RAGE, Macrophages & HDL Biology

描述（由申请方提供）：高密度脂蛋白（HDL）在体内发挥多种血管保护作用。HDL促进过量胆固醇从外周组织流出，通过运输到肝脏排泄，抑制氧化应激和炎症， 增强内皮功能。在小鼠和人类受试者中，AGE受体（AGE）表达在非糖尿病和糖尿病动脉粥样硬化的血管和免疫细胞中上调。在非糖尿病和糖尿病低密度脂蛋白受体（LDLR）基因敲除小鼠和载脂蛋白Apoe基因敲除小鼠中的研究表明，阻断配体-受体相互作用可显著抑制动脉粥样硬化。在本申请中，我们将首次将晚期糖基化终产物受体（HDL）的作用与HDL的生物学联系起来，这是由三个新发现引起的：首先，与表达HDL的BMDM相比，来自非糖尿病或糖尿病小鼠的HDL缺陷型骨髓衍生巨噬细胞（BMDM）显示出显著增加的胆固醇流出ApoA 1和HDL 2。平行地，两种关键胆固醇转运蛋白Abca 1和Abcg 1的mRNA水平在无β-内酰胺酶的BMDM中分别比野生型BMDM高112倍和>250倍。当siRNA降低THP-1人巨噬细胞中的ApoA 1表达时，与用scramble siRNA处理相比，导致显著更高的胆固醇流出ApoA 1和HDL 2。第二，在miR-33缺失的BMDM中，Mir 33的水平显著低于在野生型小鼠对照BMDM中发现的水平; THP-1巨噬细胞中miR-33表达的敲低导致MIR 33水平的高度显著降低。有趣的是，Mir 33“宿主基因”（Srebf 2）的mRNA和蛋白质水平在RAGE表达的BMDM与无RAGE的BMDM之间没有差异，表明Mir 33的RAGE依赖性调节的独特机制。第三，在寻求确定巨噬细胞吞噬减弱组织损伤性炎症反应的机制时，我们发现BMDM与AGEs配体孵育以RAGE依赖性方式增强促炎性“M1”型巨噬细胞标志物的表达并降低“M2”型巨噬细胞标志物的表达。我们假设，胆固醇通过降低关键胆固醇转运蛋白的表达来干扰HDL代谢的益处，其后果包括：（1）。减少胆固醇从动脉粥样硬化斑块中的去除，导致动脉粥样硬化的加速进展和减少消退;和（2）.维持支持RAGE依赖性信号转导机制的富含胆固醇的膜特性，从而增强“M1”巨噬细胞极化和巨噬细胞迁移。我们将在本申请中使用体外和体内方法来测试这些概念，以辨别细胞因子影响Mir 33/Abca 1/Abcg 1和巨噬细胞胆固醇流出、极化和迁移的调节以及体内逆转胆固醇转运和动脉粥样硬化的分子机制。总之，这项工作将揭示新的治疗策略，以增强HDL功能和心血管健康。

DESCRIPTION (provided by applicant): High density lipoprotein (HDL) exerts multiple vascular-protective effects in vivo. HDL promotes excess cholesterol efflux from peripheral tissues via transport to the liver for excretion, suppresses oxidative stress and inflammation, and enhances endothelial function. In murine and human subjects, receptor for AGE (RAGE) expression is upregulated in vascular and immune cells in non-diabetic and diabetic atherosclerosis. Studies in non-diabetic and diabetic LDL receptor (LDLR) null mice and apolipoprotein Apoe null mice revealed that blockade of ligand-RAGE interaction resulted in significant suppression of atherosclerosis. In this application, we will link for the first time ke roles for the receptor for advanced glycation end products (RAGE) to the biology of HDL prompted by three novel discoveries: first, RAGE deficient bone marrow derived macrophages (BMDMs) from non-diabetic or diabetic mice displayed significantly increased cholesterol efflux to ApoA1 and to HDL2 compared to RAGE expressing BMDMs. In parallel, mRNA levels for the two key cholesterol transporters, Abca1 and Abcg1, were H2-fold and >250-fold higher in RAGE null BMDMs vs. wild- type BMDMs, respectively. When RAGE expression was reduced by siRNAs in THP-1 human macrophages, significantly higher cholesterol efflux to ApoA1 and HDL2 resulted compared to treatment with scramble siRNAs. Second, in RAGE null BMDMs, levels of Mir33 were significantly lower than those found in wild-type mice control BMDMs; knockdown of RAGE expression in THP-1 macrophages resulted in highly significant reduction in MIR33 levels. Intriguingly, mRNA and protein levels of the Mir33 "host gene" (Srebf2) did not differ between RAGE-expressing vs. RAGE null BMDMs, suggesting unique mechanisms of RAGE-dependent regulation of Mir33. Third, in seeking to identify the mechanisms by which macrophage RAGE attenuates tissue-damaging inflammatory responses, we discovered that incubation of BMDMs with RAGE ligand AGEs enhanced expression of pro-inflammatory "M1" type macrophage markers and reduced expression of "M2" type macrophage markers in a RAGE-dependent manner. We hypothesize that RAGE interferes with the benefits of HDL metabolism by reducing expression of key cholesterol transporters, consequences of which include: (1). reduced removal of cholesterol from atherosclerotic plaques leading to accelerated progression and reduced regression of atherosclerosis; and (2). maintenance of cholesterol-rich membrane properties that support RAGE-dependent signal transduction mechanisms and thereby potentiate "M1" macrophage polarization and macrophage migration. We will test these concepts in this application using in vitro and in vivo approaches to discern the molecular mechanisms by which RAGE impacts regulation of Mir33/Abca1/Abcg1 and macrophage cholesterol efflux, polarization and migration and in vivo, reverse cholesterol transport and atherosclerosis. Taken together, this work will uncover novel therapeutic strategies to enhance HDL function and cardiovascular health.