Lipoproteins and Inflammation in the Development of Diabetic Complications

描述（由申请人提供）： 在过去的几年里，我们已经清楚地表明，在两个大型糖尿病队列的1型和2型糖尿病（DCCT/EDIC和VADT队列），高水平的含有氧化LDL（oxLDL）和晚期糖化终产物修饰LDL（AGE-LDL）的免疫复合物（IC）与冠状动脉疾病（CAD）相关1- 4，并可以强烈预测CAD的发展和进展的早期阶段的1型糖尿病3。我们还在包括许多晚期CAD患者的VADT队列中显示，IC中高水平的丙二醛修饰（MDA）-LDL能够预测急性心血管疾病（CVD）事件4。然而，当循环IC含有高水平的MDA-LDL和高水平的oxLDL或AGE-LDL时，遭受急性事件的风险降低4，导致我们得出结论，IC携带的修饰的脂蛋白在斑块进展/稳定性和斑块不稳定性中发挥不同的调节作用。我们的机制研究支持上述假设。OxLDL IC诱导巨噬细胞转化为泡沫细胞5 -7，导致促存活基因上调8，并诱导促炎细胞因子释放10。OxLDL IC还诱导肾小球系膜细胞增加胶原蛋白的产生12。相反，MDA-LDL IC诱导巨噬细胞凋亡，刺激巨噬细胞和主动脉内皮细胞表达基质金属蛋白酶（MMPs），并减少系膜细胞中胶原基因的表达。假设和具体目标：基于我们的临床和体外初步数据，我们假设循环IC中LDL的主要修饰类型（MDA-LDL、oxLDL或AGE-LDL）对斑块进展/不稳定有显著影响。我们推测，当MDA是IC携带的LDL的主要修饰时，巨噬细胞对这些IC的摄取诱导细胞凋亡，增加MMPs的释放，并增加胶原酶活性，从而导致斑块不稳定。相反，当oxLDL是IC中LDL的主要修饰时，这些IC的摄取诱导巨噬细胞存活并刺激促炎介质和生长因子的释放，从而有助于斑块扩张/稳定。此外，我们假设，包括MDA-LDL IC和MMP的生物标志物组将比常规CVD生物标志物组更能够强烈预测患有急性CVD事件的高风险患者。为了验证上述假设，我们提出了三个目标。第一个目的是比较暴露于IC（MDA-LD IC和oxLDL IC）的巨噬细胞的激活模式，这些IC对巨噬细胞存活和凋亡的影响，以及所涉及的途径。在第二个目标中，我们将测量已知与来自VADT队列的患者样本中的斑块不稳定性相关的新型生物标志物，其中先前已经测量了MDA-LDL IC和常规CVD生物标志物。我们将确定包括MDA-LDL IC、MMP-1和MMP-9在内的一组药物是否具有显著更高的预测能力，以识别急性心肌梗死高危患者。 MI.最后，在第三个目标中，我们将在动脉粥样硬化和2型糖尿病的动物小鼠模型上确定，通过小鼠IgM抗oxLDL和IgM抗MDA-LDL治疗来阻断/减少oxLDL IC和MDA-LDL IC对Fc受体的参与是否会阻止斑块的进展和不稳定性。研究方法：细胞分离和培养、脂蛋白的分离和修饰、抗体分离、蛋白质阵列、细胞活力/凋亡测定、rtPCR、蛋白质印迹、酶活性测定、基因表达的免疫组织化学分析和动脉粥样硬化病变的组织学分析。与VA健康和意义的相关性：定义不同修饰的LDL-IC诱导的细胞活化、存活和凋亡差异的机制可能有助于定义新的治疗靶点和新的预防策略，从而减少2型糖尿病患者心血管并发症的进展和急性血管事件的发生。

DESCRIPTION (provided by applicant): In the past few years we have clearly demonstrated in two large diabetic cohorts of type 1 and type 2 diabetes (DCCT/EDIC and VADT cohorts) that high levels of immune complexes (IC) containing oxidized LDL (oxLDL) and advanced glycated end-products-modified LDL (AGE-LDL) are associated with coronary artery disease (CAD) 1-4 and can strongly predict the development and progression of CAD in the early stages of type 1 diabetes3. We have also shown in the VADT cohort, which includes many patients with advanced CAD, that high levels of malondialdehyde-modified (MDA)-LDL in IC are able to predict acute cardiovascular disease (CVD) events4. However, when circulating IC contain both high levels of MDA-LDL and high levels of oxLDL or AGE-LDL the risk to suffer an acute event is reduced4 leading us to conclude that the modified lipoproteins carried by IC play a differential modulating role in plaque progression/stability and plaque destabilization. Our mechanistic studies support the above hypothesis. OxLDL IC induces the transformation of macrophages into foam cells5-7, lead to upregulation of pro-survival genes8 and induces the release of pro-inflammatory cytokines10. OxLDL IC also induces increased collagen production by mesangial cells12. In contrast, MDA-LDL IC induces macrophage apoptosis, stimulate the expression of matrix metalloproteinases (MMPs) by macrophages and by aortic endothelial cells, and reduce the expression of collagen genes in mesangial cells. Hypothesis and Specific Aims: Based on our clinical and in vitro preliminary data, we hypothesize that the type of predominant modifications of LDL in circulating IC (MDA-LDL, oxLDL or AGE-LDL) has a significant impact in plaque progression/destabilization. We postulate that, when MDA is the predominant modification of the LDL carried by IC, the uptake of those IC by macrophages induces cell apoptosis, increased release of MMPs, and increased collagenase activity, thus contributing to plaque destabilization. In contrast, when oxLDL is the predominant modification of LDL in IC, the uptake of these IC induce macrophage survival and stimulate the release of pro-inflammatory mediators and growth factors, thus contributing to plaque expansion/stability. Furthermore we postulate that a biomarker panel including MDA-LDL IC and MMPs will be more able to strongly predict patients at high risk to suffer acute CVD events than a panel of conventional CVD biomarkers. To test the above hypotheses we propose three aims. The first aim will compare the activation patterns of macrophages exposed to IC (MDA-LD IC and oxLDL IC), the effect of these IC on the survival and apoptosis of macrophages, and the pathways involved. In the second aim we will measure novel biomarkers known to be associated with plaque instability in patient samples from the VADT cohort in which MDA-LDL IC and conventional CVD biomarkers have been previously measured. We will determine whether a panel including MDA-LDL IC, MMP-1 and MMP-9 will have a significantly higher predictive power to identify patients at high risk for acute MI. Finally in the third aim we will determine, on an animal mouse model of atherosclerosis and type 2 diabetes whether blocking/reducing engagement of Fc�Rs by oxLDL IC and MDA-LDL IC by treatment with mouse IgM anti-oxLDL and IgM anti-MDA-LDL will prevent plaque progression and instability. Methods: Cell isolation and culture, separation and modification of lipoproteins, isolation of antibodies, protein arrays, cell viability/apoptosis assays, rtPCR, western blots, enzyme-activity assays, immunohistochemical analysis of gene expression and histologic analysis of atherosclerotic lesions. Relevance to VA Health and Significance: The definition of mechanisms responsible for the differences in cell activation, survival and apoptosis induced by different modified LDL-IC may allow to define new therapeutic targets and new prevention strategies that will curtail the progression of cardiovascular complications and the occurrence of acute vascular events in patients with type 2 diabetes.