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41 5
I N H m r r I O N OF P R O D U C T I O N O F MONOCYTE/MACROPHAGE-DERIVED ANGIOGENIC A C T I V H T BY O X Y G E N F R E E - R A D I C A L S C A V E N G E R S Alisa E. Koch -~., Michael Clio*, James C. Burrows', Peter I. Polverini#, and S. Joseph Leibovi~ + Departments of Medicine', Pathology*, and Basic Sciences* Northwestern University Medical and Dental Schools, and V.A. Lakeside Medical Center~To whom all correspondence should be addressed ABSTRACt We showed previously that thiol-containing compounds inhibited the production of macrophage-mediated angiogenic activity. Since thiol-containing compounds may act on macrophages by affecting activation and inhibiting the production of oxygen free-radicals, we studied the effects of oxygen free-radical scavengers on production of angiogenic activity by elicited mouse peritoneal macrophages and lipopolysaccharide stimulated normal human monocytes. Monocyte/macrophage ccmditioned media were potently angiogenic when assayed in rat corneas, while conditioned media from oxygen free-radical scavenger-treated cells were not. The inhibitory effect of oxygen free-radical scavengers was due to a direct effect on monocyte/macrophage production of angiogenic activity but was not due solely to a decrease in the production of the macrophage-derived angiogenic cytokine tumor necrosis factor-a. We conclude that oxygen free-radical scavengers are potent inhibitors of the production of macrophage-mediated angiogenic activity. INTRODUC~ON Angiogenesis, the growth and proliferation of new blood vessels, is an important feature of wound repair and tumor growth (Leibovich, 1984, Clark et al., 1976, Thakral et al., 1976, Polverini et al., 1977, Folkman, 1985, Kulka et al., 1955, Brown et al., 1980, Jackson et al., 1989, Polverini, and Leibovich, 1984, Koch et al., 1986a). Macrophages (mSs) mediate angiogenesis in many diverse processes such as neoplastic growth and rheumatoid arthritis (Leibovich, 1984, Koch et al., 1986, Koch et al., 1988). We have shown, using normal human monocytes (mos), that cellular activation with agents such as lipepolysaccharide (LPS) or transforming growth factor-l~ is required for mos to express the angiogenic phenotype (Koch et al., 1988, Wiseman, et al. 1988). We have shown previously that the gold containing anti-rheumatic compounds, gold sodium thiomalate and auranofin, which have the common property of containing a thiol moiety, are potent inhibitors of the production of m~b-derived angiogenic activity (MDAA)(KOChet al., 1988a). This inhibitory effect appears to be due to the thiol rather than the gold content of these compounds (Koch et al., 1991). These 0309-1651/92/050415-11/$03.00/0
© 1992 Academic Press Ltd
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Cell Biology International Reports, VoL 16, No. 5, 1992
studies showed that glutathione and cysteine in their reduced but not oxidized forms exerted this inhibitory effect, suggesting that the reducing properties of the thiols might be involved. Since anti-rheumatic gold compounds, which are thiol containing, act as activated oxygen free-radical scavengers (FRSs) and inhibit superoxide release from mcks, we have examined whether FRSs might inhibit the production of angiogenic activity by mononuclear phagocytes (Mirabelli et al., 1988, Davis et al., 1983). We show that FRSs are potent inhibitors of the production of MDAA. METHODS
Premmffon ofm6 cond/t/oned medhun for em/uafon/n die rat cornea/b/oassay_ for ane/oeenes/s. Eight week old Balb/c mice were injected with Brewer's thioglycollate broth (Difco, Baltimore, MD), and their elicited peritoneal m~s were harvested four daysJater as previously described (Meltzer, 1981). M~bs prepared in this manner have been shown to be potently angiogenic (Polverini et al., 1977). M~s were plated at 1 x 106 cells/ml in RPMI plus 10% fetal calf serum (FCS) + gentamicin and allowed to adhere to 75 mm culture flasks or 100 mm tissue culture dishes (Costar, Cambridge, MA) for one hour at 37°C in an incubator gassed with 5 % CO2 and 95 % air. After adherence, the culture medium was replaced by an equal volume of RPMI1640 + 0.5% FCS + gentamicin with the following FRSs for 30 minutes: 1,1,3,3tetramethyl-2-thiourea, D-mannitol, catalase, or urea (Sigma, St. Louis, MO). The concentrations selected were based on those shown by Kobayashi, et al to inhibit maximally phorbol myristate acetate stimulated TNF-8 production by human T cell hybridomas (Kohayashi et al., 1984), or those shown to be required to reduce both oxygen free-radical production and endothelial cell killing by human neutrophils (Varani et al., 1985). After incubation with FRSs, m~s were exposed to the activating agent lipopolysaccharide (LPS)(5 ug/ml)(phenol-extracted preparation of E. Coli 055:]35, Sigma) and the mck conditioned medium harvested after 24 hours. Cell viability was determined using trypan blue exclusion. Concentrated (20x) conditioned media were assayed using the rat corneal bioassay for angiogenesis (see below). Premmtion o f human mo conditioned medium for evaluation in the rat corne__~ bioassay for aneioeene~, Human mos were isolated from huffy coats of normal volunteers by density gradient centrifugation using Sepracell-MN (Sepratech, Corp, Oklahoma City, OK). Mos isolated in this manner were >90% pure as estimated morphologically and by esterase staining (Sigma). Mos were further purified by adherence of 1 x l0 s cells/ml (RPMI plus 10% FCS plus gentamicin) to 100 mm tissue culture dishes for 2 hours at 37°C. The adherent cells were washed with PBS (xl). RPMI + 10% FCS and
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FRSs were added. After 1/2 hour, LPS (5 ug/ml) was added and the cells incubated overnight. The cells were washed with PBS (xl) and incubated for 48 hours with RPMI + 0.5% FCS + LPS (5 ug/ml) + FRSs, and the conditioned medium concentrated (20x) and assayed in the rat cornea. In some experiments, FRSs were added to previously conditioned mo/m~ media and the media assayed in rat corneas.
Rat cornea/b/oa~av for am,/oeenes/s. Concentrated mo/m~ conditioned media (5 ul) were incorporated into an equal volume of slow-release Hydron (Interferon Sciences, New Brunswick, NJ) and allowed to dry. Hydron pellets (10ul) were implanted,aseptic~y into a pocket within the rat corne~ stroma 2 mm from the limbal vasculature ~olverini and Leihovich, 1984, Koch et al., 1986, Koch et al., 1986a, Koch et al., 1991). Corneas were examined daily for seven days using a stereomicroscope and perfused with colloidal carbon at the end of the observation period to provide a permanent record of the angiogenic response (Polverini and Leibovich, 1984, Koch et al., 1986, Koch et al., 1986a, Koch et al., 1991). Corneas were examined histologically for any evidence of non-specific inflammation. Mea.mrement o f the effect.of F R ~ on ~eneral m~b orotein svnthe.~.
Assessment of protein synthesis was done using a modification of the method of Ohta (Ohta et al., 1986). After incubation with FRSs, duplicate cultures of m~s were incubated with leucine-free RPMI for one hour at 37°C. Fifty uCi/ml [3H]-leucine (Amersham, Arlington Heights, IL) were added to 5x106 cells for a further one hour incubation. M~s (5x106/2 ml RPMI) were subsequently lysed with lml 1M sodium hydroxide and the cell lysate added to 2ml 5 % trichloracetic acid. After heating at 75°C for 30 minutes, precipitation was allowed to proceed overnight at 4°C. The precipitates were pipetted in triplicate onto glass fiber filters, washed with 95% ethanol and counted in a scintillation counter (Beckman, h'vine, CA). Ayaav for tumor necrosis factor-a fiN-F-a) and imerleuldn-IB t/L-l) in mo mediu,
-
The effects of FRSs on mo production of the angiogenic cytokines IL-1 and TNT-~ were studied. Mos were isolated as described above and incubated with RPMI-1640 + 0.5% FCS + gentamicin + LPS (5 ug/ml) with or without FRSs. After incubation at 37°C for 24 hours, the mo supernatants were harvested and assayed for TNT-~, using an enzyme linked immunosorbent assay (ELISA) (Biosource International, Westlake Village, CA). This assay is specific for the measurement of biologically active TNF-a with a minimum sensitivity of 4 pg/ml, respectively. ~ 1B was measured using an ELISA (R&D Systems, Minneapolis, MN) which has a minimum sensitivity of 4.5 pg/ml.
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RESULTS
Figure 1 shows a colloidal carbon perfused corneal mount from a rat implanted with a Hydron pellet containing conditioned medium from LPS activated human mos. A positive response in this figure is indicated by the unidirectional sustained growth of capiHa~ sprouts and hah~in loops towards the implant. New capillaries grew at approximately 0.2 mm/day. Corneas examined histologically showed no evidence of nonspecific inflammation and exhibited some stromal thickening. Figure 2 shows a rat corneal mount indicating the effect of treating murine m~bs with 1,1,3,3,tetramethyl-2-thiourea, a FRS. The lack of an angiogenic response to conditioned medium from m~s treated with this compound is shown.
Figure 1. Colloidal carbon perfused corneal mount from a rat implanted with a Hydron pellet containing mo conditioned medium (top)(x33). Directional growth of capillary loops and sprouts toward the implant can be seen.
Figure 2, Colloidal carbon perfused rat corneal mount showing a cornea implanted with conditioned medium from mos treated with 1,1,3,3 tetramethyl-2-thiourea (top)(x33). No angiogenic response is seen.
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Table 1 shows the cumulative effects of FRSs on the production of mo/m4,-derived angiogenic activity. Conditioned medium from human mos or murine m4,s incubated with sham FRS (5 mM urea) exhibited positive angiogenic responses, as did conditioned media from mo/m~s incubated with urea, which is a weak FITS. In contrast, conditioned medium from mo/m~s treated with 1,1,3,3-tetramethyl-2thiourea did not induce angiogenic responses. Similarly, conditioned medium from mo/m~s preincubated with D-mannitol or catalase did not induce angiogenic responses. To determine whether the effect of the FRSs was due to a direct effect on the m~ or to effects on the MDAA or the corneal vasculature, FRSs were added to previously conditioned m~bconditioned medium. This conditioned medium elicited a positive corneal angiogenic response, indicating a direct effect of FRSs on the mo/m~ rather than on the corneal blood vessels or the MDAA (table 1). Control m~ uptake of [3H]-leucine was compared with that of FRS-treated m~bs. Protein synthesis was not appreciably altered by treatment of the m~s with FRSs. Results of ELISAs to detect the presence of ILol and TNF-c~ in human mo supernatants are shown in Figure 3. Treatment of mos with urea, a poor FRS, did not decrease TNF-c~ production. In contrast, treatment with 1,1,3,3,-tetramethyl-2thiourea or mannitol reduced TNF-ct production by 53% and 39%, respectively. Treatment of mos with FRSs, with the exception of urea, did not decrease IL-1 production. DISCUSSION
In mo/m~s, the process of ~ u l a r activation results in both early and late activation products (Hamilton and Adams, 1987). Early products include a series of oxygen free-radicals and hydrogen peroxide, produced within a few minutes of exposure of the cells to activating agents. Following this early stage, a series of gene induction events occurs, resulting in the later production of several cytokines, including TNFo~ and IL-1. The connection between the early stages of activation, with the production of activated oxygen free-radicals, and the later stages, involving cytoldne production, is not clear. The production of oxygen free-radicals is mediated by the leukocyte respiratory burst with activation of the hexose monophosphate shunt pathway, involving a series of oxidation-reduction (redox) reactions (Fantone and Ward, 1985). Reducing agents may interfere with this pathway by disrupting the cellular redox balance, thus inhibiting production of oxygen free-radices. We have shown previously that compounds containing free-thiols (reduced glutathione, cysteine) inhibit the production of MDAA, suggesting a possible connection between the production of early and late activation products (Koch et al., 1991). In this study, we examined whether products of early activation, namely oxygen free-radicals and hydrogen peroxide, might be involved in the generation of
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angiogenic activity, by using FRSs to inactivate these products and thus inhibit their 4000
A
30OO
i
20O0
1000
B
LNTRF_ATED
~
TMT-UFEA
D-MANN,
CATALASE
UNTFEATED
UFF.A
TMT-L.IgEA D-Iv~NN.
CATALASE
60o
o
4OO
i
3OO
o
T -
200
100
0
Figure 3. A) TNF-o~ levels in mo conditioned medium determined using an ELISA. Normal human peripheral blood mos were cultured either with or without: urea (SmM), 1,1,3,3-tetramethyl-2-thiourea (TMT-urea)(SmM), D-mannitol (Dmann.)(50mM), or catalase 0000 u/ml). Results of 3 experiments, each assayed in duplicate, are shown. Results expressed as the mean + S.E. B) IL-1 levels in mo conditioned medium from two donors prepared and assayed as .in "A" above. activity. In this study, we present data that suggests that FRSs can inhibit the production of MDAA in both human and murine mononuclear phagocytes. This effect appears to be directly on the too/m#, rather than on the secreted angiogenic factor(s) or the blood vessels of the target corneas. To determine if the inhibition of MDAA production was due to the inhibition of
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production of known angiogenic mediators, we measured the production of TNF-a by human mos incubated with FRS. Leibovich et al have shown that TNF-o~ is one of the major cytokines accounting for murine peritoneal m~-derived angiogenic activity (l.eibovich et al., 1987). IL-1 has also recently been shown to be angiogenic (Mahadevan, et al., 1989). While not all FRSs decrease the amount of TNF-o~ produced by LPS activated human mos, 1,1,3,3-tetramethyl-2-thiourea,in particular, decreased TNF-ot production about 53%, while urea, a weak FRS did not. Mo IL-1 production was not decreased significantly by FRSs. The change in TNF-ot alone may not be adequate to account for the dramatic decrease in MDAA. It seems possible that inhibition of production of additional eytokines may be involved in the inhibition of production of MDAA. Alternatively, FRSs may upregulate release of angiogenic inhibitors, such as thrombospondin (Rastinejad et al., 1989, Good et al., 1990). Increased levels of this inhibitory activity may be produced by rafts in response to FRSs resulting in a net loss of demonstrable angiogenic activity. Since the angiogenic phenotype in mos/m~s has been shown to be recessive, it is possible that activation of a suppressor element with subsequent upregulation of inhibitory activity rather than production of angiogenie mediators is related to oxygen freeradical production (Polverini, 1989). Oxygen free-radicals have been implicated in mediating tissue damage in a variety of pathological lesions, such as myocardial infarctions, adult respiratory distress syndrome, and rheumatoid arthritis (Fantone and Ward, 1985, Lunec et al., 1981, Muus and Oudsten, 1979). In rheumatoid arthritis, oxygen free-radicals have also been implicated since increased levels of conjugated dienes and malonyldialdehyde (products of lipid peroxidation reactions) are found in the synovial fluid and plasma of patients with active disease (Lunec et al., 1981, Muus and Oudsten, 1979). FRSs have been reported to be effective inhibitors of tissue injury in experimental models of arthritis--as well as in patients with rheumatoid arthritis (Menander-Huber and Huber, 1977, Hirschelmann and Bekemeier, 1981). In summary, in this study we have shown that FRSs can inhibit production of m~derived angiogenic activity and that this inhibition is not totally due to inhibition of production of the angiogenic cytokine TNF-ot. It is possible that naturally occurring FRSs might be involved in inhibiting the production of MDAA in vivo. ACKNOWL~:nG~
This work was supported by an Arthritis Foundation Fellowship and Grant (A.E.K.), a Veteran's Administration Merit Review (A.E.K.), N.I.H. grants AR30692 (A.E.K.), GM29135 (S.LL.), and HL39926 (P.J.P.). This work was presented in part at The American Federation for Clinical Research Meeting, Washington, D.C., 1989 and at the American College of Rheumatology Meeting, Cincinnati, OH, 1989. We wish to thank Drs. Richard Pope, Frank Schmid, and Arthur Veis for helpful discussions and Ms. Lisa Harlow for expert technical assistance.
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TABLE I
OF SCAVENGERS OF OXYGEN FREE RADICALS ON THE
PRODUCTION OF Mo/MACROPHAGE-DERIVED ANGIOGENIC ACTIVITY Test group
Numbers of corneas assayed Number of positive angiogenic responses
Number of negative angiogenic responses
5 6
0 0
I#
6
0 0
8 4
5 7
0 1
0
4
0 0
5 4
3
0
3
0
Conditioned medium from murine peritoneal m~,s preincubated with:
Control media Urea (5 m M ) (poor scavengerof oxygen freeradicals) i,1,3,3-tetramethyl-2-thiourea (5 mM)
D-mannitol (50 raM) Catalase(3000 u/ml) Conditioned medium from human mos incubated with:
Control media Urea (5mM) (poor scavengerof oxygen free radicals) 1,1,3,3-tetramethyl-thiourea (5mM) D-mannitol (50 mM) Catalase (3000 u/ml) Conditioned medium from human mos to which FRS had been added: 1,1,3,3-tetramethyl-thiourea
(SmM) catalase 0000 units/ml)
//Denotes 1 weakly positive angiogenic response.
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REFERENC"F~ Brown, R.A., Weiss, LB., and Tomtin.qoa, I.W. (1980). Angiogenic factor from synovial fluid resembling that from tumours. Lancet: i: 682-685. Clark, R.A., Stone, R.D., Lueng, D.Y.K., Silver, D.C., and Hahn, D.C. (1976). Role of macrophages in wound healing. Surg. Forum. 2?: 16-18. Davis, P.C., John.qtoa, C.L., Mill~, C.L., and Woag, K. (1983). Effects of gold compounds on the function of phagocytic cells. II. Inhibition of superoxide radical generation by tripeptide-activated polymorphonuclear leukocytes. Arthritis Rheum. 6: 82-86. Ditl:~rd CJ., Kunert, K.J., and Tappel, A.C. (1982).,Lipid peroxidation during chronic inflammation induced in rats by Freund's adjuvant: effect of vitamin E as measured by expired pentane. Res. Commun. Chem. Pathol. Pharmacol. 37: 143-146. Fantone LC., and Ward, P.A. (1985). Polymorphonuclear leukocyte-mediated cell and tissue injury: oxygen metabolites and their relations to human disease. Human Pathol. 16: 973-978. Folkman, J. (1985). Tumor angiogenesis. Adv. Cancer Res. 43: 175-203. Good D J . , Polverini, P J . , Rastinejad, F., LeBeau, M.M., Lemons, R.S., Frazier, W.A., and Bouck, N.P. (1990). A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. (USA) 87: 6624-6628. Hamilton, T.A., and Adams, D.O. (1987). Molecular mechanisms of signal transduction in macrophages. ImmunoL Today 8: 151-158. Hirschelmann, R., and Bekemeier, H. (1981). Effect of catalase, peroxidase, superoxide dismutase and 10 scavengers of oxygen radicals in carrageenin edema and in adjuvant arthritis of rats. Experientia 37: 1313-1316. Jackson, C.t:, Garbett, P.K., Marks, R.M., Chapman, G., Sonnabemi, D.H., Potter, S.R., Brooks, P.M., and Schrieber, L. (1989). Isolation and propagation of endothelial cells derived from rheumatoid synovial vasculature. Ann. Rheum. Dis. 48: 733-736. Kobayashi, Y., M. Asada, and T. Osawa (1984). mechanisms of phorbol myristate acetate-induced lymphotoxin production by human T cell hybridoma. J. Biochem. 95: 1775-1782. Koch, A.E., Polverini, P J . , and ~ c h , SJ. (1986). Stimulation of neovascularization by human rheumatoid synovial macrophages. Arthritis Rheum. 29: 471-479. Koch, A.E., Polverini, P J . , and Leibovich, SJ. (198(m). Induction of neovascularization by activated human monocytes. L Leuk. Biol. 37: 233238. Koch, A.E., Polverini, P J . , and Leibovich, SJ. (1988). Functional heterogeneity of human rheumatoid synovial tissue macrophages. J. Rheum. 15: 1058-1063.
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Koch, A.E., M. Cho, L Bummvs, S.L Leibovich, and Polverini, P.L (1988a). Inhibition of production of macrophage-derived angiogenic activity by the anti-rheumatic agents gold sodium thiomalate and auranofm. Biochem. Biophys. Res. Commun. 154: 205-212. Koch, A.E., Burrows J., Polverini P.L, Cho M., and Leibovich, S.L (1991). Thiol-containingcompounds inhibitthe production of monocyte/macrophagederived angiogenic activity. Agents Actions. In press. Kulka, I.P., Blocking, D., Ropes, M.W., and Bauer, W. (1955). Early joint lesions of rheumatoid arthritis. Arch. Path. 59: 129-150. Leibovich, S.]. (1984). The role of macrophages, in: Hunt, T.K., Heppelston, R.B., Pines, E., and Rovee, D. (eds.) Soft and Hard Tissue Repair, pp 329351, New York: Praeger Press. Leibovich, S.J., Polverini, P.I., Shepard, H.M., W'tseman, E.M., Shively, V., and Nuseir, N. (1987). Macrophage-inducedangiogenesis, Nature 329: 630-632. Lunec ]., Halloran, S.P., White, A.G., and Dormandy, T.L. (1981). Free-radical oxidation (peroxidation) products in serum and synovial fluid in rheumatoid arthritis. J. Rheum. 8: 233-245. Mahadevan V., Hart, LR., and Lewis, G.P. (1989). Factors influencing blood supply in wound granuloma quantitiated by a new in vivo technique. Canc. Res. 49: 415-419. Menander-Huber K.B., and Huber, W. In Superoxide and Superoxide Dismutases (1977). Michelson, A.M., McCord, J.M., and Fridovich, I. (eds.) London: Academic Press. Meltzer, M. (1981). Methods for Studying Mononuclear Phagocytes, in: Adams, D.O., Edelson, P.J., and Koren, H. (eds.) New York: Academic Press. Mimbclfi, C.K., Sung, C.P., Picker, D.H., Bamard, C., Jud~, P., and Badger, A.M. 0988). Effects of metal containing compounds on superoxide release from phorbol myristate acetate stimulated murine peritoneal macrophages: inhibition by anranofin and spirogermanium. J. Rheum. 15: 1064-1069. Muus, P., Bonta, I.L., and Den Oodsten, LA., (1979). Plasma levels malonyldialdehyde, a product of cyclooxygenase dependent and independent lipid pcroxidation in rheumatoid arthritis: a correlation with disease activity. Prosatag. Med. 2: 63-65. Ohta, A., Louie, LS., and Uitto, J. (1986). Collagenase production by human mononuclear cells in culture: inhibition by gold containing compounds and other antirheumatic agents. Ann. Rheum. Dis. 45: 996-1003. Polverini, P.J., Cotran, R.S., Gimbrone, M.A. Jr., and Unanue, E.R. (1977). Activated macrophages induce neovascularization. Nature 269: 804-806. Polverini, P.L, and I_~'bovich, S.L (1984). Induction of neovascularization in vivo and endothelial cell proliferation in vitro by tumor associated macrophages. Lab. Invest. 51: 635-642.
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Polverini, P.J. (1989). In Cytokines, Vol. I., (C. Sorg, Ed.), Switzerland: S. Karger. Rastinejad, F., Polverini, P.J., and Bouck, N.P. (1989). Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56: 345-355. Thakral, K.K., Goodson, W.H., and Hunt, T.K. (1979). Stimulation of blood vessel growth by wound macrophages. J. Surg.Res. 26: 430-436. Varani, J., Frigid, S.E., Ttil, G.O., Kunkel, R.G., Ryan, U.S. and Ward, P.A. (1985). Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical. Lab. Invest. 53: 656-663. Wisenmn, D.M., Polverini, PJ., Kamp, D.W., and Leiimvich, S.J. (1988). Transforming growth factor-beta (TGF-B) is a chemoattractant for human monocytes and induces their expression of angiogenic activity. Biocbem. Biophys. Res.Commun. 157: 793-800. Paper received 04.12.91.
Revised paper accepted 22.02.92.
41 5
I N H m r r I O N OF P R O D U C T I O N O F MONOCYTE/MACROPHAGE-DERIVED ANGIOGENIC A C T I V H T BY O X Y G E N F R E E - R A D I C A L S C A V E N G E R S Alisa E. Koch -~., Michael Clio*, James C. Burrows', Peter I. Polverini#, and S. Joseph Leibovi~ + Departments of Medicine', Pathology*, and Basic Sciences* Northwestern University Medical and Dental Schools, and V.A. Lakeside Medical Center~To whom all correspondence should be addressed ABSTRACt We showed previously that thiol-containing compounds inhibited the production of macrophage-mediated angiogenic activity. Since thiol-containing compounds may act on macrophages by affecting activation and inhibiting the production of oxygen free-radicals, we studied the effects of oxygen free-radical scavengers on production of angiogenic activity by elicited mouse peritoneal macrophages and lipopolysaccharide stimulated normal human monocytes. Monocyte/macrophage ccmditioned media were potently angiogenic when assayed in rat corneas, while conditioned media from oxygen free-radical scavenger-treated cells were not. The inhibitory effect of oxygen free-radical scavengers was due to a direct effect on monocyte/macrophage production of angiogenic activity but was not due solely to a decrease in the production of the macrophage-derived angiogenic cytokine tumor necrosis factor-a. We conclude that oxygen free-radical scavengers are potent inhibitors of the production of macrophage-mediated angiogenic activity. INTRODUC~ON Angiogenesis, the growth and proliferation of new blood vessels, is an important feature of wound repair and tumor growth (Leibovich, 1984, Clark et al., 1976, Thakral et al., 1976, Polverini et al., 1977, Folkman, 1985, Kulka et al., 1955, Brown et al., 1980, Jackson et al., 1989, Polverini, and Leibovich, 1984, Koch et al., 1986a). Macrophages (mSs) mediate angiogenesis in many diverse processes such as neoplastic growth and rheumatoid arthritis (Leibovich, 1984, Koch et al., 1986, Koch et al., 1988). We have shown, using normal human monocytes (mos), that cellular activation with agents such as lipepolysaccharide (LPS) or transforming growth factor-l~ is required for mos to express the angiogenic phenotype (Koch et al., 1988, Wiseman, et al. 1988). We have shown previously that the gold containing anti-rheumatic compounds, gold sodium thiomalate and auranofin, which have the common property of containing a thiol moiety, are potent inhibitors of the production of m~b-derived angiogenic activity (MDAA)(KOChet al., 1988a). This inhibitory effect appears to be due to the thiol rather than the gold content of these compounds (Koch et al., 1991). These 0309-1651/92/050415-11/$03.00/0
© 1992 Academic Press Ltd
416
Cell Biology International Reports, VoL 16, No. 5, 1992
studies showed that glutathione and cysteine in their reduced but not oxidized forms exerted this inhibitory effect, suggesting that the reducing properties of the thiols might be involved. Since anti-rheumatic gold compounds, which are thiol containing, act as activated oxygen free-radical scavengers (FRSs) and inhibit superoxide release from mcks, we have examined whether FRSs might inhibit the production of angiogenic activity by mononuclear phagocytes (Mirabelli et al., 1988, Davis et al., 1983). We show that FRSs are potent inhibitors of the production of MDAA. METHODS
Premmffon ofm6 cond/t/oned medhun for em/uafon/n die rat cornea/b/oassay_ for ane/oeenes/s. Eight week old Balb/c mice were injected with Brewer's thioglycollate broth (Difco, Baltimore, MD), and their elicited peritoneal m~s were harvested four daysJater as previously described (Meltzer, 1981). M~bs prepared in this manner have been shown to be potently angiogenic (Polverini et al., 1977). M~s were plated at 1 x 106 cells/ml in RPMI plus 10% fetal calf serum (FCS) + gentamicin and allowed to adhere to 75 mm culture flasks or 100 mm tissue culture dishes (Costar, Cambridge, MA) for one hour at 37°C in an incubator gassed with 5 % CO2 and 95 % air. After adherence, the culture medium was replaced by an equal volume of RPMI1640 + 0.5% FCS + gentamicin with the following FRSs for 30 minutes: 1,1,3,3tetramethyl-2-thiourea, D-mannitol, catalase, or urea (Sigma, St. Louis, MO). The concentrations selected were based on those shown by Kobayashi, et al to inhibit maximally phorbol myristate acetate stimulated TNF-8 production by human T cell hybridomas (Kohayashi et al., 1984), or those shown to be required to reduce both oxygen free-radical production and endothelial cell killing by human neutrophils (Varani et al., 1985). After incubation with FRSs, m~s were exposed to the activating agent lipopolysaccharide (LPS)(5 ug/ml)(phenol-extracted preparation of E. Coli 055:]35, Sigma) and the mck conditioned medium harvested after 24 hours. Cell viability was determined using trypan blue exclusion. Concentrated (20x) conditioned media were assayed using the rat corneal bioassay for angiogenesis (see below). Premmtion o f human mo conditioned medium for evaluation in the rat corne__~ bioassay for aneioeene~, Human mos were isolated from huffy coats of normal volunteers by density gradient centrifugation using Sepracell-MN (Sepratech, Corp, Oklahoma City, OK). Mos isolated in this manner were >90% pure as estimated morphologically and by esterase staining (Sigma). Mos were further purified by adherence of 1 x l0 s cells/ml (RPMI plus 10% FCS plus gentamicin) to 100 mm tissue culture dishes for 2 hours at 37°C. The adherent cells were washed with PBS (xl). RPMI + 10% FCS and
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FRSs were added. After 1/2 hour, LPS (5 ug/ml) was added and the cells incubated overnight. The cells were washed with PBS (xl) and incubated for 48 hours with RPMI + 0.5% FCS + LPS (5 ug/ml) + FRSs, and the conditioned medium concentrated (20x) and assayed in the rat cornea. In some experiments, FRSs were added to previously conditioned mo/m~ media and the media assayed in rat corneas.
Rat cornea/b/oa~av for am,/oeenes/s. Concentrated mo/m~ conditioned media (5 ul) were incorporated into an equal volume of slow-release Hydron (Interferon Sciences, New Brunswick, NJ) and allowed to dry. Hydron pellets (10ul) were implanted,aseptic~y into a pocket within the rat corne~ stroma 2 mm from the limbal vasculature ~olverini and Leihovich, 1984, Koch et al., 1986, Koch et al., 1986a, Koch et al., 1991). Corneas were examined daily for seven days using a stereomicroscope and perfused with colloidal carbon at the end of the observation period to provide a permanent record of the angiogenic response (Polverini and Leibovich, 1984, Koch et al., 1986, Koch et al., 1986a, Koch et al., 1991). Corneas were examined histologically for any evidence of non-specific inflammation. Mea.mrement o f the effect.of F R ~ on ~eneral m~b orotein svnthe.~.
Assessment of protein synthesis was done using a modification of the method of Ohta (Ohta et al., 1986). After incubation with FRSs, duplicate cultures of m~s were incubated with leucine-free RPMI for one hour at 37°C. Fifty uCi/ml [3H]-leucine (Amersham, Arlington Heights, IL) were added to 5x106 cells for a further one hour incubation. M~s (5x106/2 ml RPMI) were subsequently lysed with lml 1M sodium hydroxide and the cell lysate added to 2ml 5 % trichloracetic acid. After heating at 75°C for 30 minutes, precipitation was allowed to proceed overnight at 4°C. The precipitates were pipetted in triplicate onto glass fiber filters, washed with 95% ethanol and counted in a scintillation counter (Beckman, h'vine, CA). Ayaav for tumor necrosis factor-a fiN-F-a) and imerleuldn-IB t/L-l) in mo mediu,
-
The effects of FRSs on mo production of the angiogenic cytokines IL-1 and TNT-~ were studied. Mos were isolated as described above and incubated with RPMI-1640 + 0.5% FCS + gentamicin + LPS (5 ug/ml) with or without FRSs. After incubation at 37°C for 24 hours, the mo supernatants were harvested and assayed for TNT-~, using an enzyme linked immunosorbent assay (ELISA) (Biosource International, Westlake Village, CA). This assay is specific for the measurement of biologically active TNF-a with a minimum sensitivity of 4 pg/ml, respectively. ~ 1B was measured using an ELISA (R&D Systems, Minneapolis, MN) which has a minimum sensitivity of 4.5 pg/ml.
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RESULTS
Figure 1 shows a colloidal carbon perfused corneal mount from a rat implanted with a Hydron pellet containing conditioned medium from LPS activated human mos. A positive response in this figure is indicated by the unidirectional sustained growth of capiHa~ sprouts and hah~in loops towards the implant. New capillaries grew at approximately 0.2 mm/day. Corneas examined histologically showed no evidence of nonspecific inflammation and exhibited some stromal thickening. Figure 2 shows a rat corneal mount indicating the effect of treating murine m~bs with 1,1,3,3,tetramethyl-2-thiourea, a FRS. The lack of an angiogenic response to conditioned medium from m~s treated with this compound is shown.
Figure 1. Colloidal carbon perfused corneal mount from a rat implanted with a Hydron pellet containing mo conditioned medium (top)(x33). Directional growth of capillary loops and sprouts toward the implant can be seen.
Figure 2, Colloidal carbon perfused rat corneal mount showing a cornea implanted with conditioned medium from mos treated with 1,1,3,3 tetramethyl-2-thiourea (top)(x33). No angiogenic response is seen.
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Table 1 shows the cumulative effects of FRSs on the production of mo/m4,-derived angiogenic activity. Conditioned medium from human mos or murine m4,s incubated with sham FRS (5 mM urea) exhibited positive angiogenic responses, as did conditioned media from mo/m~s incubated with urea, which is a weak FITS. In contrast, conditioned medium from mo/m~s treated with 1,1,3,3-tetramethyl-2thiourea did not induce angiogenic responses. Similarly, conditioned medium from mo/m~s preincubated with D-mannitol or catalase did not induce angiogenic responses. To determine whether the effect of the FRSs was due to a direct effect on the m~ or to effects on the MDAA or the corneal vasculature, FRSs were added to previously conditioned m~bconditioned medium. This conditioned medium elicited a positive corneal angiogenic response, indicating a direct effect of FRSs on the mo/m~ rather than on the corneal blood vessels or the MDAA (table 1). Control m~ uptake of [3H]-leucine was compared with that of FRS-treated m~bs. Protein synthesis was not appreciably altered by treatment of the m~s with FRSs. Results of ELISAs to detect the presence of ILol and TNF-c~ in human mo supernatants are shown in Figure 3. Treatment of mos with urea, a poor FRS, did not decrease TNF-c~ production. In contrast, treatment with 1,1,3,3,-tetramethyl-2thiourea or mannitol reduced TNF-ct production by 53% and 39%, respectively. Treatment of mos with FRSs, with the exception of urea, did not decrease IL-1 production. DISCUSSION
In mo/m~s, the process of ~ u l a r activation results in both early and late activation products (Hamilton and Adams, 1987). Early products include a series of oxygen free-radicals and hydrogen peroxide, produced within a few minutes of exposure of the cells to activating agents. Following this early stage, a series of gene induction events occurs, resulting in the later production of several cytokines, including TNFo~ and IL-1. The connection between the early stages of activation, with the production of activated oxygen free-radicals, and the later stages, involving cytoldne production, is not clear. The production of oxygen free-radicals is mediated by the leukocyte respiratory burst with activation of the hexose monophosphate shunt pathway, involving a series of oxidation-reduction (redox) reactions (Fantone and Ward, 1985). Reducing agents may interfere with this pathway by disrupting the cellular redox balance, thus inhibiting production of oxygen free-radices. We have shown previously that compounds containing free-thiols (reduced glutathione, cysteine) inhibit the production of MDAA, suggesting a possible connection between the production of early and late activation products (Koch et al., 1991). In this study, we examined whether products of early activation, namely oxygen free-radicals and hydrogen peroxide, might be involved in the generation of
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angiogenic activity, by using FRSs to inactivate these products and thus inhibit their 4000
A
30OO
i
20O0
1000
B
LNTRF_ATED
~
TMT-UFEA
D-MANN,
CATALASE
UNTFEATED
UFF.A
TMT-L.IgEA D-Iv~NN.
CATALASE
60o
o
4OO
i
3OO
o
T -
200
100
0
Figure 3. A) TNF-o~ levels in mo conditioned medium determined using an ELISA. Normal human peripheral blood mos were cultured either with or without: urea (SmM), 1,1,3,3-tetramethyl-2-thiourea (TMT-urea)(SmM), D-mannitol (Dmann.)(50mM), or catalase 0000 u/ml). Results of 3 experiments, each assayed in duplicate, are shown. Results expressed as the mean + S.E. B) IL-1 levels in mo conditioned medium from two donors prepared and assayed as .in "A" above. activity. In this study, we present data that suggests that FRSs can inhibit the production of MDAA in both human and murine mononuclear phagocytes. This effect appears to be directly on the too/m#, rather than on the secreted angiogenic factor(s) or the blood vessels of the target corneas. To determine if the inhibition of MDAA production was due to the inhibition of
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production of known angiogenic mediators, we measured the production of TNF-a by human mos incubated with FRS. Leibovich et al have shown that TNF-o~ is one of the major cytokines accounting for murine peritoneal m~-derived angiogenic activity (l.eibovich et al., 1987). IL-1 has also recently been shown to be angiogenic (Mahadevan, et al., 1989). While not all FRSs decrease the amount of TNF-o~ produced by LPS activated human mos, 1,1,3,3-tetramethyl-2-thiourea,in particular, decreased TNF-ot production about 53%, while urea, a weak FRS did not. Mo IL-1 production was not decreased significantly by FRSs. The change in TNF-ot alone may not be adequate to account for the dramatic decrease in MDAA. It seems possible that inhibition of production of additional eytokines may be involved in the inhibition of production of MDAA. Alternatively, FRSs may upregulate release of angiogenic inhibitors, such as thrombospondin (Rastinejad et al., 1989, Good et al., 1990). Increased levels of this inhibitory activity may be produced by rafts in response to FRSs resulting in a net loss of demonstrable angiogenic activity. Since the angiogenic phenotype in mos/m~s has been shown to be recessive, it is possible that activation of a suppressor element with subsequent upregulation of inhibitory activity rather than production of angiogenie mediators is related to oxygen freeradical production (Polverini, 1989). Oxygen free-radicals have been implicated in mediating tissue damage in a variety of pathological lesions, such as myocardial infarctions, adult respiratory distress syndrome, and rheumatoid arthritis (Fantone and Ward, 1985, Lunec et al., 1981, Muus and Oudsten, 1979). In rheumatoid arthritis, oxygen free-radicals have also been implicated since increased levels of conjugated dienes and malonyldialdehyde (products of lipid peroxidation reactions) are found in the synovial fluid and plasma of patients with active disease (Lunec et al., 1981, Muus and Oudsten, 1979). FRSs have been reported to be effective inhibitors of tissue injury in experimental models of arthritis--as well as in patients with rheumatoid arthritis (Menander-Huber and Huber, 1977, Hirschelmann and Bekemeier, 1981). In summary, in this study we have shown that FRSs can inhibit production of m~derived angiogenic activity and that this inhibition is not totally due to inhibition of production of the angiogenic cytokine TNF-ot. It is possible that naturally occurring FRSs might be involved in inhibiting the production of MDAA in vivo. ACKNOWL~:nG~
This work was supported by an Arthritis Foundation Fellowship and Grant (A.E.K.), a Veteran's Administration Merit Review (A.E.K.), N.I.H. grants AR30692 (A.E.K.), GM29135 (S.LL.), and HL39926 (P.J.P.). This work was presented in part at The American Federation for Clinical Research Meeting, Washington, D.C., 1989 and at the American College of Rheumatology Meeting, Cincinnati, OH, 1989. We wish to thank Drs. Richard Pope, Frank Schmid, and Arthur Veis for helpful discussions and Ms. Lisa Harlow for expert technical assistance.
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TABLE I
OF SCAVENGERS OF OXYGEN FREE RADICALS ON THE
PRODUCTION OF Mo/MACROPHAGE-DERIVED ANGIOGENIC ACTIVITY Test group
Numbers of corneas assayed Number of positive angiogenic responses
Number of negative angiogenic responses
5 6
0 0
I#
6
0 0
8 4
5 7
0 1
0
4
0 0
5 4
3
0
3
0
Conditioned medium from murine peritoneal m~,s preincubated with:
Control media Urea (5 m M ) (poor scavengerof oxygen freeradicals) i,1,3,3-tetramethyl-2-thiourea (5 mM)
D-mannitol (50 raM) Catalase(3000 u/ml) Conditioned medium from human mos incubated with:
Control media Urea (5mM) (poor scavengerof oxygen free radicals) 1,1,3,3-tetramethyl-thiourea (5mM) D-mannitol (50 mM) Catalase (3000 u/ml) Conditioned medium from human mos to which FRS had been added: 1,1,3,3-tetramethyl-thiourea
(SmM) catalase 0000 units/ml)
//Denotes 1 weakly positive angiogenic response.
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Polverini, P.J. (1989). In Cytokines, Vol. I., (C. Sorg, Ed.), Switzerland: S. Karger. Rastinejad, F., Polverini, P.J., and Bouck, N.P. (1989). Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56: 345-355. Thakral, K.K., Goodson, W.H., and Hunt, T.K. (1979). Stimulation of blood vessel growth by wound macrophages. J. Surg.Res. 26: 430-436. Varani, J., Frigid, S.E., Ttil, G.O., Kunkel, R.G., Ryan, U.S. and Ward, P.A. (1985). Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical. Lab. Invest. 53: 656-663. Wisenmn, D.M., Polverini, PJ., Kamp, D.W., and Leiimvich, S.J. (1988). Transforming growth factor-beta (TGF-B) is a chemoattractant for human monocytes and induces their expression of angiogenic activity. Biocbem. Biophys. Res.Commun. 157: 793-800. Paper received 04.12.91.
Revised paper accepted 22.02.92.
