BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 833-839

Vol. 176, No. 2, 1991 April 30, 1991

C H A R A C T E R I Z A T I O N OF A N G I O G E N I N R E C E P T O R S ON B O V I N E BRAIN C A P I L L A R Y E N D O T H E L I A L C E L L S M. Chamoux,* M. P. Dehouck, #& J. C. Fruchart, # G. Spik,* J. Montreuil,* and R. Cecchelli #&1

* Laboratoire de Chimie Biologique, Unit6 Mixte de Recherche du CNRS n ° 111,Universit6 des Sciences et Techniques de Lille Flandres-Artois, 59655 Villeneuve d'Ascq Cedex - France # SERLIA, Unit6 n ° 325 de rINSERM; Institut Pasteur, 59019 Lille Cedex et &Universit6 des Sciences et Techniques de Lille Flandres-Artois, 59655 Villeneuve d'Ascq Cedex -France Received March 4, 1991

The mitogenic effect of bovine milk angiogenin was studied on bovine brain capillary and aortic endothelial cells, s m o o t h muscle cells and fibroblasts.The proliferation of only bovine brain capillary endothelial cells was detected at c o n c e n t r a t i o n s ranging from 10 to 1,000 ng/ml, with a m a x i m u m effect at 100ng/ml.This mitogenic activity m a y be correlated with a specific binding of angiogenin which w a s d e m o n s t r a t e d only to bovine brain capillary endothelial cells. [125I]-labeled angiogenin binding was time and c o n c e n t r a t i o n dependent and saturable. Scatchard analyses of binding data showed evidence of a single class of binding sites with an apparent dissociation constant of 5.10-10 M. The m o l e c u l a r mass of the angiogenin receptor (49 kDa) was determined by ligand blotting. ©1991 Academic Press, Inc. Angiogenesis, the formation of new capillaries, is relatively infrequent in normal adult tissues, but dramatically increases in growing tumours and in several other pathological conditions. Angiogenin is one of the most potent inducers of neovascularisation, when compared to other angiogenic polypeptides recently described, such as acidic and basic fibroblast growth factors, transforming growth factor (c~ and 13) and tumour necrosis factor cc (1). First characterized by Vallee's group in conditioned medium from human colon adenocarcinoma cells HT 29 (2), angiogenin has also been recently isolated from human plasma (3), bovine milk (4) and bovine plasma (5). Human and bovine angiogenins stimulate angiogenesis "in vivo" when tested on the developing vascular system of the chick chorioallantoic membrane (2,4,5). However, no direct causal relationship between angiogenin and mitogenesis has been established. Unlike other angiogenic polypeptides, angiogenin alone has no known effect on cell proliferation but has been reported to modulate the mitogenic effect of certain conditioned media (6). Furthermore, it has been reported that angiogenin does not appear as a growth factor for 1 To whom correspondence should be addressed. Abbreviations: BAAE, bovine aortic arch endothelial cells; BBCE, bovine brain capillary endothelial cells; BSA, bovine serum albumin; bFGF, basic fibroblast growth factor ; PBS, phosphate buffered saline ; PRI, placenta ribonuclease inhibitor ; RNase A, ribonuclease A; DMEM, Dulbecco's modified Earle medium.

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endothelial cells. But, its high concentration in plasma suggests that angiogenin may be involved in endothelium homeostasis. Furthermore, it has been shown that angiogenin binds to endothelial cells in the presence of 100 ~tM copper (7). In the present paper, we report that bovine milk angiogenin is mitogenic "in vitro" for bovine brain capillary endothelial cells but not for aortic endothelial cells. This difference in the behaviour of large blood vessel and capillary derived endothelial cells led us to the hypothesis that it could be due to the occurrence of angiogenin receptors, hypothesis we have verified by characterizing the receptors by ligand blotting, only on bovine brain capillary endothelial cells.

EXPERIMENTAL

PROCEDURES

Materials : Angiogenin was isolated from bovine milk (8) and the purity was determined by reverse phase chromatography in a PLRP-S column (Polymer Laboratory), giving a single peak. Human recombinant angiogenin was produced in Escherichia coli and purified as described (9). Human placenta ribonuclease inhibitor was obtained from Promega and bovine pancreatic ribonuclease A from Sigma Chemical Co. Protein iodination : Angiogenin and RNase A were labeled with 125-I (625 MBq of 125-I/gg iodine) (Amersham International) using Iodo-Gen (Pierce Chemical Co.). Iodination was performed according to the manufacturer's instructions. The unbound iodine was removed by gel filtration on a Sephadex G25 PD10 column (Pharmacia) equilibrated with the incubation buffer.The specific radioactivity varied from 15,000 to 30,000 cpm/ng. Cell cultures : Bovine brain capillary endothelial (BBCE) cells, bovine aortic arch endothelial (BAAE) cells, smooth muscle cells from bovine aorta, were isolated and cultured as described (10). Human fibroblasts were grown from primary culture of the skin of a healthy subject. Dede Chinese Hamster lung fibroblasts CCL39 were obtained from the American Type Culture Collection and subcultured for less than 20 passages. Cell growth m e a s u r e m e n t s : BBCE cells or BAAE cells (104 cells/35 ram) were seeded in 6 well plates (Coming) coated with gelatin and exposed to 2 ml of DMEM supplemented with 15 % calf serum, 50 gg/ml gentamicin and 1 ng/ml of bFGF or various concentrations of bovine milk angiogenin, or human recombinant angiogenin diluted in 10 pJ of DMEM containing 0.5 % bovine serum albumin. Medium was changed and angiogenin added every 2 days. After 5 days, triplicate wells were trypsinized and the cell number was determined using a Coulter counter. M e a s u r e m e n t of DNA synthesis : After growing 2 days or 7 days in DMEM supplemented with 15 % calf serum, 2 mM glutamine, 50 ~tg/ml gentamycin and bFGF (1 ng/ml), quiescent sparse or quiescent confluent BBCE cells (Go phase) were obtained after 48 h of growth in the absence of serum and bFGF according to Bouche et al. (11). At different times after stimulation by 100 ng/ml angiogenin, cells in serum-free medium were pulse-labeled for 1 h with [methyl-3H] thymidine (Amersham; 5p.Ci/ml). The rate of DNA synthesis was measured by the determination of [3H] cpm incorporated into trichloroacetic acid-insoluble material. Binding assays : BBCE cells and BAAE cells were seeded as described above and incubated for 5 days in DMEM supplemented with 15 % calf serum, 50 ~tg/ml gentamicin and 1 ng/ml bFGF. At day 6, cells were incubated for 2 h at 37°C in serum free DMEM containing 10 mM Hepes and 0.5 % BSA. Binding assays were then conducted at 4°C in 700 ~tl DMEM containing 10 mM Hepes, pH 7.4, 0.5 % BSA and 15 ng/ml [125I]-angiogenin or [125I]-RNAse. Non-specific binding was determined by incubating the cells with the same incubation medium to which a 100-fold excess of unlabeled angiogenin was added. At the end of the incubation, the cells were washed 3 times with PBS, once with 0.6 M NaC1 in PBS and 3 times again with the PBS. The cells were then solubilized in 1 ml of 0.1 M NaOH. The radioactivity was recorded in a LKB 1282 Compu Gamma Counter. Saturation experiments were carried out with increasing concentrations of labeled and unlabeled angiogenin and were analyzed according to the method of Scatchard (12). The effect of 100 gM Cu++ and 1 nM PRI were carried out in the same way. Each value is the mean of triplicate determinations. Ligand assays: BBCE cells and BAAE cells cultures of 5 days were scraped in cold TrisHC1 50 mM pH 6,5 containing 1 mM phenylmethylsulfonyl fluoride and centrifuged at 1,000 g for 10 min at 4°C. Membrane proteins were extracted in the same buffer containing 1 mM diisopropyl fluoride phosphate and 1% Triton X-100 at 4°C for 30 min, and centrifuged at 14,000 834

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g for 10 min. Solubilized proteins (80 btg) were subjected to SDS-PAGE on 5-15 %gels (13) in sample buffer containing only 1 % SDS. Proteins were transferred to nitrocellulose (14). The nitrocellulose transferts were blocked for 2 h with Tris buffered saline (Tris-HC1 10 mM pH 8, NaC1 50 mM, CaC12 2 raM) containing l0 % BSA. The nitrocellulose transferts were incubated for 2 h with the Tris buffered saline supplemented either with [ 125I]-angiogenin (30 ng/ml) in the absence of unlabeled angiogenin or with a 100-fold excess of unlabeled angiogenin, and autoradiographed at -70 ° C for 3 days.

RESULTS

AND DISCUSSION

Cell growth measurements : Bovine angiogenin was analyzed for mitogenic activity on endothelial cells. As shown in Fig. 1, bovine angiogenin induces only the proliferation of BBCE cells at concentrations ranging from 10 to 1,000 ng/ml with a maximum effect at 100 ng/ml, while it has no effect on the proliferation of BAAE cells submitted to the same culture conditions, in spite of the presence of angiogenin in calf serum (30-80 ng/ml) (5). The same results (not shown) were obtained with human recombinant angiogenin. In contrast, bFGF (1 ng/ml) stimulates both BAAE and BBCE cell proliferation. Like in the chorioallantoic membrane assay described (15), in our experiments, the effect of a mixture of angiogenin (100 ng/ml) with bFGF (1 ng/ml) was neither additive nor synergistic. In addition, no mitogenic effect was found for angiogenin with non-endothelial cells like bovine smooth muscle cells, human fibroblasts and CCL39 fibroblasts (data not shown). In order to demonstrate the direct mitogenic action of angiogenin, sparse BBCE cells (Go phase) were obtained after 48 h of growth in the absence of serum and angiogenin. Addition of 100 ng/ml angiogenin induces the transition from Go to G1 phase of the cell cycle, and stimulates

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Effect of bovine angiogenin concentration on the proliferation of bovine brain capillary endothelial (BBCE) cells and bovine aortic arch endothelial (BAAE) cells. The 100 % was calculated for 486,000 cells/dish and 410,000 cells/dish for BBCE and BAAE, respectively, and obtained in the presence of 1 ng/ml of bFGF. Standard deviation varied less than 10 % of the mean values. 835

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cells entering the S phase within 3 h by incorporation of labeled thymidine (not shown).No incorporation of thymidine was obtained with confluent cells. Our data are consistent with the previously published observations (16) which showed that purified tumor angiogenesis factor "TAF" enhances proliferation of capillary, but not aortic, endothelial cells "in vitro", Futhermore, it had been shown (17), "in vivo", that contrast enhancement of intracranial tumours, which reflected tumour neovascularisation, is dependent primarily on the proliferation of capillaries. On the contrary, our results are not consistent with those reported in the literature which stated that angiogenin was not mitogenic for endothelial cells (1, 6, 18). This apparent discrepancy might be related to the different methods used for the preparation of capillary endothelial cells. In fact, most of them involve collagenase digestion of microvessels (19). So we cannot exclude the possibility that after such treatment, migrating and proliferating cells might be derived from the endothelial wall of arterioles and venules which contaminate the capillaries. These contaminations may lead to erroneous interpretation, since several authors have shown that capillary endothelial cells differ from the endothelial cells of large arteries in their responses to growth and migration stimuli (20,21) and in their biochemical and functional characteristics (22). Absence of the use of enzymes in our isolation procedure excludes the possibility that we are dealing with endothelial cells derived from large vessels, because only endothelial cell islands emerging from identifiable capillaries were cloned (10). Recent studies have shown that human angiogenin stimulates intracellular diacylglycerol formation and prostacyclin secretion in endothelial cells at concentrations that induce response in the chorioallantoic membrane assay (23, 24). These observations suggest that angiogenin might act via a specific cell-membrane receptor.

Binding assays : In order to determine if the difference in the mitogenic response to bovine angiogenin could be due to the presence of receptors on bovine endothelial cells, we examined the binding of [125I]-labeled bovine angiogenin to cultured endothelial cells. As shown in Fig. 2, binding of bovine [125I]-angiogenin to BBCE cells reached an equilibrium after 1 h of incubation at 4°C while under the same experimental conditions, no significant cell-associated

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Time course of bovine [125I]-angiogenin (15 ng/ml) binding to BBCE and BAAE cells at 4°C. Triplicate determinations varied less than 5 % of the mean values. 836

Vot. 176, No. 2, 1991

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2

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Concentration dependence of the binding of [125I]-angiogenin to BBCE cells for 1 h at 4°C. Insert : Scatchard analysis of the binding data to BBCE cells. Triplicate determinations varied less than 5 % of the mean values (B = Bound ; F = Free).

radioactivity was observed with BAAE cells, and smooth muscle cells or fibroblasts (data not shown). Fig.3 shows that bovine [125I]-angiogenin reached saturation binding in the presence of B B C E cells. This saturation binding emphasized the concept that the interaction of [125I]_ angiogenin with a specific receptor has been detected. The discrepancy between the saturation binding (I5 ng/mt)and the maximum effect on the proliferation (t00 ng/ml) could be explained in different ways: l- Binding was determined at 4°C after I h, while proliferation was measured at 37°C after 48 h; 2- Binding occurs in the absence of serum. Furthermore, trypsin treatment of BBCE cells abolished the angiogenin binding demonstrating that a membrane protein-angiogenin interaction was involved. When transformed to Scatchard plots, the total binding shows evidence of a single class of binding sites with an apparent dissociation constant of 0.5 nM. The average number of angiogenin molecules associated per cell was about 11,000. To detect potential artefact binding due to the high pI (>9.5) (2) of angiogenin, binding experiments with [125I]-RNase A and BBCE cells were carried out under the same conditions but showed no cell specific interaction (data not shown). When placenta ribonuclease inhibitor , a tight-binding inhibitor of the angiogenic activity of human angiogenin (25), was preincubated with an equimolar ratio of bovine [1251]-angiogenin, the cell associated radioactivity decreased by 65 % . This result could be correlated with the inhibition of 60 % of the mitogenic activity of angiogenin when PRI was added at the same concentration in the incubation medium. Recent studies (7) have demonstrated that incubation of human angiogenin in the presence of 100 ~tM Cu ++ induces a several fold increase in the specific binding of angiogenin to calf pulmonary artery endothelial cells. Under our con~tions, addition of 100 gM Cu ++ increased the binding of [125I]-labeled angiogenin to BBCE cells by about 25 % but did not induce the binding to BAAE ceils. These results differ from those of Badet et al. (7) and remain to be further clarified. Angiogenin cell binding sites appeared to be related to celt density, since binding assays at 4°C on BBCE cells cultured for 5, 7 and 9 days revealed a decrease in angiogenin molecules associated per cell : 11,000; 5,000 and 880 respectively as the ceil density increased. The 837

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A

B

C

200

116 66 44

29 Fig .4.

Ligand blotting of [125I]-angiogenin to solubilized membrane proteins from BBCE (lanes A and B) and BAAE (lane C). The nitrocellulose transferts of solubilized membrane proteins were incubated for 2 h with 30 ng/ml of [125I]-angiogenin without unlabeled angiogenin (lanes A and C) or with a 100-fold excess of unlabeled angiogenin (lane B) and autoradiographed at -70°C for 3 days.

regulation of receptors by cell density has been reported for growth factors such as FGF (26) ; transforming growth factor 93 (27) and was considered to reflect their involvement in the growth related functions. L i g a n d blotting: Binding of [125I]-angiogenin was also studied to solubilized membrane proteins isolated from BBCE and BAAE, separated by S D S - P A G E and transferred to nitrocellulose. The direct ligand blotting allowed to identify in BBCE extract, by autoradiography, a labeled component with an apparent molecular mass of 49 kDa (Fig. 4). Competition binding assayed by introducing a 100-fold excess of unlabeled angiogenin indicated an inhibition of the binding of [ 125I]-angiogenin. No binding of [ 125I]-angiogenin to solubilized membranes isolated from BAAE was observed confirming therefore the results obtained on whole cells.

CONCLUSION These findings suggest that the rnitogenic activity of bovine angiogenin on BBCE cells can be correlated with the occurrence of specific membrane receptors of 49 kDa on this type of cell. So, bovine and human angiogenins stimulate capillary endothelial cell proliferation and could be considered as "direct" angiogenic factors. Furthermore, their effect on the proliferation of brain capillary endothelial cells at 100 ng/ml and the high concentration in the human plasma (60-150 ng/ml) (3) and in bovine milk (200-500 ng/ml) (4) suggest that angiogenin may be involved in endothelium homeostasis.

A c k n o w l e d g m e n t s : This work was supported in part by the Universit6 des Sciences et Techniques de Lille Flandres-Artois, the Centre National de la Recherche Scientifique (U.M.R. n°l 11, Director Prof. A. Verbert), the Institut National de la Sant6 et la Recherche MEdicale (U 325, Director Prof. J.C. Fruchart) and the Institut Pasteur of Lille. We are very grateful to Dr. B.L. Vallee for helpful discussion and suggestions, and to the Institut de Biotechnologie de Vitry (Rh6ne-Poulenc Sant6) for their generous gift of human recombinant angiogenin. 838

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REFERENCES

1. Folkman, J., and Klagsbrun, M. (1987) Science 235,442-447. 2. Fett, J.W., Strydom, D.J., Lobb, R.R., Alderman, E.M., Bethune, J.L., Riordan, J.F., and Vallee, B.L. (1985) Biochemistry 24, 5480-5486. 3. Shapiro, R., Strydom, D.J., Olson, K.A., and Vallee, B.L. (1987) Biochemistry 26, 5141-5146. 4. Maes, P., Damart, D., Rommens, C., Montreuil, J., Spik, G., and Tartar, A. (1988) FEBS- Lett. 241, 41-45. 5. Bond, M., and Vallee, B.L. (1988) Biochemistry 27, 6286-6287. 6. Heath, W.F., Moore, F., Bicknell, R., and Vallee, B.L. (1989) Proc. Natl. Acad. Sci. USA 86, 2718-2722. 7. Badet, J., Soncin, F., Guitton, J.D., Lamare, O., Cartwright, T., and Barritault, D. (1989) Proc. Natl. Acad. ScL USA 86, 8427-8431. 8. Spik, et al. (19-11-1989) International extension of french patent n° 8715984. 9. Den6fle, P., Kovarik, S., Guitton, J.D., Cartwright, T., and Mayaux, J.F. (1987)Gene 56, 61-70. 10. M6resse, S., Dehouck, M.P., Delorme, P., Bensa'id, M., Tauber, J.P., Delbart, C., Fruchart, J.C., and Cecchelli, R. (1989) J. Neurochem. 53, 1363-1371. 11. Bouche, G., Gas, N., Prats, H., Baldin, V., Tauber, J.P., Teissie, J., and Amalric, F. (1987) Proc. Natl. Acad. Sci. USA 84, 6770-6774. 12. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660-672. 13. Laemmli, U.K. (1970) Nature 227, 680-682. 14. Towbin, T.T., St~thelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350- 4354. 15. Fett, J.W., Bethune, J.L., and Vallee, B.L. (1987) Biochem. Biophys. Res. Commun. 146, 1122-1131. 16. Keegan, A., Hill, C., Kumar, S., Phillips, P., Shor, A., and Weiss, J. (1982) J. Cell Sci. 55, 261-272. 17. Zagzag, D., Goldenberg, H., and Brem, S. (1989) Amer. J. Roentgenol. 153, 141-146. 18. Klagsbrun, M. (1987) Current Communications in Molecular Biology ed., Cold Spring Harbor Laboratory, New York, 1-12. 19. Joo, F. (1985) Neurochem. Int. 7, 1-25. 20. Zetter, B.R. (1980) Nature 285, 41-43. 21. Folkman, J., Haudenschild, C., and Zetter, B.R. (1979) Proc. Natl. Acad. Sci.USA 76, 5217-5221. 22. Zetter, B.R. (1988) Endothelial cells, Vol II, Una Ryan Ed., CRC Press Inc., Boca Raton, Florida, 63-79. 23. Bicknell, R., and Vallee, B.L. (1988) Proc. Natl. Acad. Sci. USA 85, 5961-5965. 24. Bicknell, R., and Vallee, B.L. (1989) Proc. Natl. Acad. Sci. USA 86, 1573-1577. 25. Shapiro, R., and Vallee, B.L. (1987) Proc. Natl. Acad. Sci. USA 84, 2238-2241. 26. Neufeld, G., and Gospodarowicz, D.J. (1985) J. Biol. Chem. 260, 13860-13868. 27. Rizzino, A., Kazakoff, P., Ruff, E., Kuszynski, C., and Nebersick, J. (1988) Cancer Res. 48, 4266-4271.

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Characterization of angiogenin receptors on bovine brain capillary endothelial cells.

The mitogenic effect of bovine milk angiogenin was studied on bovine brain capillary and aortic endothelial cells, smooth muscle cells and fibroblasts...
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