Shin-ichiro Mita

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.

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 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.

Effects of cilnidipine on nitric oxide and endothelin-1 expression and extracellular signal-regulated kinase in hypertensive rats

We evaluated the effects of cilnidipine, a long-acting Ca(2+) channel antagonist, on endothelial nitric oxide synthase (eNOS), preproendothelin-1 and endothelin ETA receptor expression in the left ventricle, and evaluated the relations between these effects and coronary microvascular remodeling and extracellular signal-regulated kinases belonging to one subfamily of mitogen-activated protein kinases in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Cilnidipine (DOCA-cilnidipine, 1 mg/kg/day, subdepressor dose) or vehicle (DOCA-vehicle) was given after induction of DOCA-salt hypertension for 5 weeks. The eNOS mRNA and protein expression in the left ventricle was significantly lower in DOCA-vehicle than in control rats and significantly higher in DOCA-cilnidipine than in DOCA-vehicle rats. Preproendothelin-1 and endothelin ETA receptor expression levels and phospho-p42/p44 extracellular signal-regulated kinase activities were significantly increased in DOCA-vehicle compared with control rats and significantly suppressed in DOCA-cilnidipine compared with DOCA-vehicle rats. DOCA-vehicle rats showed a significant increase in the wall-to-lumen ratio, perivascular fibrosis and myocardial fibrosis, with all these parameters being significantly improved by cilnidipine. These results led us to conclude that phospho-p42/p44 extracellular signal-regulated kinase activities may contribute to the coronary microvascular remodeling of DOCA rats and that protective effects of cilnidipine on cardiovascular remodeling may be at least in part mediated by an increased eNOS expression and a decreased endothelin-1 and endothelin ETA receptor expression in the left ventricle.

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).