Wataru Koguchi

Effects of olmesartan on Apelin/APJ and Akt/endothelial nitric oxide synthase pathway in Dahl rats with end-stage heart failure.

Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function, and the apelin/APJ pathway seems to have opposing physiological role to the renin-angiotensin system. We investigated whether angiotensin II receptor blocker olmesartan could improve cardiac function associated with apelin/APJ and Akt/endothelial nitric oxide synthase (eNOS) pathway in Dahl salt-sensitive hypertensive (DS) rats with end-stage heart failure using NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME). High salt-loaded DS rats were treated with (1) vehicle, (2) olmesartan, and (3) olmesartan plus L-NAME for 7 weeks. Decreased end-systolic elastance and percent fractional shortening in failing rats were significantly ameliorated by olmesartan. Increased atherosclerosis and vascular remodeling and fibrosis factors such as procollagen type I and III and fibronectin expression in DS rats were inhibited by olmesartan. Downregulation of apelin and APJ expression and phosphorylation of Akt and eNOS in failing rats were significantly increased by olmesartan. In addition, administration of L-NAME completely abrogated the olmesartan-mediated improvement of cardiac function and remodeling, and apelin/APJ expression and Akt/eNOS phosphorylation. These findings suggest that olmesartan may improve cardiac dysfunction and remodeling associated with apelin/APJ and Akt/eNOS pathway in DS rats with end-stage heart failure.

Cardioprotective Effects of Pitavastatin on Cardiac Performance and Remodeling in Failing Rat Hearts

BACKGROUND Activation of phosphatidylinositol 3-kinase (PI3K)-Akt signaling by statins increases the activity of endothelial nitric oxide synthase (eNOS). We investigate whether statins (pitavastatin) improve cardiac function and remodeling via eNOS production associated with the PI3K-Akt signaling pathway, Rho-kinase (ROCK) pathway, and the development of oxidative stress in Dahl salt-sensitive (DS) hypertensive rats with heart failure (DSHF). METHODS Pitavastatin (3 mg/kg per day), or pitavastatin plus specific PI3K inhibitor, wortmannin (1 mg/kg per day), or wortmannin alone were administered from the age of 11-18 weeks. Age-matched male Dahl salt-resistant (DR) rats served as a control group. RESULTS Decreased end-systolic elastance (Ees) and percent fractional shortening (%FS) in failing rats was significantly ameliorated by pitavastatin, but not pitavastatin plus wortmannin or wortmannin alone. Upregulation of eNOS and Akt phosphorylation by pitavastatin was suppressed by pitavastatin plus wortmannin or wortmannin alone. Pitavastatin effectively inhibited the vascular lesion formation such as medial thickness and perivascular fibrosis, but not pitavastatin plus wortmannin or wortmannin alone. Activated RhoA and myosin light chain phosphorylation and RhoA, ROCK expression was inhibited by pitavastatin or a specific ROCK inhibitor, Y-27632, and downregulated eNOS expression and Akt phosphorylation was ameliorated by Y-27632. Increased expression of NAD(P)H oxidase subunits and activated p65 nuclear factor (NF)-kappaB, p44/p42 extracellular signal-regulated kinases and its downstream effector p90 ribosomal S6 kinase phosphorylation in failing rat hearts was inhibited by pitavastatin. CONCLUSIONS These findings suggest that pitavastatin may improve cardiac function and remodeling via eNOS production associated with the PI3K-Akt signaling pathway, the ROCK pathway and oxidative stress.

Effect of Eplerenone on Endothelial Progenitor Cells and Oxidative Stress in Ischemic Hindlimb

BACKGROUND We have demonstrated that angiotensin II receptor blocker (ARB) improved endothelial progenitor cells (EPCs) dysfunction through the antioxidative mechanism. Therefore, we investigate whether the selective mineralocorticoid receptor (MR) antagonist eplerenone improves EPCs function in rat hindlimb ischemia. METHODS Unilateral hindlimb ischemia was surgically induced in Wistar rats. After induced ischemia, rats received eplerenone (30 mg/kg/day), valsartan (3 mg/kg/day), or vehicle for 3 weeks. Peripheral blood mononuclear cells were isolated, subjected to flow cytometric analysis to determine the number of circulating EPCs, cultured to assay EPC colony formation, and subjected to a migration chamber assay to evaluate EPCs migration. RESULTS Blood perfusion by laser Doppler image was significantly higher in eplerenone than in vehicle. Capillary density by isolectin B4 stained of ischemic muscle was significantly increased in eplerenone compared with vehicle. Eplerenone significantly increased the number, colony formation, and migration of EPCs. Levels of endothelial nitric oxide synthase (eNOS) and angiogenic factor such as vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2) protein expression by western blot were significantly higher in eplerenone than in vehicle. Eplerenone significantly decreased the NAD(P)H oxidase p22(phox), p47(phox), gp91(phox) and MR expression and expression of aldosterone effector kinase serum and glucocorticoid-induced protein kinase 1 (Sgk1). These effects of eplerenone are similar extent as valsartan. CONCLUSIONS This study showed that eplerenone improves the proliferation and function of EPCs in rat hindlimb ischemia, suggesting that eplerenone may provide a novel and effective therapeutic strategy for the repair of cardiovascular diseases.

275 EFFECT OF EPLERENONE OR VALSARTAN ON ENDOTHELIAL PROGENITOR CELLS IN ISCHEMIC HINDLIMB

Objectives: We investigate whether the selective mineralocorticoid receptor (MR) antagonist eplerenone or valsartan improves endothelial progenitor cells (EPCs) function in rat hindlimb ischemia. Design and Methods: Unilateral hindlimb ischemia was surgically induced in Wistar rats. After induced ischemia, rats received eplerenone (30 mg/kg/day), valsartan (3 mg/kg/day), or vehicle for 3 weeks. Peripheral blood mononuclear cells were isolated, subjected to flow cytometric analysis to determine the number of circulating EPCs, cultured to assay EPC colony formation, and subjected to a migration chamber assay to evaluate EPCs migration. Results: Blood perfusion by Laser Doppler Image was significantly higher in eplerenone than in vehicle. Capillary density by Isolectin-B4 stained of ischemic muscle was significantly increased in eplerenone compared with vehicle. Eplerenone significantly increased the number, colony formation and migration of EPCs. Levels of endothelial nitric oxide synthase and angiogenic factor such as vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 protein expression by Western blot were significantly higher in eplerenone than in vehicle. Eplerenone significantly decreased the NAD(P)H oxidase p22phox, p47phox, gp91phox and MR expression and expression of aldosterone effector kinase Sgk1. These effects of eplerenone are similar extent as valsartan. Conclusions: These findings suggest that eplerenone and valsartan improves the proliferation and function of EPCs in rat hindlimb ischemia, and that eplerenone and valsartan may provide a novel and effective therapeutic strategy for the repair of cardiovascular diseases.

285 VASOPROTECTIVE EFFECTS OF PITAVASTATIN ON ENDOTHELIAL PROGENITOR CELLS IN ISCHEMIC HINDLIMB

Objectives: We investigate whether pitavastatin improves endothelial progenitor cells (EPCs) function via Rho-kinase, nitric oxide synthase (NOS), and PI3kinase/Akt pathway in rat hindlimb ischemia. Design and Methods: Unilateral hindlimb ischemia was surgically induced in Wistar rats. After induced ischemia, rats received pitavastatin (P: 1 mg/kg/day), P plus L-NAME (NOS inhibitor) (PL: 100 mg/L), P plus fasudil (Rho-kinase inhibitor) (PF: 100 mg/kg/day), P plus wortmannin (PI3kinase inhibitor) (PW: 1 mg/kg/day), or vehicle for 3 weeks. Peripheral blood mononuclear cells were isolated, subjected to flow cytometric analysis to determine the number of circulating EPCs, and cultured to assay EPC colony formation. Results: Blood perfusion by Laser Doppler Image was significantly higher in group P and PF than in group PL and PW. Capillary density by Isolectin-B4 stained of ischemic muscle was significantly increased in group P and PF compared with group PL and PW. Group P and PF significantly increased the number of colony formation of EPCs, but not group PL and PW. Levels of endothelial NOS (eNOS) and angiogenic factor such as VEGF, angiopoietin-1, and angiopoietin-2 protein expression by Western blot were significantly higher in group P and PF than in group PL and PW. Conclusions: These findings suggest that pitavastatin may improve the proliferation and function of EPCs via Rho-kinase, eNOS, and PI3kinase/Akt pathway in rat hindlimb ischemia.

880 EFFECT OF COMBINATION THERAPY WITH BONE-MARROW MONONUCLEAR CELLS IMPLANTATION AND FASUDIL ON ANGIOGENESIS IN MYOCARDIAL ISCHEMIA

Objectives: The present study was to investigate whether combination therapy with bone marrow mononuclear cells (MNCs) and Rho-kinase inhibitor, fasudil, shows to enhance angiogenesis and ameliorates cardiac function compared with MNCs alone in a canine model of chronic myocardial infarction. Design and Methods: Immediately after aspiration of BM, a model of chronic myocardial ischemia was created by LAD ligation in adult beagles, and then BM-MNCs or medium alone (control) was injected into the LAD risk area. Fasudil was administered by orally (30 mg/kg/day) in combination therapy with BM-MNCs and fasudil. Four weeks later, end-systolic elastance (Ees) was measured by the pressure-volume loop using conductance catheter, and pathological findings, and immunohistochemistry (ant-VWF, CD31, and alfa-SMA antibody) were measured for angiogenesis. Results: At 4 weeks, MNCs group decreased infarct sizes, increased capillary density, and improved Ees compared to control-group. Combination therapy with MNCs and fasudil shows to enhance angiogenesis, circulating CD34-positive cells by FACS, and ameliorates cardiac function compared with MNCs alone. Conclusions: These findings suggest that fasudil enhances neovascularization and improves cardiac function, which may be mediated by in part by endothelial progenitor cells mobilization.

448 CARDIOPROTECTIVE EFFECT OF A COMBINATION OF RHO-KINASE INHIBITOR AND P38 MAPK INHIBITOR ON CARDIOVASCULAR REMODELING IN DAHL RATS

Objectives: Rho-kinase plays a critical role in various cellular functions. p38 mitogen-activated protein kinase (p38 MAPK) plays a central role in the inflammatory cytokine response to immune challenge. We evaluated the effects of a combination of fasudil, a Rho-kinase inhibitor, and FR167653, a p38 MAPK inhibitor, on cardiovascular remodeling, inflammation, and oxidative stress in Dahl salt-sensitive hypertensive (DS) rats. Design and Methods: DS and Dahl salt-resistant (DR) rats were fed a high-salt diet at 6 weeks of age. Vehicle, fasudil (100 mg/kg per day), FR167653 (2 mg/kg per day), and combination of fasudil and FR167653 were administered to 6-week-old DS rats for 5 weeks. Results: At age of 11 weeks, DS rats in the left ventricle were characterized by increased myocardial fibrosis, phosphorylation of p38 MAPK, and myosin phosphatase targeting subunit (MYPT-1), and NAD(P)H oxidase p22phox, p47phox, gp91phox, tumor necrosis factor-&agr; and interleukin-1&bgr; expression compared with DR rats. Fasudil improved cardiovascular remodeling, inflammation, NAD(P)H oxidase subunits, and phosphorylation of p38 MAPK and MYPT-1. FR167653 also similarly ameliorated these indices but not MYPT-1 phosphorylation. Compared with either agent alone, a combination of fasudil and FR167653 was more effective for the improvement of myocardial damage, inflammation and oxidative stress. Conclusions: These findings suggest that Rho-kinase and p38 MAPK pathway may play a pivotal role in the ventricular hypertrophy, and that a combination of Rho-kinase inhibitor and p38 MAPK inhibitor may provide a potential therapeutic target in hypertension with cardiovascular remodeling.

889 CARDIOPROTECTIVE EFFECT OF APELIN-13 ON CARDIOVASCULAR REMODELING IN END-STAGE HEART FAILURE

Objectives: Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function. Recently, we and other investigator demonstrated that reduction in myocardial apelin/APJ expression might play a critical role in experimental models with end-stage heart failure. Therefore, we evaluate whether exogenous apelin infusion restores apelin/APJ expression and improves cardiac function in failing heart of Dahl salt-sensitive hypertensive (DS) rats. Design and Methods: High salt-loaded DS rats were treated with vehicle and pyroglutamylated apelin-13 (Pyr-AP13; 200 &mgr;g/kg per day, ip) from the age of 11 to 18 weeks. Results: Decreased end-systolic elastance (Ees) and percent fractional shortening in failing rats was significantly ameliorated by Pyr-AP13. Pyr-AP13 effectively inhibited the vascular lesion formation and suppressed inflammation factors such as tumor necrosis factor-&agr; and interleukin-1&bgr; protein expression. Downregulation of apelin and APJ expression, and phosphorylation of eNOS at Ser1177 and Akt at Ser473 in failing rats was significantly increased by Pyr-AP13. Upregulation of NAD(P)H oxidase p22phox, p47phox, and gp91phox in DS rats was significantly suppressed by Pyr-AP13. Conclusions: These findings suggest that exogenous apelin-13 infusion may ameliorate cardiac dysfunction and remodeling and restore apelin/APJ expression in DS rats with end-stage heart failure. Thus, apelin-13 may have significant therapeutic potential in the treatment of end-stage heart failure.