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Aims Although distinct DNA methylation patterns have been reported, its localization and roles remain to be defined in heart failure. We investigated the cellular and subcellular localization of DNA methylation and its pathophysiological significance in human failing hearts. Methods and results Using left ventricular (LV) endomyocardial biopsy specimens from 75 patients with dilated cardiomyopathy (DCM; age: 58 ± 14 years old, %female: 32%) and 20 patients without heart failure (controls; age: 56 ± 17 years old, %female: 45%), we performed immunohistochemistry and immunoelectron microscopy for methylated DNA, 5‐methylcytosine (5‐mC). We next investigated possible relations of the incidence of 5‐mC‐positive (%5‐mC+) cardiomyocytes with clinicopathological parameters. Immunopositivity for 5‐mC was detected in the cardiomyocytes and other cell types. The %5‐mC+ cardiomyocytes was significantly greater in DCM hearts than in controls (57 ± 13% in DCM vs. 25 ± 12% in controls, P < 0.0001). The localization of 5‐mC immunopositivity in cardiomyocyte nuclei coincided well with that of heterochromatin, as confirmed by immunoelectron microscopy. Substantial DNA methylation was also observed in interstitial non‐cardiomyocytes, but the incidences did not differ between control and DCM hearts (39 ± 7.9% in DCM vs. 41 ± 10% in controls, P = 0.4099). In DCM patients, the %5‐mC+ cardiomyocytes showed a significant inverse correlation with LV functional parameters such as heart rate (r = 0.2391, P = 0.0388), end‐diastolic pressure (r = 0.2397, P = 0.0397), and ejection fraction (r = −0.2917, P = 0.0111) and a positive correlation with LV dilatation (volume index at diastole; r = 0.2442, P = 0.0347; and volume index at systole; r = 0.3136, P = 0.0062) and LV hypertrophy (mass index; r = 0.2287, P = 0.0484)—that is, LV remodelling parameters. No significant correlations between DNA methylation and the histological parameters of the biopsies, including cardiomyocyte hypertrophy, fibrosis, and inflammatory cell infiltration, were noted. Conclusions The present study revealed increased nuclear DNA methylation in cardiomyocytes, but not other cell types, from DCM hearts, with predominant localization in the heterochromatin. Its significant relations with LV functional and remodelling parameters imply a pathophysiological significance of DNA methylation in heart failure.

We encountered an unfamiliar finding during electron microscopic examination of an endomyocardial biopsy obtained from a 55-year-old woman suffering from heart failure due to dilated phase hypertrophic cardiomyopathy. Many cardiomyocytes contained large vacuoles that were mainly empty except for small amounts of amorphous substrate. These were not autophagic vacuoles, as they lacked limiting membranes. Six years later, we encountered similar histological findings in three successive biopsies sourced from another hospital. They were obtained from a 77-year-old man with hypertrophic cardiomyopathy, a 28-year-old woman with endocardial fibrosis, and a 33-year-old man with dilated cardiomyopathy. This biopsy was the second for the endocardial fibrosis patient, and her first biopsy showed no vacuoles within cardiomyocytes. Close inspection of the procedures revealed that in all of these cases the fixed biopsy specimens were carried to the hospital from other institutes using a refrigerated courier service. We then fixed rat heart tissues, froze them once, and processed them for electron microscopy. In that experiment, we were able to reproduce the vacuolar cardiomyocytes, thereby demonstrating it to be a laboratory artifact. We therefore want to emphasize to physicians not to freeze biopsy specimens and not to use a refrigerated courier service for their transport.

Background The purpose of the present study was to evaluate the tissue characteristics of plaques with moderate or mild stenosis in the inner and outer curvature of the left anterior descending artery (LAD) using integrated backscatter intravascular ultrasound. Methods We evaluated 66 plaques with moderate stenosis (plaque burden >50% but ≤75%) and 49 plaques with mild stenosis (plaque burden >30% but ≤50%) in 66 patients undergoing percutaneous intervention to the LAD. All plaques were >10 mm away from any side branch or previously implanted stents. We divided vessel cross-sections into four quadrants (inner curvature, outer curvature, clockwise lateral side, and counterclockwise lateral side) using the septal branch as a landmark for the inner curvature. We averaged relative lipid area, relative fibrous area, and relative calcified area in minimal lumen area (MLA), three cross-sections proximal to the site of MLA, and three cross-sections distal to the site of MLA. Results In plaques with moderate stenosis, the relative lipid area in the inner curvature was significantly greater than in the outer curvature and lateral sides, whereas there was no significant difference in plaques with mild stenosis. Conclusion The present study provides new findings that lipid pool is clustered in the inner curvature and fibrous tissue is clustered in the outer curvature of plaques with moderate stenosis in non-branching LAD lesions.

Although OPC-28326, 4-( N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionyl-aminobenzoyl) piperidine hydrochloride monohydrate, was developed as a selective peripheral vasodilator with α2-adrenergic antagonist properties, it also reportedly exhibits angiogenic activity in an ischemic leg model. The purpose of this study was to examine the effect of OPC-28326 on the architectural dynamics and function of the infarcted left ventricle during the chronic stage of myocardial infarction. Myocardial infarction was induced in male C3H/He mice, after which the mice were randomly assigned into two groups: a control group receiving a normal diet and an OPC group whose diet contained 0.05% OPC-28326. The survival rate among the mice ( n = 18 in each group) 4 wk postinfarction was significantly greater in the OPC than control group (83 vs. 44%; P < 0.05), and left ventricular remodeling and dysfunction were significantly mitigated. Histologically, infarct wall thickness was significantly greater in the OPC group, due in part to an abundance of nonmyocyte components, including blood vessels and myofibroblasts. Five days postinfarction, Ki-67-positive proliferating cells were more abundant in the granulation tissue in the OPC group, and there were fewer apoptotic cells. These effects were accompanied by activation of myocardial Akt and endothelial nitric oxide synthase. Hypoxia within the infarct issue, assessed using pimonidazole staining, was markedly attenuated in the OPC group. In summary, OPC-28326 increased the nonmyocyte population in infarct tissue by increasing proliferation and reducing apoptosis, thereby altering the tissue dynamics such that wall stress was reduced, which might have contributed to a mitigation of postinfarction cardiac remodeling and dysfunction.

Inflammatory bowel disease (IBD) severely affects the quality of life of patients. At present, there is no clinical solution for this condition; therefore, there is a need for innovative therapies for IBD. Hepatocyte growth factor (HGF) exerts various biological activities in various organs. However, a clinically applicable and effective HGF-based therapy for IBD has yet to be developed. In this study, we examined the therapeutic effect of injecting an adenoviral vector encoding the human HGF gene (Ad.HGF) into the hindlimbs of mice with dextran sodium sulfate (DSS)-induced colitis. Plasma levels of circulating human HGF (hHGF) were measured in injected mice. The results showed that weight loss and colon shortening were significantly lower in Ad.HGF-infected mice as compared to control (Ad.LacZ-infected) colitic mice. Additionally, inflammation and crypt scores were significantly reduced in the entire length of the colon, particularly in the distal section. This therapeutic effect was associated with increased cell proliferation and an antiapoptotic effect, as well as a reduction in the number of CD4+ cells and a decreased CD4/CD8 ratio. The levels of inflammatory, as well as Th1 and Th2 cytokines were higher in Ad.HGF-infected mice as compared to the control colitic mice. Thus, systemically circulating hHGF protein, produced by an adenovirally transduced hHGF gene introduced at distal sites in the limbs, significantly ameliorated DSS-induced colitis by promoting cell proliferation (i.e., regeneration), preventing apoptosis, and immunomodulation. Owing to its clinical feasibility and potent therapeutic effects, this method may be developed into a clinical therapy for treating IBD.

We investigated the effect of cardiac-targeting erythropoietin (EPO)-encapsulated liposomes with sialyl LewisX (SLX) on myocardial infarct (MI) size, left ventricular (LV) remodeling and function, and its molecular mechanism for repairing infarcted myocardium. In rabbits, MI was induced by 30 min of coronary occlusion followed by reperfusion. EPO-encapsulated liposomes with SLX (L-EPO group), EPO-encapsulated liposomes without SLX (L-EPO without SLX group), liposomes with SLX without EPO (L group), or saline (saline group) were intravenously administered immediately after MI. MI sizes and numbers of microvessels were assessed 14 days after MI. Prosurvival proteins and signals were assessed by Western blot analysis 2 and 14 days after MI. Confocal microscopy and electron microscopy showed the specific accumulation of liposomes with SLX in the infarcted myocardium. MI and cardiac fibrosis areas were significantly smaller in the L-EPO group than in the other groups. LV function and remodeling were improved in the L-EPO group. The number of CD31-positive microvessels was significantly greater in the L-EPO group than in the other groups. Higher expressions of EPO receptors, phosphorylated (p)Akt, pERK, pStat3, VEGF, Bcl-2, and promatrix metalloproteinase-1 were observed in the infarct area in the L-EPO group than in the other groups. EPO-encapsulated liposomes with SLX selectively accumulated in the infarct area, reduced MI size, and improved LV remodeling and function through activation of prosurvival signals and by exerting antifibrotic and angiogenic effects. EPO-encapsulated liposomes with SLX may be a promising strategy for active targeting treatment of acute MI.

The Joint European Society of Cardiology-American College of Cardiology Foundation-American Heart Association-World Heart Federation Task Force for the Redefinition of Myocardial Infarction recommends cardiac troponin (cTn)-T as a first-line biomarker, and suggests the use of the 99th percentile of a reference population with acceptable precision (i.e. a coefficient of variance ≤10%) as a cut-off for the diagnosis of acute myocardial infarction. Recently developed troponin assays fulfill this analytical precision. While conventional cTnT assays have often been used as a positive or negative categorical variable, stepwise rises in high sensitivity (Hs)-cTnT in patients presenting with chronic heart failure (HF) have been associated with a progressive increase in the incidence of cardiovascular events. Similar observations have been made in the general population. Hs-cTnT at baseline and during follow-up is a powerful predictor of cardiac events in patients with HF and in the general population. Whether it is the ideal biomarker remains to be confirmed, however. We review the potential contributions of TnT assays in the assessment of risk of HF, in HF, and in myocardial diseases that cause HF.

Summary Background The ambulatory treatment of advanced heart failure (HF) with intermittent infusions of inotropes or natriuretic peptide chosen immediately before each infusion has not been described. Methods Between May 2005 and July 2009, we treated 11 patients presenting with advanced HF, who received a total of 369 infusions of carperitide, olprinone, dopamine, or dobutamine, once or twice weekly. The pharmaceutical was selected before each infusion based on the systolic blood pressure (BP). Results Carperitide, olprinone, and catecholamines were administered to 8 (73 infusions of 0.030 ± 0.004 μg/kg/min for 3.3 ± 0.8 h), 4 (18 infusions of 0.070 ± 0.017 μg/kg/min for 3.3 ± 0.5 h), and 6 patients (278 infusions of 3.6 ± 1.9 μg/kg/min for 2.8 ± 1.0 h), respectively. No adverse effect requiring cessation of infusion was observed. Over a mean follow-up of 29.3 ± 28.8 months (range 2–104), 4 patients died, all from cardiac causes. The Kaplan–Meier cumulative survival rate was 69.3% at 20 months (median follow-up). Compared with the pre-infusion period, the duration and number of hospitalizations for management of HF were decreased by 73.9% ( p = 0.017), and 51.9% ( p = 0.007), respectively, during the treatment period, and the overall medical costs by 56.9% ( p = 0.021). Conclusions In this study population, intermittent drug infusions selected from inotropes or natriuretic peptide based on the baseline systolic BP significantly decreased the length and number of hospitalizations and costs, without increasing mortality. These results indicate that intermittent infusions might be one of the therapeutic options in advanced HF.

Background— There still remain safety concerns on surgical procedures after coronary drug-eluting stents (DES) implantation, and optimal management of perioperative antiplatelet therapy (APT) has not been yet established. Methods and Results— During 3-year follow-up of 12 207 patients (DES=6802 patients and bare-metal stent [BMS] only=5405 patients) who underwent coronary stent implantation in the CREDO-Kyoto registry cohort-2, surgical procedures were performed in 2398 patients (DES=1295 patients and BMS=1103 patients). Surgical procedures (early surgery in particular) were more frequently performed in the BMS group than in the DES group (4.4% versus 1.9% at 42-day and 23% versus 21% at 3-year, log-rank P =0.0007). Cumulative incidences of death/myocardial infarction (MI)/stent thrombosis (ST) and bleeding at 30 days after surgery were low, without differences between BMS and DES (3.5% versus 2.9%, P =0.4 and 3.2% versus 2.1%, P =0.2, respectively). The adjusted risks of DES use relative to BMS use for death/MI/ST and bleeding were not significant (hazard ratio: 1.63, 95% confidence interval: 0.93 to 2.87, P =0.09 and hazard ratio: 0.6, 95% confidence interval: 0.34 to 1.06, P =0.08, respectively). The risks of perioperative single- and no-APT relative to dual-APT for both death/MI/ST and bleeding were not significant; single-APT as compared with dual-APT tended to be associated with lower risk for death/MI/ST (hazard ratio: 0.4, 95% confidence interval: 0.13 to 1.01, P =0.053). Conclusions— Surgical procedures were commonly performed after coronary stent implantation, and the risk of ischemic and bleeding complications in surgical procedures was low. In patients selected to receive DES or BMS, there were no differences in outcomes. Perioperative administration of dual-APT was not associated with lower risk for ischemic events.

SummaryIn industrialized countries, chronic heart failure (HF) is a major illness and cause of death. However, because of the paucity of specific clinical manifestations of HF, its early diagnosis and management might be challenging. Therefore, biochemical markers of HF are now being closely scrutinized. An ideal biochemical marker should be a prognostic indicator, should assist in the early diagnosis, reflect the therapeutic response, and help grading the risk associated with each stage of HF. This review summarizes our current understanding of biochemical markers of HF.

Long-term safety and efficacy of drug-eluting stents remains controversial. The CREDO-Kyoto registry cohort-2 is a physician-initiated non-company sponsored multi-center registry enrolling consecutive patients undergoing first coronary revascularization in 26 centers in Japan. We compared 3-year outcome between patients treated with sirolimus-eluting stent (SES) only (5092 patients) and bare-metal stent (BMS) only (5405 patients). SES-use as compared with BMS-use was associated with significantly lower adjusted risk for all-cause death [hazard ratio (HR) [95% confidence interval (CI)] 0.72 (0.59–0.87), P = 0.0007], which was mainly driven by the reduction in non-cardiac death [HR (95% CI) 0.64 (0.48–0.85), P = 0.002]. The risk of cardiac death [HR (95% CI) 0.82 (0.63–1.07), P = 0.15], myocardial infarction [HR (95% CI) 0.73 (0.51–1.03), P = 0.07] and definite stent thrombosis [HR (95% CI) 0.62 (0.35–1.09), P = 0.1] was not different between the two groups. Despite longer duration of thienopyridine administration, SES-use was associated with significantly lower risk for bleeding [HR (95% CI) 0.75 (0.6–0.95), P = 0.02] and similar risk for stroke [HR (95% CI) 1.0 (0.75–1.34), P = 1.0]. The risk for target-lesion revascularization (TLR) was markedly lower in the SES group [HR (95% CI) 0.42 (0.36–0.48), P < 0.0001]. The direction and magnitude of the effect of SES relative to BMS in patients presenting acute myocardial infarction (AMI) were similar to those in patients presenting otherwise. In conclusion, SES-use as compared with BMS-use was associated with marked reduction of TLR without any increases in death, myocardial infarction, stent thrombosis, stroke and bleeding in real world clinical practice regardless of clinical presentation including AMI.

Previous studies have shown inconsistent results regarding the effects of concomitant use of clopidogrel and proton pump inhibitors (PPI) on cardiovascular outcomes. We sought to evaluate the clinical impact of PPI-use in patients treated with thienopyridines after percutaneous coronary intervention (PCI) in a large Japanese observational database. Among 12446 patients discharged alive on thienopyridines (ticlopidine 90.4% and clopidogrel 9.6%), 3223 patients were treated with PPIs and 9223 patients without PPI at the time of hospital discharge. The PPI group included more patients with co-morbidities than the non-PPI group. The adjusted hazard ratio (HR) of PPI-use for a composite of cardiovascular death, myocardial infarction, and stroke was 1.26 (95% confidence interval (CI) 1.09-1.47, p = 0.002). The adjusted HR of PPI-use for bleeding was 1.26 (95% CI 1.05-1.52, p = 0.013). Cardiovascular and bleeding outcomes were not different among the three groups receiving three different types of PPI. The negative effect of PPI on cardiovascular outcome was consistently seen in both drug-eluting stent (DES) [HR 1.31 (95% CI 1.07-1.6, p = 0.0097)] and non-DES strata [HR 1.25 (95% CI: 0.99-1.57, p = 0.057)] (Interaction p = 0.79) despite the fact that the duration of thienopyridine administration was significantly longer in patients receiving DES. In conclusion, cardiovascular outcomes after PCI were significantly worse in patients with PPI than in patients without PPI in the Japanese real clinical practice. However, the observed poorer cardiovascular outcome in patients receiving PPI was most likely to be related to residual confounding and seemed not causally related to attenuation of antiplatelet effect of thienopyridine through interaction with PPI.

Granulocyte colony-stimulating factor (G-CSF) is a potent angiogenic factor. We hypothesized that G-CSF-immersed gelatin hydrogel microspheres (G-CSF-GHMs) injected into the ischemic legs might continuously release a small amount of G-CSF to locally stimulate angiogenesis without unfavorable systemic effects. Just after ligation of the right femoral artery of BALB/c mice, recombinant human G-CSF (100-μg/kg)-immersed GHM was injected into the right hindlimb muscles; the controls included a saline-injected group, an intramuscularly injected G-CSF group, a subcutaneously injected G-CSG group, and an empty GHM-injected group. Eight weeks later, improvement of blood perfusion to the ischemic limb was significantly augmented in the G-CSF-GHM group compared with any of the control groups. Despite there being no increase in the serum concentration of G-CSF, in peripheral granulocytes, or in circulating endothelial progenitor cells, not only capillary but also arteriolar density was significantly increased in this group. Next, we started treatment with G-CSF-GHM 4 weeks after ligation to examine whether the treatment is effective if performed during the chronic stage of ischemia. The late treatment was also found to effectively improve blood flow in the ischemic leg. In conclusion, G-CSF-GHM administration is suggested to be a promising and readily usable approach to treating peripheral artery disease, applicable even during the chronic stage.

Aims Autophagy is activated in cardiomyocytes in ischaemic heart disease, but its dynamics and functional roles remain unclear after myocardial infarction. We observed the dynamics of cardiomyocyte autophagy and examined its role during postinfarction cardiac remodelling. Methods and results Myocardial infarction was induced in mice by ligating the left coronary artery. During both the subacute and chronic stages (1 and 3 weeks postinfarction, respectively), autophagy was found to be activated in surviving cardiomyocytes, as demonstrated by the up-regulated expression of microtubule-associated protein-1 light chain 3-II (LC3-II), p62 and cathepsin D, and by electron microscopic findings. Activation of autophagy, specifically the digestion step, was prominent in cardiomyocytes 1 week postinfarction, especially in those bordering the infarct area, while the formation of autophagosomes was prominent 3 weeks postinfarction. Bafilomycin A1 (an autophagy inhibitor) significantly aggravated postinfarction cardiac dysfunction and remodelling. Cardiac hypertrophy was exacerbated in this group and was accompanied by augmented ventricular expression of atrial natriuretic peptide. In these hearts, autophagic findings (i.e. expression of LC3-II and the presence of autophagosomes) were diminished, and activation of AMP-activated protein kinase was enhanced. Treatment with rapamycin (an autophagy enhancer) brought about opposite outcomes, including mitigation of cardiac dysfunction and adverse remodelling. A combined treatment with bafilomycin A1 and rapamycin offset each effect on cardiomyocyte autophagy and cardiac remodelling in the postinfarction heart. Conclusion These findings suggest that cardiomyocyte autophagy is an innate mechanism that protects against progression of postinfarction cardiac remodelling, implying that augmenting autophagy could be a therapeutic strategy.

Objectives We examined the effect of asialoerythropoietin (asialoEPO), a nonerythrogenic derivative of erythropoietin (EPO), on renal dysfunction-associated heart failure. Background Although EPO is known to exert beneficial effects on cardiac function, the clinical benefits in patients with chronic kidney disease are controversial. It remains to be addressed whether previously reported outcomes were the result of relief of the anemia, adverse effects of EPO, or direct cardiovascular effects. Methods Mice underwent 5/6 nephrectomy to cause renal dysfunction. Eight weeks later, when renal dysfunction was established, anemia and cardiac dysfunction and remodeling were apparent. Mice were then assigned to receive saline (control), recombinant human erythropoietin (rhEPO) at 5,000 IU (714 pmol)/kg, or asialoEPO at 714 pmol/kg, twice/week for 4 weeks. Results Although only rhEPO relieved the nephrectomy-induced anemia, both rhEPO and asialoEPO significantly and similarly mitigated left ventricular dilation and dysfunction. The hearts of rhEPO- or asialoEPO-treated mice showed less hypertrophy, reflecting decreases in cardiomyocyte hypertrophy and degenerative subcellular changes, as well as significant attenuation of fibrosis, leukocyte infiltration, and oxidative deoxyribonucleic acid damage. These phenotypes were accompanied by restored expression of GATA-4, sarcomeric proteins, and vascular endothelial growth factor and decreased inflammatory cytokines and lipid peroxidation. Finally, myocardial activation was observed of extracellular signal-regulated protein kinase and signal transducer and activator of transcription pathways in the treated mice. Conclusions EPO receptor signaling exerts direct cardioprotection in an animal model of renal dysfunction-associated heart failure, probably by mitigating degenerative, pro-fibrosis, inflammatory, and oxidative processes but not through relief of anemia.

Lindera strychnifolia (Tendai-Uyaku), a medicinal plant, has long been used for the treatment of cardiac, renal and rheumatic diseases in Japan. We investigated the effect of Lindera strychnifolia on systolic blood pressure, cardiac function, and plasma noradrenaline levels in rats. Spontaneously hypertensive rats (SHR) were given free access to water or extract solution of Lindera strychnifolia, which was extracted with a ratio of 10 g Lindera strychnifolia roots/20 ml water. Systolic blood pressure was measured by using a tail-cuf sphygmomanometer twice a week from 10 to 30 weeks of age, and compared to the age-matched Wistar Kyoto rats (WKY) as a control group. At 30 weeks of age, heart function was measured by echocardiography and blood samples were taken for detection of plasma noradrenaline levels, and rats were then sacrificed. Systolic blood pressure gradually increased from 10 to 30 weeks of age in the SHR group, while it did not change in the WKY group. In the Lindera-treated SHR group, the increase in systolic blood pressure was significantly attenuated from 21 to 30 weeks of age. Echocardiography showed a significant increase in ejection fraction in the Lindera-treated SHR group (60.4 ± 7.8%) as compared to the SHR group (39.7 ± 23.4%). Plasma noradrenaline levels were significantly decreased in Lindera-treated SHR group compared to the SHR group. These results suggest that Lindera strychnifolia has an anti-hypertensive effect and improves cardiac function in spontaneous hypertensive rats. These effects may be related to the decrease in plasma noradrenaline levels by Lindera strychnifolia.

We investigated whether postinfarct treatment with oxytocin (OT) improves left ventricular (LV) function and remodeling via cardiac repair of myocardial ischemia-reperfusion injury.Experiments were performed with 30 minutes of coronary occlusion and 2 or 14 days of reperfusion rabbit model of myocardial infarction. LV function and remodeling were significantly improved in the OT group. The infarct size was significantly reduced in the OT group. The number of CD31-positive microvessels was increased significantly in the OT group. There were no Ki67-positive myocytes in either group. The expression of the OT receptor, phosphorylated (p)-Akt protein kinase, p-extracellular signal-regulated protein kinase, p-enodthelial NO synthase, p-signal transducer and activator of transcription 3, vascular endothelial growth factor, B-cell lymphoma 2, and matrix metalloproteinase-1 (MMP-1) were markedly increased in the OT group days 2 and 14 post myocardial infarction.Postinfarct treatment with OT reduces myocardial infarct size and improves LV function and remodeling by activating OT receptors and prosurvival signals and by exerting antifibrotic and angiogenic effects through activation of MMP-1, endothelial NO synthase, and vascular endothelial growth factor. These findings provide new insight into therapeutic strategies for ischemic heart disease.

Objectives The purpose of this study was to elucidate the usefulness of integrated backscatter (IBS) transesophageal echocardiography (TEE) for the evaluation of atrial degeneration and clarify whether atrial degeneration predicts the occurrence of atrial fibrillation (AF). Background One of the causes of AF is pathological degeneration of the left atrium (LA). However, there is no appropriate method to evaluate degeneration of the LA in the clinical setting. Methods The IBS images were acquired with TEE with a 4- to 7-MHz transducer. The IBS values were calculated as the average power of the backscattered signal from regions of interest (ROI). In the pathological study, we measured IBS values of 21 left atrial specimens obtained from 10 autopsied hearts. Relative interstitial area in the ROI was automatically calculated by a personal computer. In the clinical study, we measured IBS values of the entire LA wall at 5-mm intervals (except the posterior wall) in 42 patients (18 non-AF patients, 14 paroxysmal AF patients, and 10 chronic AF patients). Each IBS value was color-coded to construct 3-dimensional maps. Results There was a weak correlation between the relative interstitial area and IBS values (r = 0.45, p = 0.038). Average corrected IBS values of total voxels in color-coded maps in the AF group (24.4 ± 6.4 dB) and the paroxysmal AF group (23.9 ± 9.6 dB) were significantly greater than those in the non-AF group (15.6 ± 7.4 dB, p = 0.007), whereas there was no significant difference in LA diameter between the paroxysmal AF group (39.4 ± 6.5 mm) and the non-AF group (36.7 ± 5.5 mm). Conclusions With IBS-TEE, we can identify an increase in atrial degeneration that might predict the occurrence of AF before LA dilation.

BACKGROUND Acarbose, an antidiabetic drug, is an alpha-glucosidase inhibitor that can inhibit glucose absorption in the intestine. A recent large-scale clinical trial, STOP-NIDDM, showed that acarbose reduces the risk of myocardial infarction. We examined whether acarbose reduces myocardial infarct size and investigated its mechanisms. METHODS AND RESULTS Rabbits were fed with 1 of 2 diets in this study: normal chow, 30 mg acarbose per 100 g chow for 7 days. Rabbits were assigned randomly to 1 of 4 groups: control (n = 10), acarbose (n = 10), acarbose + 5HD (n = 10, intravenous 5 mg/kg of 5-hydroxydecanoate), and 5HD (n = 10, intravenous 5 mg/kg of 5HD). Rabbits then underwent 30 minutes of coronary occlusion followed by 48-hour reperfusion. Postprandial blood glucose levels were higher in the control group than in the acarbose group. The infarct size as a percentage of the left ventricular area at risk was reduced significantly in the acarbose (19.4% +/- 2.3%) compared with the control groups (42.8% +/- 5.4%). The infarct size-reducing effect of acarbose was abolished by 5HD (43.4% +/- 4.7%). Myocardial interstitial 2,5-dihydroxybenzoic acid levels, an indicator of hydroxyl radicals, increased during reperfusion after 30 minutes of ischemia, but this increase was inhibited in the acarbose group. This was reversed by 5HD. CONCLUSION Acarbose reduces myocardial infarct size by opening mitochondrial KATP channels, which may be related to the prevention of postprandial hyperglycemia and hydroxyl radical production.

To address whether adult rat ventricular cardiomyocytes (ARVCs) exposed to oxidant stress die via apoptosis (secondarily by necrosis) or primarily by necrosis, we exposed ARVCs to hydrogen peroxide (H2O2; 0.1-100 microM) for up to 24 h and then compared them with isoproterenol-induced apoptotic and Triton X-induced necrotic controls. Cellular shrinkage preceded plasma membrane disruption, reflected by trypan blue uptake in ARVCs exposed to lower concentrations of H2O2 (1 microM; an apoptotic pattern), but the order was reversed in cells exposed to higher concentrations of H2O2 (1 microM; a necrotic pattern). DNA fragmentation, caspase-3 activation, mitochondrial membrane potential preservation, and ATP preservation were all apparent in ARVCs treated with low H2O2 (0.5 microM), but not in those treated with high H2O2 (10 microM). In addition, electron microscopy revealed unique morphology in H2O2-treated ARVCs; i.e., the nuclei had a homogeneous ground glass-like appearance that was never accompanied by chromatin condensation. Apparently, high concentrations of H2O2 caused primary necrosis in ARVCs, whereas low concentrations induced biochemically comparable apoptosis, although the latter did not satisfy the morphological criteria of apoptosis. These findings caution against the use of oxidant stress, H2O2 in particular, as an inducer of apoptosis in ARVCs.

Objectives We hypothesized that erythropoietin (EPO)-immersed gelatin hydrogel microspheres (GHM) injected into ischemic legs might continuously release a small amount of EPO to locally stimulate angiogenesis without unfavorable systemic effects. Background EPO is a potent angiogenic factor, but its use for relieving ischemic organs is limited because of the untoward systemic erythrogenic effect and its short half-life in plasma. Methods The right femoral arteries of BALB/c mice were ligated. Recombinant human EPO (5,000 IU/kg)-immersed GHM was injected into the right hind limb muscles (n = 12); the control groups included a saline-injected group (n = 12), an EPO-injected group (n = 8), and an empty GHM-injected group (n = 8). Results Eight weeks later, improvement of blood perfusion to the ischemic limb was significantly augmented in the EPO-GHM group compared with any of the control groups. There was no increase in the hemoglobin level, nor was there any increase in endothelial progenitor cells. However, capillary and arteriolar densities were significantly increased in this group. Although the treatment did not affect the levels of vascular endothelial growth factor or interleukin-1 beta, it up-regulated the EPO receptor and matrix metalloproteinase-2 and activated the downstream signaling of Akt and also endothelial nitric oxide synthase in ischemic limbs, which might have been associated with the evident angiogenic and arteriogenic effects in the present system. Conclusions The present drug delivery system is suggested to have potential as a novel noninvasive therapy for ischemic peripheral artery disease.

We hypothesized that therapy, composed of antiapoptotic soluble Fas (sFas) gene transfer, combined with administration of the cardioprotective cytokine granulocyte colony-stimulating factor (G-CSF), would markedly mitigate cardiac remodeling and dysfunction following myocardial infarction (MI). On the 3rd day after MI induced by ligating the left coronary artery in mice, four different treatments were initiated: saline injection (Group C, n = 26); G-CSF administration (Group G, n = 27); adenoviral transfer of sFas gene (Group F, n = 26); and the latter two together (Group G+F, n = 26). Four weeks post-MI, Group G+F showed better survival than Group C (96 vs. 65%, P < 0.05) and the best cardiac function among the four groups. In Group G, the infarct scar was smaller and less fibrotic, whereas in Group F the scar was thicker, without a reduction in area, and contained abundant myofibroblasts and vascular cells; Group G+F showed both phenotypes. G-CSF exerted a beneficial effect on infarct tissue dynamics through antifibrotic and proliferative effects on granulation tissue; however, it also exerts an adverse proapoptotic effect that leads to thinning of the infarct scar. sFas appeared to offset the latter drawback. In vitro study using cultured myofibroblasts derived from the infarct tissue revealed that G-CSF increased proliferating activity of those cells accompanying activation of Akt and signal transducer and activator of transcription 3, while accelerating Fas-mediated apoptosis with increasing Bax-to-Bcl-2 ratio. The results suggest that combined use of G-CSF administration and sFas gene therapy is a potentially powerful tool against post-MI heart failure.

1. In the present study, we examined the effects of inhibiting transforming growth factor (TGF)-beta in a mouse model of diabetic nephropathy. 2. An adenovirus harbouring the gene encoding soluble TGF-beta type II receptor (Ad.CAG-sTbetaRII), a competitive inhibitor of TGF-beta, was injected into hindlimb muscles (systemic delivery) of mice 5 weeks after the induction of diabetes with streptozotocin. The control group was injected with an adenovirus encoding the LacZ gene (Ad-LacZ). 3. Five weeks after administration, anti-TGF-beta gene therapy was found to have had no effect on renal function, albuminuria or glucose metabolism in mice with diabetic nephropathy. Nonetheless, this gene therapy did significantly reduce fibrosis in both glomeruli and renal tubules. These effects were accompanied by attenuation of the increased expression of alpha-smooth muscle actin normally seen in kidneys of diabetic mice and better preservation of glomerular cell numbers, although the thickness of the glomerular capillary basement membrane was unchanged. The plasma concentration of soluble TGF-beta type II receptor peaked on Day 7 after treatment, but was undetectable by Day 14. Moreover, a second treatment with Ad.CAG-sTbetaRII failed to prolong the interval of gene product expression in the blood. 4. The present anti-TGF-beta gene therapy showed a significant antifibrotic effect in a model of diabetic nephropathy, but failed to improve renal function. The inadequacy of the observed effect is likely due to the relatively short interval of gene product expression. This problem will have to be overcome if gene therapies for slowly progressing diseases, like diabetic nephropathy, are to be realised.

Many claims have been made regarding the therapeutic efficacy of acupuncture. However, most controlled clinical studies have been limited to treatment of pain-related disorders and do not provide objective, quantifiable data for analysis. Traditional acupuncture has been applied to chronic obstructive pulmonary disease (COPD). However, only a few studies have been performed to determine the efficacy of this treatment. This study was conducted to determine whether a combination of traditional acupuncture treatment and conservative treatment for COPD improves dyspnea on exercise.This was a prospective trial with matched-pair parallel groups of patients from the departments of respiratory internal medicine of Gifu University of Medicine, Meiji University of Oriental Medicine, and Gifu Red Cross Hospital, Japan. Thirty patients were divided into the acupuncture group (n = 15) and the control group (n = 15). The control group received conservative treatment with medication only. The acupuncture group received acupuncture treatments once a week for 10 weeks, in addition to conservative treatment with medication. The main outcome measure was the Modified Borg dyspnea scale after the 6-minute walk test.The acupuncture group had significantly better results on the Borg scale than the control group after 10 weeks (2.2 +/- 2.7 versus 6.4 +/- 3.4, p = 0.0001, 95% confidence interval, -5.10 to -2.35, paired t-test). The 6-minute walk distance and oxygen saturation at the minimum rate improved significantly in the acupuncture group compared with the control group.This study demonstrated that acupuncture contributed to the reduction of COPD-related dyspnea on exercise in 15 matched-pair parallel subjects.

Objectives This study sought to determine whether initial medical therapy (MT) only or percutaneous coronary intervention plus medical therapy (PCI+MT) is better for patients with low-risk stable coronary artery disease (CAD) indicated for intervention in Japan. Background Several multicenter studies have suggested that in the above patients, an initial management strategy of PCI+MT does not reduce the long-term risk of cardiovascular events more effectively than initial MT only. Methods We conducted a randomized comparative study (JSAP [Japanese Stable Angina Pectoris] study) in the previously mentioned patients. Results The patients were randomized to PCI+MT (n = 192) or initial MT only group (n = 192), and the patient characteristics were very similar in the 2 groups. During the 3.3-year follow-up, there was no significant difference in the cumulative death rate between PCI+MT (2.9%) and MT (3.9%). However, the cumulative risk of death plus acute coronary syndrome was significantly smaller in PCI+MT. Conclusions In stable low-risk CAD, PCI+MT may improve long-term prognosis more effectively than MT.

During the progression of heart failure (HF), phases of chronic compensation and acute decompensation alternate and the clinical status worsens during the acute phase. At the present time, few studies have focused their attention on the cycles of compensated and decompensated phases from the perspective of myocyte injury. We hypothesize that persistently increased serum cTn concentrations during chronic compensated HF identify patients likely to need multiple admissions to the hospital for management of acute cardiac decompensation, worsening their long-term prognosis by causing further myocyte injury during the acute phase. In patients with acute cardiac decompensation, myocyte injury observed within hours or days has a long-term predictive value, and the acute surge of myocyte injury occurring in the acutely decompensated phase might be an important therapeutic target from the perspective of myocyte preservation. Clinical trials that limit myocyte injury during acutely decompensated as well as during chronic compensated HF are warranted.

Although beneficial effects of granulocyte colony-stimulating factor (G-CSF) have been demonstrated on post-myocardia infarction (MI) process, the mechanisms and feasibility are not fully agreed yet. We investigated effects of a long-term treatment with a low-dose G-CSF started 1 day after the onset of MI, on post-infarction process. One day after being made MI by left coronary ligation, mice were given G-CSF (10 microg/kg/day) for 4 weeks. The G-CSF treatment resulted in a significant mitigation of cardiac remodelling and dysfunction. In the G-CSF-treated hearts, the infarcted scar was smaller with less fibrosis and abundant vessels while in the non-infarcted area, hypertrophic cardiomyocytes with attenuated degenerative changes and reduced fibrosis were apparent. These effects were accompanied by activation of signal transducer and activator of transcription 3 (STAT3) and Akt and also by up-regulation of GATA-4, myosin heavy chain and matrix metalloproteinases-2 and -9. Apoptosis of cardiomyocytes appeared insignificant at any stages. Parthenolide, a STAT3 inhibitor, completely abolished the beneficial effects of G-CSF on cardiac function and remodelling with loss of effect on both anti-cardiomyocyte degeneration and anti-fibrosis. In contrast, wortmannin, an Akt inhibitor, did not affect G-CSF-induced benefits despite cancelling vessel increase. In conclusion, treatment with G-CSF at a small dose but for a long duration beneficially affects the post-infarction process possibly through STAT3-mediated anti-cardiomyocyte degeneration and anti-fibrosis, but not through anti-cardiomyocyte apoptosis or Akt-mediated angio-genesis. The findings may also imply a more feasible way of G-CSF administration in the clinical settings.

Proteinuria and hypertension are predictors of poor renal outcome in chronic glomerulonephritis (CGN). At the same level of blood pressure (BP) control, we evaluated which is superior, dual blockade of the rennin–angiotensin system (RAS) with both angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II type 1 (AT-1) receptor blockade (ARB) or single blockade of ARB to reduce proteinuria and to preserve renal function in patients with CGN. In this prospective, parallel, open study of 86 patients with CGN, we compared the effects on proteinuria and renal functions of 36 months with comparable blood pressure (BP) control achieved by candesartan cilexetil (candesartan, 4–12 mg/day) or benazepril hydrochrolide (benazepril, 2.5–10 mg/day) with candesartan (4 mg/day). Aiming at BP 125/75 mmHg or less, the dose of candesartan (single blockade) or benazepril (dual blockade) was increased. Dual blockade decreased proteinuria more than single blockade with ARB (−42.3 vs. −60.5%, P

Candesartan has been reported to produce nitric oxide (NO) and to decrease oxidative stress in animal studies. We investigated candesartan's effect on the production of NO and oxidative stress as well as on carotid intima-media thickness (IMT) in hypertensive patients. One-hundred age-matched hypertensive patients were enrolled into an angiotensin II receptor blocker (ARB) group (n=50) or a non-ARB group (n=50). The ARB group was treated with candesartan 8 mg and, when needed, Ca channel blockers, angiotesin-converting enzyme (ACE) inhibitors, and/or beta-blockers. The non-ARB group was treated with drugs other than ARB. Carotid IMT was assessed by echocardiography before and 12 and 24 months after treatment. The urine levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an indicator of oxidative stress, and the serum levels of NOx, an indicator of NO, were measured. Blood pressure decreased to below 140/90 mmHg to the same extent in both groups. Carotid IMT decreased significantly in the ARB group, but not in the non-ARB group, at 12 and 24 months after treatment. The urine levels of 8-OHdG decreased significantly at 6 and 12 months after treatment in the ARB group but did not decrease in the non-ARB group. The serum levels of NOx increased significantly at 6 and 12 months after treatment in the ARB group but not in the non-ARB group. In conclusion, candesartan decreases carotid IMT by enhancing NO production and decreasing oxidative stress in patients with hypertension.

Although amlodipine, a long-acting L-type calcium channel blocker, reportedly prevents left ventricular remodeling and dysfunction after myocardial infarction, the mechanism responsible is not yet well understood. Myocardial infarction was induced in mice by ligating the left coronary artery. Treatment of mice with amlodipine (10 mg·kg−1·day−1), beginning on the third day postinfarction, significantly improved survival and attenuated left ventricular dilatation and dysfunction 4 wk postinfarction compared with treatment with saline or hydralazine. Although infarct sizes did not differ among the groups, the infarcted wall thickness was greater and the infarct segment length was smaller in the amlodipine-treated group, and cellular components, including vessels and myofibroblasts, were abundant within the infarcted area. Ten days postinfarction (the subacute stage), the proliferation of granulation tissue cells in the infarcted area was similar among the groups, but the incidence of apoptosis was significantly lower in the amlodipine-treated group, where Bad, a proapoptotic Bcl-2 family protein, was significantly phosphorylated (inactivated). Calcineurin, which dephosphorylates (activates) Bad, was upregulated in infarcted hearts, but its levels were significantly reduced by amlodipine treatment. In vitro, Fas stimulation augmented calcineurin activity and induced apoptosis among infarct tissue-derived myofibroblasts; both of those effects were strongly inhibited by amlodipine, two other calcium channel blockers (verapamil or nifedipine), and two calcineurin inhibitors (cyclosporin A or FK-506). Amlodipine inhibits Fas-mediated granulation tissue cell apoptosis in infarcted hearts, possibly by attenuating the activities of calcineurin and Bad. These findings may provide new insight into the mechanism by which calcium channel blockers attenuate postinfarction cardiac remodeling and dysfunction.

It is not well-known yet how granulocyte colony-stimulating factor (G-CSF) affects nonischemic cardiomyopathy, though its beneficial effects on acute myocardial infarction are well-established. We hypothesize that G-CSF beneficially might affect nonischemic cardiomyopathy through the direct cardioprotective effects. Here, we show that a single injection of doxorubicin (DOX, 15 mg/kg) induced left ventricular dilatation and dysfunction in mice within 2 weeks, and that these effects were significantly attenuated by human recombinant G-CSF (100 microg/kg/day for 5 days). G-CSF also protected hearts against DOX-induced cardiomyocyte atrophy/degeneration, fibrosis, inflammatory cell infiltration and down regulation of GATA-4 and sarcomeric proteins, myosin heavy chain, troponin I and desmin, both in vivo and in vitro. Cardiac cyclooxygenase-2 was upregulated and G-CSF receptor was downregulated in DOX-induced cardiomyopathy, but both of those effects were largely reversed by G-CSF. No DOX-induced apoptotic effects were seen, nor were there any changes in tumor necrosis factor-alpha or transforming growth factor-beta1 levels. Among downstream mediators of G-CSF receptor signaling, DOX-induced cardiomyopathy involved inactivation of extracellular signal-regulated protein kinase (ERK); the ERK inactivation was reversed by G-CSF. Inhibition of ERK activation, but not cyclooxygenase-2 inhibition, completely abolished beneficial effect of G-CSF on cardiac function. G-CSF did not promote differentiation of bone marrow cells into cardiomyocytes according to the experiment using green fluorescent protein-chimeric mice, and inhibition of CXCR4+ cell homing using AMD3100 did not diminish the effect of G-CSF. Finally, G-CSF was also effective when administered after cardiomyopathy was established. In conclusion, these findings imply the therapeutic usefulness of G-CSF mainly through restoring ERK activation against DOX-induced nonischemic cardiomyopathy.

Background In patients with end-stage renal disease, angiotensin II type 1A receptor (AT1) blockade attenuates the associated cardiac dysfunction. We investigated the molecular signaling mediating that effect. Methods and Results We used 5/6 nephrectomy to induce significant renal dysfunction in AT1 knockout (AT1KO) and wild-type mice (WT). Twelve weeks after nephrectomy, WT showed significant left ventricular dilation and dysfunction that were accompanied by cardiomyocyte hypertrophy, fibrosis, and reduced capillary density. All of these effects were significantly mitigated in AT1KO. Nephrectomy led to upregulation of myocardial expression of AT1, transforming growth factor-β1 (TGF-β1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and phosphorylated Akt (p-Akt), and also led to increased oxidative damage in cardiomyocytes. In AT1KO, TGF-β1, TIMP-1, oxidative damage levels were lower, whereas MMPs and p-Akt levels were higher. Treating nephrectomized WT mice with valsartan (an AT1 blocker), but not hydralazine, improved cardiac function and altered molecular signaling in a manner similar to that seen in AT1KO mice. Notably, AT1 expression was downregulated in valsartan-treated but not hydralazine-treated hearts. Conclusions These findings provide novel insight into the mechanism underlying the beneficial effects of AT1 blockade on cardiac function in a model of renal dysfunction–associated heart failure.

Angiotensin converting enzyme inhibitors (ACE-Is) and angiotensin II receptor blockers (ARBs) are frequently used for the treatment for glomerulonephritis and diabetic nephropathy because of their albuminuria- or proteinuria-reducing effects. To many patients who are nonresponsive to monotherapy with these agents, combination therapy appears to be a good treatment option. In the present study, we examined the effects of the addition of an ARB (losartan) followed by titration upon addition and at 3 and 6 months (n=14) and the addition of an ACE-I followed by titration upon addition and at 3 and 6 months (n=20) to the drug regimen treatment protocol in type 2 diabetic patients with nephropathy for whom more than 3-month administration of an ACE-I or the combination of an ACE-I plus a conventional antihypertensive was ineffective to achieve a blood pressure (BP) of 130/80 mmHg and to reduce urinary albumin to

Porcine pancreatic elastase (PPE), which induces emphysema via apoptosis, was administered to wild-type and Fas-deficient (lpr) mice. On days 3 and 28 after administration, the mean linear intercepts within lung tissues were significantly higher in PPE-treated wild-type and lpr mice than in control mice, though there were no significant differences between the PPE-treated groups. Likewise, the numbers of TUNEL-positive cells were increased in the lungs of PPE-treated wild-type and lpr mice, and again the effect was similar in the two PPE-treated groups. These findings suggest that apoptosis associated with PPE-induced emphysema is not mediated via the Fas/Fas-ligand pathway.

QT dispersion has been reported to increase in patients with essential hypertension, and abnormal QT dispersion is associated with arrythmias and sudden cardiac death. However, whether change in QT dispersion is related to oxidative stress is unclear. We examined the effect of the angiotensin II receptor blocker valsartan on QT dispersion and the relationship between oxidative stress and QT dispersion in patients with essential hypertension. Hypertensive patients whose systolic blood pressure (SBP) was more than 140 mmHg and/or diastolic blood pressure (DBP) was more than 90 mmHg were treated with valsartan. Blood pressure was measured once a month for 6 months. The difference between the maximal and minimal QT intervals within a 12-lead surface ECG was measured and QT dispersion and QTc dispersion corrected by heart rate were obtained before and 6 months after treatment. Left ventricular mass (LVM) assessed by echocardiography was obtained at baseline and 6 months after treatment. Venous blood samples were obtained at baseline and 6 months after treatment to measure serum levels of lipoperoxidation (LPO) and type I and III procollagen. Treatment with valsartan significantly decreased SBP and DBP. QTc dispersion decreased significantly 6 months after treatment with valsartan as compared to the baseline values. Valsartan treatment did not affect the LVM. Valsartan significantly decreased the abnormally high LPO levels. The changes in QTc dispersion were positively correlated with changes in the serum levels of LPO and with changes in DBP. The correlation between changes in LPO and QTc dispersion was more close than that between changes in DBP and QTc dispersion. In conclusion, antihypertensive therapy with valsartan reduces QTc dispersion and this may be related to the ability of valsartan to reduce oxidative stress in patients with essential hypertension.

1. Intratracheal instillation of bleomycin induces a condition in rabbits that serves as a useful model of human pulmonary fibrosis. Bleomycin-induced production of reactive oxygen species leads to acute lung inflammation and induction of apoptosis, which is followed by pulmonary fibrosis at a later chronic stage. In the present study, we tested whether edaravone, a free radical scavenger, would suppress bleomycin-induced acute pulmonary inflammation. 2. Rabbits were divided into three groups (n = 10 in each): (i) a bleomycin-treated group, which received intratracheal instillation of 2 mg/kg bleomycin; (ii) a bleomycin + edaravone group, which received a 10 day regimen of daily intravenous injections of edaravone (3 mg/kg per day) beginning 3 days before bleomycin instillation; and (iii) a saline control group. Rabbits were killed for analysis 7 days after bleomycin administration. 3. In lung tissues from the bleomycin-treated group, marked infiltration of inflammatory cells, consisting mainly of lymphocytes, neutrophils and eosinophils, was observed. In addition, significantly increased numbers of TUNEL-positive (apoptotic) and transforming growth factor-beta-positive cells were seen. All these effects were significantly attenuated by treatment with edaravone. 4. The findings of the present study suggest that edaravone may be useful in the prevention of acute lung injury resulting from the production of reactive oxygen species.

The technical limitations of isolating target cells have restricted the utility of pluripotent embryonic stem (ES) cells. For example, early cardiac (i.e., precontractile) cells have not been isolated from ES cells. Here, we find that direct expression of reporter genes under cell-specific promoters—the currently available strategy for isolating cells lacking cell-specific surface markers—is ineffective for isolating progenitor cells. This was due to the weak activity of cell-specific promoters, particularly in ES cells at early stages. We show that adenoviral conditional targeting efficiently isolates viable ES cell-derived target cells without harmful effects. In this strategy, we employ the α-myosin heavy chain and Nkx2.5 promoter to visualize and purify efficiently differentiated and primitive cells of the cardiac lineage, respectively. While the former cells predominantly expressed sarcomeric proteins and maintained contractile function, the latter demonstrated neither of these features, but rather exhibited expression patterns characteristic of a mixture of primitive cells and cardiomyocytes. Interestingly, smooth muscle actin was predominantly expressed in the latter cells, and both functionally known and unknown genes were systematically identified, demonstrating the benefits of this system. Thus, our method facilitates molecular and cellular studies of development and ES cell-derived cell therapy.

1. In the present study, we investigated the effect of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), a free radical scavenger, on myocardial infarct (MI) size and cardiac function in an in vivo model of MI in rabbits. We further investigated the contribution of hydroxyl radicals, superoxide and nitric oxide (NO) to its effects. 2. Anaesthetized open-chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was injected with saline 10 min before reperfusion. The edaravone group (n = 10) was injected with a bolus of 3 mg/kg edaravone 10 min before reperfusion. The edaravone + N(G)-nitro-L-arginine methyl ester (L-NAME) group (n = 5) was given 10 mg/kg, i.v., L-NAME 10 min before the administration of 3 mg/kg edaravone. The L-NAME group (n = 5) was given 10 mg/kg, i.v., L-NAME 20 min before reperfusion. Infarct size was measured using the triphenyl tetrazolium chloride method and is expressed as a percentage of area at risk. Cardiac function was assessed by echocardiography 14 days after infarction. 3. In another series of experiments, rabbits were subjected to 30 min coronary occlusion and 30 min reperfusion and myocardial interstitial 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA levels, indicators of hydroxyl radical, were measured using a microdialysis technique. 4. Infarct size in the edaravone group was significantly reduced compared with that in the control group (27.4 +/- 6.8 vs 43.4 +/- 6.8%, respectively; P < 0.05). The edaravone-induced reduction of infarct size was abolished by pretreatment with L-NAME. Myocardial interstitial levels of 2,3-DHBA and 2,5-DHBA increased 20 and 30 min after ischaemia and peaked at 10 min reperfusion in the control group. Edaravone significantly inhibited the increase in 2,3-DHBA and 2,5-DHBA levels seen during reperfusion. Dihydroethidium staining showing in situ detection of superoxide was less intense in ischaemic myocardium in the edaravone-treated group compared with the control group. Edaravone improved cardiac function and left ventricular remodelling 14 days after infarction. 5. In conclusion, edaravone significantly reduces MI size and improves cardiac function and LV remodelling by decreasing hydroxyl radicals and superoxide in the myocardium and increasing the production of NO during reperfusion in rabbits.

Objectives Late treatment with erythropoietin (EPO), as well as the administration before the onset of or during the acute stage of myocardial infarction (MI), has recently been shown to mitigate post-MI heart failure. We investigated the mechanisms, including the downstream signaling pathways, for the beneficial effect of late treatment with EPO on chronic post-MI heart failure. Methods and results EPO (1500 U/kg, twice a week) was administered to mice beginning 6 weeks after induction of large MI. The EPO treatment for 4 weeks diminished left ventricular dilatation and improved function. It significantly reduced inflammatory cell infiltration and fibrosis, and increased vascular density in noninfarcted areas. The elevated levels of the inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α and transforming growth factor-β1 seen in the failing hearts were returned nearly to control levels by EPO treatment. Oxidative damage in surviving cardiomyocytes was also significantly attenuated by EPO. Expression of EPO receptor was upregulated in failing hearts, and EPO treatment led to myocardial activation of signal transducer and activator of transcription-3 (Stat3), Stat5, and Akt. These in vivo effects of EPO were confirmed in vitro in experiments that showed the anti-inflammatory and anti-oxidant effects of EPO to be mediated via Stat and Akt activation. Finally, the beneficial effects of EPO were found to persist for 4 weeks after discontinuing treatment. Conclusions It thus appears that Stat-mediated reduction of inflammation and cytokine production and Akt-mediated attenuation of oxidative stress accompany the beneficial effects of late treatment with EPO on chronic post-MI heart failure.

Objectives The purpose of the present study was to validate the diagnostic accuracy of optical coherence tomography (OCT), integrated backscatter intravascular ultrasound (IB-IVUS), and conventional intravascular ultrasound (C-IVUS) for tissue characterization of coronary plaques and to evaluate the advantages and limitations of each of these modalities. Background The diagnostic accuracy of OCT for characterizing tissue types is well established. However, comparisons among OCT, C-IVUS, and IB-IVUS have not been done. Methods We examined 128 coronary arterial sites (42 coronary arteries) from 17 cadavers; IVUS and OCT images were acquired on the same slice as histology. Ultrasound signals were obtained using an IVUS system with a 40-MHz catheter and digitized at 1 GHz with 8-bit resolution. The IB values of the ultrasound signals were calculated with a fast Fourier transform. Results Using histological images as a gold standard, the sensitivity of OCT for characterizing calcification, fibrosis, and lipid pool was 100%, 98%, and 95%, respectively. The specificity of OCT was 100%, 94%, and 98%, respectively (Cohen’s κ = 0.92). The sensitivity of IB-IVUS was 100%, 94%, and 84%, respectively. The specificity of IB-IVUS was 99%, 84%, and 97%, respectively (Cohen’s κ = 0.80). The sensitivity of C-IVUS was 100%, 93%, and 67%, respectively. The specificity of C-IVUS was 99%, 61%, and 95%, respectively (Cohen’s κ = 0.59). Conclusions Within the penetration depth of OCT, OCT has a best potential for tissue characterization of coronary plaques. Integrated backscatter IVUS has a better potential for characterizing fibrous lesions and lipid pools than C-IVUS.

Recent studies suggest transplanted bone marrow cells (BMCs) can be used to reconstitute coronary vessels and myocardium following acute myocardial infarction, thereby improving cardiac function. We sought to investigate the therapeutic potential of BMC transplantation in the treatment of nonischemic cardiomyopathy. Experimental cardiomyopathy was produced by treating rabbits for 8 weeks with doxorubicin (2 mg/kg per week). Two weeks after the treatment was finished, freshly aspirated BMCs or an equivalent volume of phosphate-buffered saline was injected directly into the left ventricular free wall. Four weeks later, heart function was examined during perfusion on a Langendorff apparatus. Left ventricular developed pressure and +/-dp/dt were significantly better in the transplantation group, among which echocardiography also showed significantly better ejection fractions. In addition, left ventricular weights as a fraction of body weight and left ventricular wall thicknesses were both lower in rabbits transplanted with BMCs than in controls. Immunohistochemical analyses carried out 2 weeks after transplantation showed no new myocardium and a very small number of endothelial cells originating from BMCs. On the other hand, immunoblotting revealed upregulated expression of transforming growth factor-beta1 and downregulated expression of matrix metalloproteinase-1 and tumor necrosis factor-alpha following BMC transplantation. In conclusion, autologous BMC transplantation into cardiomyopathic rabbit hearts ameliorates the decline in ventricular function without regenerating cardiomyocytes, most likely by altering expression of various cytokines.

This study aims to define tissue characteristics of vulnerable plaques before acute coronary syndrome (ACS) by use of integrated backscatter intravascular ultrasound (IB-IVUS).Tissue characterization of coronary plaques is possible with the use of IB-IVUS.The subjects were 140 patients with angina pectoris, and we selected 160 coronary lesions without significant stenosis for evaluation. Ultrasound signals were obtained by an IVUS system using a 40-MHz catheter.At the follow-up (30 +/- 7 months), 12 plaques caused ACS after the initial IVUS examination. Ten of the 12 plaques had IVUS parameters recorded at baseline. These 10 plaques were classified as vulnerable plaques (VP), and the other plaques were classified as stable plaques (SP; n = 143). There was no significant difference of vessel area, lumen area, and plaque area between VP and SP. However, plaque burden (60 +/- 9% vs. 52 +/- 9%; p = 0.014), eccentricity (0.70 +/- 0.10 vs. 0.55 +/- 0.17; p = 0.013), remodeling index (1.30 +/- 0.08 vs. 1.16 +/- 0.16; p = 0.006) and percentage lipid area (72 +/- 10% vs. 50 +/- 16%; p0.0001) were greater in VP than in SP. Percentage fibrous area (23 +/- 6% vs. 47 +/- 14%; p0.0001) was smaller in VP than in SP. The sensitivities, specificities, and positive predictive values of percentage fibrous area (90%, 96%, and 69%, respectively) and percentage lipid area (80%, 90%, and 42%, respectively) for classifying VP were evaluated.Tissue characteristics of VP before ACS were different from those of SP. This suggests that VP and SP as classified by IB-IVUS are useful in predicting ACS.

It has been suggested that apoptosis may be responsible for a significant amount of cardiomyocyte death during acute myocardial infarction as well as for a progressive loss of surviving cells in failing hearts. Typical apoptosis can indeed be induced in cardiomyocytes at the experimental conditions. In actual heart diseases, in contrast, there is very little direct morphological evidence of apoptosis in cardiomyocytes occurring at any stage of myocardial infarction and heart failure, despite the availability of much indirect evidence that includes detection of DNA fragmentation and apoptosis-related factors. For that reason, the potential efficacy of therapeutic intervention to prevent apoptosis remains controversial. This review will survey available data from both animals and humans to critically assess the role of cardiomyocyte apoptosis during myocardial infarction and its relevance to myocardial remodeling and during progression to heart failure. Also considered will be nonmyocyte interstitial cells, which have received less attention than myocytes despite definitive evidence of their apoptosis in the infarcted heart and recent studies suggesting that blockade of apoptosis among these cells mitigates postinfarction cardiac remodeling and heart failure. We conclude from our survey that there are many hurdles to surmount before regulation of apoptosis can be clinically applied in the treatment of myocardial infarction and heart failure.

We examined a possible preventive effect of Linderae radix (LR), the root of Lindera strychnifolia, on the progression of diabetic nephropathy. Water extract of Linderae radix (LR extract) was orally administered to the C57BL/KsJ-db/db (db/db) mice, a model of genetic diabetes, at a dose of 730 mg/kg/day for 12 week. The LR extract treatment did not affect glucose metabolism and systolic pressure. However, it resulted in a better renal function as evaluated by creatinine clearance (Ccr) and serum creatinine than the control; Ccr and serum creatinine were progressively worsened in controls (0.13+/-0.01 (l/day) and 0.69+/-0.04 (mg/dl), respectively) whereas unchanged in the treated group (0.24+/-0.03 (l/day), p

Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are cardiogenic and cardiohypertrophic growth factors. Although the therapeutic effects of IGF and HGF have been well demonstrated in injured hearts, it is uncertain whether natural upregulation of HB-EGF after myocardial infarction (MI) plays a beneficial or pathological role in the process of remodeling. To answer this question, we conducted adenoviral HB-EGF gene transduction in in vitro and in vivo injured heart models, allowing us to highlight and explore the HB-EGF-induced phenotypes. Overexpressed HB-EGF had no cytoprotective or additive death-inducible effect on Fas-induced apoptosis or oxidative stress injury in primary cultured mouse cardiomyocytes, although it significantly induced hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. Locally overexpressed HB-EGF in the MI border area in rabbit hearts did not improve cardiac function or exhibit an angiogenic effect, and instead exacerbated remodeling at the subacute and chronic stages post-MI. Namely, it elevated the levels of apoptosis, fibrosis, and the accumulation of myofibroblasts and macrophages in the MI area, in addition to inducing left ventricular hypertrophy. Thus, upregulated HB-EGF plays a pathophysiological role in injured hearts in contrast to the therapeutic roles of IGF and HGF. These results imply that regulation of HB-EGF may be a therapeutic target for treating cardiac hypertrophy and fibrosis.

Recently, ultrasound tissue characterization of the carotid arteries with an integrated backscatter (IB) analysis was shown to identify a high-risk group of atherosclerosis. To clarify whether IB ultrasound is useful in assessing arterial sclerosis as well as stiffness beta and whether IB values reflect the histological structure, we measured IB values of common carotid media in 52 subjects without coronary risk factors and in 10 patients with systemic sclerosis (SSc) in the clinical studies and 12 patients in the histological studies with a Philips Medical Systems Sonos 5500. IB values were correlated with age (r=0.69, P0.0001), intima-media thickness (r=0.72, P0.0001) and stiffness beta (r=0.80, P0.0001) in the control subjects. IB values and stiffness beta in the SSc group were greater than in an age- and sex-matched control group (IB values: 9.6+/-2.7dB versus 16.1+/-1.8dB; stiffness beta: 11.5+/-4.5 versus 20.6+/-5.6, P0.01). IB values of the media were correlated with the elastic fragmentation index (r=0.63, P=0.029) and the collagen fiber index (r=0.59, P=0.046). Measurements of IB values of carotid media are useful for non-invasively evaluating arterial sclerosis.