Abstract-Primary pulmonary hypertension is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells (VSMCs), and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have recently demonstrated that intracellular signaling pathway mediated by Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis. In the present study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of fatal pulmonary hypertension in rats. Animals received a subcutaneous injection of monocrotaline, which resulted in the development of severe pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions in 3 weeks associated with subsequent high mortality rate. The long-term blockade of Rho-kinase with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil after oral administration, markedly improved survival when started concomitantly with monocrotaline and even when started after development of pulmonary hypertension. The fasudil treatment improved pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions with suppression of VSMC proliferation and macrophage infiltration, enhanced VSMC apoptosis, and amelioration of endothelial dysfunction and VSMC hypercontraction. These results indicate that Rho-kinase-mediated pathway is substantially involved in the pathogenesis of pulmonary hypertension, suggesting that the molecule could be a novel therapeutic target for the fatal disorder. Key Words: pulmonary hypertension Ⅲ Rho-kinase Ⅲ vascular smooth muscle cells Ⅲ endothelial nitric oxide synthase Ⅲ macrophages P rimary pulmonary hypertension (PPH) is a lifethreatening disease characterized by a marked and sustained elevation of pulmonary artery pressure. The disease has no obvious causes and ultimately results in right ventricular (RV) failure and death. The pathological changes of hypertensive pulmonary arteries include endothelial injury, proliferation and hypercontraction of vascular smooth muscle cells (VSMCs), and migration of macrophages. 1-3 PPH continues to be a serious clinical problem with high morbidity and mortality. 4 In 1990s, Rho-kinase/ROK/ROCK was identified as an effector of the small GTPase Rho, 5-7 which plays an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expression. 8 -10 In a series of experimental and clinical studies, we have demonstrated that Rho-kinase-mediated pathway is substantially involved in the pathogenesis of arteriosclerosis. 11-17 These Rho-kinase-mediated alterations in blood vessels also may be involved in the pathogenesis of pulmonary hypertension (PH). In this study, we examined whether Rho-kinasemediated pathway is involved in the pathogenesis of rat model of fatal PH in vivo. Materials and MethodsThe present study was...

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The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several relaxing factors, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have previously demonstrated in animals and humans that endothelium-derived hydrogen peroxide (H 2 O 2 ) is an EDHF that is produced in part by endothelial NO synthase ( eNOS). In this study, we show that genetic disruption of all three NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]) abolishes EDHF responses in mice.The contribution of the NOS system to EDHFmediated responses was examined in eNOS ؊ / ؊ , n/eNOS ؊ / ؊ , and n/i/eNOS ؊ / ؊ mice. EDHFmediated relaxation and hyperpolarization in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased, whereas vascular smooth muscle function was preserved. Loss of eNOS expression alone was compensated for by other NOS genes, and endothelial cell production of H 2 O 2 and EDHF-mediated responses were completely absent in n/i/eNOS ؊ / ؊ mice, even after antihypertensive treatment with hydralazine. NOS uncoupling was not involved, as modulation of tetrahydrobiopterin (BH 4 ) synthesis had no effect on EDHF-mediated relaxation, and the BH 4 /dihydrobiopterin (BH 2 ) ratio was comparable in mesenteric arteries and the aorta. These results provide the fi rst evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries.

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-Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in modulating vascular tone, especially in microvessels, although its nature has yet to be elucidated. This study was designed to examine whether hydrogen peroxide (H 2 O 2 ) is an EDHF in porcine coronary microvessels with use of an electron spin resonance (ESR) method to directly detect H 2 O 2 production from the endothelium. Methods and Results-Isometric tension and membrane-potential recordings demonstrated that bradykinin and substance P caused EDHF-mediated relaxations and hyperpolarizations of porcine coronary microvessels in the presence of indomethacin and N -nitro-L-arginine. Key Words: endothelium Ⅲ endothelium-derived hyperpolarizing factor Ⅲ hydrogen peroxide Ⅲ membrane potential T he endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, nitric oxide (NO), and the still-unidentified endothelium-derived hyperpolarizing factor (EDHF). Since the first reports of the existence of EDHF, 1,2 several candidates for EDHF have been proposed, including cytochrome P-450 metabolites, 3,4 endothelium-derived K ϩ , 5,6 and gap-junctional electrical communication between endothelial cells and smooth muscle cells. 7,8 We and others have recently demonstrated that endotheliumderived hydrogen peroxide (H 2 O 2 ) is an EDHF of mouse and human mesenteric arteries, 9,10 piglet pial arterioles, 11 and human and canine coronary microvessels (in flow-induced and autoregulatory relaxation, respectively). 12,13 However, it remains to be determined whether H 2 O 2 is also an EDHF in agonist-induced relaxation of the coronary microvessels, in which EDHF plays an important role in modulating vascular tone. Moreover, endothelial production of H 2 O 2 /EDHF remains to be demonstrated by methods other than conventional tension or membrane-potential recordings. The present study was thus designed to examine whether H 2 O 2 plays a role as an EDHF in porcine coronary microvessels by using an electron spin resonance (ESR) method to directly detect endothelial production of H 2 O 2 . MethodsThis study was reviewed by the Committee on Ethics in Animal Experiments of the Kyushu University and was carried out according to the Guidelines for Animal Experiments of the Kyushu University and of the Japanese Government. Animals and Tissue PreparationWe used a total of 36 domestic male pigs (Nihon Crea, Tokyo, Japan; 2 to 3 months old and weighing 25 to 30 kg). Animals were humanely killed with a lethal dose of pentobarbital sodium, and then the right ventricular free wall was carefully removed. Epicardial right coronary arteries and distal coronary microvessels (250 to 300 m in diameter) were excised from the right ventricular free wall and

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The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived H2O2 is an EDHF in mesenteric arteries of mice and humans and in porcine coronary microvessels. However, the mechanism for the endothelial production of H2O2 as an EDHF remains to be elucidated. In this study, we tested our hypothesis that Cu,Zn-superoxide dismutase (Cu,Zn-SOD) plays a pivotal role in endothelium-dependent hyperpolarization, using control and Cu,Zn-SOD–/– mice. In mesenteric arteries, EDHF-mediated relaxations and hyperpolarizations were significantly reduced in Cu,Zn-SOD–/– mice with no inhibitory effect of catalase, while endothelium-independent relaxations and hyperpolarizations were preserved. Endothelial H2O2 production also was significantly reduced in Cu,Zn-SOD–/– mice. In Langendorff isolated heart, bradykinin-induced increase in coronary flow was significantly reduced in Cu,Zn-SOD–/– mice, again with no inhibitory effect of catalase. The exogenous SOD mimetic tempol significantly improved EDHF-mediated relaxations and hyperpolarizations and coronary flow response in Cu,Zn-SOD–/– mice. These results prove the novel concept that endothelial Cu,Zn-SOD plays an important role as an “EDHF synthase” in mice, in addition to its classical role to scavenge superoxide anions