Background-Increased activity of Rho-kinase causes hypercontraction of vascular smooth muscle and has been implicated as playing a pathogenetic role in divergent cardiovascular diseases such as coronary artery spasm. We examined whether an intracoronary infusion of fasudil, a selective Rho-kinase inhibitor, would attenuate coronary vasoconstrictor responses to acetylcholine (ACh) in patients with vasospastic angina. Methods and Results-We studied 20 consecutive patients in whom coronary artery spasm was provoked by intracoronary ACh. The patients underwent a second ACh challenge after pretreatment with intracoronary saline (nϭ5) or fasudil (nϭ15; 300 g/min for 15 minutes). Angina and coronary vasospasm were reproducibly induced by the second testing in patients who received saline. In contrast, fasudil markedly attenuated the coronary constriction induced by ACh (PϽ0.001) and prevented the occurrence of chest pain and ischemic ECG changes in all treated patients (both PϽ0.01 versus saline). Fasudil, at the dose used in this study, did not significantly change systemic hemodynamics or baseline coronary blood flow. Conclusions-Fasudil was effective in preventing ACh-induced coronary artery spasm and resultant myocardial ischemia in patients with vasospastic angina. We suggest that this Rho-kinase inhibitor may be a novel therapeutic intervention to treat ischemic coronary syndromes caused by coronary artery spasm. Key Words: angina Ⅲ ischemia Ⅲ circulation C oronary artery spasm is the underlying mechanism in a broad spectrum of ischemic heart diseases, including Prinzmetal's variant angina, unstable angina, acute myocardial infarction, and sudden cardiac death. 1 Recently, we demonstrated that increased activity of the Rho-kinasemediated pathway in vascular smooth muscle plays a central role in the genesis of enhanced vasoconstriction in animal models of hypertension 2 and coronary artery spasm. 3,4 However, it remains to be determined whether the inhibition of this Rho-kinase pathway will provide a novel therapeutic approach in patients with vasospastic disorders. Thus, in the present study, we tested our hypothesis that pretreatment with intracoronary fasudil, a potent Rho-kinase inhibitor, will inhibit coronary vasoconstrictor responses to acetylcholine (ACh) and prevent myocardial ischemia in patients with vasospastic angina. See p 1520 Methods PatientsWe studied 20 consecutive patients (10 men and 10 women; mean age, 62 years; range, 49 to 74 years) with rest angina who underwent coronary arteriography and had a positive ACh challenge. Study ProtocolsThe study protocol was approved by the Institutional Ethics Committee on Human Research, and we obtained written informed consent from each patient before the study.The protocol of our spasm provocation testing was reported previously. 5,6 Briefly, we infused graded doses of ACh (10, 30, and 100 g) into the left coronary artery. Coronary artery spasm was defined as Ͼ75% diameter reduction compared with the diameter after the infusion of intracoronary isosorbide ...

The long-term administration of N -nitro-L -arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, produces coronary vascular remodeling and myocardial hypertrophy in animals. This study used a rat model to investigate the role of angiotensin I converting enzyme (ACE) in the pathogenesis of such changes. We studied the following groups, all of which received drug treatment in their drinking water: untreated controls, and those administered L-NAME, L-NAME, and an ACE inhibitor (ACEI), and L-NAME and hydralazine. Cardiovascular structural changes and tissue ACE activities were evaluated after the first, fourth, and eighth week of treatment. In rats treated with L-NAME alone, vascular remodeling was evident at the fourth and eighth week, and myocardial hypertrophy was present at the eighth week of treatment. The vascular and myocardial remodeling were characterized by increased tissue ACE activities and immunodetectable ACE in those tissues. These changes were markedly reduced by ACEI, but not by hydralazine treatment.

Background-Monocyte infiltration into the arterial wall and its activation is the central event in atherogenesis. Thus, monocyte chemoattractant protein-1 (MCP-1) might be a novel therapeutic target against atherogenesis. We and others recently reported that blockade or abrogation of the MCP-1 pathway attenuates the initiation of atheroma formation in hypercholesterolemic mice. It remains unclear, however, whether blockade of MCP-1 can limit progression or destabilization of established lesions. Methods and Results-We report here that blockade of MCP-1 by transfecting an N-terminal deletion mutant of the MCP-1 gene limited progression of preexisting atherosclerotic lesions in the aortic root in hypercholesterolemic mice.In addition, blockade of MCP-1 changed the lesion composition into a more stable phenotype, ie, containing fewer macrophages and lymphocytes, less lipid, and more smooth muscle cells and collagen. This strategy decreased expression of CD40 and the CD40 ligand in the atherosclerotic plaque and normalized the increased chemokine (RANTES and MCP-1) and cytokine (tumor necrosis factor ␣, interleukin-6, interleukin-1␤, and transforming growth factor ␤ 1 ) gene expression. These data suggest that MCP-1 is a central mediator in the progression and destabilization of established atheroma. Key Words: gene therapy Ⅲ atherosclerosis Ⅲ leukocytes Ⅲ inflammation Ⅲ lymphocytes A therosclerosis and its complications are the major cause of death in Western countries. Atherosclerosis is now recognized to involve chronic inflammatory and immune responses. 1,2 A considerable body of evidence supports the notion that various mediators such as adhesion molecules, cytokines, and chemokines are involved in the early initiation of atherosclerotic lesions. 1,2 The precise molecular mechanism underlying later complications of atherosclerosis, however, remains unclear. Investigation of this mechanism is clinically very important, because atherosclerotic complications such as acute myocardial infarctions and stroke develop during the later stages of atherosclerosis. Recently, several groups established that the immune mediator CD40 ligand (CD40L) and its receptor CD40 are crucial not only in the initiation of atheroma formation but also in the progression and destabilization of established atheroma. 3,4 CD40 receptor binding induces production of inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs), and tissue factors in atheroma, which weakens the collagen frame of the plaque and renders it prone to rupture and thrombosis. 5,6 There are likely to be other potential mediators, however, that might contribute to the progression and destabilization of established atheroma. Conclusions-TheMonocyte chemoattractant protein-1 (MCP-1), a C-C chemokine, controls chemotaxis of mononuclear cells. 7,8 MCP-1 and its receptor (CCR2) pathway recently attracted much attention, because the MCP-1/CCR2 pathway seems to be involved in the inflammatory aspect of atherogenesis. Atheroma-forming cells (endothelial cells, smooth mu...

Abstract-Nltnc oxide (NO) plays an important role not only m the regulation of blood vessel tone, but also m the growth of vascular smooth muscle cells (VSMC) The precise mechamsm involved m the mhlbltlon of VSMC growth by NO 1s not known To further explore the effect of NO on VSMC growth, we examined the effect of NO on the expression of anglotensm II type 1 receptor (AT,-R) that Key Words: vascular smooth muscle cells n anglotensm II receptor w NO w gene transcnptlon P rohferatlon of vascular smooth muscle cells (VSMC) contnbutes to pathological changes of vascular wall such as vascular remodeling, medial hyperplasla, and neomtlmal formation associated with hypertension, atherosclerosis, and vascular qury ' In addition, VSMC produce matrix component of vascular wall such as collagen, fibronectm, and growth factors such as platelet-denved growth factor (PDGF) The matnx and growth factors also play important roles m the process of vascular remodehng and atherogenesls Therefore, understanding the mechanism of VSMC prohferatlon 1s of great mterest m vascular biology and chmcal mvestlgatlon Nltnc oxide (NO) 1s produced by a vanety of mammahan cells such as endothehum, neuron, macrophages, and VSMC fi-om L-arguune by NO synthase (NOS) *J Release of NO stimulates soluble guanylyl cyclase, lea&ng to an increase of intracellular cychc guanosine monophosphate (cGMP) level NO dilates blood vessel and inhibits prohferatlon of VSMC and platelet aggregation 3 These propemes are anti-atherogemc, and decreased NOS actnq 1s beheved to be one of the important feature of early atherogemc process ' Supplementahon of L-argmme, the precursor of NO, lessens the extent of atherosclerosis in diet-induced hypercholesterolermc rabbit 4 In vlvo transfer of type III NOS gene Into the balloon-injured artery decreased neolntlmal VSMC prohferahon 5 However, the precise mechamsm of antl-atherogemc property of NO 1s not completely understood Anglotensm (Ang) II 1s an important vasoactlve peptlde and regulates blood pressure, fluld homeostasls, and electrolyte balance by vasoconstnctlon, faclhtatlon of adrenergc nerve actlvlty, and secretion of aldosterone from the adrenal gland " Recent studies have shown that Ang II IS a growth factor of VSMC and endothehum and plays an important role m atherosclerosis The physlologcal function of Ang II IS transmitted mto target cells via its specific receptor located m the cell membrane There are two lsoforrns for Ang II receptor, which are designated as type 1 receptor (AT,-R)" and type 2 receptor (AT,-R) '" " Losartan that binds to AT,-R and PD123319 that binds to AT*-R are considered to be lsoform speclfk antagonist Although emergmg evidences suggest that AT,-R also plays an important role m the regulation of blood pressure,12 growth mhlbltlon," and apoptosa,'4 most of the cardiovascular effects of Ang II has been believed to be medlated by AT,-R"In rodents, two subtypes of AT, receptor are cloned and named AT,a and AT,b I5 Cultured VSMC express only AT,a-R, and Ang II induces PDGF-A chain, transform...

Abstract-Neointimal hyperplasia is a major cause of restenosis after coronary intervention. Because vascular injury is now recognized to involve an inflammatory response, monocyte chemoattractant protein-1 (MCP-1) might be involved in underlying mechanisms of restenosis. In the present study, we demonstrate the important role of MCP-1 in neointimal hyperplasia after cuff-induced arterial injury. In the first set of experiments, placement of a nonconstricting cuff around the femoral artery of intact mice and monkeys resulted in inflammation in the early stages and subsequent neointimal hyperplasia at the late stages. We transfected with an N-terminal deletion mutant of the human MCP-1 gene into skeletal muscles to block MCP-1 activity in vivo. This mutant MCP-1 works as a dominant-negative inhibitor of MCP-1. This strategy inhibited early vascular inflammation (monocyte infiltration, increased expression of MCP-1, and inflammatory cytokines) and late neointimal hyperplasia. In the second set of experiments, the cuff-induced neointimal hyperplasia was found to be less in CCR2-deficient mice than in control CCR2 ϩ/ϩ mice. The MCP-1/CCR2 pathway plays a central role in the pathogenesis of neointimal hyperplasia in cuffed femoral artery of mice and monkeys. Therefore, the MCP-1/CCR2 pathway can be a therapeutic target for human restenosis after coronary intervention. Key Words: remodeling Ⅲ growth substances Ⅲ inflammation Ⅲ monocytes Ⅲ gene transfer N eointimal hyperplasia is an essential stage in the development of restenosis after coronary intervention as well as atherosclerosis. 1 Therefore, studying the mechanism of neointimal hyperplasia in animals is indispensable to clarifying the underlying mechanisms and exploring the new treatment for vascular diseases. Recent evidence suggests that vascular injury may involve an inflammatory response that accelerates the recruitment and activation of monocytes through the activation of chemotactic factors including monocyte chemoattractant protein-1 (MCP-1). 2-4 MCP-1 is a potent chemotactic factor for monocytes. 5,6 Eliminating MCP-1 gene or blockade of MCP-1 signals has been shown to decrease atherogenesis in hypercholesterolemic mice. [7][8][9] However, no prior study addressed the definite role of monocytes or MCP-1-mediated signals in the development of neointimal hyperplasia after periarterial injury. Placement of nonconstricting cuff around the artery induces vascular inflammation at the early stages and subsequently causes intimal hyperplasia at the late stages. 10 -12 Wu et al 12 recently reported that angiotensin II type 1 receptor blockade attenuated vascular inflammation (monocyte infiltration and activation, upregulation of MCP-1 and inflammatory cytokines) induced by perivascular cuff placement. The latter study suggests the importance of MCP-1-mediated inflammation in mediating the formation of neointimal hyperplasia. However, no prior study has addressed the role of MCP-1 in the pathogenesis of neointimal hyperplasia after cuff placement.In the present st...

Abstract-Chronic inhibition of endothelial NO synthesis by the administration of N G -nitro-L-arginine methyl ester (L-NAME) to rats induces early vascular inflammation (monocyte infiltration into coronary vessels and monocyte chemoattractant protein-1 expression) as well as subsequent arteriosclerosis. The small GTPase Rho controls cell adhesion, motility, and proliferation and is activated by several growth factors such as angiotensin II. We investigated the effect of a specific inhibitor of Rho-kinase, Y-27632, in rats treated with L-NAME to determine the role of the Rho/Rho-kinase pathway in the development of arteriosclerosis. We found here increased activity of Rho/Rho-kinase after L-NAME administration and its prevention by angiotensin II type 1 receptor blockade. Hydralazine or lecithinized superoxide dismutase (l-SOD) did not affect Rho/Rho-kinase activity. Co-treatment with Y-27632 did not affect the L-NAME-induced increase in cardiovascular tissue ACE activity or L-NAME-induced decrease in plasma NO concentrations, but did prevent the L-NAME-induced early inflammation and late coronary arteriosclerosis. In addition, Y-27632 prevented the increased gene expression of monocyte chemoattractant protein-1 and transforming growth factor-␤1 as well as cardiac fibrosis and glomerulosclerosis. These findings suggest that increased activity of Rho/Rho-kinase pathway mediated via the angiotensin II type 1 receptor may thus be important in the pathogenesis of early vascular inflammation and late remodeling induced by chronic inhibition of NO synthesis. The beneficial effects of Rho-kinase inhibition are not mediated by restoration of NO production. The Rho-kinase pathway could be a new therapeutic target for treatment of vascular diseases. Key Words: Rho-kinase Ⅲ arteriosclerosis Ⅲ remodeling Ⅲ nitric oxide Ⅲ angiotensin R ecent studies have shown that the small GTPase Rho controls multiple cell functions-such as adhesion, proliferation, or migration through the actin cytoskeleton reorganization-or some kinase cascade activation. 1-4 Because angiotensin II 5,6 or endothelin-1 7 has been shown to activate the Rho pathway, its role in the pathogenesis of vascular disorders has become a focus of interest. Indeed, inhibitors of Rho or Rhokinase (a major target of Rho) have been shown to decrease systolic blood pressure in hypertensive rats, 8 to reduce brain infarct size after middle cerebral artery occlusion in mice, 9 and to suppress neointimal formation after balloon injury. 10 We have recently reported that chronic inhibition of nitric oxide synthesis by the administration of 78 N G -nitro-Larginine methyl ester (L-NAME) induces early inflammation (monocyte infiltration and monocyte chemoattractant protein-1 [MCP-1] expression) 11 and late cardiovascular remodeling in rats. 12 The importance of our observation is supported by the fact that the adhesion of mononuclear cells to, and their infiltration into, the blood vessel wall are assumed to be crucial early arteriosclerotic events. 13 We have further demonstrate...

Increased Ang II activity mediated via the type 1 receptor may thus be important in the pathogenesis of early cardiovascular inflammatory changes in this model. Endothelium-derived NO may decrease MCP-1 production and oxidative stress-sensitive signals by suppressing localized activity of Ang II.

Background-It remains unclear whether vascular endothelial growth factor (VEGF) is a proarteriosclerotic or an antiarteriosclerotic factor. We recently reported that long-term inhibition of nitric oxide by administering N-nitro-Larginine methyl ester (L-NAME) induces coronary vascular inflammation and arteriosclerosis. Methods and Results-We used this animal model to investigate the role of VEGF in arteriosclerosis. We blocked VEGF activity in vivo by transfecting with plasmid DNA encoding the murine soluble FLT-1 (sFLT-1) gene into thigh muscle. Soluble FLT-1 can suppress VEGF activity both by sequestering VEGF and by functioning as a dominant-negative inhibitor of VEGF receptors. We observed vascular inflammation associated with increased VEGF expression within 3 days of L-NAME administration, which was prevented by pretreatment with ACE inhibitor, angiotensin II receptor antagonist, or neutralizing monocyte chemoattractant protein-1 antibody. The sFLT-1 gene transfer attenuated the early vascular inflammation and prevented late arteriosclerosis. The sFLT-1 gene transfer also inhibited increased expression of monocyte chemoattractant protein-1 and transforming growth factor-␤, indicating creation of a positive feedback loop to cause arteriosclerosis. Conclusions-VEGF is necessary in the development of arteriosclerosis by mediating monocyte recruitment and activation in this model.