Oxidized Phospholipid-Induced Inflammation in Atherosclerosis

描述(申请人提供)：动脉粥样硬化是一种慢性炎症性疾病，其特征是单核细胞和T细胞等单核细胞的特异性渗透，而脂肪条纹病变中基本上没有中性粒细胞。动脉粥样硬化病变发展的最早步骤之一是单核细胞与血管壁内皮细胞的黏附。先前已经证明，如果用最小修饰低密度脂蛋白(MM-LDL)刺激，内皮细胞可以被激活以特异地结合单核细胞，但不能与中性粒细胞结合。这种对单个核细胞的特异性随后被观察到氧化1-palmitoyl-2-arachidonoyl-s/7-glycero-3-phosphocholine(OxPAPC)激活内皮细胞，这意味着脂质氧化产物是慢性炎症的罪魁祸首。我们假设特定的磷脂氧化产物(OxPL)触发血管炎症，并确定动脉粥样硬化和其他慢性炎症性疾病的单核细胞特异性特征。拟议的研究结果将增加我们对动脉粥样硬化是如何启动和传播的理解，并应导致治疗慢性炎症性疾病的新策略。这项建议涉及的具体目标是： 具体目标1：从结构上确定决定单核细胞特异性炎症的单个OxPL，并调查OxPL诱导的信号涉及TLR4的假设。我们将使用人脐静脉内皮细胞和分离的小鼠主动脉内皮细胞，在体外研究OxPL诱导的导致单核细胞特异性黏附的细胞内信号通路。此外，我们将检验POVPC是OxPAPC中的一个活性成分的假设，它决定了单核细胞的特异性，并确定了结构-功能关系。我们将研究TLR-4的作用以及TLR-4接头蛋白MyD88和TRIP以及各自的下游元件在OxPL介导的信号事件中的参与。 具体目的#2：探讨OxPL是体内血管炎症的触发物的假说。为了模拟OxPL在血管壁中的积累，我们将OxPL局部应用于小鼠的颈动脉，并测量趋化因子和黏附分子的表达。此外，我们将使用体外灌流的颈动脉来研究OxPL诱导的单核细胞滚动和黏附。 具体目的#3：验证一种假设，即OxPL在体内诱导特异性单个核细胞聚集需要特定的趋化因子表达模式，并涉及12脂氧合酶。利用小鼠气囊模型，我们将检测OxPL诱导的白细胞在体内的聚集，并表征白细胞亚群。我们将研究趋化因子在OxPL诱导的炎症和脂多糖诱导的炎症中的表达模式以及表达的时间进程。此外，我们还将研究12-LO在OxPL诱导的体内炎症中的作用。

DESCRIPTION (provided by applicant): Atherosclerosis is a chronic inflammatory disease, characterized by specific infiltration of monocytic cells, such as monocytes and T-cells, while neutrophils are essentially absent in the fatty streak lesion. One of the earliest steps in the development of the atherosclerotic lesion is the adhesion of monocytes to endothelial cells of the vessel wall. Previously it was demonstrated that, if stimulated with minimally modified LDL (MM-LDL), endothelial cells are activated to specifically bind monocytes, but not neutrophils. This specificity towards mononuclear cells was subsequently observed with the activation of endothelial cells by oxidized 1-palmitoyl-2-arachidonoyl-s/7-glycero-3-phosphocholine (OxPAPC), implying lipid oxidation products as culprits in chronic inflammation. We hypothesize that specific phospholipid oxidation products (OxPL) trigger vascular inflammation and determine monocyte specificity characteristic of atherosclerosis and other chronic inflammatory diseases. The outcome of the proposed studies will add to our understanding of how atherosclerosis is initiated and propagated and should lead to new strategies in the treatment of chronic inflammatory disorders. The specific aims addressed in this proposal are: Specific Aim #1: To structurally identify individual OxPL that determine monocyte specific inflammation and to investigate the hypothesis that OxPL-induced signaling involves TLR4. We will investigate intracellular signaling pathways induced by OxPL that lead to specific monocyte adhesion in vitro, using HUVEC as well as isolated murine aortic EC. Furthermore, we will test the hypothesis that POVPC is one active component in OxPAPC that determines monocyte specificity and determine structure-function relationships. We will examine the role of TLR-4 and the involvement of the TLR-4 adaptor proteins MyD88 and TRIP, as well as respective downstream elements in OxPL-mediated signalling events. Specific Aim #2: To explore the hypothesis that OxPL are triggers of vascular inflammation in vivo. To mimic accumulation of OxPL in the vascular wall, we will topically apply OxPL to carotid arteries in mice and measure chemokine and adhesion molecule expression. Furthermore, we will use ex vivo perfused carotid arteries to study OxPL-induced monocyte rolling and adhesion. Specific Aim #3: To examine the hypothesis that OxPL-induced specific mononuclear cell accumulation in vivo requires a specific chemokine expression pattern and involves 12 lipoxygenase. Using the mouse air pouch model, we will examine OxPL-induced leukocyte accumulation in vivo and characterize leukocyte subsets. We will investigate the chemokine expression pattern as well as time course of expression in OxPL-induced compared to LPS-induced inflammation. Furthermore, we will investigate the role of 12-LO in OxPL-induced inflammation in vivo.