In-stent restenosis is a novel pathobiologic process resulting from vascular smooth muscle cell (VSMC) proliferation, migration and excessive matrix production. The present study was designed to assess the activity of RhoA, a major regulator of VSMC proliferation and migration, after stenting and to determine its role in the neointimal formation. Analysis of RhoA activity in an ex vivo organ culture model of human internal mammary arteries demonstrates that stenting induced a time-dependent increase in RhoA activity (4.9 ± 0.4 vs. 1.2 ± 0.2 in control at 28 days, n = 4, p < 0.0001) associated with a concomitant decrease in p27 expression. Treatment of stented arteries with the permeant RhoA inhibitor TAT-C3 (10 µg/ml) or Rho-kinase inhibitors (Y-27632, 10 µmol/l; fasudil, 10 µmol/l) inhibited both neointimal formation and decrease in p27 expression. Rapamycin (1 and 10 nmol/l) also inhibited neointimal formation, and induced a loss of RhoA expression. The inhibitory effect of rapamycin on neointimal thickening is prevented by the dominant active form of RhoA. Our study shows that stent implantation induces maintained RhoA activation and demonstrates that the inhibitory action of rapamycin on RhoA expression plays a key role in its antirestenotic effect.

Keywords:
Artery, Restenosis, Stents, Signal transduction, Rho proteins, Rapamycin
1.
Libby P, Ganz P: Restenosis revisited – New targets, new therapies. N Engl J Med 1997;337:418–419.
2.
Landau C, Lange RA, Hillis LD: Percutaneous transluminal coronary angioplasty. N Engl J Med 1994;330:981–993.
3.
Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, Detre K, Veltri L, Ricci D, Nobuyoshi M, et al: A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. Stent Restenosis Study Investigators. N Engl J Med 1994;331:496–501.
4.
Serruys PW, de Jaegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, Emanuelsson H, Marco J, Legrand V, Materne P, et al: A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. Benestent Study Group. N Engl J Med 1994;331:489–495.
5.
Bennett MR, O’Sullivan M: Mechanisms of angioplasty and stent restenosis: Implications for design of rational therapy. Pharmacol Ther 2001;91:149–166.
6.
Lafont A, Faxon D: Why do animal models of post-angioplasty restenosis sometimes poorly predict the outcome of clinical trials? Cardiovasc Res 1998;39:50–59.
7.
Babapulle MN, Eisenberg MJ: Coated stents for the prevention of restenosis. Part II. Circulation 2002;106:2859–2866.
8.
Seasholtz TM, Majumdar M, Kaplan DD, Brown JH: Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res 1999;84:1186–1193.
9.
Halayko AJ, Solway J: Plasticity in skeletal, cardiac, and smooth muscle: Invited review: Molecular mechanisms of phenotypic plasticity in smooth muscle cells. J Appl Physiol 2001;90:358–368.
10.
Sauzeau V, Le Mellionnec E, Bertoglio J, Scalbert E, Pacaud P, Loirand G: Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res 2001;88:1102–1104.
11.
Seko T, Ito M, Kureishi Y, Okamoto R, Moriki N, Onishi K, Isaka N, Hartshorne DJ, Nakano T: Activation of RhoA and inhibition of myosin phosphatase as important components in hypertension in vascular smooth muscle. Circ Res 2003;92:411–418.
12.
Seasholtz TM, Zhang T, Morissette MR, Howes AL, Yang AH, Brown JH: Increased expression and activity of RhoA are associated with increased DNA synthesis and reduced p27(Kip1) expression in the vasculature of hypertensive rats. Circ Res 2001;89:488–495.
13.
van Nieuw Amerongen GP, van Hinsbergh VW: Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system. Arterioscler Thromb Vasc Biol 2001;21:300–311.
14.
Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S: Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997;389:990–994.
15.
Sawada N, Itoh H, Ueyama K, Yamashita J, Doi K, Chun TH, Inoue M, Masatsugu K, Saito T, Fukunaga Y, Sakaguchi S, Arai H, Ohno N, Komeda M, Nakao K: Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation 2000;101:2030–2033.
16.
Shibata R, Kai H, Seki Y, Kato S, Morimatsu M, Kaibuchi K, Imaizumi T: Role of Rho-associated kinase in neointima formation after vascular injury. Circulation 2001;103:284–289.
17.
Negoro N, Hoshiga M, Seto M, Kohbayashi E, Ii M, Fukui R, Shibata N, Nakakoji T, Nishiguchi F, Sasaki Y, Ishihara T, Ohsawa N: The kinase inhibitor fasudil (HA-1077) reduces intimal hyperplasia through inhibiting migration and enhancing cell loss of vascular smooth muscle cells. Biochem Biophys Res Commun 1999;262:211–215.
18.
Eto Y, Shimokawa H, Hiroki J, Morishige K, Kandabashi T, Matsumoto Y, Amano M, Hoshijima M, Kaibuchi K, Takeshita A: Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs. Am J Physiol Heart Circ Physiol 2000;278:H1744–H1750.
19.
Matsumoto Y, Uwatoku T, Oi K, Abe K, Hattori T, Morishige K, Eto Y, Fukumoto Y, Nakamura K, Shibata Y, Matsuda T, Takeshita A, Shimokawa H: Long-term inhibition of Rho-kinase suppresses neointimal formation after stent implantation in porcine coronary arteries: Involvement of multiple mechanisms. Arterioscler Thromb Vasc Biol 2004;24:181–186.
20.
Guérin P, Rondeau F, Grimandi G, Heymann MF, Heymann D, Pillet P, Al Habash O, Loirand G, Pacaud P, Crochet D: Neointimal hyperplasia after stenting in a human mammary artery organ culture. J Vasc Res 2004;41:46–53.
21.
Sauzeau V, Le Jeune H, Cario-Toumaniantz C, Smolenski A, Lohmann SM, Bertoglio J, Chardin P, Pacaud P, Loirand G: Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 2000;275:21722–21729.
22.
Ren XD, Kiosses WB, Schwartz MA: Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 1999;18:578–585.
23.
Poon M, Marx SO, Gallo R, Badimon JJ, Taubman MB, Marks AR: Rapamycin inhibits vascular smooth muscle cell migration. J Clin Invest 1996;98:2277–2283.
24.
Weber JD, Hu W, Jefcoat SC Jr, Raben DM, Baldassare JJ: Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27. J Biol Chem 1997;272:32966–32971.
25.
Tanner FC, Yang ZY, Duckers E, Gordon D, Nabel GJ, Nabel EG: Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res 1998;82:396–403.
26.
Braun-Dullaeus RC, Ziegler A, Bohle RM, Bauer E, Hein S, Tillmanns H, Haberbosch W: Quantification of the cell-cycle inhibitors p27 and p21 in human atherectomy specimens: Primary stenosis versus restenosis. J Lab Clin Med 2003;141:179–189.
27.
Fleming IN, Elliott CM, Exton JH: Differential translocation of rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J Biol Chem 1996;271:33067–33073.
28.
Numaguchi K, Eguchi S, Yamakawa T, Motley ED, Inagami T: Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments. Circ Res 1999;85:5–11.
29.
Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE: Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J Cell Biol 1993;123:741–747.
30.
Renshaw MW, Toksoz D, Schwartz MA: Involvement of the small GTPase rho in integrin-mediated activation of mitogen-activated protein kinase. J Biol Chem 1996;271:21691–21694.
31.
Gregory CR, Huie P, Billingham ME, Morris RE: Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Its effect on cellular, growth factor, and cytokine response in injured vessels. Transplantation 1993;55:1409–1418.
32.
Gallo R, Padurean A, Jayaraman T, Marx S, Roque M, Adelman S, Chesebro J, Fallon J, Fuster V, Marks A, Badimon JJ: Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle. Circulation 1999;99:2164–2170.
33.
Sousa JE, Costa MA, Abizaid A, Abizaid AS, Feres F, Pinto IM, Seixas AC, Staico R, Mattos LA, Sousa AG, Falotico R, Jaeger J, Popma JJ, Serruys PW: Lack of neointimal proliferation after implantation of sirolimus-coated stents in human coronary arteries: A quantitative coronary angiography and three-dimensional intravascular ultrasound study. Circulation 2001;103:192–195.
34.
Suzuki T, Kopia G, Hayashi S, Bailey LR, Llanos G, Wilensky R, Klugherz BD, Papandreou G, Narayan P, Leon MB, Yeung AC, Tio F, Tsao PS, Falotico R, Carter AJ: Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model. Circulation 2001;104:1188–1193.
35.
Sun J, Marx SO, Chen HJ, Poon M, Marks AR, Rabbani LE: Role for p27(Kip1) in vascular smooth muscle cell migration. Circulation 2001;103:2967–2972.
36.
Nourse J, Firpo E, Flanagan WM, Coats S, Polyak K, Lee MH, Massague J, Crabtree GR, Roberts JM: Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 1994;372:570–573.
37.
Hirai A, Nakamura S, Noguchi Y, Yasuda T, Kitagawa M, Tatsuno I, Oeda T, Tahara K, Terano T, Narumiya S, Kohn LD, Saito Y: Geranylgeranylated rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells. J Biol Chem 1997;272:13–16.
38.
Gingras AC, Raught B, Sonenberg N: Regulation of translation initiation by FRAP/mTOR. Genes Dev 2001;15:807–826.
39.
Sisto T, Isola J: Incidence of atherosclerosis in the internal mammary artery. Ann Thorac Surg 1989;47:884–886.
40.
Hodgson JM, Marshall JJ: Direct vasoconstriction and endothelium-dependent vasodilation. Mechanisms of acetylcholine effects on coronary flow and arterial diameter in patients with nonstenotic coronary arteries. Circulation 1989;79:1043–1051.
41.
Reddy KG, Nair RN, Sheehan HM, Hodgson JM: Evidence that selective endothelial dysfunction may occur in the absence of angiographic or ultrasound atherosclerosis in patients with risk factors for atherosclerosis. J Am Coll Cardiol 1994;23:833–843.
© 2005 S. Karger AG, Basel
2005
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.