The indazole derivative YD-3 inhibits thrombin-induced vascular smooth muscle cell proliferation and attenuates intimal thickening after balloon injury

Summary

Proliferation of vascular smooth muscle cells (VSMCs) is postulated to be one of the key events in the pathogenesis of atherosclerosis and restenosis. We investigated whether YD-3, a lowmolecular weight, non-peptide compound, could modulate proliferation of VSMCs in vitro and restenosis after balloon angioplasty in vivo. We examined the effect of YD-3 on thrombininduced VSMC proliferation by [³H]thymidine incorporation assay. The data demonstrated that YD-3 inhibited VSMC proliferation in a concentration-dependent manner. To define the mechanisms of YD-3 action, we found that YD-3 showed a profound inhibition on thrombin-induced Ras and ERK1/2 activities by using Western blotting analysis. Furthermore, oral administration of YD-3 exhibited a marked reduction in neointimal thickness using the carotid injury model in rats. Using immunochemical detection, our experiments also revealed that YD-3 significantly suppressed expression of the PAR-1 receptor, and markedly inhibited PAR-1-activating peptide (SFLLRN)-induced VSMC proliferation in a concentration-dependent manner. These results suggest that YD-3 inhibits thrombin-induced VSMC growth via the Rasand ERK1/2-mediated signaling pathway. Moreover, YD-3 also shows a developmental potential in the treatment of atherosclerosis and restenosis after vascular injury.

Keywords

YD-3 - vascular smooth muscle cells - thrombin - restenosis - signal transduction

^* Chieh-Yu Peng and Shiow-Lin Pan contributed equally to this work

• References

• 1 Eidt JF, Allison P, Noble S. et al. Thrombin is an important mediator of platelet aggregation in stenosed canine coronary arteries with endothelial injury. J Clin Invest 1989; 84: 18-27.
• 2 McNamara CA, Sarembock IJ, Gimple LW. et al. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993; 91: 94-8.
• 3 Vu TK, Hung DT, Wheaton VI. et al. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-68.
• 4 Ishihara H, Connolly AJ, Zeng D. et al. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 1997; 386: 502-6.
• 5 Kahn ML, Zheng YW, Huang W. et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-4.
• 6 Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci USA 1999; 96: 11023-7.
• 7 Fuster V, Badimon L, Badimon JJ. et al. The pathogenesis of coronary artery disease and the acute coronary syndromes (1). N Engl J Med 1992; 326: 242-50.
• 8 Libby P, Schwartz D, Brogi E. et al. A cascade model for restenosis. A special case of atherosclerosis progression. Circulation 1992; 86: 11147-52.
• 9 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362: 801-9.
• 10 Davies MG, Hagen PO. Pathobiology of intimal hyperplasia. Br J Surg 1994; 81: 1254-69.
• 11 Bauters C, Isner JM. The biology of restenosis. Prog Cardiovasc Dis 1997; 40: 107-16.
• 12 Takada M, Tanaka H, Yamada T. et al. Antibody to thrombin receptor inhibits neointimal smooth muscle cell accumulation without causing inhibition of platelet aggregation or altering hemostatic parameters after angioplasty in rat. Circ Res 1998; 82: 980-7.
• 13 Wilcox JN, Rodriguez J, Subramanian R. et al. Characterization of thrombin receptor expression during vascular lesion formation. Circ Res 1994; 75: 1029-38.
• 14 Nelken NA, Soifer SJ, O’Keefe J. et al. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest 1992; 90: 1614-21.
• 15 Wu CC, Huang SW, Hwang TL. et al. YD-3, a novel inhibitor of protease-induced platelet activation. Br J Pharmacol 2000; 130: 1289-96.
• 16 Wu CC, Hwang TL, Liao CH. et al. Selective inhibition of protease-activated receptor 4dependent platelet activation by YD-3. Thromb Haemost 2002; 87: 1026-33.
• 17 Wu CC, Hwang TL, Liao CH. et al. The role of PAR4 in thrombin-induced thromboxane production in human platelets. Thromb Haemost 2003; 90: 299-308.
• 18 Cheung WM, D’Andrea MR, AndradeGordon P. et al. Altered vascular injury responses in mice deficient in protease-activated receptor-1. Arterioscler Thromb Vasc Biol 1999; 19: 3014-24.
• 19 Pan SL, Guh JH, Huang YW. et al. Inhibition of Ras-mediated cell proliferation by benzyloxybenzaldehyde. J Biomed Sci 2002; 09: 622-30.
• 20 Cobb MH, Hepler JE, Cheng M. et al. The mitogen-activated protein kinases, ERK1 and ERK2. Semin Cancer Biol 1994; 05: 261-8.
• 21 Lowes VL, Ip NY, Wong YH. Integration of signals from receptor tyrosine kinases and g protein-coupled receptors. Neurosignals 2002; 11: 5-19.
• 22 Lin CC, Shyr MH, Chien CS. et al. Thrombinstimulated cell proliferation mediated through activation of Ras/Raf/MEK/MAPK pathway in canine cultured tracheal smooth muscle cells. Cell Signal 2002; 14: 265-75.
• 23 Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest 1983; 49: 327-33.
• 24 Rivard A, Andres V. Vascular smooth muscle cell proliferation in the pathogenesis of atherosclerotic cardiovascular diseases. Histol Histopathol 2000; 15: 557-71.
• 25 Walz DA, Anderson GF, Ciaglowski RE. et al. Thrombin-elicited contractile responses of aortic smooth muscle. Proc Soc Exp Biol Med 1985; 180: 518-26.
• 26 Hatton MW, Moar SL, Richardson M. Deendothelialization in vivo initiates a thrombogenic reaction at the rabbit aorta surface. Correlation of uptake of fibrinogen and antithrombin III with thrombin generation by the exposed subendothelium. Am J Pathol 1989; 135: 499-508.
• 27 Freedman JE. Thrombin, thrombomodulin, and extracellular signal-regulated kinases regulating cellular proliferation. Circ Res 2001; 88: 651-3.
• 28 Schieffer B, Drexler H, Ling BN. et al. G protein-coupled receptors control vascular smooth muscle cell proliferation via pp60csrc and p21ras. Am J Physiol 1997; 272: C2019-30.
• 29 Jones SM, Kazlauskas A. Growth factordependent signaling and cell cycle progression. FEBS Lett 2001; 490: 110-6.
• 30 Luttrell LM. Activation and targeting of mitogen-activated protein kinases by G-proteincoupled receptors. Can J Physiol Pharmacol 2002; 80: 375-82.
• 31 Campbell SL, Khosravi-Far R, Rossman KL. et al. Increasing complexity of Ras signaling. Oncogene 1998; 17: 1395-413.
• 32 Ayllon V, Rebollo A. Ras-induced cellular events (review). Mol Membr Biol 2000; 17: 65-73.
• 33 Wagner AC, Williams JA. Low molecular weight GTP-binding proteins: molecular switches regulating diverse cellular functions. Am J Physiol 1994; 266: G1-14.
• 34 Chen LB, Buchanan JM. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc Natl Acad Sci U S A 1975; 72: 131-5.
• 35 Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res 1985; 56: 139-45.
• 36 Macfarlane SR, Seatter MJ, Kanke T. et al. Proteinase-activated receptors. Pharmacol Rev 2001; 53: 245-82.