Takashi Shiroto

Protective Role of Endogenous Erythropoietin System in Nonhematopoietic Cells Against Pressure Overload–Induced Left Ventricular Dysfunction in Mice

Background— Erythropoietin (Epo) receptors (EpoRs) are expressed in the heart. We have recently demonstrated that the endogenous Epo-EpoR system plays an important protective role in myocardial ischemia in mice and humans. In the present study, we tested our hypothesis that the endogenous Epo-EpoR system in nonhematopoietic cells also plays a protective role against pressure overload-induced cardiac dysfunction in vivo. Methods and Results— Transgene-rescued EpoR-null mutant mice (EpoR−/−rescued) that express EpoR exclusively in the hematopoietic cells were subjected to transverse aortic constriction (TAC). At 1 week after TAC, left ventricular weight and lung weight were significantly increased in EpoR−/−rescued mice compared with wild-type mice, although the fibrotic area was comparably increased after TAC in the 2 genotypes. In the EpoR−/−rescued mice with TAC, left ventricular end-diastolic diameter was significantly increased, left ventricular fractional shortening was significantly decreased, and survival rate was significantly decreased compared with wild-type mice with TAC. Phosphorylation of STAT3 at 5 hours and 1 week after TAC and that of p38 at 5 hours after TAC were significantly increased in wild-type mice but not in EpoR−/−rescued mice. Vascular endothelial growth factor protein expression and capillary density in left ventricular myocardium were significantly decreased in EpoR−/−rescued mice with TAC compared with wild-type mice with TAC. Conclusions— These results suggest that the endogenous Epo-EpoR system in the nonhematopoietic cells plays an important protective role against pressure overload-induced cardiac dysfunction in vivo.

Role of Rho-Kinase in the Pathogenesis of Coronary Hyperconstricting Responses Induced by Drug-Eluting Stents in Pigs In Vivo

OBJECTIVES This study examined whether the Rho-kinase pathway is involved in the pathogenesis of coronary hyperconstricting responses induced by drug-eluting stents (DES) in pigs in vivo. BACKGROUND Recent studies showed that coronary vasoconstricting responses are enhanced at the edge of coronary segments implanted with DES compared with bare-metal stents (BMS) in humans. We have previously shown that the activated Rho-kinase pathway plays a central role in the molecular mechanism of coronary vasospasm in animals and humans. METHODS Human coronary artery smooth muscle cells (hCASMCs) were coincubated with various concentrations of paclitaxel (10(-9) to 10(-6) mol/l, corresponding levels reported in DES-implanted arterial tissue) for 24 h. A paclitaxel-eluting stent (PES), sirolimus-eluting stent (SES), and BMS were randomly implanted in the left coronary arteries in pigs for 4 weeks. RESULTS In hCASMCs, paclitaxel significantly enhanced Rho-kinase expression and activity. In a porcine model, coronary vasoconstricting responses to serotonin (10 and 100 microg/kg intracoronary administration) were significantly enhanced at the PES site compared with the BMS site (45+/-4% vs. 30+/-3%; p<0.01; n=12 each), and were abolished by hydroxyfasudil (90 and 300 microg/kg intracoronary administration), a selective Rho-kinase inhibitor. The PES enhanced inflammatory responses and microthrombus formation at the stent edge, where immunoreactivities for Rho-kinase expression and activity were increased. In organ chamber experiments, serotonin-induced contractions were significantly enhanced in rings from the PES edge site compared with the BMS edge site. The SES also caused similar coronary hyperconstricting responses to serotonin in vivo. CONCLUSIONS These results suggest that the Rho-kinase pathway plays an important role in the pathogenesis of DES-induced coronary hyperconstricting responses.

Cardiac shock wave therapy ameliorates left ventricular remodeling after myocardial ischemia–reperfusion injury in pigs in vivo

ObjectivesLeft ventricular (LV) remodeling after acute myocardial infarction (AMI) is associated with a poor prognosis and an impaired quality of life. We have shown earlier that low-energy extracorporeal cardiac shock wave (SW) therapy improves chronic myocardial ischemia in pigs and humans and also ameliorates LV remodeling in a pig model of AMI induced by permanent coronary ligation. However, in the current clinical setting, most of the patients with AMI receive reperfusion therapy. Thus, in this study we examined whether our SW therapy also ameliorates LV remodeling after myocardial ischemia–reperfusion (I/R) injury in pigs in vivo. MethodsPigs were subjected to a 90-min ischemia and reperfusion using a balloon catheter and were randomly assigned to two groups with or without SW therapy to the ischemic border zone (0.09 mJ/mm2, 200 pulses/spot, 9 spots/animal, three times in the first week) (n=15 each). ResultsFour weeks after I/R, compared with the control group, the SW group showed significantly ameliorated LV remodeling in terms of LV enlargement (131±9 vs. 100±7 ml), reduced LV ejection fraction (28±2 vs. 36±3%), and elevated left ventricular end-diastolic pressure (11±2 vs. 4±1 mmHg) (all P<0.05, n=8 each). The SW group also showed significantly increased regional myocardial blood flow (−0.06±0.11 vs. 0.36±0.13 ml/min/g, P<0.05), capillary density (1.233±31 vs. 1.560±60/mm2, P<0.001), and endothelial nitric oxide synthase activity (0.24±0.03 vs. 0.41±0.05, P<0.05) in the ischemic border zone compared with the control group (n=7 each). ConclusionThese results indicate that our SW therapy is also effective in ameliorating LV remodeling after myocardial I/R injury in pigs in vivo.

Characterization of heart failure patients with mid-range left ventricular ejection fraction—a report from the CHART-2 Study

The new category of heart failure (HF), HF with mid‐range left ventricular ejection fraction (LVEF) (HFmrEF), has recently been proposed. However, the clinical features of HFmrEF, with reference to HF with preserved LVEF (HFpEF) and HF with reduced LVEF (HFrEF) in the same HF cohort, remain to be fully examined.

Long-term treatment with nifedipine suppresses coronary hyperconstricting responses and inflammatory changes induced by paclitaxel-eluting stent in pigs in vivo: possible involvement of Rho-kinase pathway

AIMS Accumulating evidence indicates that coronary vasoconstricting responses are enhanced at the edges of coronary segment implanted with a drug-eluting stent (DES) compared with a bare-metal stent (BMS) in humans. We have recently demonstrated that Rho-kinase pathway plays an important role in DES-induced coronary hyperconstricting responses associated with inflammatory changes in pigs in vivo. This study examined whether long-term treatment with calcium channel blocker suppresses DES-induced coronary hyperconstricting responses in pigs in vivo. METHODS AND RESULTS Paclitaxel-eluting stent (PES) and a BMS were randomly implanted in the left coronary arteries in male domestic pigs with and without long-acting nifedipine (NIF, 4 mg/kg/day) for 4 weeks (n = 7 each). Coronary vasomotion was evaluated by quantitative coronary angiography at least 24 h after withdrawal of NIF to avoid its direct effects on coronary vasomotion. In the control group (without NIF), coronary vasoconstricting responses to serotonin (10 and 100 µg/kg, i.c.) were significantly enhanced at the PES site compared with the BMS site (P = 0.009), which were abolished by hydroxyfasudil (90 and 300 µg/kg, i.c.), a selective Rho-kinase inhibitor. The PES-induced vasoconstricting responses were significantly inhibited in the NIF group (P = 0.019). Histological examination showed that inflammatory cell accumulation and microthrombus formation were enhanced at the PES site compared with the BMS site (P < 0.05), both of which were significantly suppressed by NIF associated with reduced Rho-kinase expression and activity (P < 0.05). CONCLUSION These results indicate that long-term treatment with NIF suppresses PES-induced coronary abnormalities partly through Rho-kinase pathway inhibition in vivo.

Eicosapentaenoic acid reduces ischemic ventricular fibrillation via altering monophasic action potential in pigs

Although high intake of n-3 fatty acids is associated with reduced mortality of patients with ischemic heart disease, especially reduction in sudden cardiac death (SCD), the detailed mechanisms remain to be elucidated. Thus, the present study was designed to examine whether long-term treatment with eicosapentaenoic acid (EPA), a major component of n-3 fatty acids, reduces ischemia-induced ventricular fibrillation (VF) in pigs in vivo, and if so, what molecular mechanisms are involved. Male pigs were treated with either a control chow (control group) or a control chow plus EPA (600 mg/kg/day, PO, EPA group) for 3 weeks and were subjected to myocardial ischemia for 90 min (n=8 each) with measurement of the monophasic action potential (MAP), as a marker of ventricular electrophysiological activities. The EPA treatment significantly attenuated the occurrence of VF (control 5.1±1.7 vs. EPA 1.5±0.8 times/animal, P<0.05) and markedly reduced the mortality (control 50% vs. EPA 0%, P<0.05), with the attenuation of MAP duration shortening during ischemia (control -28.1±3.0% vs. EPA -18.2±1.4%, P<0.05). These beneficial effects of EPA were abolished by pre-treatment with cromakalim, a K(ATP) channel opener (0.3 μg/kg/min, IC). Furthermore, EPA significantly inhibited the mRNA and protein expression of Kir6.2, a major component of sarcolemmal K(ATP) channels, in both the ischemic region and non-ischemic regions. These results indicate that long-term treatment with EPA reduces ischemia-induced VF and SCD in pigs in vivo, for which attenuation of MAP duration shortening may be involved.

Association of Adventitial Vasa Vasorum and Inflammation With Coronary Hyperconstriction After Drug-Eluting Stent Implantation in Pigs In Vivo

BACKGROUND The importance of adventitial inflammation has been implicated for the pathogenesis of coronary artery disease. However, the roles of adventitial changes in drug-eluting stent (DES)-induced coronary hyperconstriction remain largely unknown. In the present study, this issue in pigs in vivo with a special reference to adventitial vasa vasorum (VV) formation and Rho-kinase activation, a central mechanism of coronary vasospasm, was examined. METHODS AND RESULTS Each animal received a sirolimus-eluting stent (SES) and a biolimus A9-eluting stent (BES), one in the left anterior descending and another in the left circumflex coronary arteries in a randomized manner (n=18). After 1, 3 and 6 months, coronary vasomotion was examined. At 1 month, coronary vasoconstriction to serotonin was significantly enhanced at the SES edges as compared with the BES edges (SES, 52±7% vs. BES, 22±3%, P<0.01), which was equally prevented by a selective Rho-kinase inhibitor, hydroxyfasudil. A significant difference in vasoconstriction between SES and BES was sustained for 6 months. A micro-CT showed VV augmentation at the SES site, extending to the proximal and distal edges. Immunostainings demonstrated that VV formation, macrophage infiltration in the adventitia and Rho-kinase expressions/activation were significantly enhanced at the SES edges as compared with the BES edges. CONCLUSIONS The DES with durable polymers enhances VV formation and inflammation in the adventitia, associating with the pathogenesis of DES-induced coronary hyperconstriction through Rho-kinase activation in pigs in vivo.

Fast ignition realization experiment with high-contrast kilo-joule peta-watt LFEX laser and strong external magnetic field

A petawatt laser for fast ignition experiments (LFEX) laser system [N. Miyanaga et al., J. Phys. IV France 133, 81 (2006)], which is currently capable of delivering 2 kJ in a 1.5 ps pulse using 4 laser beams, has been constructed beside the GEKKO-XII laser facility for demonstrating efficient fast heating of a dense plasma up to the ignition temperature under the auspices of the Fast Ignition Realization EXperiment (FIREX) project [H. Azechi et al., Nucl. Fusion 49, 104024 (2009)]. In the FIREX experiment, a cone is attached to a spherical target containing a fuel to prevent a corona plasma from entering the path of the intense heating LFEX laser beams. The LFEX laser beams are focused at the tip of the cone to generate a relativistic electron beam (REB), which heats a dense fuel core generated by compression of a spherical deuterized plastic target induced by the GEKKO-XII laser beams. Recent studies indicate that the current heating efficiency is only 0.4%, and three requirements to achieve higher efficiency of the fast ignition (FI) scheme with the current GEKKO and LFEX systems have been identified: (i) reduction of the high energy tail of the REB; (ii) formation of a fuel core with high areal density using a limited number (twelve) of GEKKO-XII laser beams as well as a limited energy (4 kJ of 0.53-μm light in a 1.3 ns pulse); (iii) guiding and focusing of the REB to the fuel core. Laser–plasma interactions in a long-scale plasma generate electrons that are too energetic to efficiently heat the fuel core. Three actions were taken to meet the first requirement. First, the intensity contrast of the foot pulses to the main pulses of the LFEX was improved to >109. Second, a 5.5-mm-long cone was introduced to reduce pre-heating of the inner cone wall caused by illumination of the unconverted 1.053-μm light of implosion beam (GEKKO-XII). Third, the outside of the cone wall was coated with a 40-μm plastic layer to protect it from the pressure caused by imploding plasma. Following the above improvements, conversion of 13% of the LFEX laser energy to a low energy portion of the REB, whose slope temperature is 0.7 MeV, which is close to the ponderomotive scaling value, was achieved. To meet the second requirement, the compression of a solid spherical ball with a diameter of 200-μm to form a dense core with an areal density of ∼0.07 g/cm2 was induced by a laser-driven spherically converging shock wave. Converging shock compression is more hydrodynamically stable compared to shell implosion, while a hot spot cannot be generated with a solid ball target. Solid ball compression is preferable also for compressing an external magnetic field to collimate the REB to the fuel core, due to the relatively small magnetic Reynolds number of the shock compressed region. To meet the third requirement, we have generated a strong kilo-tesla magnetic field using a laser-driven capacitor-coil target. The strength and time history of the magnetic field were characterized with proton deflectometry and a B-dot probe. Guidance of the REB using a 0.6-kT field in a planar geometry has been demonstrated at the LULI 2000 laser facility. In a realistic FI scenario, a magnetic mirror is formed between the REB generation point and the fuel core. The effects of the strong magnetic field on not only REB transport but also plasma compression were studied using numerical simulations. According to the transport calculations, the heating efficiency can be improved from 0.4% to 4% by the GEKKO and LFEX laser system by meeting the three requirements described above. This efficiency is scalable to 10% of the heating efficiency by increasing the areal density of the fuel core.

Low-Intensity Pulsed Ultrasound Enhances Angiogenesis and Ameliorates Left Ventricular Dysfunction in a Mouse Model of Acute Myocardial Infarction

Objective— Left ventricular (LV) remodeling after acute myocardial infarction still remains an important issue in cardiovascular medicine. We have recently demonstrated that low-intensity pulsed ultrasound (LIPUS) therapy improves myocardial ischemia in a pig model of chronic myocardial ischemia through enhanced myocardial angiogenesis. In the present study, we aimed to demonstrate whether LIPUS also ameliorates LV remodeling after acute myocardial infarction and if so, to elucidate the underlying molecular mechanisms involved in the beneficial effects of LIPUS. Approach and Results— We examined the effects of LIPUS on LV remodeling in a mouse model of acute myocardial infarction, where the heart was treated with either LIPUS or no-LIPUS 3 times in the first week (days 1, 3, and 5). The LIPUS improved mortality and ameliorated post–myocardial infarction LV remodeling in mice. The LIPUS upregulated the expression of vascular endothelial growth factor, endothelial nitric oxide synthase, phosphorylated ERK, and phosphorylated Akt in the infarcted area early after acute myocardial infarction, leading to enhanced angiogenesis. Microarray analysis in cultured human endothelial cells showed that a total of 1050 genes, including those of the vascular endothelial growth factor signaling and focal adhesion pathways, were significantly altered by the LIPUS. Knockdown with small interfering RNA of either &bgr;1-integrin or caveolin-1, both of which are known to play key roles in mechanotransduction, suppressed the LIPUS-induced upregulation of vascular endothelial growth factor. Finally, in caveolin-1–deficient mice, the beneficial effects of LIPUS on mortality and post–myocardial infarction LV remodeling were absent. Conclusions— These results indicate that the LIPUS therapy ameliorates post–myocardial infarction LV remodeling in mice in vivo, for which mechanotransduction and its downstream pathways may be involved.

Flash Kα radiography of laser-driven solid sphere compression for fast ignition

Time-resolved compression of a laser-driven solid deuterated plastic sphere with a cone was measured with flash Kα x-ray radiography. A spherically converging shockwave launched by nanosecond GEKKO XII beams was used for compression while a flash of 4.51 keV Ti Kα x-ray backlighter was produced by a high-intensity, picosecond laser LFEX (Laser for Fast ignition EXperiment) near peak compression for radiography. Areal densities of the compressed core were inferred from two-dimensional backlit x-ray images recorded with a narrow-band spherical crystal imager. The maximum areal density in the experiment was estimated to be 87 ± 26 mg/cm2. The temporal evolution of the experimental and simulated areal densities with a 2-D radiation-hydrodynamics code is in good agreement.