Chemokines in Healing Myocardial Infarcts

描述（由申请人提供）。趋化因子在梗死心肌中诱导，可能在炎性白细胞浸润、纤维组织沉积和心脏修复中发挥作用。单核细胞化学引诱蛋白(MCP)-1是一种强效的单核细胞化学引诱蛋白，在梗死中迅速上调，可能有助于梗死愈合。CXC趋化因子干扰素- γ诱导蛋白(IP)-10是一种弱的单核细胞趋化剂，但具有抗纤维化特性的强效血管抑制剂，在梗死心脏中被显著诱导，可能在调节梗死血管生成和纤维化中起作用。本研究将探讨MCP-1和IP-10在心肌梗死愈合中的作用。特异性目的1将阐明MCP-1在梗死愈合中的功能作用，在小鼠心肌梗死模型中使用基因靶向动物。MCP-1可能影响造血细胞（主要是单核细胞）以及常驻心肌细胞（成纤维细胞和内皮细胞）。为了检验MCP-1信号在梗死的常驻细胞和血源性细胞中的相对重要性，将使用缺乏CCR2 （MCP-1受体）的动物，在过继转移WT或CCR2 -/-骨髓细胞后创建嵌合小鼠。为了解剖梗死中巨噬细胞活化和细胞因子表达抑制的机制，我们将测试两个有趣的假设：a) MCP-1选择性地招募一个特定的单核细胞亚群[被确定为CCR2 hi/fraktalkine receptor (CX3CR1) lo]，该亚群对炎症介质的激活更敏感；b) MCP-1可能直接调节巨噬细胞的活化、分化和基因表达。这些问题将通过CX3CR1GFP/+小鼠的体内实验（其中GFP表达决定了血液中两个不同的单核细胞亚群）和分离的小鼠单核细胞和巨噬细胞的体外研究来解决。特异性目的2将探讨IP-10在心肌梗死愈合中的作用。初步实验表明，IP-10作为白细胞化学引诱剂可能不太重要。但在梗死区清除和纤维蛋白临时基质形成之前，可能在防止过早成纤维细胞积累和血管生成方面起关键作用，这是支持纤维组织沉积所必需的。这一假设将在IP-10和CXCR3缺陷小鼠中得到验证；此外，IP-10/CXCR3相互作用在浸润梗死的常驻细胞和造血细胞中的相对重要性将通过使用WT和CXCR3 -/-骨髓细胞过继转移的嵌合小鼠进行检测。具体目的3将探讨MCP-1和IP-10对分离的心脏成纤维细胞和梗死肌成纤维细胞纤维组织沉积的相反作用的机制。研究MCP-1和IP-10对心肌成纤维细胞和梗死心肌成纤维细胞增殖、迁移和活化的影响。将从MCP-1 （CCR2和MCP-1 KO）和IP-10 （CXCR3和IP-10 KO）信号缺陷小鼠中分离梗死肌成纤维细胞，将其表型、动力、增殖活性和基因表达与WT梗死肌成纤维细胞进行比较。通过基因微阵列分析，我们发现了IP-10在抑制成纤维细胞粘附、增殖和迁移相关基因中的新作用。因此，我们将研究IP-10对局灶黏附组装和拆卸、成纤维细胞迁移和增殖的影响。了解趋化因子在梗死愈合中的作用可能会发现新的治疗靶点，旨在优化心脏修复和减少不良重构。

DESCRIPTION (provided by applicant. Chemokines are induced in the infarcted myocardium and may play a role in inflammatory leukocyte infiltration, fibrous tissue deposition, and cardiac repair. Monocyte Chemoattractant Protein (MCP)-1, a potent mononuclear cell chemoattractant is rapidly upregulated in infarcts and may contribute to infarct healing. The CXC chemokine Interferon-gamma inducible Protein (IP)-10, a weak mononuclear cell chemoattractant, but potent angiostatic agent with anti-fibrotic properties, is markedly induced in the infarcted heart and may have a role in regulating infarct angiogenesis and fibrosis. This proposal will examine the role of MCP-1 and IP-10 in healing myocardial infarcts. Specific aim 1 will elucidate the functional role of MCP-1 in infarct healing, using gene-targeted animals in a murine model of myocardial infarction. MCP-1 may affect hematopoietic cells (predominantly mononuclear cells) as well as resident myocardial cells (fibroblasts and endothelial cells). In order to examine the relative importance of MCP-1 signaling in resident and blood-derived cells of the infarct, animals deficient in CCR2, the MCP-1 receptor, will be used to create chimeric mice after adoptive transfer of WT or CCR2 -/- bone marrow cells. In order to dissect the mechanisms responsible for suppressed macrophage activation and cytokine expression in the infarct we will test two intriguing hypotheses: a) that MCP-1 selectively recruits a specific monocvte subset [identified as CCR2 hi/fraktalkine receptor (CX3CR1) lo] that is more responsive to activation by inflammatory mediators, and b) that MCP-1 may directly regulate macrophage activation, differentiation and gene expression. These questions will be addressed using in vivo experiments with CX3CR1GFP/+ mice (in which GFP expression determines two distinct monocyte subpopulations in the bloodstream) and in vitro studies with isolated murine monocytes and macrophages. Specific aim 2 will investigate the role of IP-10 in healing mvocardial infarcts. Preliminary experiments suggest that IP-10 may be less important as a leukocyte chemoattractant. but may have a crucial role in preventing premature fibroblast accumulation and angiogenesis before the infarcted area is debrided and a fibrin-based provisional matrix, necessary to support fibrous tissue deposition is formed. This hypothesis will be tested using IP-10 and CXCR3 deficient mice; in addition the relative importance of IP-10/CXCR3 interactions in resident and hematopoietic cells infiltrating the infarct will be examined using chimeric mice with adoptive transfer of WT and CXCR3 -/- bone marrow cells. Specific aim 3 will explore the mechanisms responsible for the opposing effects of MCP-1 and IP-10 on fibrous tissue deposition using isolated cardiac fibroblasts and infarct myofibroblasts. The effects of MCP-1 and IP-10 on proliferation, migration and activation of cardiac fibroblasts and infarct myofibroblasts will be studied. Infarct myofibroblasts will be isolated from mice with defective MCP-1 (CCR2 and MCP-1 KO) and IP-10 (CXCR3 and IP-10 KO) signaling: their phenotvpe, motilitv, proliferative activity and gene expression will be compared with WT infarct myofibroblasts. Using gene microarray analysis, we identified novel actions of IP-10 in repression of genes associated with fibroblast adhesion, proliferation and migration. Accordingly we will study the effects of IP-10 on focal adhesion assembly and disassembly, fibroblast migration and proliferation. Understanding the role of chemokines in infarct healing may identify novel therapeutic targets aiming at optimizing cardiac repair and decreasing adverse remodeling.