Keiko Morikawa

Long-Term Inhibition of Rho-Kinase Suppresses Angiotensin II–Induced Cardiovascular Hypertrophy in Rats In Vivo Effect on Endothelial NAD(P)H Oxidase System

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.

Long-Term Treatment With a Rho-Kinase Inhibitor Improves Monocrotaline-Induced Fatal Pulmonary Hypertension in Rats

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.

Electron spin resonance detection of hydrogen peroxide as an endothelium-derived hyperpolarizing factor in porcine coronary microvessels

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 (H2O2) is an EDHF in porcine coronary microvessels with use of an electron spin resonance (ESR) method to directly detect H2O2 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&ohgr;-nitro-l-arginine. The contribution of H2O2 to the EDHF-mediated responses was demonstrated by the inhibitory effect of catalase and by the relaxing and hyperpolarizing effects of exogenous H2O2. Endothelial production of H2O2 was quantified in bradykinin- or substance P–stimulated intact blood vessels by ESR spectroscopy. Tiron, a superoxide scavenger that facilitates H2O2 formation, enhanced bradykinin-induced production of H2O2, as well as the EDHF-mediated relaxations and hyperpolarizations. By contrast, cytochrome P-450 inhibitors (sulfaphenazole or 17-octadecynoic acid) or a gap junction inhibitor (18&agr;-glycyrrhetinic acid) failed to inhibit the EDHF-mediated relaxations. Involvement of endothelium-derived K+ was not evident in experiments with ouabain plus Ba2+ or exogenous K+. Conclusion—These results provide ESR evidence that H2O2 is an EDHF in porcine coronary microvessels.

Crucial role of nitric oxide synthases system in endothelium-dependent hyperpolarization in mice

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 (H2O2) 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 EDHF-mediated responses was examined in eNOS−/−, n/eNOS−/−, and n/i/eNOS−/− mice. EDHF-mediated 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 H2O2 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 (BH4) synthesis had no effect on EDHF-mediated relaxation, and the BH4/dihydrobiopterin (BH2) ratio was comparable in mesenteric arteries and the aorta. These results provide the first evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries.

Inhibition of Renin-Angiotensin System Ameliorates Endothelial Dysfunction Associated With Aging in Rats

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 A2/prostaglandin H2 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. Conclusions—These results demonstrate that long-term inhibition of the renin-angiotensin system ameliorates endothelial dysfunction associated with aging through the inhibition of the synthesis of COX-2–derived vasoconstricting factors and superoxide anions.

Influence of diabetes mellitus, hypercholesterolemia, and their combination on EDHF-mediated responses in mice

The endothelium synthesizes and releases several vasodilator substances, including vasodilator prostaglandins, NO, and EDHF. NO-mediated relaxations are reduced by various risk factors, such as diabetes mellitus and hypercholesterolemia. However, it remains to be elucidated whether EDHF-mediated relaxations also are reduced by those factors and their combination. In this study, we addressed this point in mice. We used small mesenteric arteries from control, diabetic (streptozotocin-induced), apolipoprotein-E-deficient (ApoE−/−), and diabetic ApoE−/− mice. In control mice, endothelium-dependent relaxations to acetylcholine were largely mediated by EDHF. This EDHF-mediated component was slightly reduced in diabetic mice, preserved in ApoE−/− mice, and markedly reduced in diabetic ApoE−/− mice with an increase in NO-mediated component and a negative contribution of indomethacin-sensitive endothelium-derived contracting factor (EDCF). Endothelium-independent relaxations to sodium nitroprusside or NS1619, a direct opener of calcium-activated K channels, were attenuated in ApoE−/− and diabetic ApoE−/− mice. Endothelium-dependent hyperpolarizations were significantly reduced in diabetic mice, preserved in ApoE−/− mice, and again markedly reduced in diabetic ApoE−/− mice. These results indicate that hypercholesterolemia alone minimally affects the EDHF-mediated relaxations, and diabetes mellitus significantly attenuated the responses, whereas their combination markedly attenuates the responses with a compensatory involvement of NO and a negative contribution of EDCF.

Roles of Endothelial Oxidases in Endothelium-derived Hyperpolarizing Factor Responses in Mice

The endothelium synthesizes and releases several vasodilator substances, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDHF in animals and humans and that superoxide anions derived from endothelial nitric oxide synthases (NOSs) system are an important precursor for EDHF/H2O2 in mice. There are several intracellular sources of superoxide anions other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, and mitochondrial electron transport chain. In this study, we examined the possible role of endothelial oxidases other than NOSs in the EDHF-mediated responses. In angiotensin II-infused mice, both EDHF-mediated relaxations and hyperpolarizations to acetylcholine were significantly reduced, nitric oxide-mediated relaxations were rather enhanced, and vascular smooth muscle responses were preserved. Antihypertensive treatment normalized blood pressure but failed to improve EDHF-mediated responses in those mice. Acute inhibition of endothelial oxidases other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, or mitochondrial electron transport chain, had no inhibitory effects on EDHF-mediated responses. Furthermore, in p47phox-knockout mice, EDHF-mediated responses were unaltered. These results suggest that endothelial oxidases other than NOSs are not involved in EDHF/H2O2 responses in mice, suggesting a specific link between endothelial NOSs system and EDHF responses under physiological conditions.

Important role of superoxide dismutase in EDHF-mediated responses of human mesenteric arteries.

The endothelium synthesizes and releases several vasodilator substances, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have identified hydrogen peroxide (H2O2) as an EDHF in mouse and human mesenteric arteries and porcine coronary microvessels. We also have recently demonstrated that Cu,Zn-SOD plays an important role in EDHF synthesis in mouse mesenteric arteries. However, it remains to be determined whether SOD also plays an important role in EDHF-mediated responses of human arteries. In this study, we addressed this point in human mesenteric arteries. We used small mesenteric arteries of patients who underwent gastrectomy operations. Isometric tensions and membrane potentials were recorded in the presence of indomethacin and Nω-nitro-L-arginine to inhibit the synthesis of prostacyclin and NO, respectively. Pretreatment with Tiron, a cell-permeable SOD-mimetic, significantly enhanced the EDHF-mediated relaxations and hyperpolarizations to bradykinin, and this effect was abolished by catalase, indicating that this enhancing effect was achieved by H2O2. By contrast, Tiron did not affect endothelium-independent relaxations, indicating that the enhancing effect of Tiron is not caused by the enhancement of vascular smooth muscle responses. These results indicate that SOD plays an important role in EDHF-mediated relaxations and hyperpolarizations of human mesenteric arteries.

Sustained Elevation of Serum Cortisol Level Causes Sensitization of Coronary Vasoconstricting Responses in Pigs In Vivo A Possible Link Between Stress and Coronary Vasospasm

Vasospastic angina is induced by stress, for which cortisol secreted by activated hypothalamic/pituitary/adrenal axis may play an important role. However, direct evidence for this notion is still lacking. In this study, we examined whether sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses in pigs in vivo and, if so, whether Rho-kinase, which we found is a key molecule of coronary vasospasm, is involved. Oral administration of cortisol (20 mg/kg per day) increased its serum level to that seen in restraint stress in pigs. Thus, we examined coronary vasomotor responses in the following 4 groups: (1) control (without cortisol); (2) cortisol (20 mg/kg per day, PO) for 9 days; (3) cortisol plus RU38486 (a glucocorticoids receptor antagonist, 10 mg/kg per day, PO) for 9 days; and (4) cortisol for 9 days followed by 6-week withdrawal. Coronary angiography showed that intracoronary serotonin caused coronary hyperconstriction and reduction in coronary blood flow associated with ischemic ECG changes (coronary vasospasm) in only the cortisol group. All of these responses were abolished by hydroxyfasudil, a specific Rho-kinase inhibitor, in vivo. Organ chamber experiments demonstrated that serotonin concentration-dependently caused hypercontractions of coronary vascular smooth muscle associated with Rho-kinase activation (as evidenced by the enhanced phosphorylation of myosin binding subunit, a substrate of Rho-kinase) in only the cortisol group. All of these responses were again inhibited by hydroxyfasudil in vitro. These results indicate that sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses through Rho-kinase activation, suggesting the link between stress and coronary vasospasm.

Prostacyclin does not inhibit rho-kinase: An implication for the treatment of pulmonary hypertension

Primary pulmonary hypertension continues to be a fatal disease. We have recently demonstrated that long-term inhibition of Rho-kinase, an effector of the small GTPase Rho, is effective for the treatment of pulmonary hypertension (PH) in rats and humans. Prostacyclin has been clinically used for the treatment of PH with moderate success. However, it remains to be examined whether Rho-kinase inhibition is involved in its beneficial effects on PH. In an ELISA assay, neither prostacyclin nor its oral analogue, beraprost sodium, inhibited Rho-kinase even at higher concentrations (10−7 to 10−5 M, 100 to 10,000 times higher than their clinical concentrations), whereas specific Rho-kinase inhibitors, fasudil and hydroxyfasudil, markedly (∼95%) inhibited the Rho-kinase activity at 10−5 M (near their clinical concentrations). Beraprost sodium did not significantly suppress serotonin-induced vascular smooth muscle cell (VSMC) contractions or Rho-kinase activity of the rat aorta without endothelium, as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, whereas hydroxyfasudil markedly suppressed the VSMC contractions and Rho-kinase activity. These results indicate that prostacyclin lacks direct inhibitory effect on Rho-kinase and suggest that combination therapy with prostacyclin and a Rho-kinase inhibitor could exert further beneficial effects on PH.