京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18162号 / 医博第3882号 / 新制||医||1003(附属図書館) / 31020 / 京都大学大学院医学研究科医学専攻 / (主査)教授 福原 俊一, 教授 佐藤 俊哉, 教授 坂田 隆造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Myocardial infarction (MI) frequently occurs in patients with diabetes resulting in higher mortality and morbidity than non-diabetic patients. We and others have shown that bone marrow-derived endothelial progenitor cells (BM-EPCs) promote cardiac neovascularization and attenuate ischemic injury in animal models. Lately, emerging evidence supports that exosomes (Exo), a family of extracellular vesicles, mediate stem cell therapy by carrying cell-specific biological cargo and by inducing signaling via transferring of bioactive molecules to target cells. Despite promising results of stem cells/Exo in preclinical studies, autologous cell-based therapies yielded modest clinical results, suggesting cellular/Exo reparative function may be compromised by the presence of comorbid diseases including complications associated with diabetes. Recent studies suggest that epigenetic mechanisms, such as histone and DNA modifications for gene silencing, promote diabetes-induced vascular complication. Therefore, we hypothesized that diabetic EPCs produce exosomes of altered and dysfunctional content that compromise their reparative function in ischemic heart disease via epigenetic alterations. We collected EPC-Exo from non-diabetic (db/+) and diabetic (db/db) mice and examined their reparative effect in vitro and on permanent left anterior descending (LAD) coronary artery ligation and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo. Our data demonstrated that compared to non-diabetic EPC-Exo, diabetic EPC-Exo promoted neonatal rat cardiomyocyte cell apoptosis under hypoxic stress and repressed endothelial tube formation and cell survival. In vivo studies revealed that non-diabetic EPC-Exo treatments improved cardiac function and remodeling while diabetic EPC-Exo significantly depressed cardiac function, reduced capillary density, increased fibrosis in the permanent LAD ligation MI injury. Moreover, in the I/R MI model, we found that non-diabetic EPC-Exo mediated cardio-protection was lost compared with diabetic-EPC-Exo, and diabetic-EPC-Exo increased immune cell infiltration, infarcted area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac), a gene activating histone modification, expression was decreased in mouse cardiac endothelial cells (MCECs) treated with db/db EPC-Exo compared with db/+ EPC-Exo, suggesting diabetic EPC-Exo inhibits endothelial cell gene expression. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that diabetic EPC-Exo reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in MCECs. Moreover, we found that a small molecular inhibitor of HDACs, valproic acid (VPA), effectively prevented diabetic EPC-Exo-medicated H3K9Ac reduction, indicating VPA may rescue the beneficial gene expression and cell function. Taken together, our results provide evidence that diabetic EPC-Exo reparative function is impaired in the ischemic heart and this may be through HDACs-mediated H3K9Ac downregulation leading to inhibition of beneficial genes in recipient cardiac endothelial cells. Reversing diabetic EPC-Exo function by treating with HDAC inhibitors may provide a new path for autologous exosome therapy for myocardial repair in diabetic patients. However, questions still remain on what the content change of stem cell-derived exosome under diabetic condition is.Emerging evidence support a key role of variety of stem /progenitor cell-secreted Exo as a pivotal paracrine entity to mitigate cardiovascular injury. Beside EPC-, cortical bone stem cell (CBSC)-, and cardiac stem/progenitor cell (CPC)- derived Exo are adequate to enhance cardiac repair and regeneration after injury. As widely acknowledged, the comorbidities such as hyperglycemia is a characteristic of diabetes and a major driving factor in CVD. The functional role of stem/progenitor cell- derived Exo and molecular signature of their secreted Exo cargo under hyperglycemic conditions remain elusive. Therefore, we hypothesize that hyperglycemic stress causes transcriptome changes in stem/progenitor cell- derived Exo that may compromise their reparative function. To identify the content change in Exo under hyperglycemia, we performed an unbiased Exo transcriptome signatures from 3 different aforementioned stem/progenitor cells by next generation exosome RNA sequencing (RNA-seq). The results indicated that the size and number of Exo were not changed from 3 stem/progenitor cells between normal and high glucose groups. Furthermore, analysis revealed differential expression of variety of RNA species in Exo and the portions of different RNA were change under hyperglycemia. Specifically, we identified 241 common-dysregulated mRNAs, 21 ncRNAs and 16 miRNAs in three stem cell-derived Exo. Based on mRNA data, Gene Ontology (GO) revealed that potential function of common mRNAs mostly involved in metabolism and transcriptional regulation. We also provided the detail information of these non-annotated ncRNAs and the potential mRNA targets by miRNA-mRNA prediction. This study not only provides potential candidates for individual stem cell types but also identifies common genes in response to hyperglycemia. These reference data are critical for future biological studies and application of stem/progenitor cell-derived Exo in ischemic heart or other diseases to prevent the adverse effects of hyperglycemia-induced stem/progenitor cell- derived Exo dysfunction. / Biomedical Sciences / Accompanied by five Microsoft Excel files: 1) Supplementary Table 1 2) Supplementary Table 2 3) Supplementary Table 3 4) Supplementary Table 4 5) Supplementary Table 5

Background: Patients with pre-existing vascular disease are known to have worse outcomes after acute myocardial infarction (AMI). However, there is limited data for outcomes stratified by type and number of vascular territories involved. Methods: Using the Nationwide Inpatient Sample (2015–2017), we examined outcomes of AMI in patients with pre-existent vascular disease stratified by number as well as types of diseased beds including all five major vascular sites: cardiac, cerebrovascular, renal, aortic and peripheral vascular disease (PVD). Multivariable logistic regression was used to determine the adjusted odds ratios (aOR) of adverse outcomes and invasive procedure utilization. Results: Out of 2,184,614 AMI admissions, 49.7% had pre-existent vascular disease. The odds of major adverse cardiovascular and cerebrovascular events (MACCE), mortality, ischemic stroke and major bleeding incrementally increased and was highest in those with ≥3 vascular sites involved (aOR for MACCE 1.16, CI 1.13–1.19; mortality 1.3, CI 1.26–1.34; stroke 1.15, CI 1.1–1.2; major bleeding 1.21, CI 1.16–1.25). Amongst those with a single pre-existent diseased vascular bed, the adjusted odds of MACCE appeared to be higher in those with PVD (1.28, CI 1.26–1.31), aortic disease (1.24, CI 1.19–1.29), and cerebrovascular disease (1.22, CI 1.2–1.25). Patients with pre-existent vascular disease had a lower overall likelihood of undergoing invasive revascularization procedures. Conclusions: Approximately half of the population presenting with AMI have pre-existent vascular disease. There is an incremental increase in adverse outcomes with increasing number of diseased vascular beds, with further differences in outcomes and utilization of invasive procedures based on sub-types of sites involved.

Aims: There are limited data on the management and outcomes of chronic liver disease (CLD) patients presenting with acute myocardial infarction (AMI), particularly according to the subtype of CLD. Methods: Using the Nationwide Inpatient Sample (2004-2015), we examined outcomes of AMI patients stratified by severity and sub-types of CLD. Multivariable logistic regression was performed to assess the adjusted odds ratios (aOR) of receipt of invasive management and adverse outcomes in CLD groups compared with no-CLD. Results: Of 7 024 723 AMI admissions, 54 283 (0.8%) had a CLD diagnosis. CLD patients were less likely to undergo coronary angiography (CA) and percutaneous coronary intervention (PCI) (aOR 0.62, 95%CI 0.60-0.63 and 0.59, 95%CI 0.58-0.60, respectively), and had increased odds of adverse outcomes including major adverse cardiovascular and cerebrovascular events (1.19, 95%CI 1.15-1.23), mortality (1.30, 95%CI 1.25-1.34) and major bleeding (1.74, 95%CI 1.67-1.81). In comparison to the non-severe CLD sub-groups, patients with all forms of severe CLD had the lower utilization of CA and PCI (P <.05). Among severe CLD patients, those with alcohol-related liver disease (ALD) had the lowest utilization of CA and PCI; patients with ALD and other CLD (OCLD) had more adverse outcomes than the viral hepatitis sub-group (P <.05). Conclusions: CLD patients presenting with AMI are less likely to receive invasive management and are associated with worse clinical outcomes. Further differences are observed depending on the type as well as severity of CLD, with the worst management and clinical outcomes observed in those with severe ALD and OCLD.

Excerpt: Dr Meier and colleagues1 present intriguing data that individuals with a first acute myocardial infarction (AMI) were less likely than matched controls to have used tetracycline antibiotics or quinolones in the previous 3 years. The authors raise the possibility that organisms susceptible to these antibiotics may be involved in the pathogenesis of coronary heart disease. However, several methodological limitations lead to other possible explanations for the observed associations.

Ischemic cardiomyopathies, such as myocardial infarction (MI), are a leading cause of heart failure in both men and women throughout the world. Despite timely intervention post-MI, the loss of viable myocardium can lead to global remodeling and loss of function in many patients due to the limited regenerative potential of heart tissue. Thus, there is a critical need to better understand the repair mechanisms involved and to develop new preventative and reparative therapies for treating MI and preventing progression to heart failure. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite of glycolysis and the main precursor of advanced glycation end-products (AGEs), which can cause oxidative stress and wound healing delay. MG was shown to play an important causative role in the cellular changes, adverse remodeling and functional loss of the infarcted heart. This suggests MG as a target for therapy to restore cell-ECM signaling, inhibit oxidative stress and improve cardiac function post-MI. The aim of this PhD project was to develop new biomaterial therapies that can reduce the effects of MG, decrease oxidative stress, enhance electrical conductivity and improve cardiac contractility and function post-MI. There were three primary objectives: 1) To develop an injectable antioxidant and hydrogel system for minimizing the effects of MG and promoting cardiac repair post-MI; 2) To synthesize a nanoparticle system for targeted delivery of Glyoxalase-1 (Glo1) enzyme to cardiac tissue for reducing the accumulation of MG, limiting adverse remodeling and preserving cardiac function following MI; and 3) To design a sprayable nano-therapeutic that uses surface engineered custom designed multi-armed peptide grafted nanogold for on-the-spot coating of infarcted myocardial surface for increasing contractility of the myocardium post-MI. In the first study, a fisetin-loaded collagen type I hydrogel (fisetin-HG) was injected intramyocardially in mice at 3h post-MI, and compared to fisetin-alone, hydrogel-alone, or saline treatment. The fisetin-HG treatment increased the level of glyoxalase-1 (the main MG-metabolizing enzyme), reduced MG-AGE accumulation, and decreased oxidative stress in the MI heart, which was associated with smaller scar size and improved cardiac function. Treatment with fisetin-HG also promoted neovascularization and increased the number of pro-healing macrophages in the infarct area, while reducing the number of pro-inflammatory macrophages. The second study revealed that when delivered intravenously at 3h post-MI, our Glo1-loaded nanoparticles specifically targeted the damaged cardiac tissue, led to improved cardiac function, protected cell viability and limited infarct expansion by reducing oxidative stress post-MI. Lastly, the third study showed that, when applied at 1-week post-MI, the sprayed nanogold treatment remained at the treatment site for at least 28 days with no significant off-target organ infiltration. Our results demonstrated a remarkable increase in cardiac function, muscle contractility, and myocardial electrical conductivity post-MI. Overall, these findings show that reducing MG levels through both increased activity of Glo1 and direct MG scavenging as well as increasing cardiac contractility may be a promising approach to limit adverse cardiac remodeling, prevent damage, and preserve the function of the infarcted heart

Ataxia-telangiectasia mutated kinase (ATM), a serine/threonine kinase primarily located in the nucleus, is typically activated in response to DNA damage. Individuals with mutations in ATM gene develop a disease called Ataxia telangiectasia (AT). These individuals are more susceptible to ischemic heart disease and metabolic disorder. Our lab has previously shown that ATM plays a critical role in β-adrenergic receptor (β-AR) - and myocardial infarction (MI)-stimulated myocyte apoptosis and cardiac remodeling. This study tested the hypothesis that ATM plays a critical role in cardiac autophagy and glucose metabolism following MI and ischemia, respectively. Early during MI (4 hours after its onset) and 4 hours post-treatment with ATM inhibitor KU-55933, ATM deficiency resulted in autophagic impairment in the heart and in cardiac fibroblasts, respectively. Such autophagic changes in the heart and in cardiac fibroblasts associated with the activation of GSK-3β and mTOR, and inactivation of Akt and AMPK. ATM deficiency also augmented autophagy in the infarct region of the heart 28 days post-MI as well as in cardiac fibroblasts treated with ATM inhibitor KU-55933 for 24 hours. Autophagic changes in the infarct region during ATM deficiency associated with enhanced Akt, Erk1/2, and mTOR activation. Additionally, the lack of ATM accelerated glycolysis and gluconeogenesis and augmented TCA cycle metabolism under non-ischemic conditions. Following a 20 minute global ischemic period, the glycolytic pathway, not the gluconeogenic pathway, was down-regulated during ATM deficiency which was found to be associated with alterations in TCA cycle metabolism. Such metabolic rearrangements associated with changes in the phosphorylation of Akt, GSK-3β, and AMPK alongside alterations in Glut4 protein expression. Thus, ATM deficiency impairs autophagy early after the onset of MI and in cardiac fibroblasts treated with ATM inhibitor KU-55933 for 4 hours. In contrast, ATM deficiency appears to augment autophagy late post-MI in the infarct region of the heart and in cardiac fibroblasts treated with ATM inhibitor KU-55933 for 24 hours. Lack of ATM alters glucose and TCA cycle metabolism with and without ischemia. Such findings implicate ATM as a key player in autophagic changes in the heart in response to MI as well as in glucose metabolism under non-ischemic and ischemic conditions.

Transplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR- 1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti-fibrotic protein, will enhance cardiac myocyte differentiation, inhibit native cardiac apoptosis, and reduce fibrosis in the infarcted myocardium. Furthermore, we assess levels of associated pro- (caspase-3, PTEN) and anti-(Akt) apoptotic proteins as well as a pro-fibrotic protein (MMP-9) in the post-MI and cell transplanted heart. microRNAs (miRs) have emerged as critical regulators of various physiological processes including development, differentiation, metabolism, and death. Indeed, miR- 1 plays an integral role in early cardiac development in Drosophila and mice as well as mediates differentiation of cardiac myocytes in vitro. To that end, we generated ES cells overexpressing miR-1 (miR-1-ES cells), transplanted them into the infarcted myocardium, and evaluated their impact on cardiac myocyte differentiation, myocardial repair, and left ventricular dysfunction post-MI. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared to ES cell and culture media transplanted hearts. Assessment of apoptosis revealed overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-Akt and inhibition of caspase-3, PTEN, and superoxide anion production. A significant reduction iv in interstitial and vascular fibrosis was quantified in miR-1-ES and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1- ES cell and ES cell groups was observed. Finally mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls. Our data suggests miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI ultimately giving rise to enhanced cardiac repair, regeneration, and function. Next, we assessed the role of miR-1-ES cells in a chronic model of MI as research has shown that apoptosis occurs not only hours but months following ischemia. 4 weeks following transplantation into the infarcted myocardium, we provide evidence demonstrating reduced cardiac apoptosis in miR-1-ES cell transplanted hearts compared to respective controls. Moreover, we show significant elevation of p-Akt levels and diminished PTEN levels in hearts transplanted with miR-1-ES cells as determined by enzyme-linked immunoassays. Finally, using echocardiography, we reveal mice receiving miR-1-ES cell transplantation post-MI had significantly improved cardiac function compared with animals transplanted with ES cell and culture media. Our data suggests that miR-1, when overexpressed in transplanted ES cells, has the capacity to inhibit apoptosis long term while attenuating contractility loss. In addition to enhancing cardiac-specific donor cell differentiation, improving the efficacy by which stem cells promote cell survival and repair in the host myocardium is imperative in the pursuit of refining and optimizing stem cell therapy. To that end, we overexpressed TIMP-1, an endogenous inhibitor of apoptosis and fibrosis, in ES cells (TIMP-1-ES cells), transplanted them into infarcted myocardium, and evaluated their v impact on adverse cardiac remodeling. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to assess apoptosis, fibrosis, and heart function. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase in p-Akt activity compared with ES cells or culture media controls. Interstitial and vascular fibrosis was significantly decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key pro-fibrotic protein, was significantly reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP- 1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells. Overall, our data suggest that genetic manipulation of ES cells following transplantation in the infarcted heart enhances cardiac myocyte differentiation, inhibits apoptosis and fibrosis as well as improves cardiac function.