ETAETB receptor antagonist Bosentan inhibits neointimal development in collared carotid arteries of rabbits

Life Sciences, Vol. 63, No. 18. pp. PL Z59-266, 1998 Copyright Q 1998 Elsevier Science Inc. Printed in the USA. All rights reserved om-3205/98 $19.00 + .oo

PI1 s0024-3205(98)00437-8

ELSEVIER

PhXRM4COLOGY LETTERS Accelerated Communication

ETA/ETa RECEPTOR ANTAGONIST BOSENTAN INHIBITS NEOINTIMAL DEVELOPMENT IN COLLARED CAROTID ARTERTES OF RABBITS

Giuseppe Marano’, Sergio Palazzesil, Paola Bemucci’, Mauro Grigioni* , Roberto Formigari**, Luigi Ballerini* *

Laboratorio di Farmacologia (5) and Laboratorio di Ingegneria Biomedica (*), Istituto Superiore di Sanitl; Ospedale Bambino Ged (**); Dipartimento di Medicina Sperimentale e Patologia, Universita La Sapienza 6, Rome, Italy (SubmittedJune 11, 1998; acceptedJuly 13, received in tinal form August19, 1998)

1998;

The contribution of endothelin to the genesis of neointimal development in collared rabbit carotid arteries, a widely accepted model of atherosclerosis, was investigated. Three sets of rabbits were studied. In the first group, a non-occlusive, biologically inert silastic collar was positioned around the right carotid artery of the rabbit. In another group, the application of the collar was accompanied by endothelial denudation via a Fogarty arterial balloon catheter, while the third group of animals underwent only endothelial denudation. After two weeks, intimal hyperplasia of a similar degree was observed in all groups. The administration of the nonselective ETAiETr, receptor antagonist Bosentan, significantly reduced both the neointimal area and the intimalmedia area ratio in all groups. However, the beneficial effects of Bosentan were less pronounced in balloon injured vessels than in collared ones. The results of the present study indicate that i) endothelin has a key role in the development of intimal hyperplasia following arterial collaring, ii) the contribution of endothelin to intimal hyperplasia is greater in collared arteries that in balloon injured ones, and iii) the nonselective ETA/ETB receptor antagonists are potential tools for the prevention of intimal hyperplasia. 0 1998 Elsevier Science Inc.

Abstract

Key WO&Y: intimal

hyperplasia,

collared

carotid

arteries,

rabbit,

bosentan,

endothelin,

endothelium

Introduction

Intimal hyperplasia is considered one of the major components of vascular proliferative disorders such as atherosclerosis and restenosis following procedures of myocardial revascularization. Various growth factors have been hypothesized to be involved in the mechanisms responsible for the genesis of intimal hyperplasia. Recently, it has been suggested that endothelin (ET), a potent vasoconstrictor, could importantly contribute to the development of intimal hyperplasia. In fact, endothelin was shown to act as a growth promoter of vascular smooth muscle cells both in vitro and in vivo (l-5), and an increased content of ET-l in previously denuded carotid arteries was reported (6,7). Furthermore, although the role of the ET receptor subtypes in the development of intimal hyperplasia after endothelial removal needs still to be clarified (6,8), the administration of ET receptor antagonists was able to inhibit intimal hyperplasia formation in the rat balloon injury Corresponding. Author: Giuseppe Marano MD, Laboratorio di Farmacologia, Istituto Superiore di San&a, Viale Regina Elena 299,00161 Roma, Italy; phone +39 6 4990 2395; fax +39 6 4938 7104; E-mail: [email protected]

PL-260

Endothelin in Collared Carotid Arteries

Vol. 63, No. 18, 19!I8

model (3,9,10) and in a porcine coronary stent model (11). However, to the best of our knowledge, the contribution of endothelin to the development of intimal hyperplasia in experimental models such as the collared artery model, where the endothelium is preserved (12), has never been investigated. We therefore investigated whether endothelin contributes to the genesis of intimal lesion under experimental conditions which preserve the endothelium and contemporarily induce the development of intimal hyperplasia (12). To that purpose, we evaluated the in vivo effects of the administration of the nonselective ET*/ETn receptor antagonist Bosentan, on intimal hyperplasia induced by the positioning of a hollow silastic collar around the common carotid artery (12). Furthermore, we also investigated the effects of endothelin receptor blockade on the development of intimal hyperplasia after removal of endothelium by balloon, with the aim of verifying whether i) there were significant differences in the contribution of endothelin to the neointima formation between the two models, and ii) the entity of intimal hyperplasia after removal of endothelium could be enhanced by collaring. Materials and Methods Animals. Thirty-six male New Zealand white (NZW) rabbits (Charles River, Italy), weighing 2.5 3.0 kg, were studied. The animals were individually caged in a room with artificially controlled temperature (21-24°C) and a 12h light - 12h dark cycle. Water and food were provided ad libitum. Animal care and use followed the directive of the Council of European Communities (86/609/EEC). Animal model. Anesthesia was induced intramuscularly with ketamine (10 mg/kg) and midazolam (0.1 mg/kg), and orotracheai intubation was attempted immediately after loss of consciousness. If the rabbit did not chew when this was attempted, the level of unconsciousness was judged to be sufficient. A cuffed endotracheal tube of 3.0 mm I.D., (D.A.R., Mirandola, Italy) was inserted into the trachea, and connected to a respiratory pump (mod. 6025, Ugo Basile, Italy), set at a tidal volume of 10 ml/kg. The ventilatory rate was adjusted to 30-35 breaths/mm (0.50-0.58 Hz) to keep end-tidal CO2 between 35 and 37 mmHg. End-tidal anaesthetic and carbon dioxide levels were continuously monitored (Gas Monitor, 5250 RGM, Ohrneda, England). Pancuronium bromide (0.1 mg/kg intravenously) was injected to induce muscle relaxation and facilitate ventilation mechanically. Anesthesia was maintained with end-tidal 1.8% isoflurane (1 MAC) in a gaseous mixture of nitrous oxide (NZO) and oxygen (02) (50/50% v/v). Body temperature and lead II of the electrocardiogram (ECG) were monitored continuosly. A 24-gauge catheter needle was inserted percutaneously into the marginal ear vein and an intravenous flow control system (DIAL-A-FLO, Abbott) was attached to it. Lactated Ringer’s solution was infused at 4 ml/kg/h throughout the study. In a first series of experiments consisting of 2 groups of 7 rabbits each (A and B), both carotid arteries were exposed surgically, and the right carotid artery was enclosed by a non-occlusive, biologically inert silastic collar of 1.0 cm length and an internal diameter of 3.5 mm (volume of about 96 mm3). In all experiments the contralateral carotid from the same animal was used as a control and was surgically manipulated in an identical fashion and for the same time as the carotid enclosed by the collar (sham-operated control). In a second series of experiments, 2 groups of 5 rabbits each (C and D) were anesthetized as above, and the bifurcation of both common carotid arteries were exposed surgically. Thereafter, a 3F Fogarty arterial balloon catheter was inserted in the right common carotid artery and advanced into the aortic arch. The balloon was inflated with a fixed volume of fluid, and slowly pulled through the carotid artery to the bifircation. The procedure was repeated three times to ensure complete removal of the endothelium. Thereafter, the right external carotid artery was ligated, and the right common carotid artery was enclosed by a non-occlusive, biologically inert silastic collar of length and internal diameter equal to that above described. In all experiments the contralateral carotid

Vol. 63, No. 18, 1998

Endothelin in Collared Carotid Arteries

PL-261

from the same animal was used as a control and was surgically manipulated in an identical fashion and for the same time as the denuded carotid artery (sham-operated control). In a third series of experiments, 2 groups of 6 rabbits each (E and F) were anesthetized as above. In both groups the right common carotid artery was denuded, whereas the contralateral carotid was used as a control and was surgically manipulated in an identical fashion and for the same time as the disendothelized carotid artery (sham-operated control). The B, D and F groups of rabbits were treated with Bosentan (Ho&an-La Roche, Basel, Switzerland) at the dosage of 10 mg/kg/day iv., whereas 4 C and E groups received the equivalent volume of vehicle, beginning 1 day before surgery and continuing for 14 days until necropsy. The dosage protocol chosen for Bosentan was based on in vitro and in vivo studies (13). In addition, preliminary in vivo studies performed by this laboratory before the beginning of the protocol, indicated that Bosentan at the dose of 10 mg/‘kddie, significantly (p < 0.01) inhibited the pressor responses to big ET-l (0.1 nmol/kg) with a long duration of action. Therefore, the dosage of Bosentan appeared to be sufficient to antagonize the endothelin binding to ETA receptors in vivo. Data from literature (13) also indicates that Bosentan at the dose of 10 mgn
rate, thus suggesting

PL-262

Endothelin in Collared Carotid Arteries

Vol. 63, No. 18, 1998

in arterial blood pressure. At harvest, all the carotid arteries were patent and did not show any abnormality on gross examination, except for a diffuse periadventitial fibrous thickening in the region surrounded by the collar. In the first experimental protocol, groups A and B carotid arteries showed a severe (group A) to very mild (group B) degree of intimal hyperplasia in the region which had been surrounded by the collar (Fig. LA, IB). Intimal hyperplasia consisted of a neo-growth of spindle-shaped cells, identified as smooth muscle cells because of their positive staining for actine, and an extracellular matrix lined by a continuous layer of endothelial cells, either flat or swollen. Neither intimal inflammatory infiltrates, nor medial lesions were observed. The carotid segments which were not enclosed in the collar, did not show any intimal lesion. In the sham-operated left carotid arteries, no noticeable changes could be detected (Fig. 2). On histomorphometric analysis, both the neointimal area and the intima/media area ratio were significantly lower in Bosentan-treated rabbits (group B) than in vehicle-treated animals (group A) (respectively 0.03 f 0.007 mm2 vs 0.14 + 0.02 mm2, p< 0.01, and 13% f 2% vs 29% k 4%, pcO.01) (Fig 3). In the second experimental protocol, intimal hyperplasia was present in both C and D group carotid arteries, with morphological features similar to those observed in A and B groups. Morphometric analysis showed significant differences in neointimal thickening between C group and D group. Both the neointimal area and the intima/media area ratio were significantly lower in group D carotid arteries than in group C (0.08 _+0.003 mm2 vs 0.13 f 0.01 mm2, pcO.05 and 18% k 0.7% vs 25% f 2%, p
Discussion

Our study was conducted in an animal model of intimal hyperplasia that allowed us to study the role of endothelin under experimental conditions which preserve the endothelium, and at the same time stimulate the proliferative activity of vascular smooth muscle cells. The most important finding of this study was that endothelin contributes importantly to the development of intimal hyperplasia. Although the importance of endothelin in the development of intimal hyperplasia in the balloon injury model has already been highlighted (3,9,10), we are able for the first time to report that the involvement of endothelin in the development of intimal hyperplasia can also occur in endothelialized arterial tracts. We obviously do not know which type of stimulus is involved in the release of endothelin from the endothelium at present. However, taking into account that ischemia of the arterial wall (12) and changes in hemodynamic conditions (15) were reported after the collaring occurred, we speculate that these conditions are responsible for the release of endothelin in the present model.

Vol. 63, No. 18, 1998

Endothelin in Collared Carotid Arteries

PL-263

1B

Fig. 1. Intimal thickening in collared carotid arteries without (A) and with (B) Bosentan administration. (A) The artery shows severe intimal hyperplasia consisting of smooth muscle cells lined by a continuous layer of endothelial cells. (B) A mild degree of intimal thickening is present in Bosentan-treated group. The arrow head points to the internal elastic membrane separating the intima from the media. Haematoxylin and eosin, 25X

PL-264

Endothelin in Collared Carotid Arteries

Vol. 63, No. 18, 1998

Fig. 2. A sham-operated carotid artery from Group A (Collared arteries). The picture shows neither medial nor intimal changes. The arrow head points to the internal elastic membrane. Haematoxylin and eosin, 25X

Fig. 3 Collar-induced intimal hyperplasia was reduced by Bosentan (Group B). Bosentan also reduced intimal hyperplasia in other groups (groups D and F) even if in a less pronounced manner. Rabbit carotid arteries were treated with vehicle of Bosentan (open bars) or Bosentan (filled bars) for 14 days. Sham-operated groups are shown as horizontal hatch bars. * P < 0.01 Bosentan treated groups vs Vehicle treated groups; # P < 0.05 Collared artery treated with Bosentan vs Other groups treated with Bosentan.

Vol. 63, No. 18, 1998

Endothelin

in Collared Carotid Arteries

PL-265

A tiuther significant result of our study was that Bosentan induced a greater decrease in neointimal formation in collared arteries than in balloon injured ones, from which we conclude that the amount of endothelin released in collared arteries is greater than that involved in balloon injured arteries. In addition, we also conclude that in de-endothelialized vessels, collaring does not contribute significantly to neointima formation, because collaring did not influence the magnitude of intimal lesions after removal of the endothelium. A final comment is also warranted regarding some possible limitations of our study. First, we used a nonselective ETA/ET* receptor antagonist. Because several endothelin receptor subtypes have been described based on in vitro and in vivo pharmacological evidence (8, 16-19) we do not know how much the blockade of ETs receptors, and in particular the ETB receptor that mediates vasoconstriction, contributes to the decrease of neointimal lesion formation. Studies of in situ hibridization performed in the rat balloon injury model have reported an increased expression of mRNA for ETA but not for ETB in the developing lesions (3). However, it cannot be ruled out that the blockade of the ETn receptor that mediates vasoconstriction may contribute to a reduction in the development of intimal hyperplasia, although establishing the relative contribution of the two receptors to the neointima formation was not among the objectives of our study. Second, although one can hypothesize that changes in the endothelial production of endothelin contribute to the neointimal development in collared arteries, the exact cellular sites of these changes were not identified by specific studies (i.e in situ hybridization). In conclusion, the results of the present study indicate that i) endothelin has a key role in the development of intimal hyperplasia following arterial collaring, ii) the contribution of endothelin to intimal hyperplasia is greater in collared arteries that in balloon injured ones, and iii) the nonselective ET.&TB receptor antagonists are potential tools for the prevention of intimal hyperplasia. Acknowledgments The authors wish to thank Ms. Daniela Ferraro for her excellent technical assistance References 1. Y HIRATA, Y TAGAGI, Y FUKUDA, F MARUMO. Atherosclerosis a:225-228 (1989) 2. S EGUCHI, Y HIRATA, M IHARA, M YANO, F MARUMO. FEBS Lett m:243-246 (1992) 3. P FERRER M VALENTINE, T JENKINS-WEST, H WEBER, NL GOLLER, SK DURHAM, CJ MOLLOY, S MORELAND. J Cardiovasc Pharmacol26:908-915 (1995) 4. JD TRACHTENBERG, S SUN, ET CHOI, AD CALLOW, US RYAN. J Cardiovasc Pharmacol22 (suppl8):S355-359 (1993) 5. SA DOUGLAS, C LOUDEN, EH OHLSTEIN. J Cardiovasc Pharmacol22 (suppl 8):S371-373 (1993) 6. H AZUMA, H HAMASAKI, Y MIMI, T TERADA, 0 MATSUBARA. Am J Physiol 267: H2259H2267 (1994) 7. TJ RESINK, AWA HAHN, T SCOTT-BURDEN, J POWELL, E WEBER FR BUHLER. Biochem Byophys Res Commun 168: 1303-13 10 (1990) 8. S KARNE, CK JAYAWICKREME, MRLERNER. J. Biol. Chem. =:19126-19133 (1993) 9. SA DOUGLAS, C LOUDEN, LM VICKERY-CLARK, BL STORER T HART, GZ FEUERSTEIN, JD ELLIOT, EH OHLSTEIN. Circ Res E: 190-197 (1994). 10.M TSUJINO, Y HIRATA, S EGUCHI, T WATANABE, F CHATANI, F MARUMO. Life Sci 56 :PL-449-454 (1995) 11. CJ MCKENNA SE BURKE, TJ OPGENORTH, RJ PADLEY, LJ CAMRUD, AR CAMRUD, J JOHNSON, PJ CARLSON, A LERMAN, DR Jr HOLMES, RS SCHWARTZ. Circulation 9: 2551-2556 (1998)

PL-266

Endothelin in Collared Carotid Arteries

Vol. 63, No. 18, 1998

12.RFG BOOTH, JF MARTIN, AC HONEY, DG HASSALL, JE BEESLEY, S MONCADA. Atherosclerosis 76: 257-268 (1989) 13.AC YONG, G TOWNLEY, GW. Clin Exp Pharmacol Physiol u:215-217 (1992) 14.M CLOZEL, V BREU, GA GRAY, B KALINA, B-M LOFFLER, K BURRI, J-M CASSAL, G HIRTH, M MULLER, W mIDHART, H RAMUZ. J Pharmacol Exp Ther =:228-235 (1994) 15.Y NIIMI, H AZUMA, K HIRAKAWA. Am. J. Physiol. 266: H1348-1356 (1994) 16.S EGUCHI, M HIRATA, M IHARq M YANO, F MARUMO. FEBS Lett. 302: 243-246 (1992) 17.M IHARA, K NOGUCHI, T SAEKI, T FUKUDA, S TSUCHlDA, S KIMuRq T FUKAMI, K ISHIKAW~ M NISHIKIBE, M YANO. Life Sci. 2:247-255 (1992) 18.S EMORI, Y HIRATA, K KANNO, K OHTA, S EGUCHI, T IMAI, M SHICHIRI, F MARUMO. Biochem. Biophys. Res. Commun. m:228-235 (1991) 19.A INOUE, M YANAGISAWA, S KlMUR4, Y KASUYA T MIYAUCHI, K GOTO,T MA&XI. Proc. Natl. Acad. Sci. USA j&2863-2867 (1989)