THE CHEMOKINE RECEPTOR CXCR-2 IN ATHEROSCLEROSIS

描述(改编自《调查者摘要》)：多种机制运作 以促进巨噬细胞在动脉粥样硬化内膜内的聚集。这 拟议的工作部分源于其他人的观察，包括这一发现 在MCP-1受体CCR2缺陷的apoE基因敲除小鼠中， 动脉粥样硬化减轻，但病变的大小和复杂性仍在进展 从5周到13周。此外，体外单核细胞CCR2的表达也被抑制 斑块中可能存在的几种炎性介质，或通过 向巨噬细胞分化。因此，他们提出了其他机制 在促进巨噬细胞在体内的保留和扩张方面发挥了作用 动脉粥样硬化病变。根据他们以前的工作，他们发现 内膜巨噬细胞表达趋化因子受体CXCR2和CXCR2 配体、GROAlpha和IL-8。初步数据还表明，白细胞 小鼠CXCR2同源物mIL-8RH缺乏，早期进展受阻 病变内巨噬细胞丰富，病变巨噬细胞滞留减少。他们 因此，假设特定的CXCR2效应在皮损内起中介作用 募集的巨噬细胞的生长、炎症分化和激活。 它们的具体目标如下： 使用两种动物，定义CXCR2介导的动脉粥样硬化事件的序列 模型系统。他们将首先将受辐射的LDLR-/-小鼠移植到骨骼中 MIL-8/CXCR2-/-或对照小鼠的骨髓。老鼠 将被喂以食物或高胆固醇饮食以确定mIL-8RH在 轻度与重度高胆固醇血症引起的动脉粥样硬化。第二，评估 MIL-8RH在白细胞以外的细胞中的作用，他们将研究LDLR-/-， 杂交获得mIL-8RH-/-小鼠。早期单核细胞进入，脂肪条纹 两组的形成和病变进展将在3点到30点进行研究 几周。他们将同时检测病变mIL-8RH和巨噬细胞标志物 包括每组小鼠的MOMA2。他们还将比较时间和 巨噬细胞空间聚集与mIL-8RH、KC/GROα、JE/MCP-1的表达 和CCR2，oxLDL表位的出现和脂质沉积。 验证CXCR2表达通过促进巨噬细胞发挥作用的假设 在早期动脉粥样硬化病变中的滞留，部分是通过增加 巨噬细胞β1整合素介导的黏附。首先，他们将确定是否 LDLR-/-、mIL-8RH+/+对病变单核细胞的滞留作用大于LDLR-/-， MIL-8RH-/-小鼠。为此，他们将分离、标记(使用[~3H]-甘油)和 将骨髓单核细胞注入高脂饮食的LDLR-/-小鼠。迁移 标记的单核细胞在体内的滞留将在主动脉病变中进行测量。 分别于输液后第2、4、7、14天。然后，他们将检验这一假设 MIL-8RH-/-巨噬细胞的广泛性细胞培养黏附缺陷 小鼠与β1整合素活性降低有关，β1整合素结合 纤维连接蛋白(VLA-4、VLA-5)。他们还将研究CXCR2介导的 人外周血单核细胞的特异性中和黏附 整合素和整合素配体的抗体。他们还将测试 巨噬细胞CXCR2表达是巨噬细胞基质必需的假说 入侵。 验证CXCR2通过影响巨噬细胞黏附而表达的假设 介导巨噬细胞分化为明显的促动脉粥样硬化炎症 表型。在这里，他们将进一步研究人类外周血单核细胞来源 巨噬细胞与小鼠野生型和mIL-8RH-/-骨髓来源的巨噬细胞 在体外正常表达CXCR2的条件下。他们会 具体观察CXCR2配体、GROAlpha和IL-8对 巨噬细胞增殖及JE/MCP-1的表达。他们还将确定 如果CXCCR2介导的黏附调节apoE的产生，低密度脂蛋白的氧化， 和胆固醇酯的积累。最后根据具体的结果进行了分析 目的2，他们将检验VLA-5和VLA-4通过 在CXCR2配体的刺激下，调节其中的一个或多个活动。他们 还会将这些结果与鼠标的功能相关联 特定目标1的动脉粥样硬化病变(即体内相关性研究 CXCR2表达与增殖细胞核抗原、氧化型低密度脂蛋白抗原及清除剂的关系 受体表达)。

DESCRIPTION (Adapted from Investigator's Abstract): Multiple mechanisms operate to promote macrophage accumulation within the atherosclerotic intima. This proposed work stems in part from observations by others, including the finding that in apoE knockout mice which are deficient in the MCP-1 receptor CCR2, atherosclerosis is diminished, but lesion size and complexity still progress from 5-13 weeks. Also monocyte CCR2 expression in vitro is attenuated by several inflammatory mediators which can be in the plaque, or by differentiation to macrophages. They therefore propose that other mechanisms are at play in promoting macrophage retention and expansion within the atherosclerotic lesions. Base on their previous work, they have found that intimal macrophages express the chemokine receptor CXCR2, and the CXCR2 ligands, GROalpha, and IL-8. Initial data also indicate that leukocyte deficiency of the mouse CXCR2 homologue, mIL-8RH, arrested progression of early macrophage-rich lesions and decreased retention of lesion macrophages. They therefore hypothesize that specific CXCR2 effects mediate the intra-lesional growth, inflammatory differentiation and activation of recruited macrophages. Their specific aims are as follows: Define the sequence of atherogenic events mediated by CXCR2, using two animal model systems. They will first transplant irradiated LDLR-/- mice with bone marrow from mIL-8/ CXCR2-/- or control mice under gnotobiotic conditions. Mice will be fed a chow or high cholesterol diet to define the role of mIL-8RH in mild vs. severe hypercholesterolemia-induced atherosclerosis. Second, to assess the role of mIL-8RH in cells other than leukocytes, they will study LDLR-/-, mIL-8RH-/- mice derived by crossbreeding. Early monocyte ingress, fatty streak formation, and lesion progression will be studied in both groups at 3 to 30 weeks. They will concurrently detect lesion mIL-8RH and macrophage markers including MOMA2 in each group of mice. They will also compare temporal and spatial macrophage accumulation, expression of mIL-8RH, KC/GROalpha, JE/MCP-1 and CCR2, the appearance of oxLDL epitopes and lipid deposition. Test the hypothesis that CXCR2 expression acts by promoting macrophage retention in early atherosclerotic lesions, and does so in part by increasing macrophage beta1 integrin-mediated adhesion. First they will determine if retention of lesion monocytes is greater in LDLR-/-, mIL-8RH+/+ than LDLR-/-, mIL-8RH-/- mice. To do so they will isolate, label (using [3H]-glycerol) and infuse bone marrow monocytes into LDLR-/- mice on a high fat diet. Migration and retention of labeled monocytes in vivo will be measured in aortic lesions at 2,4,7, and 14 days post infusion. They will then test the hypothesis that the generalized cell culture adhesion defect of macrophages from mIL-8RH-/- mice is associated with decreased activation of beta1 integrins, which bind fibronectin (VLA-4, VLA-5). They will also study CXCR2-mediated effects on adhesion of human peripheral blood monocytes using specific neutralizing antibodies for integrins and integrin ligands. They will also test the hypothesis that macrophage CXCR2 expression is necessary for macrophage matrix invasion. Test the hypothesis that CXCR2 expression, via effects on macrophage adhesion, mediates macrophage differentiation to a distinct, pro-atherogenic inflammatory phenotype. Here they will further study human peripheral monocyte-derived macrophages and mouse wild type and mIL-8RH-/- bone marrow derived macrophages under conditions where CXCR2 is normally expressed in vitro. They will specifically look at the effects of the CXCR2 ligands, GROalpha, and IL-8 on macrophage proliferation and expression of JE/MCP-1. They will also determine if CXCCR2-mediated adhesion modulates the production of apoE, oxidation of LDL, and accumulation of cholesteryl ester. Finally based on the results of Specific Aim 2, they will test the hypothesis that VLA-5, and VLA-4 activation, via stimulation by CXCR2 ligands, regulate one or more of these activities. They will also correlate these results with the features of the mouse atherosclerotic lesions of Specific Aim 1 (i.e. in vivo correlation studies between CXCR2 expression and PCNA, oxidized LDL antigens, and scavenger receptor expression).