American Jounal of Pathology, Vol. 138, No. 2, Februwy 1991

Copright © American Association of Pathologists

Tumor Necrosis Factor Induces Apoptosis (Programmed Cell Death) in Normal Endothelial Cells In Vitro Bernard Robaye,* Roger Mosselmans,t Walter Fiers,t Jacques E. Dumont,* and Paul Galandt From the Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire (IRJBHN)* and the Laboratoire de Cytologie et de Canckrologie Experimentale, t Faculte de Medecine, Free University of Brussels (ULB.), Brussels, and the Laboratory of Molecular Biologyv, State University of

Ghent, Ghent, Belgium

Tumor necrosis factor (TNF) is cytotoxic for many tumoral cell lines, whereas normal cells generally are considered resistant to this action. This study shows that this cytokine causes massive death of bovine endothelial cells in primary culture in a concentration- and time-dependent manner. Dying cells exhibit all the ultrastructural changes and the internucleosome cleavage of DNA associated with apoptosis or programmed cell death.' This is the first report clearly showing a direct toxicity of TNF on endothelial cells and demonstrating that this results from the induction of the program of apoptotic death. Our observation raises the possibility that hemorrhagic necrosis in vivo, after treatment with T7N, might involve a direct cytocidal action on endothelial cells of the tumor neovasculature. (Am 1 Pathol 1991, 138:447-453)

Tumor necrosis factor (TNF) is a cytokine secreted by macrophages and activated T cells.1'2 In vitro, most tumoral cell lines are sensitive to its cytotoxic/cytostastic effect. In vivo, this cytokine is also able to induce hemorrhagic necrosis of certain transplantable mouse and human tumors.' It has been suggested that the latter effect might reflect predominantly an action of TNF, alone or in combination with other monokines, on tissue factorlike procoagulant activity, resulting in intravascular thrombus formation,56 especially in the newly formed microcirculation in the tumor.7 We considered the possibility that TNF might present some direct toxic action on the endothelium in the tumor vasculature. Indeed, if no clear pic-

ture emerges from the literature as to a direct toxicity of TNF on endothelial cells,810 some indirect evidences suggest the possibility that the cytokine might alter their viability and morphology.9,11 To test this hypothesis directly, we investigated the effect of recombinant human TNF (rhTNF) in vitro on primary cultures of bovine aortic endothelial cells. The data reported here show that rhTNF induces massive death of endothelial cells, the dying cells exhibiting the ultrastructural and biochemical features that characterize apoptosis. It is suggested that this action of TNF might be, at least partially, responsible for its necrogenic effect on tumors in vivo.

Materials and Methods Materials The rhTNF used in this study was provided by Dr. J. Tavernier (Biogent, Belgium). The preparation contained 16 mg/ml of protein (specific activity: 7 107 U/mg of protein) and was contaminated by 3.2 ng/ml of endotoxin. Anti-hTNF monoclonal antibody (TNFbl1) was a gift of Dr. M. D. de Baets (University of Limburg, Maastricht, The Netherlands) and was used at a dilution of 1:100 (15 ,ug IgG/ml).

Preparation of Cells Bovine endothelial cells were obtained as previously

described,12 from aortas of freshly slaughtered cows. Work performed within the framework of "Sciences de la Vie" Actions from the Belgian Ministry for Scientific Podicy and supported by grants from the Foundation for Scientific Research, the Fondation Van Buuren, the Fondation Rose et Jean Hoguet, the Banque Nationale de Belgique, the Association contre le Cancer (Belgium), and the Fonds pour la Recherche

Cancrokogique (CGER). Accepted for publication September 26,1990. Address reprint requests to Bemard Robaye, IRIBHN, Faculte de Medecine, Free University of Brussels (U.L.B.), 808, Route de Lennik, Bat.

C-B-1070, Brussels, Belgium. 447

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Briefly, the cells were collected from the aorta by collagenase treatment, seeded on 1 00-mm Petri dishes, and incubated for 3 days at 37°C under an atmosphere of 5% C02/95% air in the following selective medium: Minimum Essential Medium (MEM) D-valine (80%, vlv), fetal calf serum (20%, v/v), 2 mmoVI (millimolar) glutamine, 100 U/ml penicillin, 100 ,ug/ml streptomycin, and 2.5 ,ug/ml amphotericin B. When the cultures formed confluent monolayers, the cells were detached by trypsin-ethylene dinitrilo tetraacetic acid (EDTA) treatment (1 mg/ml-1 mmolA). The culture was pursued and all experiments were achieved under an atmosphere of 5% C02/95% air in the following complete medium: Dulbecco's Modified Eagle Medium (DMEM) (60%, v/v), Ham's F-1 2 (20%, v/v), fetal calf serum (20%, v/v) with the same concentration of penicillin, streptomycin, and amphotericin B. For each experiment, the cells of one aorta were used between the second and the fifth passage.

Microscopic Study We seeded 105 cells in 35-mm Petri dishes and allowed them to attach for 48 hours. The medium then was replaced by fresh medium containing or not containing rhTNF at a dose of 2000 U/ml. At specified times, the cells were fixed and embedded as previously described for microscopic examination.13 Ultrathin section, stained with uracyl acetate and lead citrate, were examined in a Philips 301 electron microscope (Philips, Eindhoven, The Netherlands). After our discovery that a proportion of the cells were apoptotic (see Results), further quantification of the frequency of apoptotic cells was performed at light microscopy.

Endothelial cells (5 x 1 04) were seeded in 1 ml of complete culture medium in 35-mm Petri dishes. Two days later, the cells were incubated with or without 2000 U/ml of rhTNF. At specified times, the dishes were rinsed with 1 ml of DMEM medium, fixed for 30 minutes with methanol, and air dried. Before examination, the cells were stained with hematoxylin and eosin. An average of 1000 cells were counted per Petri dish in a blind fashion and the results expressed as the percentage of apoptotic cells counted in rhTNF dishes minus the percentage of apoptotic cells observed in control dishes. Initial stages of apoptosis were identified readily by the dense masses of chromatin and the condensed basophilic cytoplasm. Later stages of the process, such as fragmentation of the cytoplasm to form apoptotic bodies, also were observed.

in 1 00-mm Petri dishes, cultured for 24 hours in 5 ml of complete medium, and incubated in the same medium supplemented with 33 ,uCi/ml of [32P]thymidine (Amersham, Brussels, Belgium) for an additional 24 hours. The cells were then washed twice with 5 ml of DMEM and incubated in complete medium with or without 2000 U/ml of rhTNF. After 8 hours of incubation, the cells were rinsed with DMEM and lysed with a lysis buffer containing 5 mmol/I TRIS-HCI pH 8.0,20 mmol/l EDTA pH 7.4 and 1% Triton X-1 00. The lysate was centrifuged at 27,000g for 20 minutes to separate the intact chromatin (pellet) from the fragmented DNA (supernatant).15 The supernatant was extracted sequentially for 15 minutes with equal volumes of phenol, phenol:chloroform (1:1), and chloroform: isoamyl alcohol (24:1), precipitated in two volumes of 100% ethanol, 0.1 mol/l (molar) NaCI at -20°C overnight, and resuspended in 40 ,ul of 10 mmol/l TRIS-HCI and 1 mmolA EDTA pH 7.4. Before electrophoresis for 4 hours at 160 V in 2% agarose, the samples were submitted to a RNAse treatment (100 ,ug/ml) for 3 hours at 37°C. The gel was fixed in 7% (w/v) trichloroacetic acid for 30 minutes (one change) and dried onto 3-mm Whatman paper. Autoradiograph of the gel was obtained on beta-Max hyperfilm (Amersham).

DNA Fragmentation Assay For this assay (a modification of the protocol described by Duke et al16), 5 x 104 cells were seeded in 1 ml of complete culture medium in 35-mm Petri dishes. The next day the cells were incubated for 24 hours in 1 ml of the same medium supplemented with 3 ,uCi/ml of [3H]thymidine (Amersham). They were then rinsed twice with 1 ml of DMEM and incubated for specified times in the complete culture medium with or without rhTNF. At the end of the incubation, the medium was retained. The cells were lysed in 600 ,ul of lysis buffer (see above). The lysate was maintained on ice for 10 minutes in microfuge tubes (Treff Lab, Degersheim, Switzerland) and was centrifuged at 1 5,000g for 30 minutes. The supematant was removed and the pellet was dried and dissolved in 100 ,ul of Soluene-350 (Packard Instruments, Warrenville, RI). The radioactivity in the culture medium, the 1 5,000g supernatant, and the pellet was counted on a Canberra Packard Tri-Carb 1600 CA (Packard Instruments). Specific fragmentation due to rhTNF was calculated using the formula16: % DNA fragmentation =

DNA Electrophoresis We used the method described by Jones et al14 with some modifications. Briefly, 2.5 x 1 05 cells were seeded

(dpm frag, TNF) - (dpm frag, control) 100, (dpm total) - (dpm frag, control) in which dpm frag = number of desintegrations per

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minute (dpm) in the incubation medium plus the dpm in the 15,000g supernatant, and dpm total = dpm frag plus the dpm in the 15,000g pellet.

51Cr Release Assay For this assay, 5 x 1 0 cells were seeded in 1 ml of complete culture medium in 35-mm Petri dishes. The next day the cells were incubated for 4 hours in 1 ml of the same medium supplemented with 20 ,uCi/ml of 51 Cr (Amersham). They were then rinsed twice with 1 ml of DMEM and incubated for 24 hours in 1 ml of complete culture medium with or without rhTNF. At different times, aliquots of the medium were counted on a Canberra Packard Minaxi gamma counter (Parckard Instruments). The percentage of specific release was calculated by the

formula16: % specific 51Cr release

- CPmcontrol =CPMTNF Cpmtotal - Cpmcontrol

where cpmTNF = counts per minute (cpm) in the culture medium of rhTNF-treated cells, cpmsntroi = cpm in the culture medium of control cells, and cpmtotaj = cpm in the lysate of cells at time 0 of the incubation treated 30 minutes with 0.5% Triton X-1 00 at 40C.

Determination of Cell Proliferation For determination of endothelial cell proliferation, we counted the number of cells per Petri dish the day after cell seeding and after a 24-hour incubation in complete culture medium. The cells were rinsed twice with 1 ml of DMEM, detached by trypsin-EDTA treatment (see above), and counted using a hematocymeter.

hours or more revealed morphologic alterations of endothelial cells that were typical of the various steps of apoptotic death.1718 Evidence for apoptosis included appearance of compact masses of chromatin in the nucleus (Figure 1), various stages of nuclear fragmentation, condensation of the cytoplasm, blebbing of the cell surface (Figure 2), segregation of cytoplasmic organelles and of dense chromatin into membrane-bound vacuoles (Figure 3), and phagocytosis of apoptotic bodies by normallooking cells (Figure 4). The presence of many apoptotic bodies undergoing secondary necrosis was also noted (not shown). In one experiment, counting at the light microscope showed an incidence of 9.4% of apoptotic cells due to rhTNF after 4 hours of incubation. This percentage reached to 22% at 8 hours and decreased later: 14% of apoptotic cells at 24 hours and 12.6% at 48 hours (Figure 6). No differences were noted between control and rhTNF-treated cells before 4 hours of incubation.

Endothelial Cell DNA Fragmentation Induced by rhTNF From a biochemical point of view, apoptosis is characterized by the stimulation of an endogenous endonuclease that cleaves the nuclear DNA at the linker regions between nucleosomes, resulting in the production of oligonucleosome fragments.1116,18 The electrophoretic pattern (Figure 5) of the DNA extracted from the 27,000g supernatant of 2000 U/ml rhTNF-treated cells indeed showed the expected discrete fragments of molecular weights corresponding to multiples of about 180 base pairs.

Quantification of DNA Fragmentation Prostacyclin Release Endothelial cells (5 x 1 04) were seeded in 35-mm Petri dishes as described above. After a 48-hour incubation, they were incubated for 8 hours with the indicated concentration of rhTNF in 1 ml of DMEM. The medium was then collected and the amount of released prostacyclin was determined by the radioimmunoassay of its degradation product, prostaglandin 6-keto-F1a.12

Results Effect of rhTNF on Endothelial Cells: Microscopic Study As shown in Figures 1 to 4, ultrastructural analysis of endothelial cells incubated with 2000 U/mI of rhTNF for 4

The determination of DNA fragmentation (quantitatively correlated with the proportion of apoptotic cells15) allows us to evaluate the relative incidence of the observed phenomenon. The time course study of DNA fragmentation in endothelial cells incubated with 2000 U/ml rhTNF showed (Figure 6) that significant DNA cleavage (11 %) was first seen after 4 hours of incubation in the presence of rhTNF, reached near 20% after 8 hours, and 40% when the cells were treated for 48 hours. The slope of the time curve was attenuated between 8 and 48 hours of incubation when compared to its 2- to 8-hour value. The maximal DNA fragmentation at 48 hours varied from 40% to 58% in different experiments. DNA fragmentation can be correlated with the proportion of apoptotic cells between 0 and 8 hours (Figure 6). For late times, the two curves diverge as expected due to the fact that contrary to DNA fragmentation, morphologic detection of apoptotic cells does

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4 Figures 1 to 4. Thefigures illustrate all the classical stages of apoptosis of endothelial cells exposed to rhTNFfor 4 hours and more, up to 24 hours, the longest period investigated Features of apoptosis shown are sharply delineated masses of condensed chromatin (Figure 1, 2:cm), distension of the rough endoplasmic reticulum (Figure 1: rer) and loss of microvilli; formation of apoptotic bodies (asterisks), ie, segregation of areas of the cytoplasm in condensed masses, containing mitochondria (arrows), rough endoplasmic reticulum (arrowheads) and lipid droplets (Li) (Figures 2 and 3) and nuclear fragments, with dense masses of chromatin (Figure 3.cm); phagocytosed apoptotic bodies, in healthy-looking cells (Figure 4) (magnification: Figure 1: X 7400; Figure 2: X8500; Figure 3: x5000; Figure 4: x 3750).

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The day after seeding, we counted 91,983 ± 18,727 cells per Petri dish; 24 hours later we found 247,500 + 13,258 cells per dish. This demonstrates active cell proliferation under our experimental conditions. Recombinant human TNF (2000 U/ml) had only a small effect on 51Cr release (3%) from endothelial cells after 24 hours of incubation. Table 1 shows that significant DNA fragmentation (7%) already was detected when the cells were incubated for 8 hours with 20 U/mI of rhTNF. This effect was concentration dependent with maximal response at 2000 U/ml concentration. A rhTNF concentration of 20,000 U/ ml did not increase further DNA fragmentation (data not shown). In two of the five experiments performed, a maximal effect already was obtained at 200 U/ml. This concentration-dependent curve is similar to that of rhTNFstimulated phosphorylation of 27-kd proteins of endothelial cells,19 but 200 U/ml of rhTNF were necessary to enhance the release of prostacyclin from these cells (Table 1).

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Recombinant TNF preparations may be contaminated with some endotoxin, known to be toxic for endothelial cells at a concentration of 150 ng/ml.20 In this study, the final concentration of contaminating endotoxin was 5.7 x 10-6 ng/ml, which seems negligible. Heating of rhTNF (700C, 1 hour) is known to destroy the biologic activity of TNF but not of endotoxin.9 We tested the effect of such a treatment on the ability of rhTNF to induce DNA fragmentation in endothelial cell cultures. The results in Table 2 show that this treatment completely abolished the DNA fragmentation response. Table 2 shows further that the rhTNF cytotoxic effect on endothelial cells also was sup-

not integrate values from all early periods: phagocytosed cells, detached, or secondary lysed cells were not scored as apoptotic cells. At 4 and 8 hours of incubation, the rhTNF-specific released radioactivity was predominantly collected from the 15,000g supernatant (87% and 83%, respectively). Later the cleaved DNA was recovered in the incubation medium with only 20% (at 24 hours) and 1% (at 48 hours) in the cell supernatant. These kinetics correlate with morphologic findings of an early apoptosis followed later by secondary necrosis and membrane disruption. Figure 6. Kinetic of rhTNF induction of apoptosis in endothelial cells. O-O: % ofDNA fragmentation in rhTNF treated cells; the values are mean (±SD) of triplicate measurements of one eperiment offour. *-*: % of apoptotic cells due to rhTNF, counted at light microscopy. An average of 1000 cells were blindly counted per Petn dish (one disb per time studied).

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Table 1. Effect of rbTNF Concentrations on DNA Fragmentation and Prostacyclin Release in

Endothelial Cells rhTNF U/ml 0 2 20 200 2000

% DNA fragmentation

-0.3 ± 7.2 ± 19.1 ± 30.3 ±

1 0.9 0.7 0.8

Prostacyclin release pg/dish 2250 ± 2310 ± 2640 ± 4910 ± 7900 ±

200 420 460 510 460

To test DNA fragmentation, the cells were incubated 8 hours in presence of rhTNF in 1 ml of complete culture medium; for determination of prostacyclin release, cells from the same preparation were incubated in 1 ml of DMEM alone. Average values (±SD) of triplicate measurements are taken from one representative experiment of five performed.

pressed in the presence of monoclonal anti-rhTNF antibody at a dilution of 1:100.

Discussion Our observations show that rhTNF causes the death of normal bovine aortic endothelial cells in culture and that this occurs not by direct necrosis of cells but rather by a process that involves the DNA fragmentation and the ultrastructural changes characteristic of apoptosis. The findings that heat and monoclonal anti-hTNF antibody abolished this effect indicate that this action of rhTNF is not due to the endotoxin contamination of our preparation. Apoptosis is a cell death mechanism involved in different physiologic conditions, such as embryogenesis, 18,21 hormone deprivation,22'23 homeostasis of the immune system,24 and also in immunologically mediated cell killing.24 Based on a time-lapse cinematographic study, a previous report concluded that sensitive tumor cells can respond to TNF in vitro by necrosis lysis or by apoptosis, depending on the cell line.25 The present work constiTable 2. Effect of Heat and Monoclonal Antibody TNF Treatment on rhTNF Ability to Induce DNA Fragmentation in Endotbelial Cells % DNA Treatment fragmentation 21.4 + 1.4 rhTNF (2000 U/ml) 1.0 ± 0.5 700C heated rhTNF rhTNF (2000 U/ml) 19.8 ± 1.35 -0.3 ± 5 Ab/100 + TNF rhTNF was incubated 1 hour at 700C before the test or monoclonal anti-hTNF antibody diluted 1/100 (15jLg/ml) was present during the 8-hour incubation with rhTNF (2000 U/ml). The results are mean ± SD from two representative experiments, each with triplicate measurements.

tutes the first report that TNF can induce apoptosis of normal cell type. The fact that the TNF cytotoxicity concerns endothelial cells deserves peculiar attention. Indeed the interactions between TNF and endothelial cells seem to be complex and multiple, leading notably to promote inflammation and coagulation (for review, see Pober JS, Cotran RS-6). Obviously tumor growth requires neovascularization and an action of TNF on the integrity of this vascularization is important. It has been observed that TNF is angiogenic when injected into the rabbit cornea,10 but Sato et a19 reported that TNF can inhibit tumor cell-mediated angiogenesis. This paradox may be explained by the fact that vessels of the tumor neovasculature are influenced by factors that distinguish their endothelium from that of normal vessels.27 Furthermore quiescence or proliferation of the cells may imply differences in susceptibility and type of response to TNF action. We must notice that our endothelial cells have been treated with TNF when in active proliferation. In vitro, toxic, and/or cytostatic effect of TNF on endothelial cells has been previously reported,10,28 31 but this effect was late (seen after 24 hours or more of incubation) and was dependent on growth factors, endotoxin, or interferon. This delayed toxic effect of TNF might be a late consequence of the early apoptosis described in this work. We show here that lower concentrations of TNF are necessary for the toxic effect and for 27-kd proteins phosphorylation than for prostacyclin generation. This observation supports the physiologic relevance of the TNF apoptotic effect. The reported observation that tumor perfusion with heparin did not suppress TNF-induced hemorrhagic necrosis in situ already suggested that coagulation might not be necessary for this action.2 Our data showing that TNF is able to trigger 'programmed cell death' in endothelial cells correlate with the hypothesis that tumor regression after in vivo administration of TNF may indeed be due, at least partly, to direct cytocidal action on endothelial cells of the tumor neovasculature, which could lead to a secondary intravascular coagulation.

Acknowledgments The authors thank S. Pirotton for helpful suggestions and for critical reading of this article, Dr. J. M. Boeynaems for stimulating discussion, and Dr. M. Parmentier for useful advice. They thank Dr. J. Tavemier (Biogent, Belgium) for providing rhTNF and Dr. M. D. de Baets (University of Limburg, Maastricht, The Netherlands) for providing anti-hTNF monoclonal antibody.

References 1. Matthews N: Tumor necrosis factor from the rabbit. 11. Pro-

duction by monocytes. Br J Cancer 1978, 38:31f0315

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2. Steffen M, Oltman OG, Moore MAS: Simultaneous production of Tumor necrosis factor-a and lymphotoxin by normal T cells after induction with IL-2 and anti-T3. J Immunol 1988, 140:2621-2624 3. Carswell EA, Old W, Kassel RL, Green S, Fiore N, Williamson B: An endotoxin-induced serum factor that causes necrosis of tumors. Proc NatI Acad Sci USA 1975, 72:36663670 4. Le J, Vilcek J: Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Laboratory Invest 1987, 56:234-248 5. Bevilacqua MP, Rober JS, Majeau GR, Fiers W, Cotran R, and Gimbrone MA Jr: Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: Characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci USA 1986, 83:4533-4537 6. Nawroth PP, Stem DM: Modulation of endothelium cell hemostatic properties by tumor necrosis factor. J Exp Med

1986,163:740-745 7. Shimomura K, MandaT, Mukumoto S, Kobayashi K, Nakano K, Mori J: Recombinant human tumor necrosis factor-a: Thrombus formation is a cause of anti-tumor activity. Int J Cancer 1988, 41:243-247 8. Cavender DE, Edelbaum D, Ziff M: Endothelial cell activation induced by tumor necrosis factor and lymphotoxin. Am J

Pathol 1989,134:551-559 9. Sato N, Goto T, Haranaka K, Satomi N, Nariuchi H, ManoHirano Y and Sawasaki Y: Actions of tumor necrosis factor on cultured vascular endothelial cells: Morphologic modulation, growth inhibition, and cytotoxicity. J Natl Can Inst 1986, 76:1113-1121 10. Frater-Schroder M, Riseau W, Hallmann R, Gantschi P, Bohlen P: Tumor necrosis factor type a, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc Natl Acad Sci USA 1987, 84:5277-5281 11. Schuger L, Varani J, Marks RM, Kunkel SL, Johnson KJ, Ward PA: Cytotoxicity of tumor necrosis factor-a for human umbilical vein endothelial cells. Laboratory Invest 1989, 61:62-68 12. Van Coevorden A, Boeynaems JM: Physiological concentration of ADP stimulates the release of prostacyclin from bovine aortic endothelial cells. Prostaglandins 1984, 27:615-626 13. Mosselmans R, Hepbum, A, Dumont JE, Fiers W, Galand P: Endocytic pathway of recombinant murine tumor necrosis factor in L-929 cells. J Immunol 1988,141:3096-3100 14. Jones DP, McConkey DJ, Nicotera P, Orrenius S: Calciumactivated DNA fragmentation in rat liver nuclei. J Biol Chem 1989, 264:6398-6403 15. Wyllie AH: Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980, 284:555-56 16. Duke RC, Chervenak R, and Cohen JJ: Endogenous endonuclease-induced DNA fragmentation: An early event in

cell-mediated cytolysis. Proc Natl Acad Sci USA 1983, 80:6361-6365 17. Kerr JFR, Wyllie AH, Currie AR: Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972, 26:239-257 18. Wyllie AH: Cell death: A new classification separating apoptosis from necrosis. In Bowen ID, Lockshin RA, eds. Cell Death in Biology and Pathology. London, Chapman and Hall, 1981, pp 9-34 19. Hepbum A, Demolle D, Boeynaems J-M, Fiers W, Dumont JE: Rapid phosphorylation of a 27kDa protein induced by tumor necrosis factor. Biochem Biophys Res Commun 1987, 227:175-178 20. Harlan JM, Harker LA, Reidy MA, Gajdusek CM, Schwartz SM, Striker GE: Lipopolysaccharide-mediated bovine endothelial cell injury in vitro. Lab Invest 1983, 13:813-818 21. Allen TD: Ultrastructural aspects of cell death. In Potten CS, ed. Perspectives on Mammalian Cell Death. Oxford, Science Publication, 1987, pp 39-65 22. Araki S, ShimadaY, Kaji K, Hayashi H: Apoptosis of vascular endothelial cess by fibroblast growth factor deprivation. Biochem Biophys Res Commun 1990,168:1194-1200 23. Nawaz S, Lynch MP, Galand P, Gerchenson LE: Hormonal regulation of cell death in rabbit uterin epitheluim. Am J Pathol 1987, 127:51-59 24. Duvall E, Wyllie AH: Death and the cell. Immunol Today 1986,7:115-119 25. Laster SM, Wood, JG, Gooding LR: Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol 1988,141, 2629-2634 26. Pober JS, Cotran RS: Cytokines and endothelial cell biology. Physiological Reviews 1990, 70:427-451 27. Nawroth P, Handley D, Matsueda G, De Waal R, Gerlach H, Blohm D, Stern D: Tumor necrosis factor/cachectin-induced intravascular fibrin formation in Meth A fibrosarcomas. J Exp Med 1988, 168:637-647 28. Schweigerer L, Malerstein B, Gospodarowicz D: Tumor necrosis factor inhibits the proliferation of cultured capillary endothelial cells. Biochem Biophys Res Commun 1987, 143:997-1004 29. Kahaled MB, Smith EA, Soma Y, Leroy EC: Effect of lymphotoxin and tumor necrosis factor on endothelial and connective tissue cell growth and function. Clinic Immunol Immunopath 1988, 49:261-272 30. van de Wiel P, Pieters RHH, van der Pijl A, Bloksma N: Synergic action between tumor necrosis factor and entotoxins or poly(A.U) on cultured bovine endothelial cells. Cancer Immunol Immunother 1989, 29:23-28 31. Segusa Y, Ziff M, Welkovich L, Cavender D: Effect of inflammatory Cytokines on Human Endothelial Cell Proliferation. J Cell Physiol 1990,142:488-495 32. Watanabe N, Niitsu Y, Umeno H, Kuriyama H, Neda H, Yamauchi N, Maeda M, Urushizaki I: Toxic effect of tumor necrosis factor on tumor vasculature in mice. Cancer Res 1988, 48:2179-2183