京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22314号 / 医博第4555号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 Shohab YOUSSEFIAN, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Original published work submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Medicine (Internal Medicine) 18 October, 2017. / The very first coronary artery balloon angioplasty is reported to have been performed by Gruntzig in 1977.1 Subsequently to this, over the past 40 years, there have been significant advances in coronary angiography and intervention. Coronary artery interventional techniques have evolved and improved significantly. There have been considerable device developments, new generation stents and novel antiplatelet therapy which have all proved to reduce the incidence of the primary periprocedural complications associated with percutaneous coronary intervention (PCI). [No abstract provided. Information taken from introduction] / LG2018

Myocardial infarction (MI), a late-stage event of many cardiovascular diseases (CVD), results in cardiomyocyte death, myeloid cell recruitment to promote cellular debris removal and excessive cardiac remodeling affecting architecture and function, which can ultimately lead to heart failure. Currently, the use of biomaterials to intervene on the hostile post-MI environment and promote myocardial healing is being investigated to restore cardiac function. It has been shown that an injectable collagen matrix improves cardiac repair by altering macrophage polarization, reducing cell death and enhancing angiogenesis, leading to a reduction in infarct size and improved cardiac function when delivered at 3 hours post-MI. MicroRNAs (miRNA) “fine tune” gene expression by negatively regulating the translational output of target messenger RNA (mRNA). As such, miRNAs present interesting therapeutic opportunities for the treatment of MI. However, the delivery of miRNA mimics and/or inhibitors can be complicated by degradation and off target effects. The objectives of this thesis were to determine how the matrix may regulate endogenous miRNAs and to explore the biomaterial’s ability to deliver therapeutic miRNAs. It was shown that matrix treatment of MI mouse hearts resulted in altered expression of 119 miRNAs, some of which had functions linked to the beneficial effects of matrix treatment. Of particular interest, miR-92a was down-regulated within the infarct and peri-infarct cardiac tissue 2 days after matrix treatment (delivered at 3-hours post-MI) compared to PBS treatment. In in vitro cultures, the matrix down-regulated miR-92a levels in macrophages but did not significantly alter miR-92a expression in endothelial cells, circulating angiogenic cells or fibroblasts. In addition, using an in vitro model system, it was shown that the matrix may have the potential to deliver functional therapeutic miRNAs to cells; however further experimental optimisation is required to confirm these results. Therefore, collagen matrix treatment may be a promising approach to regulate and/or deliver miRNAs for protecting the myocardial environment and improving function of the infarcted heart.

Background: Transplantation of marrow stromal cells (MSC) has been shown to improve heart perfusion and cardiac function after ischemia. Erythropoietin (EPO) is capable of inducing angiogenesis and inhibiting cell apoptosis. The aim of this study was to investigate the effect of EPO on the therapeutic potency of MSC transplantation in a rat model of myocardial infarction. Methods: MSC viability was detected by MTT andflow cytometry following culture in serum-free medium for 24 h with or without EPO. Release of vascular endothelial growth factor (VEGF) by MSC incubated with different doses of EPO was assayed using ELISA. Immediately after coronary ligation, autologous MSC (3 × 10 6 cells) were injected into the ischemic myocardium (MSC and MSC-EPO groups). EPO (3,000 U/kg body weight) was injected daily for 3 consecutive days starting 1 day prior to ligation. The same EPO dose was also injected for consecutive 3 days starting 15 days after surgery (EPO and MSC-EPO groups). Control animals were injected saline solution for the same time period. Cardiac function was assessed by echocardiography 2 and 21 days after surgery, respectively. Western blot and immunohistological assessments were performed to examine the effects of treatments. Results: In vitro, EPO inhibited MSC apoptosis induced by serum-free medium and increased vascular endothelial growth factor (VEGF) release by MSC. In vivo, cardiac infarct size was significantly smaller, cardiac function significantly improved, and capillary density obviously higher in the MSC and EPO groups than in the control group. Combined treatment with EPO infusion and MSC transplantation demonstrated a further decrease in infarct size, a further improvement in cardiac function, and a further increase in capillary density compared with MSC or EPO alone. Furthermore, a higher ratio of phosphorylated Akt to total Akt was measured by Western blot; Bcl-2 was upregulated and Bax was downregulated by immunohistochemistry in the MSC-EPO group compared to the other three groups. Conclusion: Transplantation of MSC combined with EPO infusion is superior to MSC monotherapy for angiogenesis and cardiac function recovery.

We postulated that combining erythropoietin (EPO) infusion with bone marrow mesenchymal stem cells (MSC) delivery may give better prognosis in a rat infarcted heart. Acute myocardial infarction (MI) model was developed by coronary artery ligation. Animals were grouped (n = 18) to receive intramyocardial injection of 30 μl saline solution without cells (EPO and control groups) or with 3 × 106 MSC from transgenic green fluorescent protein (GFP)+ male mice (MSC and MSC-EPO groups). The animals received either 5000 U/kg body weight EPO (EPO and MSC-EPO groups) or saline solution (MSC and control groups) for 7 days after MI. Cardiac functions were measured by echocardiography and cardiac tissue was harvested for immunohistological studies 3 weeks after surgery. We observed regeneration of MSC in and around the infarcted myocardium in MSC and MSC-EPO groups. Capillary density was markedly enhanced with significantly smaller infarct size and reduced fibrotic area in MSC-EPO group as compared with other three groups. A smaller left ventricular (LV) diastolic dimension and a higher LV fractional shortening were observed in MSC-EPO group than in other three groups. Transplantation of MSC combined with cytokine EPO is superior to either of the monotherapy approach for angiomyogenesis and cardiac function recovery.

Experiments were performed to test the postulate that exercise training (ExT) improves the blunted renal excretory response to acute volume expansion (VE), in part, by normalizing the neural component of the volume reflex typically observed in chronic heart failure (HF). Diuretic and natriuretic responses to acute VE were examined in sedentary and ExT groups of rats with either HF or sham-operated controls. Experiments were performed in anesthetized (Inactin) rats 6 wk after coronary ligation surgery. Histological data indicated that there was a 34.9 ± 3.0% outer and 42.5 ± 3.2% inner infarct of the myocardium in the HF group. Sham rats had no observable damage to the myocardium. In sedentary rats with HF, VE produced a blunted diuresis (46% of sham) and natriuresis (35% of sham) compared with sham-operated control rats. However, acute VE-induced diuresis and natriuresis in ExT rats with HF were comparable to sham rats and significantly higher than sedentary HF rats. Renal denervation abolished the salutary effects of ExT on renal excretory response to acute VE in HF. Since glomerular filtration rates were not significantly different between the groups, renal hemodynamic changes may not account for the blunted renal responses in rats with HF. Additional experiments confirmed that renal sympathetic nerve activity responses to acute VE were blunted in sedentary HF rats; however, ExT normalized the renal sympathoinhibition in HF rats. These results confirm an impairment of neurally mediated excretory responses to acute VE in rats with HF. ExT restored the blunted excretory responses as well as the renal sympathoinhibitory response to acute VE in HF rats. Thus the beneficial effects of ExT on cardiovascular regulation in HF may be partly due to improvement of the neural component of volume reflex. Copyright © 2006 the American Physiological Society.

Objective: Osteopontin (OPN), increased in the heart following myocardial infarction (MI), plays an important role in post-MI remodeling. Angiogenesis, an important feature of tissue repair, begins in the infarcted myocardium within 3 days post-MI. Here, the authors studied the role of OPN in myocardial angiogenesis using wild-type (WT) and OPN knockout (KO) mice. Results: Measurement of angiogenic response using Griffonia simplicifolia lectin-1 (GSL-1) staining indicated reduced capillary density in the infarcted region of the OPN KO hearts as compared to WT hearts 7 and 14 days post-MI. Arteriolar density was lower in OPN KO hearts 14 days post-MI. The number of CD31 positive cells was also lower in the infarcted region of the OPN KO hearts as compared to WT hearts 14 days post-MI. In contrast, capillary and arteriolar densities in the noninfarcted regions of OPN KO and WT hearts were not significantly different. In vivo myocardial angiogenesis measured using Matrigel implantation in the left ventricular myocardium indicated significant decrease in the percentage of vessel-like areas in the OPN KO vs. WT hearts. Furthermore, in vitro Matrigel tube formation assay demonstrated a significant decrease in total tube length in cardiac microvascular endothelial cells (CMECs) isolated from OPN KO hearts as compared to CMECs from WT hearts. Treatment of OPN KO CMECs with purified OPN protein significantly increased total tube length, while bovine serum albumin had no effect. Conclusion: Lack of OPN impairs myocardial angiogenic response, leading to adverse remodeling post-MI.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNɣ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis. Hoyer, Naxerova, et al. generate a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. They find that local injury activates macrophages in remote organs and that these adaptations were damaging or protective in different settings.

The Joint Commission in a joint effort with the Centers of Medicare and Medicaid Services (CMS) has established certain "core measures" by which hospital performance is measured. One of these is the measure for patients with ST-elevation myocardial infarction (STEMI) recommending percutaneous coronary intervention within 90 minutes of presentation to the Emergency Department in institutions that are able to provide this service. This recommendation does not take into account the long-term use of clopidogrel that is recommended by the American College of Cardiology and American Heart Association for patients that are treated with coronary stents. The purpose of this study was to evaluate outcomes of providing a short course of clopidogrel versus a prescription alone for clopidogrel to uninsured patients experiencing STEMI who were treated with a bare metal stent. After conducting a cost-benefit analysis, a policy was approved that provided uninsured STEMI patients with clopidogrel at discharge rather than a prescription. A social worker evaluated patients to determine if they met criteria and arranged for medication delivery to the patient’s bedside. A retrospective chart review for all patients who presented to the Emergency Department during two different time frames (before and after policy implementation) was conducted to evaluate if providing clopidogrel decreased readmissions. Data were collected on over a 15-month period of time before and after the clopidogrel policy implementation to allow for evaluation of 90-day readmissions with repeat STEMI. Data were analyzed using chi-square cross tabulation and T-test for independent samples. A total of 201 charts were reviewed: 100 from the pre-intervention group and 101 from the post-intervention group. Demographic characteristics of age, gender and insurance status iv were not statistically different between groups. The mean age for the control group was 59.1 (+ 13.8) years and 58.9 (+ 13.6) years for the intervention group. Twenty percent of the patients were uninsured. Five uninsured patients were readmitted with STEMI prior to the intervention compared to two patients in the intervention group (p = .191). The admissions for the preintervention patients occurred in the first 30 days after discharge compared to 31-60 days in the post-intervention group. All of the patients who were readmitted were assessed to be noncompliant with treatment. Additionally, a transition to increased use of bare metal stents in STEMI patients from 23.1% pre-intervention to 67.4% post-intervention was noted (p < .001). Although no differences were found in readmission rates, fewer readmissions for STEMI were noted after the intervention. The small number of patients who were readmitted with STEMI likely accounted for this finding, and additional monitoring of readmission rates is warranted. Despite provision of the clopidogrel, adherence remains an issue and needs to be addressed. During the intervention, physicians were encouraged to consider the financial and social resources of individual STEMI patients presenting to the Emergency Department to help identify patients that would be less likely to adhere to antiplatelet therapy. In those believed to be at high risk for non-adherence, primarily due to inability to purchase the relatively expensive medication clopidogrel, many physicians chose to insert bare metal stents rather than drugeluting stents to take advantage of the shorter course of clopidogrel required post procedure. Provision of a 30-day course of clopidogrel and aspirin was a major part of this effort to decrease recurrent myocardial infarction in this at-risk population. A few patients eligible for the clopidogrel were not provided the medication if they were admitted to a nursing unit where staff members were not familiar with the policy; revisions to the policy to ensure medication is provided to all eligible patients will be made. Providing clopidogrel to patients who experience v STEMI may improve adherence and thereby decrease readmissions as a result of repeat STEMI due to subacute thrombus formation. Patients who experience STEMI continue to be vulnerable after STEMI. Programs that provide medication to patients should be expanded within this facility and to other hospital systems to encompass all patients who are treated for STEMI. Multi-disciplinary collaboration is necessary in developing and implementing a program that will address care for this.