Lipoproteins and Inflammation in the Development of Diabetic Complications

糖尿病并发症发生过程中的脂蛋白和炎症

• 批准号: 9275407
• 负责人: MARIA F LOPES-VIRELLA
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-10-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275407
• 关键词: Acute; Address; Age; Animal Model; Animals; Antibodies; Antigen-Antibody Complex; Apolipoprotein E; Apoptosis; Apoptotic; Arterial Fatty Streak; Atherosclerosis; Binding; Biological Assay; Biological Markers; Blood Circulation; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cell Culture Techniques; Cell Separation; Cell Survival; Cells; Chronic; Clinical; Collagen; Collagen Gene; Complications of Diabetes Mellitus; Coronary Arteriosclerosis; Data; Development; Diabetes Mellitus; Disease Progression; Endothelial Cells; Event; Fc Receptor; Gelatinase B; Gene Expression; Gene Expression Profiling; Genes; Goals; Growth; Growth Factor; Health; Histologic; Human; Immunoglobulin G; Immunoglobulin M; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Insulin-Dependent Diabetes Mellitus; Interstitial Collagenase; Knockout Mice; Lead; Lipoproteins; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Malondialdehyde; Matrix Metalloproteinases; Measures; Mediating; Metalloproteases; Metalloproteinase Gene; Methods; Modification; Mus; Non-Insulin-Dependent Diabetes Mellitus; Pathogenesis; Pathway interactions; Patients; Pattern; Play; Predictive Value; Prevention strategy; Process; Production; Protein Array; Risk; Risk Factors; Role; Sampling; Serum; Testing; Therapeutic; Up-Regulation; Western Blotting; anti-IgM; base; biomarker panel; cardiovascular disorder risk; cohort; collagenase; cytokine; diabetic; enzyme activity; high risk; inhibitor/antagonist; low density lipoprotein inhibitor; macrophage; mesangial cell; mouse model; new therapeutic target; novel; novel marker; overexpression; oxidized low density lipoprotein; prevent; public health relevance; uptake

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.

描述（由申请人提供）： 在过去的几年中，我们在两个大型 1 型和 2 型糖尿病队列（DCCT/EDIC 和 VADT 队列）中清楚地证明，含有氧化 LDL (oxLDL) 和高级糖化终产物的高水平免疫复合物 (IC)修饰的 LDL (AGE-LDL) 与冠状动脉疾病 (CAD) 1-4 相关，可以强有力地预测 1 型糖尿病早期 CAD 的发生和进展3。 VADT 队列包括许多晚期 CAD 患者，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 中同时含有高水平的 MDA-LDL 时。 LDL 和高水平的 oxLDL 或 AGE-LDL 会降低发生急性事件的风险4，这使我们得出结论，IC 携带的修饰脂蛋白在斑块进展/稳定性和斑块中发挥着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，导致促生存基因8 的上调，并诱导促炎细胞因子的释放10。相反，MDA-LDL IC 诱导巨噬细胞凋亡，刺激巨噬细胞和主动脉表达基质金属蛋白酶 (MMP)。假设和具体目标：根据我们的临床和体外初步数据，我们对循环 IC 中 LDL 的主要修饰类型（MDA-LDL、oxLDL 或 AGE）感兴趣。 -LDL）对斑块进展/不稳定具有显着影响，当 MDA 是 IC 携带的 LDL 的主要修饰时，巨噬细胞摄取这些 IC 会诱导细胞凋亡， MMP 释放增加，胶原酶活性增加，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 的摄取会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/稳定性。即使我们假设包含强 MDA-LDL IC 和 MMP 的生物标志物组也比 CVD 组更能够预测患有急性 CVD 事件高风险的常规患者为了检验上述假设，我们提出了三个目标：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，以及在第二个目标中，我们将测量来自 VADT 队列的患者样本中已知与斑块不稳定相关的新生物标志物，其中 MDA-LDL IC 和传统 CVD 生物标志物之前已被测量。低密度脂蛋白集成电路， MMP-1和MMP-9将具有显着更高的预测能力来识别急性高风险患者 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是否通过使用小鼠 IgM 抗 oxLDL 和 IgM 抗治疗来阻断/减少 oxLDL IC 和 MDA-LDL IC 与 Fc�R 的结合。 -MDA-LDL 将防止斑块进展和不稳定。方法：细胞分离和培养、脂蛋白分离和修饰、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以及动脉粥样硬化病变的组织学分析与 VA 健康的相关性和意义：不同修饰 LDL- 诱导的细胞活化、存活和凋亡差异的机制的定义。 IC 可能有助于确定新的治疗目标和新的预防策略，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。