Investigation of therapeutic potential of exogenous ubiquitin following myocardial ischemia/reperfusion injury

缺血性心脏病(IHD)是退伍军人死亡的主要原因。心肌梗塞(MI) IHD的严重后果，通常可归因于心肌梗死的有害影响 缺血再灌注(I/R)损伤。心脏炎症和细胞外基质沉积(瘢痕 形成)是心肌梗死后心脏重塑的重要事件。靶向治疗方法 这些事件可能会给心肌梗塞患者带来希望。这项建议调查了治疗的潜力。 泛素(UB；一种小分子量蛋白质，通常与标记蛋白质有关 蛋白酶体降解)在心肌缺血和I/R损伤后的心肌重构中的作用。这个 提出的建议是基于我们的新观察，即外源UB在β中起到保护作用- 肾上腺素能受体刺激的心肌重塑。在人THP1白血病细胞中，CXCR4是 被确认为胞外UB的受体。我们的初步数据使用-i)全球心脏模型 缺血/再灌注(I/R；朗宁多夫模型)；以及ii)活体小鼠心肌I/R损伤 外源性UB对I/R损伤后心肌重构的保护作用UB治疗 缩小梗塞面积，减少心脏炎症反应，改善心功能3天 在体外，UB治疗抑制了中性粒细胞的迁移，增强了中性粒细胞的吞噬活性。 巨噬细胞。在成人心脏成纤维细胞中，UB处理激活了ERK1/2，并增加了表达 血管内皮生长因子-A(VEGF-A)的表达。UB与CXCR4和CXCR4的免疫共沉淀 拮抗作用可阻断细胞外UB对ERK1/2活化和VEGF-A表达的影响。UB 治疗增强α-平滑肌肌动蛋白(肌成纤维细胞分化的标志)的表达 和胶原凝胶收缩活性，同时减少成纤维细胞迁移到 并抑制胎牛血清刺激的细胞增殖。这些观察结果引导我们提出了假设 外源性UB很可能通过CXCR4发挥作用，调节心脏炎症反应和 细胞外基质生物学通过影响中性粒细胞、巨噬细胞和 成纤维细胞，从而在缺血和I/R后的心肌重构中发挥心脏保护作用 受伤。目的1在体内研究外源性UB对心肌细胞的治疗作用 心肌缺血和I/R损伤后的重构。目标2将在体内检测CXCR4的作用 CXCR4拮抗剂、Ub突变体和CXCR4拮抗剂对外源性Ub心肌保护作用的调节 细胞类型特异性CXCR4基因敲除小鼠。目标3将研究细胞和分子机制 外源性UB通过何种途径影响心脏炎症反应和细胞外基质生物学 协调I/R后的心脏保护反应。本项目的创新之处在于调研 外源性UB对心肌缺血后心肌重构的治疗作用 和I/R损伤(临床相关模型)。调查外源性UB作用的拟议研究 在心脏炎症和细胞外基质方面，生物学可能会发现治疗的新策略 来自IHD的。

Ischemic heart disease (IHD) is a leading cause of death among veterans. Myocardial infarction (MI) is a serious outcome of IHD, and is generally attributable to the detrimental effects of myocardial ischemia/reperfusion (I/R) injury. Cardiac inflammation and extracellular matrix deposition (scar formation) are essential events in the cardiac remodeling post-MI. Therapeutic approaches targeting these events may hold promise for patients with MI. This proposal investigates the therapeutic potential of ubiquitin (UB; a small molecular weight protein typically associated with tagging proteins for proteasomal degradation) in myocardial remodeling following myocardial ischemia and I/R injury. The proposal is based on our novel observations that exogenous UB plays a protective role in β- adrenergic receptor-stimulated myocardial remodeling. In human THP1 leukemia cells, CXCR4 is identified as a receptor for extracellular UB. Our preliminary data using - i) isolated heart model of global ischemia/reperfusion (I/R; Langendorff model); and ii) in vivo myocardial I/R injury in mice now suggest a protective role of exogenous UB in myocardial remodeling following I/R injury. UB treatment decreased infarct size, reduced cardiac inflammatory response and improved heart function 3 days post-I/R. In vitro, UB treatment inhibited migration of neutrophils, and enhanced phagocytic activity of macrophages. In adult cardiac fibroblasts, UB treatment activated ERK1/2, and increased expression of vascular endothelial growth factor-A (VEGF-A). UB co-immunoprecipitated with CXCR4, and CXCR4 antagonism negated the effects of extracellular UB on ERK1/2 activation and VEGF-A expression. UB treatment augmented α-smooth muscle actin (a marker for myofibroblast differentiation) expression and collagen gel contractile activity of fibroblasts, while decreasing migration of fibroblasts into the wound area and inhibiting FBS-stimulated cell proliferation. These observations led us to hypothesize that exogenous UB, most likely acting via CXCR4, modulates cardiac inflammatory response and extracellular matrix biology by affecting phenotype and/or function of neutrophils, macrophages and fibroblasts, thereby playing a cardioprotective role in myocardial remodeling following ischemia and I/R injury. Aim 1 will investigate, in vivo, the therapeutic potential of exogenous UB in myocardial remodeling following myocardial ischemia and I/R injury. Aim 2 will examine, in vivo, the role of CXCR4 in modulation of cardioprotective effects of exogenous UB using CXCR4 antagonist, UB mutants and cell type-specific CXCR4 knockout mice. Aim 3 will investigate the cellular and molecular mechanisms by which exogenous UB affects cardiac inflammatory response and extracellular matrix biology to coordinate post-I/R cardioprotective response. The Innovation of this project lies in the investigation of therapeutic potential of exogenous UB in myocardial remodeling following myocardial ischemia and I/R injury (clinically relevant model). The proposed studies investigating the role of exogenous UB in cardiac inflammation and extracellular matrix biology may uncover novel strategies for the treatment of IHD.