Divergent effects of estrogen and nicotine on Rho-kinase expression in human coronary vascular smooth muscle cells.

Recent studies have demonstrated that up-regulated Rho-kinase plays an important role in the pathogenesis of coronary arteriosclerosis and vasospasm. We have shown that inflammatory stimuli, such as angiotensin II and interleukin-1beta, up-regulate Rho-kinase expression and activity in human coronary vascular smooth muscle cells, for which intracellular signal transduction mediated by protein kinase C and NF-kappaB is involved. Here, we show that estrogen down-regulates while nicotine up-regulates Rho-kinase and that nicotine counteracts the inhibitory effect of estrogen on angiotensin II-induced Rho-kinase expression. Furthermore, we demonstrated that the intracellular signal transduction of the inhibitory effect of estrogen is mediated by an estrogen receptor. These results demonstrate that inflammatory stimuli up-regulate Rho-kinase, for which estrogen (mediated by an estrogen receptor) and nicotine exert divergent inhibitory and stimulatory effects on the Rho-kinase expression, respectively, and may explain in part why the incidence of arteriosclerotic and vasospastic disorders is increased in postmenopausal women and smokers.

[Inhibition of Rho-kinase by fasudil preventing anginal attacks associated with spastic angina: a case report].

An 86-year-old woman was admitted with unstable angina pectoris. Plain old balloon angioplasty (POBA) was performed for 90% stenosis at segment 7 of the left coronary artery with concomitant treatment with nitrate, calcium antagonists, and nicorandil. Five days after POBA, she again suffered chest pain at rest with ST depression by electrocardiography, despite increased doses of calcium-antagonist and nicorandil. Coronary arteriography showed no evidence of restenosis (50%) at the POBA site. The involvement of coronary artery spasm was considered and intravenous treatment with a Rho-kinase inhibitor, fasudil, was started, which resulted in disappearance of the anginal attacks. She refused to continue the fasudil treatment on day 5, which resulted in reappearance of anginal attacks. Third coronary angiography showed a 90% restenosis at POBA site and percutaneous coronary intervention was again performed. This case suggests that a Rho-kinase inhibitor is potentially effective to prevent anginal attacks in spastic angina.

Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells.

Recent studies have demonstrated that upregulated Rho-kinase plays an important role in the pathogenesis of arteriosclerosis and vasospasm in both animals and humans. However, little is known about the molecular mechanism(s) involved in the Rho-kinase upregulation. Since inflammatory mechanisms have been implicated in the pathogenesis of arteriosclerosis and vasospasm, we examined whether inflammatory stimuli upregulate Rho-kinase in vitro and in vivo. In cultured human coronary vascular smooth muscle cells (hcVSMC), inflammatory stimuli, such as angiotensin II and interleukin-1beta, increased Rho-kinase expression (at both mRNA and protein levels) and function (as evaluated by the extent of the phosphorylation of the ERM (the ezrin/radixin/moesin) family, substrates of Rho-kinase) in a time- and concentration-dependent manner. The expression of Rho-kinase was inhibited by blockades of protein kinase C (PKC) (by either GF109253 or prolonged treatment with phorbol myristate acetate for 24 h) and an adenovirus-mediated gene transfer of dominant-active Ikappa-B, suggesting an involvement of PKC and NF-kappaB in the intracellular signal transduction pathway for the Rho-kinase expression. Furthermore, coronary vascular lesion formation (characterized by medial thickening and perivascular fibrosis) induced by a long-term administration of angiotensin II was markedly suppressed in NF-kappaB(-/-) mice with reduced expression and activity of Rho-kinase in vivo. These results indicate that the expression and function of Rho-kinase are upregulated by inflammatory stimuli (e.g. angiotensin II and IL-1beta) in hcVSMC with an involvement of PKC and NF-kappaB both in vitro and in vivo.

Long-term inhibition of Rho-kinase suppresses left ventricular remodeling after myocardial infarction in mice.

BACKGROUND: Rho-kinase has been implicated as an important regulator of inflammatory responses mediated by cytokines and chemokines. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined whether long-term blockade of Rho-kinase suppresses LV remodeling in a mouse model of MI in vivo. METHODS AND RESULTS: Mice underwent ligation of the left coronary artery and were treated with a Rho-kinase inhibitor, fasudil (100 mg x kg(-1) x d(-1) in tap water), for 4 weeks, starting 1 day after the surgery. At 4 weeks, LV infarct size was histologically comparable between the 2 groups. LV cavity dilatation and dysfunction evaluated by echocardiography were significantly suppressed in the fasudil group (P<0.05, n=15 to 28). The beneficial effects of fasudil were accompanied by suppression of cardiomyocyte hypertrophy and interstitial fibrosis (both P<0.01, n=6). The expression of inflammatory cytokines, including transforming growth factor (TGF)-beta2, TGF-beta3, and macrophage migration inhibitory factor, was upregulated in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (both P<0.05, n=10 to 11). Rho-kinase activity as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, was significantly increased in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (P<0.05, n=5). CONCLUSIONS: These results indicate that Rho-kinase is substantially involved in the pathogenesis of LV remodeling after MI associated with upregulation of proinflammatory cytokines, suggesting a therapeutic importance of the molecule for the prevention of post-MI heart failure.

Remnant lipoproteins from patients with sudden cardiac death enhance coronary vasospastic activity through upregulation of Rho-kinase.

OBJECTIVE: Sudden cardiac death (SCD) still remains a serious problem. We have previously shown that remnant-like particles (RLP) are the major risk factor for SCD and that Rho-kinase plays a central role in the molecular mechanism of coronary vasospasm. In this study, we examined whether RLP from patients with SCD upregulate Rho-kinase associated with an enhanced coronary vasospastic activity. METHODS AND RESULTS: We isolated RLP and non-RLP in very-low-density lipoprotein (VLDL) fraction from SCD patients without coronary stenosis. We performed in vivo study in which we treated the coronary artery with RLP or non-RLP fraction at the adventitia in pigs. After 1 week, intracoronary serotonin caused marked coronary hyperconstriction at the segment treated with RLP fraction but not with non-RLP fraction (P<0.001, n=6), and hydroxyfasudil, a selective Rho-kinase inhibitor, dose-dependently inhibited the spasm in vivo. In organ chamber experiments, serotonin caused hypercontraction of vascular smooth muscle cells (VSMC) from RLP-treated segment, which was significantly inhibited by hydroxyfasudil (P<0.001, n=6). In cultured human coronary VSMC, the treatment with RLP significantly enhanced the expression and activity of Rho-kinase (P<0.05, n=6). CONCLUSIONS: These results indicate that RLP from SCD patients upregulate Rho-kinase in coronary VSMC and markedly enhance coronary vasospastic activity.

Long-term treatment with a specific Rho-kinase inhibitor suppresses cardiac allograft vasculopathy in mice.

Cardiac allograft vasculopathy (CAV) continues to be a major cause of late graft failure after cardiac transplantation. We have demonstrated that Rho-kinase, an effector of the small GTPase Rho, plays an important role in the pathogenesis of arteriosclerosis. In this study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of CAV using a specific Rho-kinase inhibitor and a dominant-negative Rho-kinase. Hearts from AKR mice were heterotopically transplanted to C3H/He (allograft) or AKR mice (isograft), and the effects of long-term oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil, on CAV were examined at 2 and 4 weeks after the transplantation. Coronary remodeling in the allografts characterized by intimal thickening and perivascular fibrosis was dose-dependently suppressed in the fasudil group compared with the control group (P<0.01, n=9 to 10). The inhibitory effects of hydroxyfasudil were mimicked by in vivo gene transfer of dominant-negative Rho-kinase (P<0.05, n=4). Among the proinflammatory cytokines examined, those of macrophage migration inhibitory factor, interferon-gamma, and transforming growth factor-beta1 were upregulated in the control group and were dose-dependently inhibited in the fasudil group (P<0.01, n=5). Vascular inflammation in the allografts, as evidenced by accumulation of inflammatory cells (macrophages and T cells), was also significantly inhibited in the fasudil group (P<0.05, n=5 to 10). These results indicate that long-term treatment with fasudil suppresses CAV in mice, suggesting that Rho-kinase is an important therapeutic target for the prevention of CAV.

Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system.

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.

Acute vasodilator effects of HMG-CoA reductase inhibitors: involvement of PI3-kinase/Akt pathway and Kv channels.

3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase inhibitors (statins) have several non-lipid-lowering actions; however, characteristics of their acute vasodilator effects remain to be elucidated. In this study, acute vasodilator effects of statins were examined in isolated rat blood vessels. After incubation with cerivastatin (1 microM) for 2 hours, acetylcholine-induced endothelium-dependent relaxations were enhanced in the rat aorta. This effect was abolished by a nitric oxide synthase (NOS) inhibitor, L-NNA, and by a PI3 kinase inhibitor, LY294002. Western blot analysis showed that the extent of phosphorylation of Akt, an active form of Akt, was increased by cerivastatin while it was reduced by LY294002, suggesting an involvement of PI3 kinase/Akt-dependent activation of endothelial NOS. At higher concentrations (1-300 microM), both cerivastatin and fluvastatin, but not pravastatin, directly relaxed the blood vessels, regardless of the presence or absence of the endothelium. These relaxations were abolished by KCl and were significantly inhibited by an inhibitor of Kv channel, 4-aminopyridine. These results indicate that multiple mechanisms are involved in the acute vasodilator effects of statins, including augmentation of nitric oxide-mediated endothelium-dependent relaxations through the PI3 kinase/Akt pathway and endothelium-independent relaxations via Kv channel-mediated smooth muscle hyperpolarizations. These acute vasodilator effects of statins may account, at least in part, for their beneficial effects on cardiovascular diseases associated with impaired organ blood flow.

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.

Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase.

BACKGROUND: Hypoxia-induced pulmonary hypertension is a major cause of morbidity and mortality. Hypoxia induces pulmonary vasoconstriction, in part, by decreasing endothelial nitric oxide synthase (eNOS) expression. The mechanism by which hypoxia decreases eNOS expression is not known but may involve Rho-kinase-induced actin cytoskeletal changes in vascular endothelial cells. METHODS AND RESULTS: To determine whether hypoxia regulates eNOS expression through Rho-kinase, we exposed human saphenous and pulmonary artery endothelial cells to hypoxia (3% O2) with and without a Rho-kinase inhibitor, hydroxyfasudil (0.1 to 100 micromol/L), for various durations (0 to 48 hours). Hypoxia increased Rho-kinase expression and activity by 50% and 74%, decreased eNOS mRNA and protein expression by 66+/-3% and 57+/-5%, and inhibited eNOS activity by 48+/-9%. All of these effects of hypoxia on eNOS were reversed by cotreatment with hydroxyfasudil. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase or Rho-kinase by overexpression of dominant-negative Rho-kinase reversed hypoxia-induced decrease in eNOS expression. Indeed, disruption of the actin cytoskeleton, the downstream target of Rho-kinase, by cytochalasin D also upregulated eNOS expression. Hypoxia reduced eNOS mRNA half-life from 22+/-2 to 13+/-2 hours, which was reversed by cotreatment with hydroxyfasudil. However, neither hypoxia nor hydroxyfasudil had any effects on eNOS gene transcription. CONCLUSIONS: These results indicate that hypoxia-induced decrease in eNOS expression is mediated by Rho-kinase and suggest that Rho-kinase inhibitors may have therapeutic benefits in patients with hypoxia-induced pulmonary hypertension.