Kohtaro Yoshida

Critical role of Rho-kinase pathway for cardiac performance and remodeling in failing rat hearts

OBJECTIVES Rho and Rho-kinase play a critical role in the regulation of cellular functions such as proliferation and migration. To elucidate the molecular mechanisms that regulate cardiac function and cardiovascular remodeling, we determined whether the signaling pathway through Rho is involved in Dahl salt-sensitive hypertensive rats with congestive heart failure (CHF) using a specific Rho-kinase inhibitor, Y-27632. METHODS Y-27632 was administered from the left ventricular hypertrophy stage (11 weeks) to the CHF stage (18 weeks) for 7 weeks. The left ventricular end-systolic pressure-volume relationship (contractility: E(es)) was evaluated using a conductance catheter. RESULTS Downregulated E(es) in the CHF stage was significantly ameliorated by Y-27632 treatment. Increased RhoA protein, Rho-kinase gene expression and myosin light chain phosphorylations in CHF rats were suppressed by Y-27632. Upregulated proto-oncogene c-fos gene expression in CHF rats was decreased by inhibiting Rho-kinase. In contrast, Y-27632 showed no effect on upregulated extracellular signal-regulated kinases (ERK) and p70S6 kinase phosphorylations, which were reported to be involved in protein synthesis. In the CHF stage, Y-27632 effectively inhibited vascular lesion formation such as medial thickness and perivascular fibrosis. CONCLUSIONS These results suggest that differential activation of the Rho-Rho-kinase and the ERK-p70S6 kinase pathways may play a critical role in CHF, and the Rho-Rho-kinase pathway is involved in the pathogenesis of cardiac dysfunction and cardiovascular remodeling. Thus, inhibition of the Rho-kinase pathway may be at least a potential therapeutic strategy for CHF.

Cardioprotective Mechanisms of Eplerenone on Cardiac Performance and Remodeling in Failing Rat Hearts

Aldosterone may play a pivotal role in the pathophysiology of heart failure. To elucidate the beneficial cardioprotective mechanism of eplerenone, a novel selective aldosterone blocker, we hypothesized that eplerenone stimulates endothelial NO synthase (eNOS) through Akt and inhibits inducible NO synthase (iNOS) via nuclear factor &kgr;B (NF-&kgr;B) after the development of oxidative stress and activation of the lectin-like, oxidized, low-density lipoprotein receptor 1 (LOX-1) pathway in Dahl salt-sensitive rats with heart failure. Eplerenone (10, 30, and 100 mg/kg per day) was given from the age of the left ventricular hypertrophy stage (11 weeks) to the failing stage (18 weeks) for 7 weeks. The left ventricular end-systolic pressure-volume relationship was evaluated using a conductance catheter. Decreased percentage of fractional shortening by echocardiography and end-systolic pressure-volume relationship in failing rats was significantly ameliorated by eplerenone. Downregulated eNOS expression, eNOS and Akt phosphorylation, and NOS activity in failing rats were increased by eplerenone. Upregulated expression of the mineralocorticoid receptor aldosterone synthase (CYP11B2); NAD(P)H oxidase p22phox, p47phox, gp91phox, iNOS, and LOX-1; and activated p65 NF-&kgr;B, protein kinase C&bgr;II, c-Src, p44/p42 extracellular signal-regulated kinase, and p70S6 kinase phosphorylation were inhibited by eplerenone. Eplerenone administration resulted in significant improvement of cardiac function and remodeling and upregulation of sarcoplasmic reticulum Ca2+-ATPase expression. These findings suggest that eplerenone may have significant therapeutic potential for heart failure, and these cardioprotective mechanisms of eplerenone may be mediated in part by stimulating eNOS through Akt and inhibiting iNOS via NF-&kgr;B after activation of the oxidative stress-LOX-1 pathway and signal transduction pathway.

Eplerenone Shows Renoprotective Effect by Reducing LOX-1–Mediated Adhesion Molecule, PKCε-MAPK-p90RSK, and Rho-Kinase Pathway

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) may play an important role in atherosclerosis by inducing leukocyte adhesion molecules, such as intercellular and vascular cell adhesion molecule-1 (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]). We hypothesized that eplerenone, a novel selective aldosterone blocker, produces inhibition of LOX-1–mediated adhesion molecules, suppresses mitogen-activated protein (MAP) kinase and its downstream effector p90 ribosomal S6 kinase (p90RSK) through the protein kinase C&egr; (PKC&egr;) pathway, and improves endothelial function by inhibition of Rho-kinase in the renal cortex of Dahl salt-sensitive hypertensive (DS) and salt-resistant (DR) rats. Eplerenone (10, 30, and 100 mg/kg per day) was given from the age of 6 weeks to the left ventricular hypertrophy stage (11 weeks) for 5 weeks. At 11 weeks, expression levels of LOX-1, ICAM-1, VCAM-1, and Rho-kinase were higher in DS rats than in DR rats and were decreased by eplerenone. Similarly, upregulated phosphorylation of PKC&egr;, MAP kinase, and p90RSK in DS rats was also inhibited by eplerenone. In contrast, downregulated endothelial nitric oxide synthase mRNA was increased by eplerenone to a similar degree as after treatment with Y-27632, a selective Rho-kinase inhibitor. Eplerenone administration resulted in significant improvement in glomerulosclerosis (eplerenone 10 mg, −61%; 30 mg, −78%; and 100 mg, −84% versus DS; P<0.01, respectively) and urinary protein (10 mg, −78%; 30 mg, −87%; and 100 mg, −88% versus DS; P<0.01, respectively). These results suggest that the renoprotective effects of eplerenone may be partly caused by inhibition of LOX-1–mediated adhesion molecules and PKC&egr;–MAP kinase–p90RSK pathway, and improvement in endothelial function.

Celiprolol Activates eNOS Through the PI3K-Akt Pathway and Inhibits VCAM-1 Via NF-κB Induced by Oxidative Stress

Abstract—Vascular cell adhesion molecule-1 (VCAM-1) and reactive oxygen species play critical roles in early atherogenesis, and nitric oxide (NO) is an important regulator of the cardiovascular system. Although celiprolol, a specific &bgr;1-antagonist with weak &bgr;2-agonistic action, stimulates endothelial nitric oxide synthase (eNOS) production, the mechanisms remain to be determined. Because it was recently reported that phosphatidylinositol 3-kinase (PI3K) and its downstream effector Akt are implicated in the activation of eNOS and that regulation of VCAM-1 expression is mediated via nuclear factor-&kgr;B (NF-&kgr;B), we hypothesized that celiprolol activates phosphorylation of eNOS through the PI3K-Akt signaling pathway; that celiprolol modulates VCAM-1 expression, which is associated with inhibiting NF-&kgr;B phosphorylation; and that celiprolol suppresses NAD(P)H oxidase p22phox, p47phox, gp91phox, and nox1 expression in the left ventricle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. eNOS and Akt phosphorylation upregulated by celiprolol alone were suppressed by treatment with celiprolol plus wortmannin. Increased expression of VCAM-1, p22phox, p47phox, gp91phox, nox1, activated p65 NF-&kgr;B, c-Src, p44/p42 extracellular signal-regulated kinases, and their downstream effector p90 ribosomal S6 kinase phosphorylation in DOCA rats was inhibited by celiprolol. Celiprolol administration resulted in a significant improvement in cardiovascular remodeling and suppression of transforming growth factor-&bgr;1 gene expression. In conclusion, celiprolol suppresses VCAM-1 expression because of inhibition of oxidative stress, NF-&kgr;B, and signal transduction, while increasing eNOS via stimulation of the PI3K-Akt signaling pathway and improving cardiovascular remodeling.

Cardioprotective Mechanisms of Spironolactone Associated with the Angiotensin-Converting Enzyme/Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinases, NAD(P)H Oxidase/Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1, and Rho-Kinase Pathways in Aldosterone/Salt-Induced Hypertensive Rats

Studies were performed to test the hypothesis that the angiotensin-converting enzyme (ACE)/epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinases (ERK) pathway, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase/lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) pathway, and Rho-kinase pathway contribute to the pathogenesis of aldosterone/salt-induced hypertensive rats. Wistar rats were given 1% NaCl to drink and treated with one of the following combinations for 6 weeks: vehicle; aldosterone (0.75 μg/h); aldosterone plus a mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day); aldosterone plus an ACE inhibitor, imidapril (1 mg/kg/day); aldosterone plus an NAD(P)H oxidase inhibitor, apocynin (0.5 mmol/l); and aldosterone plus an Rho-kinase inhibitor, Y-27632 (3 mg/kg/day). Upregulated expression of ACE and EGFR and p44/p42ERK phosphorylation were suppressed by spironolactone or imidapril. Upregulated NAD(P)H oxidase subunits and LOX-1 expression were inhibited by spironolactone or apocynin. Increased expression of RhoA and Rho-kinase and myosin light chain phosphorylation were decreased by spironolactone or Y-27632. Moreover, these drugs effectively inhibited the vascular lesion formation, as measured by the medial thickness and level of perivascular fibrosis, and suppressed the expression of transforming growth factor-β1, type I and III collagen, and monocyte chemoattractant protein-1 mRNA. Spironolactone may be useful as a cardioprotective agent to prevent cardiovascular remodeling via the ACE/EGFR/ERK, NAD(P)H oxidase/LOX-1, and Rho-kinase pathways.

Cardioprotective mechanisms of Rho-kinase inhibition associated with eNOS and oxidative stress-LOX-1 pathway in Dahl salt-sensitive hypertensive rats.

Objectives Rho-kinase plays a crucial role in various cellular functions. To elucidate molecular mechanisms of Rho-kinase-mediated cardiovascular remodeling in vivo, we evaluated whether a signaling pathway through Rho is involved, and whether Y-27632, a specific Rho-kinase inhibitor, stimulates endothelial nitric oxide synthase (eNOS) and suppresses the oxidative stress and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) pathway in the left ventricle of Dahl salt-sensitive hypertensive (DS) rats. Methods Y-27632 (3 mg/kg per day) or vehicle were given for 5 weeks, from age 6 weeks to a stage of left ventricular hypertrophy (11 weeks). Age-matched Dahl salt-resistant (DR) rats fed the same diet served as a control group. Results Increased left ventricular weight in the hypertrophy stage was significantly ameliorated by Y-27632. Upregulated RhoA protein, Rho-kinase gene expression and myosin light-chain phosphorylation in the hypertrophy stage were suppressed by Y-27632. Increased expression of NAD(P)H oxidase p22phox, p47phox, gp91phox and LOX-1 in DS rats were inhibited by Y-27632. Upregulated protein kinase Cϵ and p65 nuclear factor-κB phosphorylation in DS rats was reduced by Y-27632. In contrast, downregulated eNOS expression in hypertrophy stage was upregulated by Y-27632. Y-27632 effectively inhibited vascular lesion formation, such as medial thickness and perivascular fibrosis, and suppressed transforming growth factor-β1, type I and III collagen, and fibronectin gene expression. Conclusions Inhibiting the Rho-kinase pathway may play a key role in the cardioprotective effect on cardiovascular remodeling associated with eNOS and the oxidative stress-LOX-1 pathway in DS rats, and may be at least a potential therapeutic strategy for hypertension with cardiac hypertrophy.

Cardioprotective effect of angiotensin II type 1 receptor antagonist associated with bradykinin-endothelial nitric oxide synthase and oxidative stress in Dahl salt-sensitive hypertensive rats.

Objectives The interactions between eNOS or oxidative stress and bradykinin under long-term treatment of angiotensin II type 1 receptor antagonists (ATRA) remain unknown. To elucidate the molecular mechanisms of the cardioprotective effect of ATRA, we evaluated whether valsartan affects the bradykinin–eNOS and nicotinamide adenine dinucleotide (NAD(P)H) oxidase pathway. Methods After 5 weeks of feeding an 8% NaCl diet to 6-week-old Dahl salt-sensitive hypertensive (DS) rats, a distinct stage of concentric left ventricular hypertrophy (LVH) was noted. Six-week-old DS rats were treated with one of the following drug combinations for 5 weeks until the onset of LVH: vehicle; bradykinin B2 receptor antagonist FR172357 alone; high-dose hydralazine; low-dose hydralazine; high-dose valsartan; low-dose valsartan; high and low-dose valsartan plus FR172357. Age-matched Dahl salt-resistant rats fed the same diet served as controls. Results eNOS expression and activity, which was decreased in hypertrophy, was increased by high or low-dose valsartan, but not by high and low-dose valsartan plus FR172357 or FR172357 alone or high and low-dose hydralazine. The increased expression of NAD(P)H oxidase p22phox, p47phox, p67phox, and gp91phox in DS rats was suppressed by high or low-dose valsartan, but not by high or low-dose valsartan plus FR172357 or FR172357 alone or high and low-dose hydralazine. Valsartan effectively inhibited vascular lesion formation and suppressed the expression of transforming growth factor-β1, connective tissue growth factor, and type I collagen, but not valsartan plus FR172357 or FR172357 alone or high and low-dose hydralazine. Conclusion These findings suggest that valsartan may have cardioprotective effects in this model, partly associated with the bradykinin–eNOS and oxidative stress pathway.

Cardioprotective Effect of Benidipine on Cardiac Performance and Remodeling in Failing Rat Hearts

BACKGROUND We have known that endothelial nitric-oxide synthase (eNOS) and oxidative stress may play a key role in cardiac performance in failing rat hearts. However, the interactions between eNOS or oxidative stress and bradykinin (BK) under treatment of calcium channel blockers (CCBs) remain unknown. To elucidate the mechanism underlying the cardioprotective effect of long-acting dihydropyridine CCBs, we evaluated the effect of benidipine on the BK-eNOS and NAD(P)H oxidase pathway in Dahl salt-sensitive (DS) rats with heart failure. METHODS 11-week-old DS rats were treated with one of the following drug combinations for 7 weeks until the onset of the failing stage: vehicle, BK B2 receptor antagonist (FR172357 (FR)) alone, hydralazine, benidipine, and benidipine plus FR. The left ventricular end-systolic pressure-volume relationship (ESPVR) (contractility: Ees) was evaluated using a conductance catheter. RESULTS Downregulated Ees and per cent of fractional shortening (%FS) assessed by echocardiography and eNOS expression in the failing stage were both significantly increased by using benidipine; this result was not found, however, when using FR alone or hydralazine or benidipine plus FR. Upregulated expression of NAD(P)H oxidase p22phox and p47phox and lectin-like oxidized low-density lipoprotein receptor-1, and downregulated superoxide dismutase-1 (SOD-1) were significantly ameliorated by benidipine, but not by FR alone or by hydralazine or benidipine plus FR. Benidipine effectively inhibited vascular lesion formation and suppressed atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGF-beta1), but this was not the case when using FR alone or hydralazine or benidipine plus FR. CONCLUSIONS These results suggest that benidipine may be useful for cardioprotective agents in preventing the cardiac dysfunction and remodeling associated with the BK-eNOS and oxidative stress pathway.

Celiprolol inhibits mitogen-activated protein kinase and endothelin-1 and transforming growth factor-β1 gene in rats

We evaluated the cardioprotective effects of long-term treatment with celiprolol (for 5 weeks), a specific beta(1)-adrenoceptor antagonist with a weak beta(2)-adrenoceptor agonist action, on endothelin-1 and transforming growth factor (TGF)-beta(1) expression and cardiovascular remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Upregulated preproendothelin-1, endothelin ET(A) receptor, TGF-beta(1), c-fos, and type I collagen expression and extracellular signal-regulated kinase activities were suppressed by celiprolol. Celiprolol effectively inhibited vascular lesion formation such as medial thickness and perivascular fibrosis. These observations suggested that extracellular signal-regulated kinase and c-fos gene pathway may contribute to the cardiovascular remodeling of DOCA rats, and that cardioprotective effects of celiprolol on cardiovascular remodeling may be mediated, at least in part, by suppressed expression of endothelin-1 and TGF-beta(1).