Ikuko Kunihiro

Inhibition of Myosin Phosphatase by Upregulated Rho-Kinase Plays a Key Role for Coronary Artery Spasm in a Porcine Model With Interleukin-1β

BACKGROUND We recently demonstrated that the Rho-kinase-mediated pathway plays an important role for coronary artery spasm in our porcine model with interleukin-1beta (IL-1beta). In this study, we examined whether or not Rho-kinase is upregulated at the spastic site and if so, how it induces vascular smooth muscle hypercontraction. METHODS AND RESULTS Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta-bound microbeads. Two weeks after the operation, as reported previously, intracoronary serotonin repeatedly induced coronary hypercontractions at the IL-1beta-treated site both in vivo and in vitro, which were markedly inhibited by Y-27632, one of the specific inhibitors of Rho-kinase. Reverse transcription-polymerase chain reaction analysis demonstrated that the expression of Rho-kinase mRNA was significantly increased in the spastic compared with the control segment. Western blot analysis showed that during the serotonin-induced contractions, the extent of phosphorylation of the myosin-binding subunit of myosin phosphatase (MBS), one of the major substrates of Rho-kinase, was significantly greater in the spastic than in the control segment and that the increase in MBS phosphorylations was also markedly inhibited by Y-27632. There was a highly significant correlation between the extent of MBS phosphorylations and that of contractions. CONCLUSIONS These results indicate that Rho-kinase is upregulated at the spastic site and plays a key role in inducing vascular smooth muscle hypercontraction by inhibiting myosin phosphatase through the phosphorylation of MBS in our porcine model.

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.

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.

Evidence for Protein Kinase C-Mediated Activation of Rho- Kinase in a Porcine Model of Coronary Artery Spasm

Objective—We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model. However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm. Methods and Results—A segment of left porcine coronary artery was chronically treated with IL-1&bgr;–bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1&bgr;–treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5′-[&ggr;-thio]triphosphate (GTP&ggr;S), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca2+ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTP&ggr;S-induced Ca2+ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKC&dgr; isoform was activated during the hypercontraction. Conclusions—These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKC&dgr; may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.

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

&NA; 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 &mgr;M) 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 μM), 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.

Endothelial vasodilator function is preserved at the spastic/inflammatory coronary lesions in pigs.

BACKGROUND The question of whether or not endothelial vasodilator function in the spastic coronary artery is preserved is still controversial. We recently developed a porcine model in which long-term and local treatment with interleukin-1beta (IL-1beta) from the adventitial site causes coronary arteriosclerotic changes and vasospastic responses to autacoids. The aim of this study was to examine the endothelial vasodilator function in our new porcine model of the spasm both in vivo and in vitro. METHODS AND RESULTS A segment of the porcine coronary artery was aseptically wrapped with cotton mesh that held absorbed IL-1beta-bound microbeads. Two weeks after the procedure, intracoronary administration of serotonin caused coronary vasospasm at the IL-1beta-treated site (n = 10). Coronary vasodilatation to bradykinin, substance P, or an increase in coronary blood flow was preserved at the spastic site. Vasodilator responses to 3-morpholinosydnonimine (an NO donor) and nitroglycerin also were comparable between the 2 sites. The vasoconstricting response to N(G)-monomethyl-L-arginine and the extent of the augmentation of the serotonin-induced vasoconstriction were comparable between the 2 sites. Organ chamber experiments showed that endothelium-dependent relaxations to bradykinin, the calcium ionophore A23187, and even the vasospastic agonist serotonin were preserved at the spastic site, whereas contractions to serotonin were augmented at the spastic site regardless of the presence or absence of the endothelium (n = 6). Endothelium-independent relaxations to sodium nitroprusside were also preserved at the spastic site. CONCLUSIONS These results indicate that endothelial vasodilator function is preserved at the spastic site and that the spasm is caused primarily by smooth muscle hypercontraction in our porcine model.

Combination therapy with cerivastatin and nifedipine improves endothelial dysfunction after balloon injury in porcine coronary arteries

HMG-CoA reductase inhibitors and calcium channel blockers have antiatherogenic effects; however, their mechanisms remain to be elucidated. This study examined the effect of cerivastatin and/or nifedipine on the endothelial dysfunction in porcine balloon-injured coronary arteries. Normal male pigs were randomly divided into the following four groups: control, cerivastatin (1 mg/kg/d PO), nifedipine (4 mg/kg/d PO), and their combination (n = 10 each). We started the treatments 3 days before balloon injury in the proximal left coronary arteries and continued for 4 weeks after the procedure. Then, we examined endothelial vasodilator functions ex vivo in organ chambers and in vitro by Western blotting for eNOS expression. Endothelium-dependent relaxations to serotonin, but not those to bradykinin or the calcium ionophore A23187 or endothelium-independent relaxations to sodium nitroprusside, were significantly impaired by balloon injury. The monotherapy with cerivastatin or nifedipine partially improved, and their combination supernormalized the relaxations to serotonin without affecting those to bradykinin or A23187 or endothelium-independent relaxations to sodium nitroprusside. The expression of eNOS was significantly reduced by balloon injury and normalized by the combination therapy. These results indicate that the combination therapy improves endothelial dysfunction after balloon injury, in which the up-regulation of eNOS may be involved.