Background-Hypoxia-induced pulmonary hypertension is a major cause of morbidity and mortality. Hypoxia induces pulmonary vasoconstriction, in part, by decreasing endothelial nitric oxide synthase (eNOS) expression. The mechanism by which hypoxia decreases eNOS expression is not known but may involve Rho-kinase-induced actin cytoskeletal changes in vascular endothelial cells. Methods and Results-To determine whether hypoxia regulates eNOS expression through Rho-kinase, we exposed human saphenous and pulmonary artery endothelial cells to hypoxia (3% O 2 ) with and without a Rho-kinase inhibitor, hydroxyfasudil (0.1 to 100 mol/L), for various durations (0 to 48 hours). Hypoxia increased Rho-kinase expression and activity by 50% and 74%, decreased eNOS mRNA and protein expression by 66Ϯ3% and 57Ϯ5%, and inhibited eNOS activity by 48Ϯ9%. All of these effects of hypoxia on eNOS were reversed by cotreatment with hydroxyfasudil. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase or Rho-kinase by overexpression of dominant-negative Rho-kinase reversed hypoxia-induced decrease in eNOS expression. Indeed, disruption of the actin cytoskeleton, the downstream target of Rho-kinase, by cytochalasin D also upregulated eNOS expression. Hypoxia reduced eNOS mRNA half-life from 22Ϯ2 to 13Ϯ2 hours, which was reversed by cotreatment with hydroxyfasudil. However, neither hypoxia nor hydroxyfasudil had any effects on eNOS gene transcription. Conclusions-These

Intracellular signaling pathway mediated by Rho/Rho-kinase plays an important role in the regulation of vascular smooth muscle contraction and other cellular functions, including adhesion, migration, proliferation, and hypertrophy. A Rho-kinase inhibitor has been shown to lower blood pressure in hypertensive rats in vivo, however, direct evidence for the involvement of Rho-kinase in hypertensive vascular disease is still lacking. In this study, we tested our hypothesis that Rho-kinase is involved substantially in functional and structural alterations of hypertensive blood vessels in spontaneously hypertensive rats (SHR). Force measurement with isolated blood vessels demonstrated that Rho-kinase was involved significantly in the vascular hyperreactivity in SHR. Biochemical analysis showed that both the expression and the activity of Rho-kinase were augmented in blood vessels of SHR. It is important to note that the involvement of Rho-kinase in the vascular hyperreactivity preceded the development of hypertension. Finally, long-term blockade of Rho-kinase suppressed vascular lesion formation such as medial hepertrophy and perivascular fibrosis in SHR. Our results provide the first evidence that augmented expression and function of Rho-kinase plays a key role in the pathogenesis of hypertensive vascular disease, which suggests that this molecule could be regarded as a novel therapeutic target in hypertension. Key words: vascular smooth muscle cells • Ca 2 -sensitization • vascular lesion formationlthough hypertension is one of the major predisposing risk factors of cardiovascular disease, its pathogenesis still remains to be fully elucidated. It is widely accepted that hypertension is associated with the increased peripheral vascular resistance, which is determined by the functional and structural alterations of resistance arteries (1-3). During agonist-induced contraction of vascular smooth muscle cell (VSMC), phosphorylation of myosin light chain (MLC) is a crucial step for force development. The extent of MLC phosphorylation depends on the balance between the activity of Ca 2+ /calmodulin-dependent myosin light chain kinase (MLCK) and that of myosin phosphatase (MLCPh) (4). It has been demonstrated that Rho-kinase/ROKα/ROCK II (an isoform to p160ROCK/ROKβ/ROCK I) (5), which is activated A

Background-Rho-kinase has been implicated as an important regulator of inflammatory responses mediated by cytokines and chemokines. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined whether long-term blockade of Rho-kinase suppresses LV remodeling in a mouse model of MI in vivo. Methods and Results-Mice underwent ligation of the left coronary artery and were treated with a Rho-kinase inhibitor, fasudil (100 mg · kg Ϫ1 · d Ϫ1 in tap water), for 4 weeks, starting 1 day after the surgery. At 4 weeks, LV infarct size was histologically comparable between the 2 groups. LV cavity dilatation and dysfunction evaluated by echocardiography were significantly suppressed in the fasudil group (PϽ0.05, nϭ15 to 28). The beneficial effects of fasudil were accompanied by suppression of cardiomyocyte hypertrophy and interstitial fibrosis (both PϽ0.01, nϭ6). The expression of inflammatory cytokines, including transforming growth factor (TGF)-␤ 2 , TGF-␤ 3 , and macrophage migration inhibitory factor, was upregulated in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (both PϽ0.05, nϭ10 to 11). Rho-kinase activity as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, was significantly increased in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (PϽ0.05, nϭ5). Conclusions-These results indicate that Rho-kinase is substantially involved in the pathogenesis of LV remodeling after MI associated with upregulation of proinflammatory cytokines, suggesting a therapeutic importance of the molecule for the prevention of post-MI heart failure.

Abstract-Intracellular signaling pathway mediated by small GTPase Rho and its effector Rho-kinase plays an important role in regulation of vascular smooth muscle contraction and other cellular functions. We have recently demonstrated that Rho-kinase is substantially involved in angiotensin II-induced gene expressions and various cellular responses in vitro. However, it remains to be examined whether Rho-kinase is involved in the angiotensin II-induced cardiovascular hypertrophy in vivo and, if so, what mechanisms are involved. Long-term infusion of angiotensin II for 4 weeks caused hypertrophic changes of vascular smooth muscle and cardiomyocytes in rats. Both changes were significantly suppressed by concomitant oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor, hydroxyfasudil, after oral administration. Angiotensin II caused a perivascular accumulation of macrophages and Rho-kinase activation, both of which were also significantly suppressed by fasudil. Vascular NAD(P)H oxidase expression (nox1, nox4, gp91phox, and p22phox) and endothelial production of superoxide anions were markedly increased by angiotensin II, both of which were also significantly suppressed by fasudil. Thus, fasudil ameliorated the impaired endothelium-dependent relaxations caused by angiotensin II without affecting vasodilator function of vascular smooth muscle. These results provide evidence that Rho-kinase is substantially involved in the angiotensin II-induced cardiovascular hypertrophy in rats in vivo. The suppression of endothelial NAD(P)H oxidase upregulation and resultant superoxide production and the amelioration of endothelial vasodilator function may be involved in this process. 4 It has been demonstrated that Rho-kinase/ROK␣/ROCK II (an isoform to p160ROCK/ROK␤/ROCK I), 5 which is an effector of the small GTPase Rho, inhibits MLC phosphatase activity by phosphorylating its myosin-binding subunit 6,7 and thus plays a central role in agonist-induced Ca 2ϩ sensitization and hypercontraction of VSMCs. 4,5,7 We have demonstrated that Rho-kinase plays an important role in angiotensin II-induced mRNA expression of monocyte chemoattractant protein-1 8 and of plasminogen activator inhibitor-1 9 in cultured rat aortic VSMCs. We have recently demonstrated that in cultured human coronary VSMCs, the expression of Rho-kinase is enhanced by inflammatory stimuli, such as angiotensin II and interleukin-1␤ (IL-1␤). 10 However, it remains to be determined whether Rho-kinase is involved in angiotensin II-induced cardiovascular hypertrophy in vivo and, if so, what mechanisms are involved. The present study was thus designed to examine these points in rats in vivo. Materials and Methods AnimalsThis experiment was reviewed and approved by the Committee on Ethics of Animal Experiments of the Kyushu University. A total of 111 adult male WKY rats (14 to 17 weeks old, weighing 300 to 350 g) obtained from the colony at the Kyushu University were used. Animals were anesthetized with intraperitoneal pentobarbital

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Objective-Endothelial vasodilator functions are progressively impaired with aging, which may account in part for the increased incidence of cardiovascular events in elderly people. We examined what treatment could ameliorate the endothelial dysfunction associated with aging in rats. Methods and Results-Aged (12-month-old) Wistar-Kyoto rats were treated with vehicle, temocapril, CS-866 (an angiotensin II type 1 receptor antagonist), cerivastatin, or hydralazine for 2 weeks. Endothelium-dependent relaxations (EDRs) of aortas from aged rats were markedly impaired compared with EDRs of aortas from young (3-month-old) rats. Indomethacin, NS-398 (a cyclooxygenase [COX]-2 inhibitor), and SQ-29548 (a thromboxane A 2 /prostaglandin H 2 receptor antagonist) acutely restored EDRs in aged rats, suggesting an involvement of COX-2-derived vasoconstricting eicosanoids. Tiron, a superoxide scavenger, also partially improved EDRs, suggesting an involvement of superoxide. EDRs were significantly ameliorated in aged rats after long-term treatment with temocapril or CS-866 but not after treatment with cerivastatin or hydralazine. Indomethacin induced no further improvement of EDRs after treatment with temocapril or CS-866. COX-2 protein expression and superoxide production were increased in the aortas of aged rats and were also attenuated by treatment with temocapril or CS-866. Key Words: endothelium-dependent relaxation Ⅲ aging Ⅲ endothelium-derived contracting factors Ⅲ cyclooxygenase Ⅲ renin-angiotensin system T he endothelium plays an important role in modulating vascular tone by synthesizing various vasoactive substances, such as endothelium-derived relaxing factors and endothelium-derived contracting factors. 1-3 The former include NO, prostacyclin (prostaglandin I 2 ), and endotheliumderived hyperpolarizing factor (EDHF), and the latter include cyclooxygenase (COX)-derived eicosanoids, such as thromboxane A 2 (TXA 2 )/endoperoxide (prostaglandin H 2 [PGH 2 ]), endothelin-1, and superoxide anion (O 2 Ϫ ). [1][2][3] It is well known that endothelial dysfunction predisposes an individual to life-threatening cardiovascular events, such as myocardial infarction, and that impairment of endotheliumdependent relaxations (EDRs) is caused by an imbalance between relaxing and contracting factors released from the endothelium. [1][2][3] In addition to other cardiovascular risk factors (eg, hyperlipidemia, hypertension, diabetes mellitus, and smoking), aging is strongly correlated with endothelial dysfunction in animals and humans. 4 -9 However, effective treatment that ameliorates endothelial dysfunction in aged blood vessels still remains to be developed. Conclusions-TheseIt has been reported that aging-related endothelial dysfunction is caused by increased production of COX-derived endothelium-derived contracting factors 4 -6 and superoxide, 7,8 both of which counteract the vasodilating and vasculoprotective effects of NO, whereas the production of NO per se may not be so impaired with aging. 8,10 It has been reported that ACE inhibit...

Restenosis after angioplasty still remains a major problem for which neointimal formation appears to play an important role. Recent studies in vitro suggested that Rho kinase, a target protein of Rho, is important in various cellular functions. We thus examined whether Rho kinase is involved in the restenotic changes after balloon injury. In vivo gene transfer was performed immediately after balloon injury in both sides of the porcine femoral arteries with adenoviral vector encoding either a dominant negative form of Rho kinase (AdDNRhoK) or beta-galactosidase (AdLacZ) as a control. One week after the transfer, immunohistochemistry confirmed the successful gene expression in the vessel wall, whereas 2 wk after the transfer, Western blotting showed the functional upregulation of Rho kinase at the AdLacZ site and its suppression at the AdDNRhoK site. Angiography showed the development of a stenotic lesion at the AdLacZ site where histological neointimal formation was noted, whereas those changes were significantly suppressed at the AdDNRhoK site. These results indicate that Rho kinase is involved in the pathogenesis of neointimal formation after balloon injury in vivo.

Abstract-Cardiac allograft vasculopathy (CAV) continues to be a major cause of late graft failure after cardiac transplantation. We have demonstrated that Rho-kinase, an effector of the small GTPase Rho, plays an important role in the pathogenesis of arteriosclerosis. In this study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of CAV using a specific Rho-kinase inhibitor and a dominant-negative Rho-kinase. Hearts from AKR mice were heterotopically transplanted to C3H/He (allograft) or AKR mice (isograft), and the effects of long-term oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil, on CAV were examined at 2 and 4 weeks after the transplantation. Coronary remodeling in the allografts characterized by intimal thickening and perivascular fibrosis was dose-dependently suppressed in the fasudil group compared with the control group (PϽ0.01, nϭ9 to 10). The inhibitory effects of hydroxyfasudil were mimicked by in vivo gene transfer of dominant-negative Rho-kinase (PϽ0.05, nϭ4). Among the proinflammatory cytokines examined, those of macrophage migration inhibitory factor, interferon-␥, and transforming growth factor-␤1 were upregulated in the control group and were dose-dependently inhibited in the fasudil group (PϽ0.01, nϭ5