Masaya Sakamoto

Leukemia Inhibitory Factor Enhances Survival of Cardiomyocytes and Induces Regeneration of Myocardium After Myocardial Infarction

Background—Myocardial infarction (MI) is a leading cause of cardiac morbidity and mortality in many countries; however, the treatment of MI is still limited. Methods and Results—We demonstrate a novel gene therapy for MI using leukemia inhibitory factor (LIF) cDNA. We injected LIF plasmid DNA into the thigh muscle of mice immediately after inducing MI. Intramuscular injection of LIF cDNA resulted in a marked increase in circulating LIF protein concentrations. Two weeks later, left ventricular remodeling, such as infarct extent and myocardial fibrosis, was markedly attenuated in the LIF cDNA–injected mice compared with vehicle-injected mice. More myocardium was preserved and cardiac function was better in the LIF-treated mice than in the vehicle-injected mice. Injection of LIF cDNA not only prevented the death of cardiomyocytes in the ischemic area but also induced neovascularization in the myocardium. Furthermore, LIF cDNA injection increased the number of cardiomyocytes in cell cycle and enhanced mobilization of bone marrow cells to the heart and their differentiation into cardiomyocytes. Conclusions—The intramuscular injection of LIF cDNA may induce regeneration of myocardium and provide a novel treatment for MI.

Upregulation of Heat Shock Transcription Factor 1 Plays a Critical Role in Adaptive Cardiac Hypertrophy

Exercise-induced cardiac hypertrophy has been reported to have better prognosis than pressure overload-induced cardiac hypertrophy. Cardiac hypertrophy induced by exercise was associated with less cardiac fibrosis and better systolic function, suggesting that the adaptive mechanisms may exist in exercise-induced hypertrophy. Here, we showed a critical role of heat shock transcription factor 1 (HSF1), an important transcription factor for heat shock proteins, in the adaptive mechanism of cardiac hypertrophy. We examined expression of 8800 genes in the heart of exercise-induced hypertrophy model using DNA chip technique and compared with pressure overload–induced hypertrophy. Expression of HSF1 and its target molecule heat shock proteins was significantly upregulated in the heart by exercise but not by chronic pressure overload. Constitutive activation of HSF1 in the heart significantly ameliorated death of cardiomyocytes and cardiac fibrosis and thereby prevented cardiac dysfunction as well as hypertrophy induced by chronic pressure overload. Conversely, decreased activity of HSF1 in the heart promoted cardiac dysfunction in response to exercise, a load that normally leads to adaptive hypertrophy with preserved systolic function. Likewise, cardiac function was significantly impaired from the early phase of pressure overload, when HSF1 activation was inhibited. These results suggest that HSF1 plays a critical role in the transition between adaptive and maladaptive hypertrophy.

Cardiac 12/15 lipoxygenase–induced inflammation is involved in heart failure

To identify a novel target for the treatment of heart failure, we examined gene expression in the failing heart. Among the genes analyzed, Alox15 encoding the protein 12/15 lipoxygenase (LOX) was markedly up-regulated in heart failure. To determine whether increased expression of 12/15-LOX causes heart failure, we established transgenic mice that overexpressed 12/15-LOX in cardiomyocytes. Echocardiography showed that Alox15 transgenic mice developed systolic dysfunction. Cardiac fibrosis increased in Alox15 transgenic mice with advancing age and was associated with the infiltration of macrophages. Consistent with these observations, cardiac expression of monocyte chemoattractant protein 1 (MCP-1) was up-regulated in Alox15 transgenic mice compared with wild-type mice. Treatment with 12-hydroxy-eicosatetraenoic acid, a major metabolite of 12/15-LOX, increased MCP-1 expression in cardiac fibroblasts and endothelial cells but not in cardiomyocytes. Inhibition of MCP-1 reduced the infiltration of macrophages into the myocardium and prevented both systolic dysfunction and cardiac fibrosis in Alox15 transgenic mice. Likewise, disruption of 12/15-LOX significantly reduced cardiac MCP-1 expression and macrophage infiltration, thereby improving systolic dysfunction induced by chronic pressure overload. Our results suggest that cardiac 12/15-LOX is involved in the development of heart failure and that inhibition of 12/15-LOX could be a novel treatment for this condition.

Heat Shock Transcription Factor 1 Protects Cardiomyocytes From Ischemia/Reperfusion Injury

Background—Because cardiomyocyte death causes heart failure, it is important to find the molecules that protect cardiomyocytes from death. The death trap is a useful method to identify cell-protective genes. Methods and Results—In this study, we isolated the heat shock transcription factor 1 (HSF1) as a protective molecule by the death trap method. Cell death induced by hydrogen peroxide was prevented by overexpression of HSF1 in COS7 cells. Thermal preconditioning at 42°C for 60 minutes activated HSF1, which played a critical role in survival of cardiomyocytes from oxidative stress. In the heart of transgenic mice overexpressing a constitutively active form of HSF1, ischemia followed by reperfusion-induced ST-segment elevation in ECG was recovered faster, infarct size was smaller, and cardiomyocyte death was less than wild-type mice. Protein kinase B/Akt was more strongly activated, whereas Jun N-terminal kinase and caspase 3 were less activated in transgenic hearts than wild-type ones. Conclusions—These results suggest that HSF1 protects cardiomyocytes from death at least in part through activation of Akt and inactivation of Jun N-terminal kinase and caspase 3.

Evidence-based practice guideline for the treatment for diabetes in Japan 2013

To promote evidence-based diabetes treatment, the Japan Diabetes Society (JDS) published, in February 2002, our first ‘‘Evidence-based Practice Guide for the Treatment of Diabetes in Japan’’ (J Japan Diab. Soc. 45, Supple 1, 2002). Since then, revised editions have been released every 3 years. Evidence-based medicine is a practice of medicine that attempts to integrate individual physician’s clinical expertise and the best external research evidence, and apply them to solve individual patient’s problems. The physician’s clinical expertise decides whether or not the information obtained from the literature can be applied directly to the individual patient. (Sackett, D.L. et al., BMJ 312: 71, 1996). Based on this policy, this guideline is formulated with several aims: to gather evidence on diabetes treatment both in Japan and overseas, describe it in forms that are easy to understand and use, subject it to evaluation by diabetes specialists, and offer recommendations for diagnosis and treatment. Although the JDS publishes numerous guidelines related to diabetes, it makes sure that all its books including the ‘‘Treatment Guide for Diabetes’’ comply with the recommendations laid out in this guideline. The guideline won plaudits from the Appraisal of Guidelines for Research and Evaluation (AGREE), an external tool for evaluating various guidelines, for its scope and purpose, rigor of development, and clarity of presentation. The 2013 Edition of the Evidence-based Practice Guideline for the Treatment of Diabetes in Japan consists of 355 pages and 24 chapters. Each chapter has statements, explanations, abstract-tables, and references. The Englishlanguage edition was simplified for publication on our website. It features the entire Statement, plus commentaries that provide explanations of several passages. The tables, figures and references are limited only to those that are necessary. We hope this guideline will be found useful both in Japan and overseas.

Comparison of vildagliptin twice daily vs. sitagliptin once daily using continuous glucose monitoring (CGM): Crossover pilot study (J-VICTORIA study)

BackgroundNo previous studies have compared the DPP-4 inhibitors vildagliptin and sitagliptin in terms of blood glucose levels using continuous glucose monitoring (CGM) and cardiovascular parameters.MethodsTwenty patients with type 2 diabetes mellitus were randomly allocated to groups who received vildagliptin then sitagliptin, or vice versa. Patients were hospitalized at 1 month after starting each drug, and CGM was used to determine: 1) mean (± standard deviation) 24-hour blood glucose level, 2) mean amplitude of glycemic excursions (MAGE), 3) fasting blood glucose level, 4) highest postprandial blood glucose level and time, 5) increase in blood glucose level after each meal, 6) area under the curve (AUC) for blood glucose level ≥180 mg/dL within 3 hours after each meal, and 7) area over the curve (AOC) for daily blood glucose level <70 mg/dL. Plasma glycosylated hemoglobin (HbA1c), glycoalbumin (GA), 1,5-anhydroglucitol (1,5AG), immunoreactive insulin (IRI), C-peptide immunoreactivity (CPR), brain natriuretic peptide (BNP), and plasminogen activator inhibitor-1 (PAI-1) levels, and urinary CPR levels, were measured.ResultsThe mean 24-hour blood glucose level was significantly lower in patients taking vildagliptin than sitagliptin (142.1 ± 35.5 vs. 153.2 ± 37.0 mg/dL; p = 0.012). In patients taking vildagliptin, MAGE was significantly lower (110.5 ± 33.5 vs. 129.4 ± 45.1 mg/dL; p = 0.040), the highest blood glucose level after supper was significantly lower (206.1 ± 40.2 vs. 223.2 ± 43.5 mg/dL; p = 0.015), the AUC (≥180 mg/dL) within 3 h was significantly lower after breakfast (484.3 vs. 897.9 mg/min/dL; p = 0.025), and urinary CPR level was significantly higher (97.0 ± 41.6 vs. 85.2 ± 39.9 μg/day; p = 0.008) than in patients taking sitagliptin. There were no significant differences in plasma HbA1c, GA, 1,5AG, IRI, CPR, BNP, or PAI-1 levels between patients taking vildagliptin and sitagliptin.ConclusionsCGM showed that mean 24-h blood glucose, MAGE, highest blood glucose level after supper, and hyperglycemia after breakfast were significantly lower in patients with type 2 diabetes mellitus taking vildagliptin than those taking sitagliptin. There were no significant differences in BNP and PAI-1 levels between patients taking vildagliptin and sitagliptin.Trial registrationUMIN000007687

Arachidonate 12/15-lipoxygenase-induced inflammation and oxidative stress are involved in the development of Diabetic Cardiomyopathy

Diabetes affects cardiac structure and function, and it has been suggested that diabetes leads to cardiomyopathy. Arachidonate 12/15-lipoxygenase (LOX) has been suggested to play an important role in atherogenesis and heart failure. However, the role of 12/15-LOX in diabetic cardiomyopathy has not been examined. In this study, we investigated the effects of cardiac 12/15-LOX on diabetic cardiomyopathy. We created streptozotocin (STZ)-induced diabetic mice and compared them with Alox15-deficient mice. Expression of 12/15-LOX and inflammatory cytokines such as tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB were upregulated in STZ-induced diabetic hearts. Disruption of 12/15-LOX significantly improved STZ-induced cardiac dysfunction and fibrosis. Moreover, deletion of 12/15-LOX inhibited the increases of TNF-α and NF-κB as well as the production of STZ-induced reactive oxygen species in the heart. Administration of N-acetylcysteine in diabetic mice prevented STZ-induced cardiac fibrosis. Neonatal cultured cardiomyocytes exposed to high glucose conditions induced the expression of 12/15-LOX as well as TNF-α, NF-κB, and collagen markers. These increases were inhibited by treatment of the 12/15-LOX inhibitor. Our results suggest that cardiac 12/15-LOX–induced inflammation and oxidative stress are involved in the development of diabetic cardiomyopathy and that inhibition of 12/15-LOX could be a novel treatment for this condition.

Angiotensin II Type 1a Receptor Is Involved in Cell Infiltration, Cytokine Production, and Neovascularization in Infarcted Myocardium

Objective—Angiotensin II is critically involved in left ventricular remodeling after myocardial infarction. Neovascularization has been thought to prevent the development of left ventricular remodeling and deterioration to heart failure. To elucidate the role of angiotensin II in neovascularization during cardiac remodeling, we induced myocardial infarction in angiotensin II type1a receptor (AT1) knockout (KO) mice. Methods and Results—There were more vessels in the border zone of infarcted hearts of wild-type (WT) mice and AT1KO mice at 14 days after operation, compared with in the left ventricle of sham-operated mice, and the number was larger in WT mice than in AT1KO mice. Consistent with these observations, the infarcted heart of AT1KO mice expressed lower levels of matrix metalloproteinase and endothelial nitric oxide synthase activity. More inflammatory cells such as granulocytes and macrophages were infiltrated in the infarcted hearts of WT mice than AT1KO mice at 4 days. A variety of cytokines and chemokines were increased in infarcted hearts of WT and AT1KO mice, and many of them were more remarkable in WT mice than in AT1KO mice at 14 days. Conclusions—AT1 plays a critical role in inflammatory cell infiltration, cytokine production, and neovascularization in infarcted hearts.

Possible involvement of corticotropin-releasing factor receptor signaling on vascular inflammation

Based on the reported anti-inflammatory and anti-stress responses by corticotropin-releasing factor (CRF) receptor signaling, endogenous CRF receptor agonists, CRF, urocortin (UCN) I and its related peptides, may play protective roles against cardiovascular stresses via the CRF receptor signaling. Therefore, the present study was designed to evaluate the involvement of CRF receptor signaling against vascular inflammatory stress using human aortic endothelial cells (HAECs). In addition, due to the possible involvement of CRF receptor signaling in the effects of statin on endothelial cells, the effects of pitavastatin on the expression of UCN-related peptides in HAECs were also evaluated. HAECs expressed all UCNs, CRF type 1 receptor (CRF-R1), and CRF type 2 (CRF-R2)alpha and CRF-R2beta mRNAs. Real time PCR analysis revealed that UCN I mRNA was down-regulated, whereas UCN II mRNA was up-regulated by tumor necrosis factor (TNF)-alpha. Selective blockade of CRF-R1 resulted in significant increase in TNF-alpha-induced expression of vascular adhesion molecule-1 at mRNA level and E-selectin at mRNA and protein levels. Pitavastatin up-regulated UCN I mRNA without TNF-alpha, but co-incubation with pitavastatin and TNF-alpha resulted in decrease in UCN I mRNA. On the contrary, UCN II, CRF-R1, and CRF-R2 mRNAs were markedly increased by co-incubation of pitavastatin and TNF-alpha. These facts indicate that CRF-R1 signaling may have protective role against TNF-alpha-induced vascular inflammation. In addition, because of up-regulation of CRF-R1 mRNA by pitavastatin with or without TNF-alpha, CRF-R1 may be involved in the vasoprotective effects of pitavastatin.

Regulation of urocortin I and its related peptide urocortin II by inflammatory and oxidative stresses in HL-1 cardiomyocytes

Despite our knowledge on the regulation of urocortin (Ucn) I and its related peptides in the heart, the possible involvement of cardiovascular stress substances, such as cytokines or angiotensin II (Ang II), on this regulation remains to be fully elucidated. We therefore evaluated the potential role of cardiovascular stress substances on the regulation of the Ucn-corticotropin-releasing hormone (CRH) receptor system in HL-1 cardiomyocytes using a Ucn I-specific RIA, conventional reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR. Ucn I mRNA levels were shown to be up-regulated by lipopolysaccarides (LPS), tumor necrosis factor-alpha (TNF-alpha), Ang II, H(2)O(2), and pyrrolidinedithiocarbamate (PDTC). The LPS- and Ang II-induced increase in Ucn I mRNA levels was abolished by tempol. In addition, the secretion of Ucn I from HL-1 cardiomyocytes was stimulated by LPS and TNF-alpha. On the contrary, Ucn II mRNA was increased by TNF-alpha alone and Ang II with tempol, and the TNF-alpha-induced increase in Ucn II mRNA was abolished by erythromycin and PDTC. These results suggested that Ucn I mRNA may be up-regulated by oxidative stress, whereas Ucn II mRNA may be up-regulated by the activated nuclear factor-kappaB, i.e. inflammatory stress. CRH-R2 mRNA may be negatively regulated by the increase in expression of Ucn I and/or Ucn II mRNA. In conclusion, the Ucn-CRH receptor system may be regulated by two major forms of cardiac stresses, i.e. oxidative and inflammatory stress, and may play a critical role in cardiac stress adaptation in heart diseases.