cells7 and enhances angiogenesis in model in vivo systems.5'8 There is good evidence that PD-ECGF is identical to thymidine phosphorylase (TP).-14 This enzyme catalyzes the reversible phosphorylysis of thymidine to thymine and 2'-deoxy-D-ribose-1 -phosphate (dR1-P). Thymine is further metabolized to dihydrothymine, which in turn is converted to ,B-amino-iso-butyric acid (f3aBa). The phosphate group can be removed from dR1-P to form 2'-deoxy-D-ribose (D-ribose). PD-ECGF/TP does not contain the sequence of amino acids associated with secreted peptides,6 and a specific receptor protein has not been identified. The enzyme activity appears to be essential for the stimulatory effect of PDECGF/TP on angiogenesis,5'15 which might be mediated through readily diffusible metabolic products.7'11'16 We have investigated this possibility further by undertaking a systematic study of PD-ECGF/TP, the main substrate and immediate products, and more distant metabolites (thymidine-5'-monophosphate, dihydrothymine, f3aBa, D-ribose) on microvessel formation in a serum-free three-dimensional model of angiogenesis using rat aorta explants. The aim was to obtain information about the mechanism of action of PD-ECGF/TP, which might help the development of target specific antivascular therapies with particular reference to human ovarian cancer. This work was undertaken during the tenure of a studentship by D. P. Stevenson from Unipath Ltd., Bedford, United Kingdom. Accepted for publication March 13, 1998. Address reprint requests to Professor W. P. Collins, Department of Obstetrics and Gynaecology, King's College Hospital, Bessemer Road, London SE5 9PJ, UK. E-mail:
[email protected].
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Materials and Methods Recombinant human PD-ECGF was purchased from R & D Systems (Abingdon, UK). All other chemicals were obtained from Sigma (Poole, UK).
Isolation of Rat Aorta The protocol of Nicosia and Ottinetti17 was used with minor modifications.18 Female Wistar rats (1 to 3 months old) were killed by asphyxiation with carbon dioxide followed by cervical dislocation. The full length of the thoracic aorta was removed under aseptic conditions after partial dissection of the fibroadipose tissue in situ. The aorta was held at the cut ends of the tissue to avoid damaging the wall and immediately transferred to a tissue culture dish containing cold Dulbecco's modified Eagle's medium/Ham F12 medium (DMEM/HAM F12; 1:1 v/v). Any remaining adherent periadventitial fat and connective tissue were removed, and the aorta was cut into ring segments (1 mm wide); the proximal and distal 2-mm pieces were discarded. The rings were bisected to give half-ring explants, which were transferred to fresh DMEM/ HAM F12. Agarose rings were used as culture wells to set the collagen gels. Briefly, a sterile 1.5% (w/v) solution of agarose (type VII) was poured into 10-cm tissue culture dishes, and once set, agarose rings were obtained by punching two concentric circles in the agarose gel with specifically designed aluminum punches with diameters of 10 mm and 17 mm, respectively. The agarose rings were transferred to 10-cm culture dishes; each dish contained 5 rings. Collagen (50 mg; type from rat tail) dissolved overnight in 2 ml of sterile 0.1% (v/v) glacial acetic acid was diluted to a final concentration of 4.3 mg/ml with 10% (v/v) DMEM/HAM F12. The ionic strength and pH of the solution were raised simultaneously to initiate gel formation. The final collagen gel solution consisted of 4.3 mg/ml collagen, NaHCO3 (11.7 mg/ml in DMEM/HAM F12) and 1ox Eagle's Minimum Essential Medium. These solutions were stored on ice, and aliquots from each were rapidly removed and mixed in the ratio 7:2:1 (v/v/v) respectively. The pH was adjusted to physiological levels with 0.1 mol/L sodium hydroxide using phenol red as a visual indicator. The bottom of each agarose well was coated with 200 ,ul of collagen solution. Collagen gel formation occurred rapidly at 370C. The agarose wells were completely filled with collagen solution (-600 ,ll in total), and the aortic explant was transferred to the agarose well. The explant was positioned in the center of the collagen gel facing downwards; 20 ml of serum-free medium was added to each culture dish, and the dishes were stored at 370C overnight. On day 1 gels were removed from the surrounding agarose, randomly allocated to test or control groups, and allowed to float in 1 ml of test or control medium. Explant gels were incubated in a humidified incubator at 37°C (5% C02:95% 02), and the medium was changed every 2 to 3 days in all cultures. DMEM/HAM F12 was used as the serum-free growth medium. All media were supplemented with glutamine (2 mmol/L), penicillin (100
units/ml), streptomycin (100 ,ug/ml), and amphotericin B (2.5 ,ug/ml). A full characterization of the microvessel outgrowths has been reported by Nicosia and Ottinetti.17 19 Although Nicosia et a120 reported the spontaneous regression of microvessels after 14 days of culture, this phenomenon was not observed in our laboratory.
Microvessel Formation Each explant was examined on days 4, 7, 11, and 14 of culture. The number of microvessels was counted by direct observation of live explants under phase-inverted optics according to the criteria of Nicosia and Ottinetti.17 An index of microvessel length (ie, rate of growth) was obtained from video prints using a brightfield microscope attached to a video graphic printer (Sony, Weybridge, UK). A transparent template was overlaid on each video print to quantify the number of microvessels that crossed gridlines at various distances from a section of the explant edge.
PD-ECGF/TP and Related Factors Explants were cultured in DMEM/HAM F12 containing a range of PD-ECGF/TP concentrations (1 to 100 ng/ml) to produce a dose-response curve. Thymidine, thymidine5'-monophosphate, and selected metabolites from thymidine phosphorylase activity (thymine, dihydrothymine, PaBa, dR-1-P, and D-ribose) were tested. Each compound was dissolved in sterile, filtered phosphate-buffered saline and stored as a stock solution of 100 mmol/L
(thymidine, thymidine-5'-monophosphate, dihydrothymine, faBa, dR-1-P, and D-ribose) or 10 mmol/L (thymine). Aliquots of the stock solutions were added to culture medium to give a final concentration of 1 mmol/L. For dose-response studies, serial dilutions of the stock solutions were prepared in culture medium.
Statistical Analysis The distribution of values from each set of replicate determinations (control and experimental) were assessed for normality. The coefficient of variation for control replicates between 18 to 51 microvessels ranged from 5.2 to 11.6. The corresponding coefficient of variation for PDECGF-containing cultures (over the range 1 to 100 ng/ml) ranged from 5.7 to 11.0 and for thymidine containing cultures (over the range 1.0-100.0 ,umol/L) from 2.1 to 11.1. The value for the effect of each compound tested per explant was expressed as a percentage of the mean of the control replicates for that experiment, and the data are shown as the mean ± SE or SD. Student's t-test was used to evaluate the significance of differences between the results from test cultures and untreated controls and between end-points at different times in culture. A P value < 0.05 was considered to be significant.
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Results Microvessels had sprouted from the edges of the explants after 3 to 4 days of culture. The number and length of the vessels increased with time until the experiments were terminated on culture day 14. The vessels also formed branches that underwent anastomosis and formed a complex network. An example of a control explant on culture day 11 is shown in Figure 1A.
Microvessel Number PD-ECGF stimulated the production of microvessels compared with the controls. A dose-response curve is shown in Figure 2a. The effects of PD-ECGF at 50 and 100 ng/ml were significantly different from the corresponding controls. Thymidine initially stimulated the growth of microvessels. However, by day 4 there was a reduction in the mean number of vessels, and with increasing time in culture, the growth was abolished com-
pletely. A thymidine dose-response curve in the absence of exogenous PD-ECGF shows maximal growth at 1 ,umol/L and a total absence of microvessels because of cell death at 1000 gmol/L (Figure 2b). There was very little difference in the inhibitory effects between 10 and 100 ,umol/L. The degeneration of microvessels by thymidine was characterized by endothelial cell rounding, which is suggestive of apoptosis. A section of an explant illustrating this phenomenon at culture day 11 is shown in Figure 1B. The addition of PD-ECGF/TP at the minimum stimulatory dose (10 ng/ml) and thymidine (1000 ,umol/L) produced a similar pattern of microvessel degradation to that for the nucleoside alone; however, the effect was significantly reduced by culture day 4 (P < 0.01). The data by day of culture for the control and experimental explants are shown in Table 1.
Table 1. Microvessel Number per Explant under Control Conditions and in the Presence of PD-ECGF (10 ng/ml), Thymidine (1 mmol/L), and PD-ECGF plus Thymidine
Culture day
Control mean (SD)
PD-ECGF mean (SD)
Thymidine mean (SD)
PD-ECGF + thymidine mean (SD) 17.3 (4.8) 10.0 (3.2) 5.5 (2.1)
32.6 (5.4) 5.6 (2.5) 20.7 (5.9) 4.0 (t) 24.2 (8.6) 30.3 (7.5) 1.0 (t) 43.6 (15.3) 52.5 (11.9) ND 44.2 (7.8) 59.5 (21.6) ND, no microvessels detected; t. data not normally distributed because of zero values for individual replicates. 4 7 11 14
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Figure 3. Effect of thymidine phosphorylase-related metabolites on the number of microvessels from rat aorta explants on culture day 11. ,BaBa, 7 explants, control 24 ± 2 SE; Thy, thymine (18 explants, control 24 ± 2 SE); DHT, dihydrothymine (8 explants, control 29 ± 2 SE), TMP, thymidine-5'monophosphate (7 explants, control 29 + 2 SE); dR, 2'-deoxy-D-ribose (10 explants, control 29 ± 1 SE); dR-1-P, 6 explants, control 29 + 1 SE. '*P < 0.01, ***P < 0.001.
The effect of thymidine phosphorylase-related metabolites (1 mmol/L) on day 11 of culture is shown in Figure 3. Thymidine-5'-monophosphate, the precursor of thymidine in the pyrimidine catabolic pathway, significantly inhibited microvessel production of the explants in the collagen matrix from day 4 of culture. Thereafter, there was a gradual degradation of microvessels with approximately 90% having degenerated by day 14. Microvessel breakdown was characterized by endothelial cell rounding. Thymine, the enzymatic product, and dihydrothymine, a further breakdown product, had no significant effect on microvessel number. However, 3aBa (from 10 to 1000 ,umol/L) increasingly stimulated microvessel formation over the 14-day incubation period (up to 192.3 ± 22.8% of the level of growth seen in control cultures). On day 4 of culture the number of microvessels from explants in media containing dR-1-P did not differ from the controls, but from day 7 onwards it limited the level of microvessel formation to approximately 60% of the mean control value on day 14; D-ribose did not produce a significant stimulatory or inhibitory effect on microvessel number at the concentration tested.
Microvessel Length The length of microvessels (,um) per section expressed as a percentage of the mean control value (290 ± 22 SE, n = 20) on culture day 11 is summarized in Figure 4; faBa (100 ,mol/L), D-ribose (100 to 1000 ,umol/L), and PD-ECGF (10 ng/ml) significantly stimulated microvessel elongation above that seen for the explants under control conditions.
Discussion The characteristics of microvessel growth that we observed from rat aorta explants under control conditions are similar to previous descriptions.1721 This is the first
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Figure 4. Effect of thymidine phosphorylase-related metabolites on the length of microvessels per section from rat aorta explants on culture day 11 (means + SE). D-ribose, 10 video prints; dR-1-P, 30 video prints; TMP, 30 video prints; Thd, 13 video prints; Thy, 24 video prints; DHT, 9 video prints; f3aBa, 30 video prints; PD-ECGF, 20 video prints. **P < 0.01, *11P < 0.001.
report, however, of PD-ECGF/TP angiogenic-like activity in the serum-free culture system, although the factor had been shown previously to effect indices of angiogenic activity in vivo and chemotactic activity in vitro.8'22 We have also shown in the present study that high concentrations of thymidine (the substrate for thymidine phosphorylase) inhibited microvessel formation, whereas the addition of PD-ECGF to media containing high thymidine concentrations reduced the level of inhibition. It has been shown that exogenous thymidine equilibrates with intracellular thymidine23 leading to an increase in the intracellular concentration of thymidine-5'-monophosphate, a compound that also inhibited microvessel formation at high concentrations in this assay. Concomitantly, the levels of other deoxyribonucleotides may be reduced by allosteric changes in the enzyme ribonucleotide diphosphate reductase,24 which may lead to cell synchronization, decreased growth, and eventually death. The addition of exogenous PD-ECGF depletes the thymidine concentration by converting it to thymine and thereby reduces the inhibition of microvessel formation by decreasing the level of inhibition of ribonucleotide diphosphate reductase. The conversion of thymidine to thymine may also lead to an increase in the number of microvessels by the subsequent formation of ,B-amino-iso-butyric acid. Human thymidine phosphorylase activity in blood is located mainly in the platelets and appears to be the main regulator of blood thymidine homeostasis.25 Whole human platelets have been shown to induce human umbilical vein endothelial cell migration in vitro and increase DNA synthesis and cell division.26 Aggregation of human platelets at the site of wounds increases the local concentration of PD-ECGF/TP, although the protein is not released from the cell.12 PD-ECGF/TP may possibly modulate local nucleoside pool imbalances caused by tissue damage and stimulate endothelial cell migration and angiogenesis. Isolated endothelial cells have also been observed to contain intact platelets after a period of co-culture.27 This finding suggests that damaged endo-
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thelial cells in vivo may take up platelets to modulate DNA precursor pools. The resultant activity could increase cell growth and stimulate angiogenesis. However, it is still unclear how the effect is limited to endothelial cells. The production of endothelial DNA precursors may be dependent on blood thymidine homeostasis because of the unique position of the endothelium within the vasculature.27 PD-ECGF stimulation of angiogenesis may involve the production of adhesion molecules on the endothelial cell surface and the production of other angiogenic molecules such as the family of vascular endothelial cell growth factors. It is important to note that investigators who have evaluated cellular growth by labeled thymidine uptake may have produced misleading results if a high level of thymidine phosphorylase activity was present at the time of the test. The effect of PD-ECGF on the intracellular level of thymidine may explain its angiogenic-like effect, although metabolic products may also be involved in the mechanism of action. The only breakdown product of thymidine that possessed significant angiogenic-like activity was PaBa. The mechanism by which this metabolite stimulates angiogenesis is unclear, although it is possible that the molecules are transaminated within cells to succinyl CoA and fed into the citric acid cycle to provide a fuel source for increased cell mitosis (cellular proliferation is one of the component events of angiogenesis). 2'-deoxyD-ribose has been reported to stimulate angiogenesis in vivo;7 however this compound was found not to possess any significant direct effect on microvessel number in the in vitro rat aorta assay but did promote a significant increase in microvessel length at the higher doses. The difference in findings may be because of a number of reasons: 1) the absence of serum from the culture medium; 2) D-ribose may act indirectly (via other cells) in vivo to stimulate angiogenesis; or 3) the effect described previously may represent microvessel elongation. One other ribose derivative, dR-1-P was observed to inhibit angiogenesis within this system. This effect was specific to the deoxy moiety of the compound; D-ribose-1-phosphate did not significantly inhibit angiogenesis (unpublished data). It is therefore possible that the inhibition is in some way related to the deoxy forms of the ribose derivatives; a result similar to that seen for nucleosides/deoxynucleosides (unpublished data). The mechanism by which these derivatives specifically inhibit angiogenesis is not known. PD-ECGF does not possess a signal sequence and is therefore not a classical secreted protein.8 The potential mechanism of action of PD-ECGF/TP may not require secretion of the protein. The action of this factor/enzyme within one cell could affect surrounding cells within a tumor as the products/substrates can cross the cell membrane by facilitated diffusion/membrane transport.23'28 In summary, it would seem that the thymidine phosphorylase activity of PD-ECGF stimulates angiogenesis by a two-step mechanism: 1) by the reduction of thymidine (and hence thymidine-5'-monophosphate) levels within the cell, both compounds inhibit microvessel formation and 2) by the production of compounds, such as ,BaBa, which stimulate microvessel formation and growth rate.
These actions of PD-ECGF/TP affect the cellular level of positive and negative angiogenic metabolites, and this mechanism bears a striking similarity to the proposed method of angiogenesis control.2 It follows that the development of drugs to inhibit thymidine phosphorylase activity might help the management of patients before surgery to remove in situ cancers of the ovary or breast.
References 1. Koos RD: Ovarian angiogenesis. The Ovary. Edited by EY Adashi and PCK Leung. New York, Raven Press, 1993, pp 433-453 2. Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995, 1:27-31 3. Bourne TH, Reynolds K, Campbell S, Collins WP: Ultrasound screening for early ovarian cancer. Diagn Oncol 1992, 2:35-38 4. Folkman J, Shing Y: Angiogenesis. J Biol Chem 1992, 267:10931 5. Moghaddam A, Zhang H, Fan TD, Hu D, Lees VC, Turley H, Fox SB, Gatter KC, Harris AL, Bicknell R: Thymidine phosphorylase is angiogenic and promotes tumor growth. Proc NatI Acad Sci USA 1995, 92:998-1002 6. Reynolds K, Farzaneh F, Collins WP, Campbell S, Bourne TH, Lawton F, Moghaddam A, Harries AL, Bicknell R: Correlation of ovarian malignancy with expression of platelet-derived endothelial cell growth factor. J Natl Cancer Inst 1994, 86:1234-1238 7. Haraguchi M, Miyadera K, Uemura K, Sumizawa T, Furukawa T, Yamada K, Akiyama S: Angiogenic activity of enzymes. Nature 1994, 368:198-199 8. Ishikawa F, Miyazono K, Hellman U, Drexler H, Wernstedt C, Hagiwara K, Usuki K, Takaku F, Risau W, Heldin C: Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor. Nature 1989, 338:557-562 9. Barton GJ, Ponting CP, Spraggon G, Finnis C, Sleep D: Human platelet-derived endothelial cell growth factor is homologous to Escherichia cofi thymidine phosphorylase. Protein Sci 1992, 1:688-690 10. Furukawa T, Yoshimura A, Sumizawa T, Haraguchi M, Akiyama S: Angiogenic factor. Nature 1992, 356:668 11. Moghaddam A, Bicknell R: Expression of platelet-derived endothelial cell growth factor in Escherichia coi and conformation of its thymidine phosphorylase activity. Biochemistry 1992, 31:12141-12146 12. Usuki K, Saras J, Waltenberger J, Miyazono K, Pierce G, Thomason A, Heldin C: Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity. Biochem Biophys Res Commun 1992, 184:1311-1316 13. Finnis C, Dodsworth N, Pollitt CE, Carr G, Sleep D: Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity. Eur J Biochem 1993, 212:201-210 14. Sumizawa T, Furukawa T, Haraguchi M, Yoshimura A, Takeyasu A, Ishizawa M, Yamada Y, Akiyama S: Thymidine phosphorylase activity associated with platelet-derived endothelial cell growth factor. J Biochem 1993, 114:9-14 15. Miyadera K, Sumizawa T, Haraguchi M, Yoshida H, Konstanti W, Yamada Y, Akiyama S: Role of thymidine phosphorylase activity in the angiogenic effect of platelet-derived endothelial cell growth factor/ thymidine phosphorylase. Cancer Res 1995, 55:1687-1690 16. Morris PB, Ellis N, Swain JL: Angiogenic potency of nucleotide metabolites:Potential role in ischemia-induced vascular growth. J Mol Cell Cardiol 1989, 21:351-358 17. Nicosia RF, Ottinetti A: Growth of microvessels in serum-free matrix culture of rat aorta: a quantitative assay of angiogenesis in vitro. Lab Invest 1990, 63:115-122 18. Jaggers DC, Collins WP, Milligan SR: Potent inhibitory effects of steroids in an in vitro model of angiogenesis. J Endocrinol 1996, 150:457-464 19. Nicosia RF, Ottinetti A: Modulation of microvascular growth and morphogenesis by reconstitutes basement membrane gel in three-dimensional culture of rat aorta: a comparative study of angiogenesis in matrigel, collagen, fibrin, and plasma clot. In Vitro Cell Dev Biol 1990, 26:119-128
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20. Nicosia RF, Bonanno E, Smith M: Fibronectin promotes the elongation of microvessels during angiogenesis. J Cell Physiol 1993, 154:654661 21. Diglio CA, Grammas P, Giacomelli F, Weiner, J: Angiogenesis in rat aorta ring explant cultures. Lab Invest 1989, 60:523-531 22. Gruber BL, Marchese MJ, Kew R: Angiogenic factors stimulate mastcell migration. Blood 1995, 86:2488-2493 23. Wohlhueter RM, Marz R, Plagemann PG: Thymidine transport in cultured mammalian cells: kinetic analysis, temperature dependence and specificity of the transport system. Biochim Biophys Acta 1979, 553:262-283
24. O'Dwyer PJ, King SA, Hoth DF, Leyland-Jones B: Role of thymidine in biochemical modulation: a review. Cancer Res 1987, 47:3911-3919 25. Shaw T, Smillie RH, MacPhee DG: The role of blood platelets in nucleoside metabolism: assay, cellular location and significances of thymidine phosphorylase in human blood. Mutat Res 1988, 200:99-116 26. Maca RD, Fry GL, Hoak JC, Loh PT: The effects of intact platelets on cultured human endothelial cells. Thromb Res 1977, 11:715-727 27. Miyazano K, Usuki K, Heldin C: Platelet-derived endothelial cell growth factor. Prog Growth Factor Res 1991, 3:207-217 28. Berlin RD, Oliver JM: Membrane transport of purine and pyrimidine bases and nucleosides in animal cells. Int Rev Cytol 1975, 42:287-336
