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Anti-Inflammatory Effects of Beraprost Sodium, a Stable Analogue of PGI2, and Its Mechanisms
Anti-InflammatoryEffects of Beraprost Sodium,a StableAnalogueof PG12,and Its Mechanisms Yuji Ueno,HiroshiKoike,ShigeyasuAnnoh andShintaroNishio Toray Industries,Inc., Basic ResearchLaboratories 1111 Tebiro Kamakura,Kanagawa248, Japan We examined whether beraprostsodium (beraprost),a stable analogue of PGIP,has an anti-inflammatoryeffect on the permeability barrier through endothelial cells in vivo. The injection of collagen (5 pg/head) plus epinephrine (0.6 pglhead) showed time-dependently the increased Evans blue dye leakage of the lung in mice for 60 min. Beraprost significantly suppressed thisleakage dose-dependently(control; 11.26 d 1.64 pgllung, beraprostIOpg/kg; 7.49 k 1.36 pgllung, 30 pglkg; 5.33 k 0.71 pg/lung, 100 pg/kg; 5.52 k 0.79 pg/lung). Pulmonarythromboem bolism-induced Evans blue dye leakage was alsoreduced significantly by aspirin(5 mgllzg),but PGE1(170 pglkg) showed a tendency topoten tiate the edematogenicresponse. One week after the injection of same dosage of collagenplus epinephrine in mice, pulmonary thromboembolism showed the increase of wet-to-dryweightratio of the lung (normal; 3.84 k 0.01, control;3.96 k 0.04) and right ventricular hypertrophy (normal;28.2 &0.9Y0,control;32.3 &0.9?4.)compared to normal mice, Beraprostsignificantlysuppressedlung edema and hypertrophy dose-dependently,and over 30 pglkglday of beraprost,the effects were statisticallysignificant(beraprost30 pglkglday; 3.85 &0,02 and 27.8 k 1.47., 100 pglkglday; 3.85& 0.02 and 27.3& 1.1%). Beraprostsignificantlyreduced 5-hydroxytryptamine(5-HT; 17 nmol/paw)-induced rat paw edema dose-dependently(5-HT alone; 100?4.,beraprost10-13 mol/paw; 91.19 *2.22?/., 10-12mol/paw; 85. 79&4.85Y0, 10-1’mol/paw;
Addressforcorrespondence andproofs:YujiUeno,Ph.D., TorayIndustries,Inc., Basic ResearchLaboratories,1111 Tebiro Kamakura,Kanagawa248, Japan. Telephonenumber:0467-32-2111; Faxnumber:0467-32-2135 Prostaglandins 53:279-289, 1997 01997 byElsevierScienceInc. 655AvenueoftheAmericas,NewYork,NY10010
0090-6980/97/$17.00 PIIS0090-6980(97]00001-4
Anti-inflammatory Effects of Beraprost and Its .Mechanisms: Ueno et al. 78.49 23.957.). 5-HT-induced edema was also suppressed significantly by the co-injectionof (-)-isoproterenol(10-’2mollpaw), but PGE1(10” mollpaw) significantlypotentiated the edematogenic response. From these results, we propose that the anti-inflammatoryeffect of beraprost may be contributed, in part, to the permeability barrier through end othelial cells in vivo. @ 1997 by Elsevier Science Inc. Keywords: Beraprostsodium;Anti-inflammatoryeffect; Pulmonary thromboembolismjLungedemaandventricularhypertrophyj5-HT-induced edema;ProstaglandinEl.
Introduction The vascular endothelium has an important function as a selective barrier between the intraluminal compartment of the blood vessel and the subendothelial space of the vessel wall’. Recently, Warren et al.z suggested that increasing cAMP concentrations in endothelium may suppress edema by enhancing the permeability barrier in vivo. However, PGI~,a major product of arachidonic acid in endothelial cells, causes a significant increase in cAMP production of endothelial cells3, and in some cases, such as inflammation, PGIZpotentates capillarypermeabilityl,s Beraprost, a chemically stable PG12analogue,was synthesized by replacing the exoenolether structure with a cyclopenta [b] benzofuran structure. Beraprost can be administered orally and has been demonstrated to possess a similar pharmacologicalprofile to PGIT,i.e., potent platelet aggregationinhibitory effects in various animal species7’8,an antithrombotic effect in the hamster cheek pouch9,an antithromboembolitic effect in mice]o, vasodilating effects in the dog]] and increasing cAMP levels in endothelial cells12.In the present study, we examined whether beraprosthas an anti-inflammatoryeffect due to the permeability barrier through endothelial cells cmpulmonary thromboembolisminducedlung injury in mice andrat pawedema.
MaterialandMethods Measurement of Pulmonary Thromboembolism-Induced Edema in Mice
Lung
Male albino mice weighing26 to 33 g were used. A mixture of collagen (5 ~g),epinephrine(0.6 ~g)and Evans blue dye (2 ~g)diluted in saline at a volume of 100 pl was injected into the tail vein of each mouse. Beraprost (p.o.)and PGE, (iv.) were administered 15 min after the challenge of the inducer of thrombosis except for aspirin (5 rein, i.p.). The animals were killed and the lungs were removed and minced. The minced lungs were then incubated with formamide (5 ml) for 12 h at
280
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al. room temperature and 3 h at 370C. The optical density of the solution was measuredat 620 nm in a microplatereader(BIO-RAD). Measurement of Pulmonary Thromboembolism-Induced Injury in Mice
Lung
Male albino mice weighing26 to 32 g were used. A mixture of collagen (5 pg) and epinephrine (0.6 pg) diluted in saline at a volume of 100 pl was injected into the tail vein of each mouse. One day after the challenge of the inducer of thrombosis, the administration of beraprost (10, 30 and 100 Lg/kg/day, p.o.) was initiated 6 times. One week later, the lung was weighedin wet and then allowedto stand at room temperature until a stable weight (dry weight) was obtained. The ratio of the wet lung weight to dry weight was used as an index of water accumulation in the lung. The extent of right ventricular hypertrophywas assessedby the method described as follows13.After the atria were trimmed away from the heart, the right ventricular free wall (RV) was carefully separated from the left ventricle plus septum (LV + S). The percent of weight of RV to that of LV + S [RV /(LV + S) x 100]was then used as an index of the extent of right ventricularhypertrophy. Measurement of Paw Edema in Rats Male Wistar rats (170-200 g) were used. Edema was induced by a single subplantar injection of 5-hydroxytryptamine(5-HT; 17 nmol/paw)dissolved saline, into the left hind pawof the rat underlight ether anesthesia. Beraprost, (-)-isoproterenol,and PGE1 were always co-injected with 5-HT in a final volume of 0.1 ml. Paw volume was measured immediately before the injection and at selected time intervals using a hydroplethysmometer (MK-550, Muromachi Kikai, Japan). Results were expressedas the increase of paw volume (ml) calculated by subtracting the basal volume. The area under the time course curve (AUC)was calculated and the results were expressedas a percent of the inhibition or potentiation of the total edema volume comparedwith that of animals receiving 5-HT alone. List of Compounds The drugs employedwere: collagen (Niko Bioscience, Japan), epinephrine hydrochloride (Daiichiseiyaku, Japan), aspirin (Midorijuji, Japan), PGE1 (Wake, Japan), 5-HT and (-)-isoproterenol(Sigma Chemical Co., U.S.A.).
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al.
i ?
oTime (rein) FIGURE1. Time course of Evans blue dye leakage inducedby pulmonarythromboembolismin mice. A mixtureof mllagen (5 ~g), epinephrine(0.6 Kg) and Evans blue dye (2 Kg) diluted in saline at a volume of 100 PI was injected into the tail vein of each mouse. The results are expressedas mean+ S.E. of 3-9 animals.
L
Control
10
100 Aspirin PGE 30 BPS (~@kg)
FIGURE2. Effects of beraprost(BPS), aspirin (5 mglkg) and PGEI (170 ~glkg) on pulmonary thromboembolism-induced lungedema in mice. BPS and PGEI were administered15 min after collagenplusepinephrineinjection.Aspirinwas administered5 min after thrombosischallenge. Each columnrepresentsthe mean + S.E. of 3-9 animals.*: P <0.05, ●*:P <0.01 vs. control.
282
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al. Statistical Analysis Statistical analysis was done by Dunnett test after analysis of variance (ANOVA).
Results The injection of collagenplus epinephrineshowedtime-dependentlythe increased Evans blue dye leakage of the lung in mice (Figure 1). Beraprost significantly suppressedthis leakage dose-dependently.Pulmonary thromboembolism-induced dye leakage was also reduced significantly by aspirin (n = 6), but PGE1 (n = 3) showed a tendency to potentate the edematogenicresponse(Figure2). One week after the injection of collagenplus epinephrineshowedthe increasedwet-to-dryweightratio of the lung. The effect of berapro)t(n = 6, each group)on this ratio was shown in Figure 3. Beraprost’’significantly suppressedpulmonary thromboembolism-induced lung edema dose-dependently.The percent of RV / (LV + S) x 100 of the control groupwas significantly elevated comparedto normal mice as well. The inhibition of beraproston right ventricularhypertrophywas shown from a dose of 10 pg/kg/day to 100 pg/kg,and over 30 pg/kgof beraprost,the effect was statistically significant [Figure4). Beraprost (n = 6, each group)significantly reduced 5-HT-induced rat paw edema dose-dependentlyuntil 120 min after injection (Figure 5a). 5-HT-inducededemawas suppressedsignificantly by the co-injection of (-)-isoproterenol(n = 6), but PGE1 (n = 6) significantly potentiated the edematogenicresponse(Figure5b). These effects coincide with the AUC (15-120 rein; Table 1].
Discussion Recently, anti-inflammatory effects of beraprosthave been reported on However, the mechanisms for these antiseveral animal models14’15. inflammatory effects are still unclear. In the present study, we examined whether beraprosthas an anti-inflammatory effect due to the permeability barrierthroughendothelialcells in vivo. Beraprost, but not PGE1,significantly suppressedpulmonary thromboembolism-inducedEvans blue dye leakage (Figure2). PGIj and PGE, have generally been regardedas proinflammatory mediators because they are potent vasodilators16. In the previousstudy, PulmOnarYantithromboembolitic effect of beraprostin mice induced by collagen plus epinephrine (15 ~g plus 1.8 pg/head)was about 12 times stronger than that of PGE1, although beraprost showed equipotent antiplatelet effect as PGE110. It is reported that several reagents increasing intracellular cAMP reducepermeabilityin endothelialcells17’*8.
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al,
Normal ‘Control
10
30
100
BPS (@kg) FIGURE3. Effectsof beraprost(BPS) on wet-to-dryweight ratioof the lung in mice. The wet-todry weight ratiowas used as an index of water accumulationin the lung. Each column representedthe mean + S.E. of 5-8 animals.*: p c 0.05.
4
T & m +3 ~ 3 a
2 Normal Control — 10
30
100
BPS (@kg) FIGURE4.Effects of beraprost(BPS) on the rightventricularhypertrophyin mice. The percent (%) of the rightVentricularfree Wall(RV) to the left ventricleplus septum (LV + S) was used as an index of the extent of right ventricular hypertrophy.Each column represents the mean f S.E. of 5-8 animals.*: p <0.05.
284
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al.
a)
t
h 10.8-
F ; 0.6-
E g o.4Ul
o.20 0
30
6
30
60 90 Time (rein)
120
b) 1.21~ 0.8~ 0.6S w &4o.20
60
90
120
Time (rein) FIGURE5. Effectsof beraprost,PGEI and (-)-isoproterenolon 5-HT-induced rat paw edema. (a) 5-HT (17 nmollpaw)in a final volume of 0.1 ml was injectedalone in the rat paw (n; n = 8) or co-injectedwith BPS 10”’3(m;n = 6), 10”’2(.; n = 6) or 10”” (A; n = 6). (b) 5-HT was injected alone (0; n = 8) or co-injectedwith PGE1 10-” (m;n = 6) or (-)-isoproterenol10-’2 (o; n = 6) mol/paw. Paw volume was measured immediately before the injectionand at selected time intervalsusinga hydroplethysmometer. The resultsare expressedas meant S.E.
It has been establishedthat beraprostand PGIZsignificantly increase the cAMP levels of cultured human umbilical vein endothelial cells]z, but PGE1 does not2. These findings could be speculatedthat tissue specificity between endothelial cells and vascular smooth muscle cells explains the paradoxof why prostaglandinsmay enhance or inhibit inflammatory edema. Furthermore, not only vasodilation in the pulmonary circulation, but also the permeabilitybarrier throughendothelial cells via a reProstaglandins 1997:53, April
285
Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al. TABLE1. Effectsof beraprost(BPS), PGEI and (-)-isoproterenol(ISO) on 5-HT-induced rat paw edema.
n
‘/’Change
BPS
10-13 mollpaw
Drugs
6
91. 19*2.22
BPS
10-12 mol/paw
6
85.79*4.85
BPS
10-11 mol/paw
6
78. 49k3.95*
PGE1 10-11
1s0
mollpaw
1 O-i 2 mol/paw
6
116.04k3.76’
6
78.78k3.59*
The values above refer to AUC (15-120 rein). The AUC was calculated and the resultswere expressed as a percent of the inhibitionor potentiationof the total edema volume compared p <0.05 when compared to with that of animals receiving5-HT (17 nmol/paw)alone (n = 8). ● 5-HT injectedalone in the rat paw. Values are the mean* S.E.
ceptor-mediated increase in cAMP participates in pulmonary thromboembolism-inducedlung edema. In the present study, pulmonary thromboembolism showed the increase of wet-to-dryweightratio of the lung and right ventricular hypertrophy compared to normal mice (Figure 3, 4). Pulmonary edema provokedpulmonary hypertension as progressiveright ventricular hypertrophy.Beraprostsignificantly suppressedlung edema and right ventricular hypertrophydose-dependently(Figure3, 4). It has been reported that neutrophils are less responsive to agonists that activate adenylate cyclase19but whether this is true and relevant for the accumulation neutrophils in our experimental model is not known. Although beraprostinhibited the inflammatory:processat the stages of migration of polymorphonuclearleukocytes (PMNs)20,but migration of PMNs was not shown morphologicallyin the presence of platelets during 1 week after the injection of collagen plus epinephrine in the control and beraprostgroups(datanot shown).TX& has been shown to be released into the pulmonary circulation on stimulation of the arachidonic acid cascade in the isolated lungs in the absence of platelets and PMNs21. TXAZcauses an increase in paracellularpermeability by contraction of pulmonary endothlial cells and a stable PGIj analogueprevents it22.In the present model, TXAZmay attribute to lung edemawhich probablyis caused by an increase in vascular permeability. Thus, the antiedematous effect of beraprostmight be contributed to the permeability barrierthroughendothelialcells in vivo. Beraprostand (-)-isoproterenolsignificantly reduced5-HT-inducedrat paw edema, but PGE1 significantly potentiated the edematogenic response (Figure5, Table 1). Warren et al.2 suggestedthat (-)-isoproterenol 286
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Anti-inflammatory Effects of Bemprost and Its Mechanisms: Ueno et al. was a potent stimulus to cAMP production of endothelial cells and in-
hibited edema formation,but PGE, was a potent stimulus to cAMP production of vascular smooth muscle cells and promoted edema formation. Additionally, in vitro the mechanism for the endothelial permeability decreasingpropertyof isoproterenol may be related to intracellular cAMP contentn. Mizuno-Yagyu et al.24 found that PG12 caused inhibition of transport of fluorescein dextran through aortic endothelial cells because of increased formation of tight junctions due to the increase of cAMP. Beraprostalso suppressedthe thrombin-induced macromolecular permeability in cultured vascular endothelial cells throughthe elevation of its intracellular CAMP25.On the contrary, PGE1 caused breakdownof the blood-retinal barrier by functionally opening tight junctions betweenretinal vascularendothelialcells2G.It is probable that opening of tight junctions is the mechanism of PGE1-induced increase of endothlial permeability and edema formation. Thus, antiinflammatoryeffect of beraprostmay be contributedto the permeability barrierthroughendothelialcells. In conclusion, our results suggestthat beraprostinhibited inflammatory responsessuch as exudationdue to enhancingthe permeability barrier of endothelial cells in vivo. This effect of beraprostcould relevant to therapeutic effectivenessin the inflammatorydiseases.
References 1. Furie, M.B., Cramer, E.B,, Naprstek, B.L., and Silverstein, S.C. Cultured endothelial cell monolayer that restrict the transendothelialpassageof macromoleculesand electrical current. J. Cell Biol., 98: 1033-1041, 1984. 2. Warren, J.B., Wilson, A.J., Loi, R.K., and Coughlan,M.L. Opposingroles of cyclic AMP in the vascular control of edema formation. FASEB J., 7: 13941400, 1993. 3. Hopkins, N.K., and German, R.R. Regulation of endothelial cell cyclic nu-
cleotide metabolismby prostacyclin.J. Clin. Invest., 67: 540-546, 1981. 4. Komoriya,K., Ohmori,A., Kurozumi,S., andHashimoto,Y. ProstaglandinIz
5. 6.
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as a potentiator of acute inflammation in rats. Prostaglandins,15: 557-564, 1978. Williams, T.J. ProstaglandinEz prostaglandinIzand the vascular changes of inflammation.Br. J. Pharmacol.,65: 517-524, 1979. Ohno, K., Nagase, H., Matsumoto, K., Nishiyama, H., and NishioJ S. Stereoselective synthesis of 5,6,7-trinor-4,8-inter-m-phenylene-PGIjderivatives and their inhibitory activities to human platelet aggregation. Adv. ProstaglandinThromboxaneLeukotrieneRes., 15: 279-281, 1985. Umetsu, T., Murata, T., Tanaka, Y., Osada, E., and Nishio, S. Antithrombotic effect of TRK-1OO,a novel, stable PGI, analogue. Jpn. J. Pharmacol., 43: 81-89, 1987. Nishio, S., Matsuura,H., Kanai, N., Fukatsu, Y., Hirano,T., Nishikawa, N., Kameoka,K., and Umetsu, T. The in vivo andex vivo anti~plateleteffect of
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15. 16. 17. 18. 19.
20. 21. 22.
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TRK-1OO,a stable prostacyclin analog, in several species. Jpn J Pharmacol 47: 1-9, 1988. Sire, A.K., McCraw, A.P., Cleland, M.E., Nishio, S., and Umetsu, T. Effects of a stable prostacyclin analogueon platelet function and experimentallyinducedthrombosis in the microcirculation. Arzneim-Forsch/DrugRes., 35: 1816-1818, 1985. Ueno, Y., Kawashima,A., Koike, H., and Nishio, S. Effect of beraprost sodium, a stable prostacyclin analogue, on pulmonary thromboembolism in mice. Thromb. Res., 77: 193-198, 1995. Akiba, T., Miyazaki, M., and Toda, N. Vasodilator actions of TRK-100, a new prostaglandinIzanalogue.Br. J. Pharmacol.,89: 703-711, 1986. Kainoh, M., Maruyama, I., Nishio, S., and Nakadate, T Enhancement by beraprostsodium, a stable analogueof prostacyclin, in thrombomodulinexpression on membranesurface of cultured vascular endothelial cells via increase in cyclic AMP level. Biochem. Pharmacol.,41: 1135-1140, 1991. Fulton, R.M., Hutchinson, E.C., Jones, A.M. Ventricular weight in cardiac hypertrophy.Br. HeartJ., 14: 413-420, 1952. Miyata, M., Ueno, Y., Sekine, H., Ito, O., Sakuma, F., Koike, H., Nishio, S., Nishimaki, T., and Kasukawa,R. Protective effect of beraprost sodium, a stable prostacyclin analogue, in development of monocrotaline-induced pulmonaryhypertension.J. Cardiovasc:Pharmacol.,27: 20-26, 1996. Kainoh, M., Ueno, S., Endoh, T., and Nakadate, T. Inhibitory effect of beraprostsodium on formation of lipid peroxidesin ischemia and recirculation-inducedcerebralinjury. Pharmacol.Res., 28: 249-258, 1993. Williams, T.J. Interaction between prostaglandins, leukotriens and other mediatorsof inflammation.Br. Med.Bull., 39: 239-242, 1983. Casnocha, S.A., Eskin, S.G., Hall, E.R., and Mcintire, L.V. Permeability of human endothelial monolayer: effect of vasoactive agonists and cAMP. J. Appl.Physiol., 67: 1997-2005, 1989. Langeler, E.G., and Van Hinsbergh, V.W.M. Norepinephrine and iloprost improve barrier function of human e:ndothelialmonolayer: role of cAMP. Am. J. Physiol., 260: C1052-1059, 1990. Plaut, M., Marone,G., Thomas, L.L., and Liechtenstein,L.M. In: Advances in Cyclic Nucleotides Research. Cyclic nucleotides in immune responses and allergy. (P. Hamet and H. Sands, eds.] Ravan Press, New York. 1980, p.161172. Kainoh, M., Imai, R., Umetsu, T., Hattori, M., and Nishio, S. Prostacyclin and beraprostsodium as suppressorsof activated rat polymorphnuclearleukocytes. Biochem. Pharmacol.,39: 477-483, 1990. Seeger,W., Wolf, H., Stahler, G., Neuhof, H., and Roka, L. Increasedpulmonary vascular resistance and permeability due to arachidonate metabolism in isolatedrabbit lungs.Prostaglandins,23: 157-173, 1982. Morel, N.M., Petruzzo,P.P., Hechtman, H.B., and Shepro,D. Inflammatory agonists that increase microvascular permeability in vivo stimulate cultured pulmonary microvessel endothelial cell contraction. Inflammation, 14: 571-583, 1990.
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al. 23. Minnear,F.L., Demichele,M.A.A.,Moon,D.G., Rieder,C.L., andFentonII, J.W.Isoproterenol reducesthrombin-induced pulmonaryendothelialpermeabilityin vitro.Am.J. Physiol.,26: H1613-1623,1989. 24. Mizuno-Yagyu, Y., Hashida,R., Mineo,C., Ikegami,S., Ohkuma,S., and Takano,T. Effectof PG12on transcellulartransportof fluoresceindextran throughan arterialendothelialmonolayer.Biochem.Pharmacol.,22: 38093813, 1987. 25. Imai-Sasaki,R., Kainoh,M., Ogawa,Y., Ohmori,E., Asai,Y., andNakadate, T. Inhibitionbyberaprostsodiumof thrombin-induced increasein endotheIial macromolecular permeability.Prostaglandins Leukot.Essent.FattyAcids,53: 103-108,1995. 26. Vinores,S.A., Sen,H., andCampochiaro, P.A.AnAdenosineantagonistand prostaglandin E, causebreakdownof the blood-retinalbarrierby opening tightjunctionsbetweenvascularendothelialcalls.Invest.Ophthalmol.Vis. Sci.,33: 1870-1878,1992. Editor:
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ReceivedDate: 8-8-96
AcceptedDate: 11-26-96
289
Addressforcorrespondence andproofs:YujiUeno,Ph.D., TorayIndustries,Inc., Basic ResearchLaboratories,1111 Tebiro Kamakura,Kanagawa248, Japan. Telephonenumber:0467-32-2111; Faxnumber:0467-32-2135 Prostaglandins 53:279-289, 1997 01997 byElsevierScienceInc. 655AvenueoftheAmericas,NewYork,NY10010
0090-6980/97/$17.00 PIIS0090-6980(97]00001-4
Anti-inflammatory Effects of Beraprost and Its .Mechanisms: Ueno et al. 78.49 23.957.). 5-HT-induced edema was also suppressed significantly by the co-injectionof (-)-isoproterenol(10-’2mollpaw), but PGE1(10” mollpaw) significantlypotentiated the edematogenic response. From these results, we propose that the anti-inflammatoryeffect of beraprost may be contributed, in part, to the permeability barrier through end othelial cells in vivo. @ 1997 by Elsevier Science Inc. Keywords: Beraprostsodium;Anti-inflammatoryeffect; Pulmonary thromboembolismjLungedemaandventricularhypertrophyj5-HT-induced edema;ProstaglandinEl.
Introduction The vascular endothelium has an important function as a selective barrier between the intraluminal compartment of the blood vessel and the subendothelial space of the vessel wall’. Recently, Warren et al.z suggested that increasing cAMP concentrations in endothelium may suppress edema by enhancing the permeability barrier in vivo. However, PGI~,a major product of arachidonic acid in endothelial cells, causes a significant increase in cAMP production of endothelial cells3, and in some cases, such as inflammation, PGIZpotentates capillarypermeabilityl,s Beraprost, a chemically stable PG12analogue,was synthesized by replacing the exoenolether structure with a cyclopenta [b] benzofuran structure. Beraprost can be administered orally and has been demonstrated to possess a similar pharmacologicalprofile to PGIT,i.e., potent platelet aggregationinhibitory effects in various animal species7’8,an antithrombotic effect in the hamster cheek pouch9,an antithromboembolitic effect in mice]o, vasodilating effects in the dog]] and increasing cAMP levels in endothelial cells12.In the present study, we examined whether beraprosthas an anti-inflammatoryeffect due to the permeability barrier through endothelial cells cmpulmonary thromboembolisminducedlung injury in mice andrat pawedema.
MaterialandMethods Measurement of Pulmonary Thromboembolism-Induced Edema in Mice
Lung
Male albino mice weighing26 to 33 g were used. A mixture of collagen (5 ~g),epinephrine(0.6 ~g)and Evans blue dye (2 ~g)diluted in saline at a volume of 100 pl was injected into the tail vein of each mouse. Beraprost (p.o.)and PGE, (iv.) were administered 15 min after the challenge of the inducer of thrombosis except for aspirin (5 rein, i.p.). The animals were killed and the lungs were removed and minced. The minced lungs were then incubated with formamide (5 ml) for 12 h at
280
Prostaglandins 1997:53, April
Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al. room temperature and 3 h at 370C. The optical density of the solution was measuredat 620 nm in a microplatereader(BIO-RAD). Measurement of Pulmonary Thromboembolism-Induced Injury in Mice
Lung
Male albino mice weighing26 to 32 g were used. A mixture of collagen (5 pg) and epinephrine (0.6 pg) diluted in saline at a volume of 100 pl was injected into the tail vein of each mouse. One day after the challenge of the inducer of thrombosis, the administration of beraprost (10, 30 and 100 Lg/kg/day, p.o.) was initiated 6 times. One week later, the lung was weighedin wet and then allowedto stand at room temperature until a stable weight (dry weight) was obtained. The ratio of the wet lung weight to dry weight was used as an index of water accumulation in the lung. The extent of right ventricular hypertrophywas assessedby the method described as follows13.After the atria were trimmed away from the heart, the right ventricular free wall (RV) was carefully separated from the left ventricle plus septum (LV + S). The percent of weight of RV to that of LV + S [RV /(LV + S) x 100]was then used as an index of the extent of right ventricularhypertrophy. Measurement of Paw Edema in Rats Male Wistar rats (170-200 g) were used. Edema was induced by a single subplantar injection of 5-hydroxytryptamine(5-HT; 17 nmol/paw)dissolved saline, into the left hind pawof the rat underlight ether anesthesia. Beraprost, (-)-isoproterenol,and PGE1 were always co-injected with 5-HT in a final volume of 0.1 ml. Paw volume was measured immediately before the injection and at selected time intervals using a hydroplethysmometer (MK-550, Muromachi Kikai, Japan). Results were expressedas the increase of paw volume (ml) calculated by subtracting the basal volume. The area under the time course curve (AUC)was calculated and the results were expressedas a percent of the inhibition or potentiation of the total edema volume comparedwith that of animals receiving 5-HT alone. List of Compounds The drugs employedwere: collagen (Niko Bioscience, Japan), epinephrine hydrochloride (Daiichiseiyaku, Japan), aspirin (Midorijuji, Japan), PGE1 (Wake, Japan), 5-HT and (-)-isoproterenol(Sigma Chemical Co., U.S.A.).
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al.
i ?
oTime (rein) FIGURE1. Time course of Evans blue dye leakage inducedby pulmonarythromboembolismin mice. A mixtureof mllagen (5 ~g), epinephrine(0.6 Kg) and Evans blue dye (2 Kg) diluted in saline at a volume of 100 PI was injected into the tail vein of each mouse. The results are expressedas mean+ S.E. of 3-9 animals.
L
Control
10
100 Aspirin PGE 30 BPS (~@kg)
FIGURE2. Effects of beraprost(BPS), aspirin (5 mglkg) and PGEI (170 ~glkg) on pulmonary thromboembolism-induced lungedema in mice. BPS and PGEI were administered15 min after collagenplusepinephrineinjection.Aspirinwas administered5 min after thrombosischallenge. Each columnrepresentsthe mean + S.E. of 3-9 animals.*: P <0.05, ●*:P <0.01 vs. control.
282
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al. Statistical Analysis Statistical analysis was done by Dunnett test after analysis of variance (ANOVA).
Results The injection of collagenplus epinephrineshowedtime-dependentlythe increased Evans blue dye leakage of the lung in mice (Figure 1). Beraprost significantly suppressedthis leakage dose-dependently.Pulmonary thromboembolism-induced dye leakage was also reduced significantly by aspirin (n = 6), but PGE1 (n = 3) showed a tendency to potentate the edematogenicresponse(Figure2). One week after the injection of collagenplus epinephrineshowedthe increasedwet-to-dryweightratio of the lung. The effect of berapro)t(n = 6, each group)on this ratio was shown in Figure 3. Beraprost’’significantly suppressedpulmonary thromboembolism-induced lung edema dose-dependently.The percent of RV / (LV + S) x 100 of the control groupwas significantly elevated comparedto normal mice as well. The inhibition of beraproston right ventricularhypertrophywas shown from a dose of 10 pg/kg/day to 100 pg/kg,and over 30 pg/kgof beraprost,the effect was statistically significant [Figure4). Beraprost (n = 6, each group)significantly reduced 5-HT-induced rat paw edema dose-dependentlyuntil 120 min after injection (Figure 5a). 5-HT-inducededemawas suppressedsignificantly by the co-injection of (-)-isoproterenol(n = 6), but PGE1 (n = 6) significantly potentiated the edematogenicresponse(Figure5b). These effects coincide with the AUC (15-120 rein; Table 1].
Discussion Recently, anti-inflammatory effects of beraprosthave been reported on However, the mechanisms for these antiseveral animal models14’15. inflammatory effects are still unclear. In the present study, we examined whether beraprosthas an anti-inflammatory effect due to the permeability barrierthroughendothelialcells in vivo. Beraprost, but not PGE1,significantly suppressedpulmonary thromboembolism-inducedEvans blue dye leakage (Figure2). PGIj and PGE, have generally been regardedas proinflammatory mediators because they are potent vasodilators16. In the previousstudy, PulmOnarYantithromboembolitic effect of beraprostin mice induced by collagen plus epinephrine (15 ~g plus 1.8 pg/head)was about 12 times stronger than that of PGE1, although beraprost showed equipotent antiplatelet effect as PGE110. It is reported that several reagents increasing intracellular cAMP reducepermeabilityin endothelialcells17’*8.
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283
Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al,
Normal ‘Control
10
30
100
BPS (@kg) FIGURE3. Effectsof beraprost(BPS) on wet-to-dryweight ratioof the lung in mice. The wet-todry weight ratiowas used as an index of water accumulationin the lung. Each column representedthe mean + S.E. of 5-8 animals.*: p c 0.05.
4
T & m +3 ~ 3 a
2 Normal Control — 10
30
100
BPS (@kg) FIGURE4.Effects of beraprost(BPS) on the rightventricularhypertrophyin mice. The percent (%) of the rightVentricularfree Wall(RV) to the left ventricleplus septum (LV + S) was used as an index of the extent of right ventricular hypertrophy.Each column represents the mean f S.E. of 5-8 animals.*: p <0.05.
284
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Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al.
a)
t
h 10.8-
F ; 0.6-
E g o.4Ul
o.20 0
30
6
30
60 90 Time (rein)
120
b) 1.21~ 0.8~ 0.6S w &4o.20
60
90
120
Time (rein) FIGURE5. Effectsof beraprost,PGEI and (-)-isoproterenolon 5-HT-induced rat paw edema. (a) 5-HT (17 nmollpaw)in a final volume of 0.1 ml was injectedalone in the rat paw (n; n = 8) or co-injectedwith BPS 10”’3(m;n = 6), 10”’2(.; n = 6) or 10”” (A; n = 6). (b) 5-HT was injected alone (0; n = 8) or co-injectedwith PGE1 10-” (m;n = 6) or (-)-isoproterenol10-’2 (o; n = 6) mol/paw. Paw volume was measured immediately before the injectionand at selected time intervalsusinga hydroplethysmometer. The resultsare expressedas meant S.E.
It has been establishedthat beraprostand PGIZsignificantly increase the cAMP levels of cultured human umbilical vein endothelial cells]z, but PGE1 does not2. These findings could be speculatedthat tissue specificity between endothelial cells and vascular smooth muscle cells explains the paradoxof why prostaglandinsmay enhance or inhibit inflammatory edema. Furthermore, not only vasodilation in the pulmonary circulation, but also the permeabilitybarrier throughendothelial cells via a reProstaglandins 1997:53, April
285
Anti-inflammatory Effects of Beraprost and Its Mechanisms: Ueno et al. TABLE1. Effectsof beraprost(BPS), PGEI and (-)-isoproterenol(ISO) on 5-HT-induced rat paw edema.
n
‘/’Change
BPS
10-13 mollpaw
Drugs
6
91. 19*2.22
BPS
10-12 mol/paw
6
85.79*4.85
BPS
10-11 mol/paw
6
78. 49k3.95*
PGE1 10-11
1s0
mollpaw
1 O-i 2 mol/paw
6
116.04k3.76’
6
78.78k3.59*
The values above refer to AUC (15-120 rein). The AUC was calculated and the resultswere expressed as a percent of the inhibitionor potentiationof the total edema volume compared p <0.05 when compared to with that of animals receiving5-HT (17 nmol/paw)alone (n = 8). ● 5-HT injectedalone in the rat paw. Values are the mean* S.E.
ceptor-mediated increase in cAMP participates in pulmonary thromboembolism-inducedlung edema. In the present study, pulmonary thromboembolism showed the increase of wet-to-dryweightratio of the lung and right ventricular hypertrophy compared to normal mice (Figure 3, 4). Pulmonary edema provokedpulmonary hypertension as progressiveright ventricular hypertrophy.Beraprostsignificantly suppressedlung edema and right ventricular hypertrophydose-dependently(Figure3, 4). It has been reported that neutrophils are less responsive to agonists that activate adenylate cyclase19but whether this is true and relevant for the accumulation neutrophils in our experimental model is not known. Although beraprostinhibited the inflammatory:processat the stages of migration of polymorphonuclearleukocytes (PMNs)20,but migration of PMNs was not shown morphologicallyin the presence of platelets during 1 week after the injection of collagen plus epinephrine in the control and beraprostgroups(datanot shown).TX& has been shown to be released into the pulmonary circulation on stimulation of the arachidonic acid cascade in the isolated lungs in the absence of platelets and PMNs21. TXAZcauses an increase in paracellularpermeability by contraction of pulmonary endothlial cells and a stable PGIj analogueprevents it22.In the present model, TXAZmay attribute to lung edemawhich probablyis caused by an increase in vascular permeability. Thus, the antiedematous effect of beraprostmight be contributed to the permeability barrierthroughendothelialcells in vivo. Beraprostand (-)-isoproterenolsignificantly reduced5-HT-inducedrat paw edema, but PGE1 significantly potentiated the edematogenic response (Figure5, Table 1). Warren et al.2 suggestedthat (-)-isoproterenol 286
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Anti-inflammatory Effects of Bemprost and Its Mechanisms: Ueno et al. was a potent stimulus to cAMP production of endothelial cells and in-
hibited edema formation,but PGE, was a potent stimulus to cAMP production of vascular smooth muscle cells and promoted edema formation. Additionally, in vitro the mechanism for the endothelial permeability decreasingpropertyof isoproterenol may be related to intracellular cAMP contentn. Mizuno-Yagyu et al.24 found that PG12 caused inhibition of transport of fluorescein dextran through aortic endothelial cells because of increased formation of tight junctions due to the increase of cAMP. Beraprostalso suppressedthe thrombin-induced macromolecular permeability in cultured vascular endothelial cells throughthe elevation of its intracellular CAMP25.On the contrary, PGE1 caused breakdownof the blood-retinal barrier by functionally opening tight junctions betweenretinal vascularendothelialcells2G.It is probable that opening of tight junctions is the mechanism of PGE1-induced increase of endothlial permeability and edema formation. Thus, antiinflammatoryeffect of beraprostmay be contributedto the permeability barrierthroughendothelialcells. In conclusion, our results suggestthat beraprostinhibited inflammatory responsessuch as exudationdue to enhancingthe permeability barrier of endothelial cells in vivo. This effect of beraprostcould relevant to therapeutic effectivenessin the inflammatorydiseases.
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ReceivedDate: 8-8-96
AcceptedDate: 11-26-96
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