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Leukotrienes C4 and D4 promote angiogenesis via a receptor-mediated interaction
ELSEVIER
ejp European Journal of Pharmacology 258 (1994) 151-154
Short communication
Leukotrienes C 4 and g 4 promote angiogenesis via a receptor-mediated interaction Nicos E. Tsopanoglou, Eva Pipili-Synetos, Michael E. Maragoudakis
*
Department of Pharmacology, Medical School, University of Patras, Patras 261 10, Greece (Received 18 November 1993; revised MS received 25 March 1994; accepted 29 March 1994)
Abstract
The involvement of leukotrienes in angiogcnesis was investigated in the in vivo chick chorioallantoic membrane system. In this system leukotrienes C 4 and D4 promoted angiogenesis in a dose-dependent manner. Leukotriene B 4 was ineffective. The potent and selective peptidyl leukotriene receptor antagonist, SK&F 104353-Z2, abolished the angiogenic effects of leukotrienes C 4 and D4 and reduced unstimulated angiogenesis. These results indicate that leukotrienes C 4 and D4 promote angiogenesis in the chick chorioallantoic membrane via a receptor-mediated interaction.
Key words: Angiogenesis; Leukotriene; Chorioallantoic membrane; (Chick)
1. Introduction
Angiogenesis is a multi-step process of great pathophysiological significance as it is associated with proliferative states such as tumour growth, psoriasis, arthritis, inflammation and wound healing (Maragoudakis, 1993). Leukotrienes, the products of the 5-1ipoxygenase pathway of the arachidonic acid metabolism (Lewis et al., 1990), have a profound effect on a number of biological processes with as prime example the immediate hypersensitivity reactions, such as allergic asthma. Moreover, several angiogenesis-dependent diseases including arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages (Samuelsson et al., 1987). Finally, leukotrienes a p p e a r to act as growth and differentiation factors for a number of cell types (Baud et al., 1985, 1987). In view of the above and since Kanayasu et al. (1989) reported that peptidyl leukotriene C 4 stimulated tube formation in an in vitro angiogenesis system,
* Corresponding author. Tel. 61-997-638, fax 61-997-691. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 195-D
it was of interest to evaluate further the possible involvement of leukotrienes in the angiogenic process. In the present study the effects of leukotrienes B4, Ca and D 4 were examined in an in vivo angiogenesis model, the chick chorioallantoic membrane. We report here that leukotrienes C 4 and D 4 (but n o t B 4) promoted angiogenesis in a potent and specific manner.
2. Materials and methods
Collagenase type VII from Clostridium histolyticum, cortisone acetate, leukotrienes C4, B 4 and D 4 w e r e obtained from Sigma Chem. Co. (Poole, UK). S K & F 104353-Z 2 was a kind gift from Dr. J. Gleason (SmithKline Beecham PA). [U-14C]L-proline (specific activity 273 m C i / m m o l ) was obtained from New England Nuclear (Boston, MA, USA). The plastic discs used were 13 m m round tissue culture coverslips from Nunc (Naperville, IL, USA). Fresh fertilized eggs were obtained locally (Ioannina, Greece) and kept at 10°C before incubation at 37°C. The modified in vivo chick chorioallantoic membrane angiogenesis model was used as previously described (Maragoudakis et al., 1988). Briefly, fresh fertilized eggs were incubated for 4 days at 37°C after which
152
N.E. Tsopanoglou et al. / European Journal of Pharmacology 258 (1994) 151-154
a window was opened in the egg shell to expose the chick chorioallantoic membrane. The window was then covered with tape and the eggs were returned to the incubator until day 9, when the test materials were applied. The test materials or vehicle and 0.5 /xCi U-14C-labelled proline were placed on sterile plastic discs and were allowed to dry under sterile conditions. The control discs (containing vehicle and radiolabelled proline) were placed on the chick chorioallantoic membrane 1 cm away from the disc containing the test material. A sterile solution of cortisone acetate (100 /.tg/disc) was routinely incorporated on all discs in order to prevent an inflammatory response. The loaded and dried discs were inverted and placed on the chick chorioallantoic membrane, the windows were covered and the eggs were incubated until day 11, when assessment of angiogenesis took place. Biochemical evaluation of newly formed vessels was performed by determining the extent of collagenous protein biosynthesis in the chick chorioallantoic membrane lying directly under the discs (Maragoudakis et al., 1988). Briefly, the area under the disc was cut out, placed in an appropiate buffer and protein biosynthesis was stopped. Non-protein-bound radioactivity was removed by washing with trichloroacetic acid. Discs containing radioactivity were resuspended and subjected to collagenase digestion. The resulting radiolabelled tripeptides, corresponding to basement membrane collagen and other collagenous material synthesized by the chick chorioallantoic m e m b r a n e from [U-14C]L-proline, were counted and expressed as c p m / m g protein. For morphological evaluation, the eggs were treated as above in the absence of radiolabelled proline. On day 11 the eggs were flooded with 10% buffered formalin and the plastic discs were removed. A large area around the discs was cut out and placed on a glass slide and the vascular density index (expressed as number of blood vessels) was measured by the method of HarrisHooker et al. (1983).
2.1. Calculation and statistics For each egg, collagenous protein biosynthesis under the disc containing the test material or vehicle was calculated as c p m / m g protein. Collagenous protein biosynthesis, or the number of vessels, under the disc containing the test material was then expressed as percentage of that under the control disc. The results were analyzed by paired Student's t-test.
3. Results
Leukotrienes C 4 and D 4 caused a dose-dependent increase in collagenous protein biosynthesis in the chick chorioallantoic membrane, at doses ranging from
n.8 n.8
100"
*H ,el
00"
n#32
o g
n#O
"6
m n ¢J
""
60 a#8
""
I'i'i:i:i:i:
40 ?~,~,~,~,~. 20
:,~,~,~,~,~. ] - , - , , ,-,
0.001
0.01
1
100
1000
10000
LTC4 (pg/dlsc)
rim0
10(]
80
m
O.
20
0 0.001
0.01
1
100
1000
10000
LTD4 (pg/disc)
Fig. 1. Effect of leukotriene C 4 (a) and leukotriene D 4 (b) on collagenous protein biosynthesis (CPB) in the chick chorioallantoic membrane. The results are expressed as mean_+ S.E. % changes in CPB compared to that of controls. Collagenous protein biosynthesis for controls was 12600+ 1600 c p m / m g protein, n = 70. Statistical significance: * P < 0.05, ** P < 0.02 and *** P < 0.01. Experimental details were as described in the text.
0.001-10000 p g / d i s c (Fig. la and b). The maximal increase for both leukotrienes was about 80%. It can be seen that the dose-response relationships of leukotrienes C 4 and D 4 were nearly identical, within the limits of the respective standard errors. Morphological evaluation of the area under the discs containing 100 pg of leukotriene C 4 or leukotriene D 4 showed a 32_+6.0% ( n = 8 ) and a 3 6 + 8 % (n = 7) ( P < 0.01) increase in vascular density respectively. Leukotriene B 4 on the other hand (examined within the same dose range) had no effect on either collagenous protein
N.E. Tsopanoglou et al. / European Journal of Pharmacology 258 (1994) 151-154
153
4. Discussion
100" 9080-
ns32 e,o
n.32 t,,
70o
60-
8 "6
SO"
40" m o. (J
30"
n.8 n$
20. 10" 0 LTC4
LTD4
LTC4 + SKF1043S$-Z2
LTD4 +
SKF104353-Z2
Fig. 2. Effect of SK&F 104353-Z2 on collagenous protein biosynthesis (CPB) in the chick chorioallantoic membrane. The results are expressed as mean+S.E. % change in CPB compared to that of controls. Collagenous protein biosynthesis for controls was 16400+ 1600 cpm/mg protein, n = 21. Statistical significance: * P < 0.05 and • ** P < 0.01. Experimental details were as described in the text.
biosynthesis (10 + 7.0-14 + 8.0% increase compared to controls, n = 5-8), or vascular density (4 + 6.0% increase compared to controls, n = 5 - 8 ) in the chick chorioallantoic membrane. The peptidyl leukotriene receptor antagonist, SK& F 104353-Z 2 (Hay et al., 1987), when examined alone at 1 0 / z g / d i s c , caused a small but significant drop in both collagenous protein biosynthesis (18 + 6.0% compared to controls, n = 7, P < 0.02) and vascular density (11 + 4.0% compared to controls, n = 6, P < 0.05). When S K & F 104353-Z 2 (10 /zg/disc) was combined with either leukotriene C 4 (100 p g / d i s c ) or leukotriene D 4 (100 pg/disc), the increase in collagenous protein biosynthesis caused by both compounds was greatly reduced (Fig. 2). It can be seen that the reduction of the angiogenic effect of leukotriene D 4 (to 3 + 6.0% compared to controls, n = 8) was more pronounced than that of leukotriene C a. In the presence of S K & F 104353-Z2, the remaining angiogenic effect of leukotriene C 4, although small (14 + 5.0% compared to controls, n = 8), was statistically significant ( P < 0.05). Similar results were obtained when the effect of the combination of the two leukotrienes with S K & F 104353-Z 2 ( 1 0 / z g / d i s c ) was examined on vascular density. In the presence of the antagonist, leukotriene C a caused a 17 + 4.0% (n = 6) increase in vascular density whereas the angiogenic effect of leukotriene D 4 was abolished (10 + 6.0% decrease compared to controls, n = 6).
In the present study it was shown that the peptidyl leukotrienes C 4 and D 4 promote angiogenesis in the chick chorioallantoic m e m b r a n e in a dose-dependent manner. In the same system leukotriene B 4 had no effect. The angiogenic effect of both leukotrienes was greatly reduced by the peptidyl lcukotriene receptor antagonist S K & F 104353-Z 2, which selectively inhibits leukotriene D 4 and E 4 receptor-mediated interactions (Hay et al., 1987). The fact that the effect of leukotriene C 4 was also reduced by the same antagonist suggests that in the chick chorioallantoic membrane, as in other biological systems, this leukotriene is rapidly converted to ieukotriene D 4 (Drazen and Austen, 1987). This is supported by the fact that the dose-response relationships for leukotrienes C 4 and 9 4 w e r e remarkably similar. Although conversion to leukotriene D 4 may account for most of the angiogenic activity of leukotrienc C 4, the possibility that a small part of the response was due to an interaction between leukotriene C 4 with a separate set of receptors cannot be excluded. Indeed, in the presence of S K & F 104353-Z z, some of the leukotriene C4-induced angiogenesis persisted while that of leukotriene D 4 was completely abolished. Many mechanisms could underlie the angiogenic effect of the two leukotrienes. Kanayasu et al. (1989) suggested that the angiogenic effect of leukotriene C4 in an in vitro system for evaluating angiogenesis, might be related to stimulation of cell migration. These authors, however, could not demonstrate the same effect for leukotriene D 4. It is therefore likely that the angiogenic activity of the latter requires the presence of additional cellular or extracellular components absent from in vitro tube formation systems. An obvious missing component in the in vitro system is the blood platelet. Both leukotrienes C 4 and D 4 are potent platelet activators (Crooke et al., 1987). The question then arises as to how platelets could bc involved in the promotion of the angiogenic process. Activated platelets may adhere to the vascular endothelium, increase cell permeability and initiate proliferative phenomena through the release of growth factors (Page, 1988) which are well characterized angiogenic molecules (Folkman and Shing, 1992). In conclusion, the peptidyl leukotrienes C 4 and D 4 potently and specifically promote angiogenesis in the chick chorioallantoic membrane. This process is of physiological significance since antagonizing the binding of the endogenous leukotrienes with their receptors resulted in a moderate but significant inhibition of unstimulated angiogenesis. Leukotrienes C 4 and 9 4 may therefore be added to the list of endogenous mediators involved in the angiogenic cascade.
154
N.E. Tsopanoglou et al. / European Journal of Pharmacology 258 (1994) 151-154
Acknowledgements This study was supported by grants from the Ministry of Science and Technology and the National Drug Organization (E.O.F.).
References Baud, L., J. Sraer, J. Perez, M.P. Nivez and R. Ardaillou, 1985, Leukotriene C4 binds to human glomerular epithelial cells and promotes proliferation in vitro, J. Clin. Invest. 76, 374. Baud, L., J. Perez, M. Dennis and R. Ardaillou, 1987, Modulation of fibroplast proliferation by sulfidopeptide leukotrienes: effect of indomethacin, J. Immunol. 138, 1190. Drazen, J.M. and K.F. Austen, 1987, Leukotrienes and airway responses, Am. Rev. Respir. Dis. 136, 985. Folkman, J. and Y. Shing, 1992, Angiogenesis. A minireview, J. Biol. Chem. 267(16), 10931. Harris-Hooker, S.A., C.M. Gajdusec, T.N. Wight and S.M. Schwartz, 1983, Neovascular response induced by cultured aortic endothelial cells, J. Cell. Physiol. 114, 302. Hay, D.W.P., M.R. Muccitelli, S.S. Tucker, M.L. Vickery-Clark, K.A.
Wilson, J.G. Gleason, R.F. Hall, M.A. Wasserman and T.J. Torphy, 1987, Pharmacologic profile of SK&F 104353: a novel, potent and selective peptidoleukotriene receptor antagonist in guinea pig and human airways, J. Pharmacol. Exp. Ther. 243(2), 474. Kanayasu, T., J. Nakao-Hayashi, N. Asuva, I. Morita, T.M. lshii, H. Ito and S. Murota, 1989, Leukotriene C 4 stimulates angiogensis in bovine carotid artery endothelial cells in vitro, Biochem. Biophys. Res. Commun. 159(2), 572. Lewis, R.A., K.F. Austen and R.J. Soberman, 1990, Leukotrienes and other products of the 5-1ipoxygenase pathway: biochemistry and relation to pathobiology in human disease, New Engl. J. Med. 323(10), 645. Maragoudakis, M.E., 1993, Angiogenesis, in: Annual of Cardiac Surgery, eds. M. Yakoub and J. Pepper (Current Science, London) 19. 13. Maragoudakis, M.E., M. Sarmonika and M. Panoutsakopoulou, 1988, Rate of basement membrane bio~nthesis as an index to anglogenesis, Tissue Cell 20, 531. Page, C.P., 1988, The involvement of platelets in non-thrombotic processes, Trends Pharmacol. Sci. 9, 66. Samuelsson, B., S.-E. Dahlen, J.A. Lindgren, C.A. Rouzel and C.N. Sehran, 1987, Leukotrienes and lipoxins: structure, biosynthesis and biological effects, Science 237, 1171.
ejp European Journal of Pharmacology 258 (1994) 151-154
Short communication
Leukotrienes C 4 and g 4 promote angiogenesis via a receptor-mediated interaction Nicos E. Tsopanoglou, Eva Pipili-Synetos, Michael E. Maragoudakis
*
Department of Pharmacology, Medical School, University of Patras, Patras 261 10, Greece (Received 18 November 1993; revised MS received 25 March 1994; accepted 29 March 1994)
Abstract
The involvement of leukotrienes in angiogcnesis was investigated in the in vivo chick chorioallantoic membrane system. In this system leukotrienes C 4 and D4 promoted angiogenesis in a dose-dependent manner. Leukotriene B 4 was ineffective. The potent and selective peptidyl leukotriene receptor antagonist, SK&F 104353-Z2, abolished the angiogenic effects of leukotrienes C 4 and D4 and reduced unstimulated angiogenesis. These results indicate that leukotrienes C 4 and D4 promote angiogenesis in the chick chorioallantoic membrane via a receptor-mediated interaction.
Key words: Angiogenesis; Leukotriene; Chorioallantoic membrane; (Chick)
1. Introduction
Angiogenesis is a multi-step process of great pathophysiological significance as it is associated with proliferative states such as tumour growth, psoriasis, arthritis, inflammation and wound healing (Maragoudakis, 1993). Leukotrienes, the products of the 5-1ipoxygenase pathway of the arachidonic acid metabolism (Lewis et al., 1990), have a profound effect on a number of biological processes with as prime example the immediate hypersensitivity reactions, such as allergic asthma. Moreover, several angiogenesis-dependent diseases including arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages (Samuelsson et al., 1987). Finally, leukotrienes a p p e a r to act as growth and differentiation factors for a number of cell types (Baud et al., 1985, 1987). In view of the above and since Kanayasu et al. (1989) reported that peptidyl leukotriene C 4 stimulated tube formation in an in vitro angiogenesis system,
* Corresponding author. Tel. 61-997-638, fax 61-997-691. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 195-D
it was of interest to evaluate further the possible involvement of leukotrienes in the angiogenic process. In the present study the effects of leukotrienes B4, Ca and D 4 were examined in an in vivo angiogenesis model, the chick chorioallantoic membrane. We report here that leukotrienes C 4 and D 4 (but n o t B 4) promoted angiogenesis in a potent and specific manner.
2. Materials and methods
Collagenase type VII from Clostridium histolyticum, cortisone acetate, leukotrienes C4, B 4 and D 4 w e r e obtained from Sigma Chem. Co. (Poole, UK). S K & F 104353-Z 2 was a kind gift from Dr. J. Gleason (SmithKline Beecham PA). [U-14C]L-proline (specific activity 273 m C i / m m o l ) was obtained from New England Nuclear (Boston, MA, USA). The plastic discs used were 13 m m round tissue culture coverslips from Nunc (Naperville, IL, USA). Fresh fertilized eggs were obtained locally (Ioannina, Greece) and kept at 10°C before incubation at 37°C. The modified in vivo chick chorioallantoic membrane angiogenesis model was used as previously described (Maragoudakis et al., 1988). Briefly, fresh fertilized eggs were incubated for 4 days at 37°C after which
152
N.E. Tsopanoglou et al. / European Journal of Pharmacology 258 (1994) 151-154
a window was opened in the egg shell to expose the chick chorioallantoic membrane. The window was then covered with tape and the eggs were returned to the incubator until day 9, when the test materials were applied. The test materials or vehicle and 0.5 /xCi U-14C-labelled proline were placed on sterile plastic discs and were allowed to dry under sterile conditions. The control discs (containing vehicle and radiolabelled proline) were placed on the chick chorioallantoic membrane 1 cm away from the disc containing the test material. A sterile solution of cortisone acetate (100 /.tg/disc) was routinely incorporated on all discs in order to prevent an inflammatory response. The loaded and dried discs were inverted and placed on the chick chorioallantoic membrane, the windows were covered and the eggs were incubated until day 11, when assessment of angiogenesis took place. Biochemical evaluation of newly formed vessels was performed by determining the extent of collagenous protein biosynthesis in the chick chorioallantoic membrane lying directly under the discs (Maragoudakis et al., 1988). Briefly, the area under the disc was cut out, placed in an appropiate buffer and protein biosynthesis was stopped. Non-protein-bound radioactivity was removed by washing with trichloroacetic acid. Discs containing radioactivity were resuspended and subjected to collagenase digestion. The resulting radiolabelled tripeptides, corresponding to basement membrane collagen and other collagenous material synthesized by the chick chorioallantoic m e m b r a n e from [U-14C]L-proline, were counted and expressed as c p m / m g protein. For morphological evaluation, the eggs were treated as above in the absence of radiolabelled proline. On day 11 the eggs were flooded with 10% buffered formalin and the plastic discs were removed. A large area around the discs was cut out and placed on a glass slide and the vascular density index (expressed as number of blood vessels) was measured by the method of HarrisHooker et al. (1983).
2.1. Calculation and statistics For each egg, collagenous protein biosynthesis under the disc containing the test material or vehicle was calculated as c p m / m g protein. Collagenous protein biosynthesis, or the number of vessels, under the disc containing the test material was then expressed as percentage of that under the control disc. The results were analyzed by paired Student's t-test.
3. Results
Leukotrienes C 4 and D 4 caused a dose-dependent increase in collagenous protein biosynthesis in the chick chorioallantoic membrane, at doses ranging from
n.8 n.8
100"
*H ,el
00"
n#32
o g
n#O
"6
m n ¢J
""
60 a#8
""
I'i'i:i:i:i:
40 ?~,~,~,~,~. 20
:,~,~,~,~,~. ] - , - , , ,-,
0.001
0.01
1
100
1000
10000
LTC4 (pg/dlsc)
rim0
10(]
80
m
O.
20
0 0.001
0.01
1
100
1000
10000
LTD4 (pg/disc)
Fig. 1. Effect of leukotriene C 4 (a) and leukotriene D 4 (b) on collagenous protein biosynthesis (CPB) in the chick chorioallantoic membrane. The results are expressed as mean_+ S.E. % changes in CPB compared to that of controls. Collagenous protein biosynthesis for controls was 12600+ 1600 c p m / m g protein, n = 70. Statistical significance: * P < 0.05, ** P < 0.02 and *** P < 0.01. Experimental details were as described in the text.
0.001-10000 p g / d i s c (Fig. la and b). The maximal increase for both leukotrienes was about 80%. It can be seen that the dose-response relationships of leukotrienes C 4 and D 4 were nearly identical, within the limits of the respective standard errors. Morphological evaluation of the area under the discs containing 100 pg of leukotriene C 4 or leukotriene D 4 showed a 32_+6.0% ( n = 8 ) and a 3 6 + 8 % (n = 7) ( P < 0.01) increase in vascular density respectively. Leukotriene B 4 on the other hand (examined within the same dose range) had no effect on either collagenous protein
N.E. Tsopanoglou et al. / European Journal of Pharmacology 258 (1994) 151-154
153
4. Discussion
100" 9080-
ns32 e,o
n.32 t,,
70o
60-
8 "6
SO"
40" m o. (J
30"
n.8 n$
20. 10" 0 LTC4
LTD4
LTC4 + SKF1043S$-Z2
LTD4 +
SKF104353-Z2
Fig. 2. Effect of SK&F 104353-Z2 on collagenous protein biosynthesis (CPB) in the chick chorioallantoic membrane. The results are expressed as mean+S.E. % change in CPB compared to that of controls. Collagenous protein biosynthesis for controls was 16400+ 1600 cpm/mg protein, n = 21. Statistical significance: * P < 0.05 and • ** P < 0.01. Experimental details were as described in the text.
biosynthesis (10 + 7.0-14 + 8.0% increase compared to controls, n = 5-8), or vascular density (4 + 6.0% increase compared to controls, n = 5 - 8 ) in the chick chorioallantoic membrane. The peptidyl leukotriene receptor antagonist, SK& F 104353-Z 2 (Hay et al., 1987), when examined alone at 1 0 / z g / d i s c , caused a small but significant drop in both collagenous protein biosynthesis (18 + 6.0% compared to controls, n = 7, P < 0.02) and vascular density (11 + 4.0% compared to controls, n = 6, P < 0.05). When S K & F 104353-Z 2 (10 /zg/disc) was combined with either leukotriene C 4 (100 p g / d i s c ) or leukotriene D 4 (100 pg/disc), the increase in collagenous protein biosynthesis caused by both compounds was greatly reduced (Fig. 2). It can be seen that the reduction of the angiogenic effect of leukotriene D 4 (to 3 + 6.0% compared to controls, n = 8) was more pronounced than that of leukotriene C a. In the presence of S K & F 104353-Z2, the remaining angiogenic effect of leukotriene C 4, although small (14 + 5.0% compared to controls, n = 8), was statistically significant ( P < 0.05). Similar results were obtained when the effect of the combination of the two leukotrienes with S K & F 104353-Z 2 ( 1 0 / z g / d i s c ) was examined on vascular density. In the presence of the antagonist, leukotriene C a caused a 17 + 4.0% (n = 6) increase in vascular density whereas the angiogenic effect of leukotriene D 4 was abolished (10 + 6.0% decrease compared to controls, n = 6).
In the present study it was shown that the peptidyl leukotrienes C 4 and D 4 promote angiogenesis in the chick chorioallantoic m e m b r a n e in a dose-dependent manner. In the same system leukotriene B 4 had no effect. The angiogenic effect of both leukotrienes was greatly reduced by the peptidyl lcukotriene receptor antagonist S K & F 104353-Z 2, which selectively inhibits leukotriene D 4 and E 4 receptor-mediated interactions (Hay et al., 1987). The fact that the effect of leukotriene C 4 was also reduced by the same antagonist suggests that in the chick chorioallantoic membrane, as in other biological systems, this leukotriene is rapidly converted to ieukotriene D 4 (Drazen and Austen, 1987). This is supported by the fact that the dose-response relationships for leukotrienes C 4 and 9 4 w e r e remarkably similar. Although conversion to leukotriene D 4 may account for most of the angiogenic activity of leukotrienc C 4, the possibility that a small part of the response was due to an interaction between leukotriene C 4 with a separate set of receptors cannot be excluded. Indeed, in the presence of S K & F 104353-Z z, some of the leukotriene C4-induced angiogenesis persisted while that of leukotriene D 4 was completely abolished. Many mechanisms could underlie the angiogenic effect of the two leukotrienes. Kanayasu et al. (1989) suggested that the angiogenic effect of leukotriene C4 in an in vitro system for evaluating angiogenesis, might be related to stimulation of cell migration. These authors, however, could not demonstrate the same effect for leukotriene D 4. It is therefore likely that the angiogenic activity of the latter requires the presence of additional cellular or extracellular components absent from in vitro tube formation systems. An obvious missing component in the in vitro system is the blood platelet. Both leukotrienes C 4 and D 4 are potent platelet activators (Crooke et al., 1987). The question then arises as to how platelets could bc involved in the promotion of the angiogenic process. Activated platelets may adhere to the vascular endothelium, increase cell permeability and initiate proliferative phenomena through the release of growth factors (Page, 1988) which are well characterized angiogenic molecules (Folkman and Shing, 1992). In conclusion, the peptidyl leukotrienes C 4 and D 4 potently and specifically promote angiogenesis in the chick chorioallantoic membrane. This process is of physiological significance since antagonizing the binding of the endogenous leukotrienes with their receptors resulted in a moderate but significant inhibition of unstimulated angiogenesis. Leukotrienes C 4 and 9 4 may therefore be added to the list of endogenous mediators involved in the angiogenic cascade.
154
N.E. Tsopanoglou et al. / European Journal of Pharmacology 258 (1994) 151-154
Acknowledgements This study was supported by grants from the Ministry of Science and Technology and the National Drug Organization (E.O.F.).
References Baud, L., J. Sraer, J. Perez, M.P. Nivez and R. Ardaillou, 1985, Leukotriene C4 binds to human glomerular epithelial cells and promotes proliferation in vitro, J. Clin. Invest. 76, 374. Baud, L., J. Perez, M. Dennis and R. Ardaillou, 1987, Modulation of fibroplast proliferation by sulfidopeptide leukotrienes: effect of indomethacin, J. Immunol. 138, 1190. Drazen, J.M. and K.F. Austen, 1987, Leukotrienes and airway responses, Am. Rev. Respir. Dis. 136, 985. Folkman, J. and Y. Shing, 1992, Angiogenesis. A minireview, J. Biol. Chem. 267(16), 10931. Harris-Hooker, S.A., C.M. Gajdusec, T.N. Wight and S.M. Schwartz, 1983, Neovascular response induced by cultured aortic endothelial cells, J. Cell. Physiol. 114, 302. Hay, D.W.P., M.R. Muccitelli, S.S. Tucker, M.L. Vickery-Clark, K.A.
Wilson, J.G. Gleason, R.F. Hall, M.A. Wasserman and T.J. Torphy, 1987, Pharmacologic profile of SK&F 104353: a novel, potent and selective peptidoleukotriene receptor antagonist in guinea pig and human airways, J. Pharmacol. Exp. Ther. 243(2), 474. Kanayasu, T., J. Nakao-Hayashi, N. Asuva, I. Morita, T.M. lshii, H. Ito and S. Murota, 1989, Leukotriene C 4 stimulates angiogensis in bovine carotid artery endothelial cells in vitro, Biochem. Biophys. Res. Commun. 159(2), 572. Lewis, R.A., K.F. Austen and R.J. Soberman, 1990, Leukotrienes and other products of the 5-1ipoxygenase pathway: biochemistry and relation to pathobiology in human disease, New Engl. J. Med. 323(10), 645. Maragoudakis, M.E., 1993, Angiogenesis, in: Annual of Cardiac Surgery, eds. M. Yakoub and J. Pepper (Current Science, London) 19. 13. Maragoudakis, M.E., M. Sarmonika and M. Panoutsakopoulou, 1988, Rate of basement membrane bio~nthesis as an index to anglogenesis, Tissue Cell 20, 531. Page, C.P., 1988, The involvement of platelets in non-thrombotic processes, Trends Pharmacol. Sci. 9, 66. Samuelsson, B., S.-E. Dahlen, J.A. Lindgren, C.A. Rouzel and C.N. Sehran, 1987, Leukotrienes and lipoxins: structure, biosynthesis and biological effects, Science 237, 1171.