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: 8632336
• 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/8632336
• 关键词: Acute; Address; 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; 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; Staging; Testing; Therapeutic; Up-Regulation; Western Blotting; Work; anti-IgM; base; 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; overexpression; oxidized low density lipoprotein; prevent; public health relevance; type I and type II diabetes; uptake

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 induce the transformation of macrophages into foam cells5-7, lead to upregulation of pro-survival genes8 and induce the release of pro-inflammatory cytokines10. OxLDL IC also induces increased collagen production by mesangial cells12.. In contrast, MDA-LDL IC induce 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.

在过去的几年里，我们在 1 型和 2 型糖尿病的两个大型糖尿病队列中清楚地证明了这一点 （DCCT/EDIC 和 VADT 队列）含有氧化 LDL (oxLDL) 的高水平免疫复合物 (IC) 晚期糖化终产物修饰的低密度脂蛋白 (AGE-LDL) 与冠状动脉疾病相关 (CAD)1-4，可以强有力地预测 1 型早期 CAD 的发展和进展 糖尿病3。我们还在 VADT 队列（包括许多患有晚期 CAD 的患者）中表明， 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同时含有高水平的MDA-LDL和高水平的oxLDL或 AGE-LDL 发生急性事件的风险降低了4，这使我们得出结论，修饰的脂蛋白 IC 携带的物质在斑块进展/稳定性和斑块不稳定方面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 进入泡沫细胞5-7，导致促生存基因上调8并诱导促炎物质的释放 细胞因子10. OxLDL IC 还可以诱导系膜细胞产生更多的胶原蛋白12。相比之下，MDA-LDL IC 诱导巨噬细胞凋亡，刺激基质金属蛋白酶 (MMP) 的表达 巨噬细胞和主动脉内皮细胞，并减少系膜细胞中胶原蛋白基因的表达。 假设和具体目标：根据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）具有显着影响 对斑块进展/不稳定的影响。我们假设，当 MDA 是 IC携带的LDL，巨噬细胞摄取这些IC会诱导细胞凋亡，增加释放 MMP 和胶原酶活性增加，从而导致斑块不稳定。相反，当 oxLDL是IC中LDL的主要修饰，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，从而促进斑块的形成 扩展/稳定性。此外，我们假设包括 MDA-LDL IC 和 MMP 的生物标志物组将 与一组传统 CVD 相比，能够更准确地预测患有急性 CVD 事件高风险的患者 生物标志物。 为了检验上述假设，我们提出三个目标。第一个目标是比较激活模式 巨噬细胞暴露于IC（MDA-LD IC和oxLDL IC），这些IC对巨噬细胞存活和凋亡的影响 巨噬细胞及其相关途径。在第二个目标中，我们将测量已知的新型生物标志物 与来自 VADT 队列的患者样本中的斑块不稳定性相关，其中 MDA-LDL IC 和 之前已经测量了传统的 CVD 生物标志物。我们将确定面板是否包括 MDA-LDL IC、MMP-1 和 MMP-9 将具有显着更高的预测能力来识别高水平患者 急性心肌梗死的风险。最后，在第三个目标中，我们将在动脉粥样硬化的动物小鼠模型上确定 2 型糖尿病是否可以通过治疗阻断/减少 oxLDL IC 和 MDA-LDL IC 对 Fc¿ 与小鼠 IgM 抗 oxLDL 和 IgM 抗 MDA-LDL 结合使用可防止斑块进展和不稳定。 方法：细胞分离与培养、脂蛋白分离与修饰、抗体、蛋白分离 阵列、细胞活力/凋亡测定、rtPCR、蛋白质印迹、酶活性测定、免疫组织化学 动脉粥样硬化病变的基因表达分析和组织学分析。 与 VA 健康的相关性和意义：造成差异的机制的定义 不同修饰的 LDL-IC 诱导的细胞活化、存活和凋亡可能有助于定义新的 治疗目标和新的预防策略将遏制心血管疾病的进展 2 型糖尿病患者的并发症和急性血管事件的发生。