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Angiogenesis: Modulation with opioids
Gen. Pharmac.Vol. 22, No. 6, pp. 1077-1079, 1991 Printed in Great Britain. All fights reserved
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPress pie
ANGIOGENESIS: MODULATION WITH OPIOIDS AURELIO PASl) BAOXI Q u ) RUDOLF STEINER,2 HANS-JtRG SENN,2 WALTER BgR ~ and FATHY S. MESSlHA3 t Endorphin Laboratories, Institute of Forensic Medicine, University of Zurich, CH-8028 Zurich, 2Division of Oncology, Department of Medicine C, Cantonal Hospital, St GaUen, Switzerland and 3Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX 79430, U.S.A. (Received 5 April 1991) Abstract--1. The effect of//-endorphin (~-EP) and morphine sulfate (MS), in presence and absence of naloxone (NX), on chicken chorioallantoic membrane was studied as a function of blood vessel proliferation. 2. A 50% reduction in blood vessel proliferation occurred by 10 pg of ~-EP or by 5 pg of MS per egg compared to controls. 3. An individual dose, i.e. 5 #g of//-EP, did not significantly inhibit blood vessel counts after initial 24 hr period of the drug application when given alone compared to inhibition occurring with combined use of NX. 4. NX (1/tg) did not significantly reverse the angiostatic effects of MS (10pg) or of/~-EP (5 pg). 5. The observed modulation of angiogenesis by opioids suggests involvement of/I-EP and MS in the proliferation of vascular endothelial cells. 6. This may be due to an effect of ~-EP and MS on cell-mediated immunity factors such as intefferons, interleukins and prostaglandin E2.
INTRODUCTION Opioids and their receptors have been found in a wide variety of neural and non-neural h u m a n and animal tumors (Bostwick et aL, 1987; Zagon et al., 1987; Scopsi et al., 1989). Furthermore, both endogenous and exogenous opioids, such as enkephalins and heroin, inhibited murine tumor growth (Zagon and McLaughlin, 1981; Murgo, 1985; Srisuchart et al., 1989) and prolonged survival of mice bearing Lt210 leukemia (Plotnikoff and Miller, 1983). Because the antitumorigenic effects of opioids could be reversed by naloxone (an opioid antagonist), an involvement of opioid receptors in such effects was suggested (Zagon and McLaughlin, 1981; Murgo, 1985; Faith and Murgo, 1988). The mechanisms underlying the opioid control of tumorigenesis are unclear. One proposed mode of action considers inhibitory processes of the opioids on D N A synthesis and mitosis (Zagon and McLaughlin, 1989a, b). In the present study, the effect of two potent opioids,//-EP and MS was studied on angiogenesis since tumoral growth depends on neovascularization. The chorioallantoic membrane (CAM) of the chicken embryo was the model used. MATERIALS AND METHODS Eggs Fertilized chicken eggs (Hy-lineTM, Ruedi Enzler, Switzerland) were processed using a modified form of the protocol of Barnhill and Ryan (1983). The eggs were incubated (37°C, 70% humidity) in a Covatutto 120TM incubator (M/irki, Switzerland). They were turned twice daily for the first two incubation days (ID). On the third ID, the eggs were disinfected with peracetic acid, and 2 ml of albumen were aspirated from their narrow pole with a syringe, creating an artificial air sac underlying the narrow egg
pole. To display the CAM, a window (2 cm dia) was created on ID 4 in the eggshell, over the air sac, and resealed with adhesive tape. Then, the eggs were reincubated for further nine days, short interruptions occurring at ID 7 for CAM treatment respectively daily for either vessel counting or routine observations. Agents They were applied, dissolved in 4pl of solvent, at ID 7, to a disc-shaped filter consisting of glass microfibres (3 mm dia; Whatman, England), and the disc was located on the CAM of each one of a series of five eggs. Treatment: fl-EP (Bachem, Switzerland): I, 5, 10 and 15pg (dissolved in phosphate buffered saline, PBS, pH 7.4); MS (Streuli, Switzerland): 1, 5, 10 and 50 pg (dissolved in normal saline, NS); naloxone (NX, Du Pont, Fed. Rep. Germany): alone (1 and 2 pg) and in combination (1/~g) with p-EP (5 pg) and MS (10 #g) (see Table 1). Negative controls were performed with the solvents alone. All procedures were performed under aseptic conditions. Vessel counting CAM vessels were counted in a 0.5 mm wide anulus (BarnhiU and Ryan, 1983), once daffy during the first 4 days after disc application, using a Wild M5 stereomieroscope (Leica, Switzerland) at ten-fold magnification. Statistics Vessel counts of treated and control groups were compared with the Student's t-test. The vessel counts reduction rate (RR), induced by the agents tested, was calculated with the following formula: RR = (C= -- Ct):Cc, where Cc and C~ stand for the mean vessel counts recorded in the control and the treated group. RESULTS The results are indicated in Table 1. ~ - E P caused, in comparison with control (PBS), between 24 and
1077
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AURELIOPASIet al. Table l. Opioidal effects on vesselcounts Vessel counts Dosage Agents (gg) 24 hr (%) 48 hr (%) 72 hr (%) 96 hr (%) PBS -20+3 27+3 3t+3 40__.4 BEP 1 17+2(15) 21 +3(22) 22+5(29) 33+4(18) BEP 5 21+3(-5) 19_+4"(30) 19_+5"*(39) 30_+5(25) BEP 10 16 _.+2 (20) 15 _+4** (43) 16 _+4** (48) 20 _+2** (50) BEP 15 16 _+2 (20) 15 _+3** (43) 16 _+3** (48) 20 _+3** (50) NX 1 22_+3(-10) 28_+4(-4) 38+5(-23) 41_+6(-3) NX 2 22_+4(-10) 23_+3(5) 29_+2(7) 40+4(0) BEP+ NX 5+1 12_+3'*(40) 21_+5(22) 20_+6*(36) 24_+3**(40) NS -21 _+3 26+3 32_+2 35_+2 MS 5 18_+4(14) 17+4"(35) 15_+3"*(53) 19+4"*(46) MS 10 19+3(10) 23_+5(12) 24_+5*(25) 26+5*(26) MS 50 20 _+2 (5) 24 + 4 (8) 26 _+3* (19) 27 _+4* (30) MS+NX 10+1 21_+3(0) 2 0 _ + 4 ( 2 3 ) 23_+3*(28) 26_+3*(26) CAM vesselcounts (performedat 24, 48, 72 and 96 hr after treatment)are expressedas the means (and their standard deviations) of counts done in 5 eggs. Figures in parentheses indicate reduction rates of vessel counts. Abbreviations:BEP, fl-endorphin;MS, morphine sulfate; NS, normal saline;NX, naloxone;PBS, phosphatebufferedsaline.*, ** significantlydifferent from control (*P < 0.05/**P < 0.01).
48 hr after doses of 5, 10 and 15 but not of 1 #g, significant reduction of vessel counts, i.e. significant angiostatic effects (P < 0.05 with 5 pg, P < 0.01 with either 10 or 15/~g). The corresponding vessel counts reduction rates ranged from 30 to 43%. The vessel counts were still significantly reduced (P < 0.01) in the groups with the 5, 10, and 15 g g doses 72 hr after treatment and in the group with the 10 and 15 pg doses also at the 96 hr check. MS also reduced vessel counts. A significant effect (RR: 35%, P < 0.05) was again observed between 24 and 48 hr after treatment by the 5 #g dose. This effect was even more pronounced 72 and 96 hr after treatment (RR: 53% and 46%, P < 0.01). Paradoxically, the effect of higher doses (10 and 50pg) became manifest later (72 hr after treatment), being less apparent (RR: 19-25%, P < 0.05) than with the low dose (5/~g) (RR: 53%), and it was still present 96 hr after treatment. After naloxone alone (1 and 2 #g) vessel counts were not significanty different than after PBS. Naloxone (1 #g), jointly applied with/3-EP (5/~g) potentiated significantly the angiostatic action of 13-EP 24 hr after treatment but not of MS (10#g). With the combination of N X with /3-EP and with MS, the angiostatic effect reached significancy at 7 2 h r (P < 0 . 0 5 ) and at 9 6 h r (P <0.01 with /3-EP and P < 0.05 with MS) but not at 48 hr.
angiostatic effects of MS recalls the one that this opioid had on the mitogenic activity of blood lymphocytes: the proliferation of these cells was promoted by low and inhibited by higher morphine doses (Bocchini et al., 1983). The mode of the angiostatic action of /3-EP may involve three basic mechanisms: (a) /3-EP might suppress vascular endothelial cell proliferation through inhibition (Zagon and McLaughlin, 1989a, b) of D N A synthesis and cellular mitosis; (b) /3-EP stimulates (bl) (Mandler et al., 1986) large granular lymphocytes ( L G L ) - - n a t u r a l killer cells ( N K C ) - - t o (b:) augment the interferon p r o d u c t i o n - an effect which is, in addition, significantly increased by phytohemagglutinin (PHG), or by polyinosinic polycytidylic acid (poly I:C). It is known that (b3) interferons inhibit angiogenesis in vitro (Tsuruoka et al., 1988) as well as in rico (Sidky and Borden, 1987); (c) /3-EP might inhibit (el) the inhibitory action (c:) of PGE2--itself a known angiogenesis promoter (Form and Auerbach, 1983)---on the IL-2 stimulated (c3) (Farrar, 1986) transformation (c4)
IA n4g l o g e n e s i s ,
b3',
,
a2',
",,
DISCUSSION In the present study,/3-EP exerted time and dosedependent, N X .irreversible angiostatic effects. These effects were not apparent with 1 #g. They became significant, however, at 5/~g, and peaked at 10/~g. A higher dose (15 pg) did not lead to a further increase in angiostasis. Naloxone itself was not significantly effective, and when given in combination with/3-EP, it potentiated its angiostatic effects. Also t h e anglostatic effects of MS were NX irreversible; and these effects were paradoxical in respect to the dose and the time pattern. For example, the effects appeared faster and were more pronounced by the lower (5 #g) than by the higher doses (10 and 50/~g) of MS. This inverse relationship between dose and magnitude of
C
I
DNA s nthesis]
i
.~,:,
'
lB'end°rphin I Fig. 1. Possible mechanisms of the angiostatic action of ~-EP. This figure is based on data of the literature and of the present study. More factors than those shown here might be involved as mechanisms. See the text for explanations. Solid arrows denote stimulation or production, the dashed one inhibition. Abbreviations: IFNs, interferons; IL-2, interleukin-2; LGL, large granular lymphocytes; LAK cells, lymphokine-activated killer cells; NKC, natural killer cells; T cells, T lymphocytes; PGE2, prostaglandin E2.
Angiogenesis: modulation with opioids (Kotasek et al., 1988) of T-cells to lymphokine-actirated killer cells (LAK cells) which (c5) are known (Kotasek et al., 1988) to be toxic to cultured human vascular endothelial cells. In the angiostatic effects of morphine other or additional mechanisms than those mentioned for fl-EP may be involved. This is because with fl-EP the angiostatic effects correlated directly to the dose, whereas with morphine the dose-response correlation was inverse. The described mechanisms of the angiostatic action of fl-EP suggest the possibility of the involvement of this neuropeptide and of cell-mediated immunity factors (certain lymphokines and prostaglandins) in angiostasis. This interpretation could be extended to the genesis of solid malignant tumors; in fact neovascularization is one of the critical steps involved in their establishment and growth (Folkman, 1971), and their genesis could be modulated by opioids (Zagon and McLaughlin, 1981; Murgo, 1985; Srisuchart et al., 1989). In this study, fl-EP inhibited, in a dose-dependent manner, the growth of blood vessels in vivo. In other studies, a similar effect of fl-EP on human glioma and neuroblastoma has been reported in vitro (Vonhoff and Forseth, 1982). Met-enkephalin (another endogenous opioid) has been shown to elevate the number of T helper cells and of interleukin-2 receptors of the lymphocytes in the blood of a patient (Plotnikoff et af., 1986) with acquired immune deficiency syndrome, and to induce apparent incipient healing of his Kaposi's sarcoma, a solid tumor. Kaposi's sarcoma and other diseases, such as diabetic retinopathy and neovascular glaucoma are conditions which exhibit inappropriately increased capillary growth owing to dysregulated endothelial cell proliferation (Maione et al., 1990). The angiostatic effects of fl-EP and of MS found in the present study could be viewed as a starting point of a new concept to be considered in the treatment t)f the diseases mentioned.
1079
Farrar W. L. (1986) Relationship between lymphokine and opiatergic modulation of lymphocyte proliferation. In Enkephalins and Endorphins: Stress and the Immune System (Edited by Plotnikoff N. P., Faith R. E., Murgo A. J.
and Good R. A.), pp. 241-251. Plenum Press, New York. Folkman J. (1971) Tumor angiogenesis: therapeutic implications. New Engl. J. Med. 285, 1182-I 186. Form D. M. and Auerbach R. (1983) PGE2 and angiogenesis. Proc. Soc. exp. Biol. Med. 172, 214-218. Kotasek D., Vercellotti G. M., Ochoa A. C., Bach F. H., White J. G. and Jacob H. S. (1988) Mechanism of cultured endothelial injury induced by lymphokine-activated killer cells. Cancer Res. 48, 5528-5532. Malone T. E., Gray S. G., Petro J., Hunt A. J., Bauer S. I., Carson H. F. and Sharpe R. J. (1990) Inhibition of angiogenesisby recombinant human platelet factor-4 and related peptides. Science 247, 77-79. Mandler R. N., Biddison W. E., Mandler R. and Serrate S. A. (1986) Beta-endorphin augments the cytolytic activity and interferon production of natural killer cells. J. lmmunol. 136, 934-939. Murgo A. J. (1985) Inhibition of B16-BL6 melanoma growth in mice by methionine-enkephalin.J. hath. Cancer inst. 75, 341-344. Plotnikoff N. P. and Miller G. C. (1983) Enkephalins and immunomodulators, lmmunopharmacology 5, 437--441. Plotnikoff N. P., Wybran J., Nimeh N. F. and Miller G. C. (1986) Methionine enkephalin" enhancement of T-cells in patients with Kaposi's sarcoma, AIDS and lung cancer. In Enkephalins and Endorphins: Stress and the Immune System (Edited by Plotnikoff N. P., Faith R. E., Murgo A. J. and Good R. A.), pp. 425-429. Plenum Press, New York. Scopsi L., Balslev E., Brunner N., Poulsen H. S., Andersen J., Rank F. and Larsson L. I. (1989) Immunoreactive opioid peptides in human breast cancer. Am. J. Path. 134, 473-479. Sidky Y. A. and Borden E. C. (1987) Inhibition of angiogenesis by interferons" effects on tumor- and lymphocyte-induced vascular responses. Cancer Res. 47, 5155-5161. Srisuchart B., Fuchs B. A., Sikorski E. E., Munson A. E. and Loveless S. E. (1989) Antitumor activity of enkephalin analogues in inhibiting PYB6 tumor growth in mice and immunological effects of methionine enkephalinamide. Int. J. Immunopharmac. 11, 487-500. Acknowledgements--This work was supported by the World Tsuruoka N., Sugiyama M., Tawaragi Y., Tsujimoto M., Health Organization and by the St Gallen/AppenzellLeague Nishihara T., Goto T. and Sato N. (1988) Inhibition of of the Swiss Cancer Society. We thank Mrs R. De Giuli for in vitro angiogenesis by lymphotoxin and interferonher excellent technical assistance. gamma. Biochem. biophys. Res. Commun. 155, 429-435. Vonhoff D. D. and Forseth D. (1982) Modulation of growth of human and murine tumors by human 8endorphin ~-end) (Abstract 932). Proc. Am. Ass. Cancer Res., p. 236. REFERENCES Zagon I. S. and McLaughlin P. J. (1981) Heroin prolongs survival time and retards tumor growth in mice with Bamhill R. L. and Ryan J. (1983) Biochemicalmodulation neuroblastoma. Brain Res. Bull. 7, 25-32. of angiogenesis in the chorioaUantoic membrane of the Zagon I. S. and MeLaughlin P. J. (1989a) Opioid antagonist chick embryo. J. Invest. Dermat. 81, 485-488. modulation of murine neuroblastoma: a profile of cell Bocchini G., Bonanno G. and Canevari A. (1983) Influence proliferation and opioid peptides and receptors. Brain of morphine and naloxone on human peripheral blood Res. 480, 16--28. T-lymphocytes. Drug Alcohol Depend. 11, 233-236. Bostwick D. G., Null W. E., Holmes D., Weber E., Barchas Zagon I. S. and McLaughlin P. J. (1989b) Endogenous opioid systems regulate growth of neural tumor cells in J. D. and Bensch K. G. (1987) Expression of opioid culture. Brain Res. 490, 14-25. peptides in tumors. New Engl. J. Med. 317, 1439-1443. Faith R. E. and Murgo A. J. (1988) Inhibition of pulmonary Zagon I. S., McLaughlin P. J., Goodman S. R. and Rhodes R. E. (1987) Opioid receptors and endogenous opioids in metastases and enhancement of natural killer cell activity diverse human and animal cancers. J. hath. Cancer Inst. by methionine-enkephalin. Brain Behav. Immun. 2, 79, 1059-1064. 114-122.
0306-3623/91 $3.00+ 0.00 Copyright © 1991PergamonPress pie
ANGIOGENESIS: MODULATION WITH OPIOIDS AURELIO PASl) BAOXI Q u ) RUDOLF STEINER,2 HANS-JtRG SENN,2 WALTER BgR ~ and FATHY S. MESSlHA3 t Endorphin Laboratories, Institute of Forensic Medicine, University of Zurich, CH-8028 Zurich, 2Division of Oncology, Department of Medicine C, Cantonal Hospital, St GaUen, Switzerland and 3Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX 79430, U.S.A. (Received 5 April 1991) Abstract--1. The effect of//-endorphin (~-EP) and morphine sulfate (MS), in presence and absence of naloxone (NX), on chicken chorioallantoic membrane was studied as a function of blood vessel proliferation. 2. A 50% reduction in blood vessel proliferation occurred by 10 pg of ~-EP or by 5 pg of MS per egg compared to controls. 3. An individual dose, i.e. 5 #g of//-EP, did not significantly inhibit blood vessel counts after initial 24 hr period of the drug application when given alone compared to inhibition occurring with combined use of NX. 4. NX (1/tg) did not significantly reverse the angiostatic effects of MS (10pg) or of/~-EP (5 pg). 5. The observed modulation of angiogenesis by opioids suggests involvement of/I-EP and MS in the proliferation of vascular endothelial cells. 6. This may be due to an effect of ~-EP and MS on cell-mediated immunity factors such as intefferons, interleukins and prostaglandin E2.
INTRODUCTION Opioids and their receptors have been found in a wide variety of neural and non-neural h u m a n and animal tumors (Bostwick et aL, 1987; Zagon et al., 1987; Scopsi et al., 1989). Furthermore, both endogenous and exogenous opioids, such as enkephalins and heroin, inhibited murine tumor growth (Zagon and McLaughlin, 1981; Murgo, 1985; Srisuchart et al., 1989) and prolonged survival of mice bearing Lt210 leukemia (Plotnikoff and Miller, 1983). Because the antitumorigenic effects of opioids could be reversed by naloxone (an opioid antagonist), an involvement of opioid receptors in such effects was suggested (Zagon and McLaughlin, 1981; Murgo, 1985; Faith and Murgo, 1988). The mechanisms underlying the opioid control of tumorigenesis are unclear. One proposed mode of action considers inhibitory processes of the opioids on D N A synthesis and mitosis (Zagon and McLaughlin, 1989a, b). In the present study, the effect of two potent opioids,//-EP and MS was studied on angiogenesis since tumoral growth depends on neovascularization. The chorioallantoic membrane (CAM) of the chicken embryo was the model used. MATERIALS AND METHODS Eggs Fertilized chicken eggs (Hy-lineTM, Ruedi Enzler, Switzerland) were processed using a modified form of the protocol of Barnhill and Ryan (1983). The eggs were incubated (37°C, 70% humidity) in a Covatutto 120TM incubator (M/irki, Switzerland). They were turned twice daily for the first two incubation days (ID). On the third ID, the eggs were disinfected with peracetic acid, and 2 ml of albumen were aspirated from their narrow pole with a syringe, creating an artificial air sac underlying the narrow egg
pole. To display the CAM, a window (2 cm dia) was created on ID 4 in the eggshell, over the air sac, and resealed with adhesive tape. Then, the eggs were reincubated for further nine days, short interruptions occurring at ID 7 for CAM treatment respectively daily for either vessel counting or routine observations. Agents They were applied, dissolved in 4pl of solvent, at ID 7, to a disc-shaped filter consisting of glass microfibres (3 mm dia; Whatman, England), and the disc was located on the CAM of each one of a series of five eggs. Treatment: fl-EP (Bachem, Switzerland): I, 5, 10 and 15pg (dissolved in phosphate buffered saline, PBS, pH 7.4); MS (Streuli, Switzerland): 1, 5, 10 and 50 pg (dissolved in normal saline, NS); naloxone (NX, Du Pont, Fed. Rep. Germany): alone (1 and 2 pg) and in combination (1/~g) with p-EP (5 pg) and MS (10 #g) (see Table 1). Negative controls were performed with the solvents alone. All procedures were performed under aseptic conditions. Vessel counting CAM vessels were counted in a 0.5 mm wide anulus (BarnhiU and Ryan, 1983), once daffy during the first 4 days after disc application, using a Wild M5 stereomieroscope (Leica, Switzerland) at ten-fold magnification. Statistics Vessel counts of treated and control groups were compared with the Student's t-test. The vessel counts reduction rate (RR), induced by the agents tested, was calculated with the following formula: RR = (C= -- Ct):Cc, where Cc and C~ stand for the mean vessel counts recorded in the control and the treated group. RESULTS The results are indicated in Table 1. ~ - E P caused, in comparison with control (PBS), between 24 and
1077
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AURELIOPASIet al. Table l. Opioidal effects on vesselcounts Vessel counts Dosage Agents (gg) 24 hr (%) 48 hr (%) 72 hr (%) 96 hr (%) PBS -20+3 27+3 3t+3 40__.4 BEP 1 17+2(15) 21 +3(22) 22+5(29) 33+4(18) BEP 5 21+3(-5) 19_+4"(30) 19_+5"*(39) 30_+5(25) BEP 10 16 _.+2 (20) 15 _+4** (43) 16 _+4** (48) 20 _+2** (50) BEP 15 16 _+2 (20) 15 _+3** (43) 16 _+3** (48) 20 _+3** (50) NX 1 22_+3(-10) 28_+4(-4) 38+5(-23) 41_+6(-3) NX 2 22_+4(-10) 23_+3(5) 29_+2(7) 40+4(0) BEP+ NX 5+1 12_+3'*(40) 21_+5(22) 20_+6*(36) 24_+3**(40) NS -21 _+3 26+3 32_+2 35_+2 MS 5 18_+4(14) 17+4"(35) 15_+3"*(53) 19+4"*(46) MS 10 19+3(10) 23_+5(12) 24_+5*(25) 26+5*(26) MS 50 20 _+2 (5) 24 + 4 (8) 26 _+3* (19) 27 _+4* (30) MS+NX 10+1 21_+3(0) 2 0 _ + 4 ( 2 3 ) 23_+3*(28) 26_+3*(26) CAM vesselcounts (performedat 24, 48, 72 and 96 hr after treatment)are expressedas the means (and their standard deviations) of counts done in 5 eggs. Figures in parentheses indicate reduction rates of vessel counts. Abbreviations:BEP, fl-endorphin;MS, morphine sulfate; NS, normal saline;NX, naloxone;PBS, phosphatebufferedsaline.*, ** significantlydifferent from control (*P < 0.05/**P < 0.01).
48 hr after doses of 5, 10 and 15 but not of 1 #g, significant reduction of vessel counts, i.e. significant angiostatic effects (P < 0.05 with 5 pg, P < 0.01 with either 10 or 15/~g). The corresponding vessel counts reduction rates ranged from 30 to 43%. The vessel counts were still significantly reduced (P < 0.01) in the groups with the 5, 10, and 15 g g doses 72 hr after treatment and in the group with the 10 and 15 pg doses also at the 96 hr check. MS also reduced vessel counts. A significant effect (RR: 35%, P < 0.05) was again observed between 24 and 48 hr after treatment by the 5 #g dose. This effect was even more pronounced 72 and 96 hr after treatment (RR: 53% and 46%, P < 0.01). Paradoxically, the effect of higher doses (10 and 50pg) became manifest later (72 hr after treatment), being less apparent (RR: 19-25%, P < 0.05) than with the low dose (5/~g) (RR: 53%), and it was still present 96 hr after treatment. After naloxone alone (1 and 2 #g) vessel counts were not significanty different than after PBS. Naloxone (1 #g), jointly applied with/3-EP (5/~g) potentiated significantly the angiostatic action of 13-EP 24 hr after treatment but not of MS (10#g). With the combination of N X with /3-EP and with MS, the angiostatic effect reached significancy at 7 2 h r (P < 0 . 0 5 ) and at 9 6 h r (P <0.01 with /3-EP and P < 0.05 with MS) but not at 48 hr.
angiostatic effects of MS recalls the one that this opioid had on the mitogenic activity of blood lymphocytes: the proliferation of these cells was promoted by low and inhibited by higher morphine doses (Bocchini et al., 1983). The mode of the angiostatic action of /3-EP may involve three basic mechanisms: (a) /3-EP might suppress vascular endothelial cell proliferation through inhibition (Zagon and McLaughlin, 1989a, b) of D N A synthesis and cellular mitosis; (b) /3-EP stimulates (bl) (Mandler et al., 1986) large granular lymphocytes ( L G L ) - - n a t u r a l killer cells ( N K C ) - - t o (b:) augment the interferon p r o d u c t i o n - an effect which is, in addition, significantly increased by phytohemagglutinin (PHG), or by polyinosinic polycytidylic acid (poly I:C). It is known that (b3) interferons inhibit angiogenesis in vitro (Tsuruoka et al., 1988) as well as in rico (Sidky and Borden, 1987); (c) /3-EP might inhibit (el) the inhibitory action (c:) of PGE2--itself a known angiogenesis promoter (Form and Auerbach, 1983)---on the IL-2 stimulated (c3) (Farrar, 1986) transformation (c4)
IA n4g l o g e n e s i s ,
b3',
,
a2',
",,
DISCUSSION In the present study,/3-EP exerted time and dosedependent, N X .irreversible angiostatic effects. These effects were not apparent with 1 #g. They became significant, however, at 5/~g, and peaked at 10/~g. A higher dose (15 pg) did not lead to a further increase in angiostasis. Naloxone itself was not significantly effective, and when given in combination with/3-EP, it potentiated its angiostatic effects. Also t h e anglostatic effects of MS were NX irreversible; and these effects were paradoxical in respect to the dose and the time pattern. For example, the effects appeared faster and were more pronounced by the lower (5 #g) than by the higher doses (10 and 50/~g) of MS. This inverse relationship between dose and magnitude of
C
I
DNA s nthesis]
i
.~,:,
'
lB'end°rphin I Fig. 1. Possible mechanisms of the angiostatic action of ~-EP. This figure is based on data of the literature and of the present study. More factors than those shown here might be involved as mechanisms. See the text for explanations. Solid arrows denote stimulation or production, the dashed one inhibition. Abbreviations: IFNs, interferons; IL-2, interleukin-2; LGL, large granular lymphocytes; LAK cells, lymphokine-activated killer cells; NKC, natural killer cells; T cells, T lymphocytes; PGE2, prostaglandin E2.
Angiogenesis: modulation with opioids (Kotasek et al., 1988) of T-cells to lymphokine-actirated killer cells (LAK cells) which (c5) are known (Kotasek et al., 1988) to be toxic to cultured human vascular endothelial cells. In the angiostatic effects of morphine other or additional mechanisms than those mentioned for fl-EP may be involved. This is because with fl-EP the angiostatic effects correlated directly to the dose, whereas with morphine the dose-response correlation was inverse. The described mechanisms of the angiostatic action of fl-EP suggest the possibility of the involvement of this neuropeptide and of cell-mediated immunity factors (certain lymphokines and prostaglandins) in angiostasis. This interpretation could be extended to the genesis of solid malignant tumors; in fact neovascularization is one of the critical steps involved in their establishment and growth (Folkman, 1971), and their genesis could be modulated by opioids (Zagon and McLaughlin, 1981; Murgo, 1985; Srisuchart et al., 1989). In this study, fl-EP inhibited, in a dose-dependent manner, the growth of blood vessels in vivo. In other studies, a similar effect of fl-EP on human glioma and neuroblastoma has been reported in vitro (Vonhoff and Forseth, 1982). Met-enkephalin (another endogenous opioid) has been shown to elevate the number of T helper cells and of interleukin-2 receptors of the lymphocytes in the blood of a patient (Plotnikoff et af., 1986) with acquired immune deficiency syndrome, and to induce apparent incipient healing of his Kaposi's sarcoma, a solid tumor. Kaposi's sarcoma and other diseases, such as diabetic retinopathy and neovascular glaucoma are conditions which exhibit inappropriately increased capillary growth owing to dysregulated endothelial cell proliferation (Maione et al., 1990). The angiostatic effects of fl-EP and of MS found in the present study could be viewed as a starting point of a new concept to be considered in the treatment t)f the diseases mentioned.
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Farrar W. L. (1986) Relationship between lymphokine and opiatergic modulation of lymphocyte proliferation. In Enkephalins and Endorphins: Stress and the Immune System (Edited by Plotnikoff N. P., Faith R. E., Murgo A. J.
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