(8) Vascular Biology (Arteriosclerosis)

Our research group has done a lot of research on Rho/Rho-kinase pathway, which has recently attracted much attention in various research fields, especially in the cardiovascular research field.

What is Rho-kinase?

Recent advances in molecular biology have elucidated the substantial involvement of intracellular signaling pathways mediated by small GTP-binding proteins (G proteins), such as Rho, Ras, Rab, Sarl/Arf, and Ran families. In mid 1990s, 2 Japanese groups and 1 Singapore group independently identified one of the effectors of Rho and termed it as Rho-kinase. Rho-kinase is widely expressed in the body and intervenes through agonist stimulation in the intracellular pathway of communication to participate in cell function in a variety of ways including the contraction and relaxation of the smooth muscle cells, reconstitution of the actin skeleton, the adhesion, migration, and propagation of cells, and gene expression (Figure 1). Of these, much attention has been paid to the contraction and relaxation of vascular smooth muscle of the Rho/Rho-kinase pathway.

Physiological functions in which Rho-kinase participates (vascular smooth muscle contraction and relaxation)

The phosphorylation of myosin light chain (MLC) that is determined by the balance between myosin light chain kinase (MLCK) activation and myosin light chain phosphatase (MLCPh) activation plays a crucial role in the contraction and relaxation of vascular smooth muscle cells. It is widely known that the contraction of vascular smooth muscle cells is augmented, which depends on intracellular Ca2+ concentrations. However, independent of the intracellular Ca2+ concentration, the small G protein Rho is activated via the receptor conjugate with the G protein by the stimulation from the constrictive vasoactive agent, followed by the activation of Rho-kinase, one of the effectors of Rho. The activated Rho-kinase enhances the myosin phosphatase activity by phosphorylating the myosin-binding subunit (MBS) of the enzyme and thus augments vascular smooth muscle cell contraction at a given intracellular calcium concentration (Figure 2). Therefore, Rho-kinase inhibition suppresses myosin phosphatase through phosphorylation of MBS with a resultant increase in MLC phosphorylations and hyperconstriction.

Cardiovascular Disease and Rho-kinase Inhibitors

We have demonstrated that Rho-kinase is a novel therapeutic target in a various cardiovascular diseases, including coronary artery spasms, hypertension, pulmonary hypertension, and ischemia/reperfusion injury, as well as arteriosclerotic diseases (Figure 3).

In addition, it also has been reported that the activated Rho-kinase is associated with the various disorders other than cardiovascular disease, and that many investigators have reported the beneficial effects of its inhibition. The current clinically available Rho-kinase inhibitor is only fasudil, which is an intravenous drug that suppresses cerebrovascular spasms; however, approximately 15 companies in Japan and abroad are working to develop selective Rho-kinase inhibitors with the aim for most of these to be used for cardiovascular disease.

Studies Using Swine Arteriosclerosis Models

We have demonstrated that both in vivo gene transfer of dominant-negative Rho-kinase and long-term treatment with a Rho-kinase inhibitor suppress balloon injury-induced neointimal formation in animals in vivo. Long-term treatment with MCP-1 and oxidized low-density lipoproteins (ox-LDL) causes vascular lesions characterized by neointimal formation and constrictive remodeling in porcine coronary arteries in vivo (Figure 4). Further, the chronic oral treatment with fasudil for recurrent stenosis after coronary stenting in a porcine model also suppresses this arteriosclerotic formation. It should be noted that in an IL-1beta induction model the formed arteriosclerosis lesion is also regressed by chronic administration of fasudil. Rho-kinase activity was accentuated in the lesion areas of all of these arteriosclerosis models. These results suggest that suppressing of Rho-kinase should be a new therapeutic target for arteriosclerosis as well as acute coronary syndromes such as acute myocardial infarction and unstable angina.

Study Using a Porcine Coronary Spasm Model

Coronary vasospasm plays an important role in a wide variety of ischemic heart diseases, not only in variant angina but also in other forms of angina pectoris, myocardial infarction, and sudden death. Accumulating evidence indicates that Rho-kinase is substantially involved in the pathogenesis of coronary vasospasm. Intracoronary administration of fasudil and of hydroxyfasudil markedly inhibits coronary spasm by serotonin in a porcine model with long-term treatment with IL-1beta (Figure 5). This also is the case in other porcine models of coronary spasm with long-term treatment with MCP-1 and remnant lipoproteins (from patients with sudden cardiac death). Our tension study in vitro using isolated arteries has indicated that the serotonin-induced coronary constraction and MLC phosphorylation are associated with the Rho-kinase activation. Further, mRNA levels of Rho-kinase are increased in coronary hyperconstrictive segments, in which a Rho-kinase inhibitor, Y-27632, significantly suppressed this enhancement. These results suggest that vascular smooth muscle hyperconstriction mediated by activated Rho-kinase plays a key role in patients with coronary artery spasm and that Rho-kinase inhibition is an important therapeutic strategy for vasospastic angina

Together with the Department's Circulation Group, we are currently proceeding with basic and clinical research on fasudil Rho-kinase inhibitors for pulmonary hypertension. The results from recent fundamental research show that fasudil has a very high therapeutic potential for pulmonary hypertension. In addition, we have recently demonstrated the acute beneficial effects of intravenous fasudil in patients with severe pulmonary hypertension without adverse effects. We plan to perform clinical trials with a long-term oral treatment with fasudil in patients with PAH in Japan.

Arteriosclerosis/Pulmonary Hypertension, etc.

We have done fundamental research on arteriosclerosis and inflammation (Circulation 1997, 2001, JACC2003, etc.) and consider that it is important to treat arteriosclerosis qualitatively, as well as quantitatively, as a strategy for cardiovascular diseases. In the ischemic diseaes, the improvement of vascular endothelium function, atherosclerotic plaque stability including suppression of macrophage activation and increase of collagen content, are very important for the therapeutic strategy (Circulation 2001, 2004). Rho-kinase pathway also contributes to a decline in vascular dysfunction, such as vascular spasms (ATVB 2004, J Cardivasc Pharmacol 2004, 2007, etc.). Further, we have recently demonstrated the importance of the erythropoietin receptor pathway in angiogenesis for severe ischemia (Circ Res 2007) and the vascular smooth muscle cell function by mechanical stimuli such as pulse pressure, stretch, and shear stress from our proprietary in vitro system (JCardiovasc Pharmacol 2008). Further, we demonstrated the importance of the Rho-kinase pathway and erythropoietin system also in pulmonary hypertension (Circ Res 2004, Circulation 2006, JCardiovasc Pharmacol 2006, 2007, etc.).

Publications (since 2000)

1. Satoh K, Nigro P, Matoba T, O'Dell MR, Cui Z, Shi X, Mohan A, Yan C, Abe J, Illig KA, Berk BC. Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms. Nat Med.2009;15:649-656.
2. Satoh K, Fukumoto Y, Nakano M, Ishii N, Sugamura K, Shimokawa H. Statin Ameliorates Hypoxia-Induced Pulmonary Hypertension Associated with Down-regulated Stromal Cell-Derived Factor-1. Cardiovac Res. 2009;81:226-234.
3. Satoh K, Matoba T, Suzuki J, O'Dell MR, Nigro P, Berk BC. Cyclophilin A mediates vascular remodeling by promoting inflammation and Vascular Smooth Muscle Cell proliferation. Circulation 2008;117:3088-3098.
4. Satoh K, Berk BC. Circulating smooth muscle progenitor cells: novel players in plaque stability. Cardiovasc Res. 2008;77:445-447.
5. Fukui S, Fukumoto Y, Suzuki J, Saji K, Nawata J, Tawara S, Shinozaki T, Kagaya Y, Shimokawa H. Long-term Inhibition of Rho-kinase Ameliorates Diastolic Heart Failure in Hypertensive Rats. J Cardiovasc Pharmacol. 2008 ;51:317-326.
6. Oi K, Fukumoto Y, Ito K, Uwatoku T, Abe K, Hizume T, Shimokawa H. Extracorporeal shock wave therapy ameliorates hindlimb ischemia in rabbits. Tohoku J Exp Med. 2008 ;214:151-8.
   
7. Onoue N, Nawata J, Tada T, Zhulanqiqige D, Wang H, Sugimura K, Fukumoto Y, Shirato K, Shimokawa H. Increased Static Pressure Promotes Migration of Vascular Smooth Muscle Cells: Involvement of the Rho-kinase Pathway. J Cardiovasc Pharmacol. 2008 ;51:55-61.
8. Nakano M, Satoh K, Fukumoto Y, Ito Y, Kagaya Y, Ishii N, Sugamura K, Shimokawa H. Important Role of Erythropoietin Receptor to Promote VEGF Secretion and Angiogenesis in Peripheral Ischemia in Mice. Circ Res. 2007;100:662-669.
   
9. Saji K, Fukumoto Y, Suzuki J, Fukui S, Nawata J, Shimokawa H. Colchicine, a microtubule depolymerizing agent, inhibits myocardial apoptosis in rats. Tohoku J Exp Med. 2007 ;213:139-48.
   
10. Tawara S, Fukumoto Y, Shimokawa H. Effects of Combined Therapy With a Rho-Kinase Inhibitor and Prostacyclin on Monocrotaline-Induced Pulmonary Hypertension in Rats. J Cardiovasc Pharmacol. 2007 ;50:195-200.
    
11. Shimokawa H, Rashid M. Development of Rho-kinase inhibitors for cardiovascular medicine. Trends Pharmacol Sci. 2007;28:296-302.
12. Fukumoto Y, Mohri M, Inokuchi K, Ito A, Hirakawa Y, Masumoto A, Hirooka Y, Takeshita A, Shimokawa H. Anti-ischemic effects of fasudil, a specific Rho-kinase inhibitor, in patients with stable effort angina. J Cardiovasc Pharmacol. 2007;49:117-21.
13. Fukumoto Y, Tawara S, Shimokawa H. Recent progress in the treatment of pulmonary arterial hypertension: expectation for rho-kinase inhibitors. Tohoku J Exp Med. 2007;211:309-20.
14. Jiang BH, Tawara S, Abe K, Takaki A, Fukumoto Y, Shimokawa H. Acute vasodilator effect of fasudil, a Rho-kinase inhibitor, in monocrotaline-induced pulmonary hypertension in rats. J Cardiovasc Pharmacol. 2007;49:85-9.
15. Satoh K, Kagaya Y, Nakano M, Ito Y, Ohta J, Tada H, Karibe A, Minegishi N, Suzuki N, Yamamoto M, Ono M, Watanabe J, Shirato K,Ishii N, Sugamura K, Shimokawa H.? Important Role of Endogenous Erythropoietin System to Recruit Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice.?? Circulation2006;113:1442-1450.
16. Abe K, Tawara S, Oi K, Hizume T, Uwatoku T, Fukumoto Y, Kaibuchi K, Shimokawa H. Long-term inhibition of Rho-kinase ameliorates hypoxia-induced pulmonary hypertension in mice. J Cardiovasc Pharmacol. 2006;48:280-5.
17. Hizume T, Morikawa K, Takaki A, Abe K, Sunagawa K, Amano M, Kaibuchi K, Kubo C, Shimokawa H. Sustained elevation of serum cortisol level causes sensitization of coronary vasoconstricting responses in pigs in vivo: a possible link between stress and coronary vasospasm. Circ Res. 2006;99:767-75.
18. Shimokawa H, Takeshita A. Rho-kinase is an important therapeutic target in cardiovascular medicine. Arterioscler Thromb Vasc Biol. 2005;25:1767-75.
19. Fukumoto Y, Matoba T, Ito A, Tanaka H, Kishi T, Hayashidani S, Abe K, Takeshita A, Shimokawa H. Acute vasodilator effects of a Rho-kinase inhibitor, fasudil, in patients with severe pulmonary hypertension. Heart. 2005;91:391-2.
20. Yada T, Shimokawa H, Hiramatsu O, Kajita T, Shigeto F, Tanaka E, Shinozaki Y, Mori H, Kiyooka T, Katsura M, Ohkuma S, Goto M, Ogasawara Y, Kajiya F. Beneficial effect of hydroxyfasudil, a specific Rho-kinase inhibitor, on ischemia/reperfusion injury in canine coronary microcirculation in vivo. J Am Coll Cardiol. 2005;45:599-607.
21. Abe K, Morikawa K, Hizume T, Uwatoku T, Oi K, Seto M, Ikegaki I, Asano T, Kaibuchi K, Shimokawa H. Prostacyclin does not inhibit rho-kinase: an implication for the treatment of pulmonary hypertension. J Cardiovasc Pharmacol. 2005;45:120-4.
22. Hiroki J, Shimokawa H, Mukai Y, Ichiki T, Takeshita A. Divergent effects of estrogen and nicotine on Rho-kinase expression in human coronary vascular smooth muscle cells. Biochem Biophys Res Commun. 2005;326:154-9.
23. Hiroki J, Fukumoto Y, Shimokawa H, Hirooka Y, Takeshita A. [Inhibition of Rho-kinase by fasudil preventing anginal attacks associated with spastic angina: a case report] J Cardiol. 2004;44:161-4.
24. Inokuchi K, Ito A, Fukumoto Y, Matoba T, Shiose A, Nishida T, Masuda M, Morita S, Shimokawa H. Usefulness of fasudil, a Rho-kinase inhibitor, to treat intractable severe coronary spasm after coronary artery bypass surgery. J Cardiovasc Pharmacol. 2004;44:275-7.
25. Shiotani S, Shimada M, Suehiro T, Soejima Y, Yosizumi T, Shimokawa H, Maehara Y. Involvement of Rho-kinase in cold ischemia-reperfusion injury after liver transplantation in rats. Transplantation. 2004;78:375-82.
26. Hiroki J, Shimokawa H, Higashi M, Morikawa K, Kandabashi T, Kawamura N, Kubota T, Ichiki T, Amano M, Kaibuchi K, Takeshita A. Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells. J Mol Cell Cardiol. 2004;37:537-46.
27. Hattori T, Shimokawa H, Higashi M, Hiroki J, Mukai Y, Tsutsui H, Kaibuchi K, Takeshita A. Long-term inhibition of Rho-kinase suppresses left ventricular remodeling after myocardial infarction in mice. Circulation. 2004;109:2234-9.
28. Oi K, Shimokawa H, Hiroki J, Uwatoku T, Abe K, Matsumoto Y, Nakajima Y, Nakajima K, Takeichi S, Takeshita A. Remnant lipoproteins from patients with sudden cardiac death enhance coronary vasospastic activity through upregulation of Rho-kinase. Arterioscler Thromb Vasc Biol. 2004;24:918-22.
29. Abe K, Shimokawa H, Morikawa K, Uwatoku T, Oi K, Matsumoto Y, Hattori T, Nakashima Y, Kaibuchi K, Sueishi K, Takeshit A. Long-term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats. Circ Res. 2004;94:385-93.
30. Hattori T, Shimokawa H, Higashi M, Hiroki J, Mukai Y, Kaibuchi K, Takeshita A. Long-term treatment with a specific Rho-kinase inhibitor suppresses cardiac allograft vasculopathy in mice. Circ Res. 2004;94:46-52.
31. Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Eto Y, Morishige K, Matsumoto Y, Obara K, Nakayama K, Takahashi S, Takeshita A. Evidence for protein kinase C-mediated activation of Rho-kinase in a porcine model of coronary artery spasm. Arterioscler Thromb Vasc Biol. 2003;23:2209-14.
32. 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;24:181-6.
33. Higashi M, Shimokawa H, Hattori T, Hiroki J, Mukai Y, Morikawa K, Ichiki T, Takahashi S, Takeshita A. Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system. Circ Res. 2003;93:767-75.
34. Mohri M, Shimokawa H, Hirakawa Y, Masumoto A, Takeshita A. Rho-kinase inhibition with intracoronary fasudil prevents myocardial ischemia in patients with coronary microvascular spasm. J Am Coll Cardiol. 2003;41:15-9.
35. Shimokawa H, Hiramori K, Iinuma H, Hosoda S, Kishida H, Osada H, Katagiri T, Yamauchi K, Yui Y, Minamino T, Nakashima M, Kato K. Anti-anginal effect of fasudil, a Rho-kinase inhibitor, in patients with stable effort angina: a multicenter study. J Cardiovasc Pharmacol. 2002;40:751-61.
36. Satoh S, Ikegaki I, Toshima Y, Watanabe A, Asano T, Shimokawa H. Effects of Rho-kinase inhibitor on vasopressin-induced chronic myocardial damage in rats. Life Sci. 2002;72:103-12.
37. Masumoto A, Mohri M, Shimokawa H, Urakami L, Usui M, Takeshita A. Suppression of coronary artery spasm by the Rho-kinase inhibitor fasudil in patients with vasospastic angina. Circulation. 2002;105:1545-7.
38. Shimokawa H. Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol. 2002;39:319-27.
39. Kandabashi T, Shimokawa H, Mukai Y, Matoba T, Kunihiro I, Morikawa K, Ito M, Takahashi S, Kaibuchi K, Takeshita A. Involvement of rho-kinase in agonists-induced contractions of arteriosclerotic human arteries. Arterioscler Thromb Vasc Biol. 2002;22:243-8.
40. Morishige K, Shimokawa H, Eto Y, Hoshijima M, Kaibuchi K, Takeshita A. In vivo gene transfer of dominant-negative rho-kinase induces regression of coronary arteriosclerosis in pigs. Ann N Y Acad Sci. 2001;947:407-11.
41. Masumoto A, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, Takeshita A. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension. 2001;38:1307-10.
42. Utsunomiya T, Satoh S, Ikegaki I, Toshima Y, Asano T, Shimokawa H. Antianginal effects of hydroxyfasudil, a Rho-kinase inhibitor, in a canine model of effort angina. Br J Pharmacol. 2001;134:1724-30.
    Sato S, Ikegaki I, Asano T, Shimokawa H. Antiischemic properties of fasudil in experimental models of vasospastic angina. Jpn J Pharmacol. 2001;87:34-40.
43. Ikegaki I, Hattori T, Yamaguchi T, Sasaki Y, Satoh SI, Asano T, Shimokawa H. Involvement of Rho-kinase in vascular remodeling caused by long-term inhibition of nitric oxide synthesis in rats. Eur J Pharmacol. 2001;427:69-75.
44. Shimokawa H, Morishige K, Miyata K, Kandabashi T, Eto Y, Ikegaki I, Asano T, Kaibuchi K, Takeshita A. Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo. Cardiovasc Res. 2001;51:169-77.
45. Morishige K, Shimokawa H, Eto Y, Kandabashi T, Miyata K, Matsumoto Y, Hoshijima M, Kaibuchi K, Takeshita A. Adenovirus-mediated transfer of dominant-negative rho-kinase induces a regression of coronary arteriosclerosis in pigs in vivo. Arterioscler Thromb Vasc Biol. 2001;21:548-54.
46. Mukai Y, Shimokawa H, Matoba T, Kandabashi T, Satoh S, Hiroki J, Kaibuchi K, Takeshita A. Involvement of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension. FASEB J. 2001;15:1062-4.
47. Miyata K, Shimokawa H, Kandabashi T, Higo T, Morishige K, Eto Y, Egashira K, Kaibuchi K, Takeshita A. Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in pigs in vivo. Arterioscler Thromb Vasc Biol. 2000;20:2351-8.
48. 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:H1744-50.
    Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Kawano Y, Fukata Y, Higo T, Egashira K, Takahashi S, Kaibuchi K, Takeshita A. Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1beta. Circulation. 2000;101:1319-23.
49. Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Kawano Y, Fukata Y, Higo T, Egashira K, Takahashi S, Kaibuchi K, Takeshita A. Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1beta. Circulation. 2000;101:1319-23.
50. Shimokawa H. Cellular and molecular mechanisms of coronary artery spasm: lessons from animal models. Jpn Circ J. 2000;64:1-12.

( Text by Yoshihiro Fukumoto, Shunsuke Tahara, Kimio Satoh)

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