A Novel Role for Endothelial Epsins in TNF signaling, Inflammation, and Atheroscl

描述(申请人提供)：动脉粥样硬化，血管壁增厚和硬化的过程，是心血管疾病发展的一个重要因素(在美国是发病率和死亡率的主要原因)。因此，了解动脉粥样硬化性病变进展的分子机制对人类疾病和治疗的发展具有重要意义。我们最近确定了内吞接头蛋白epsin的一个新的作用，它是一种促动脉粥样硬化和 内皮细胞中的促炎调节因子。具体地说，我们观察到，在西方饮食喂养的ApoE-/-小鼠的主动脉动脉粥样硬化中，epsins 1和2上调。为了进一步研究epins在动脉粥样硬化中的作用，我们通过将Epn1f1/f1、Epn2-/-小鼠与构成VEcad CRE的小鼠杂交，建立了内皮细胞特异性的内皮特异性内皮素缺陷ApoE-/-小鼠(ApoE-/-/EC-DKO)，然后回交并将它们培育成ApoE-/-背景。当喂养西方饮食时，与ApoE-/-/WT小鼠相比，我们发现ApoE-/-/EC-DKO小鼠对饮食诱导的动脉粥样硬化具有抵抗力。动脉粥样硬化的发展在一定程度上依赖于内皮细胞的激活，即促进免疫和炎症细胞募集的黏附分子、选择素、趋化因子和细胞因子的上调。TNFR-NF-？B信号和随后的下游转录激活是血管内皮细胞激活所必需的关键级联反应。因此，我们描述了一种研究策略来验证这一中心假说，即EPSINS通过促进TNFR-NF-？B信号转导导致炎症以及随后免疫/炎性细胞向发展中的动脉粥样硬化的募集而在动脉粥样硬化中发挥关键作用。作为支持，我们在体外报告了内皮肽缺乏对肿瘤坏死因子诱导的主动脉内皮细胞TNFR-NF-？B信号转导的影响。在体外，内皮肽缺乏的内皮细胞的激活受阻与巨噬细胞滚动/黏附的减少相对应，这是巨噬细胞募集、渗透和泡沫细胞形成的关键步骤。此外，我们有初步数据表明，在TNFR信号复合体组装中，EPSINS具有一种新的调控机制，独立于其作为内吞适配器的传统角色。因此，我们还提出了一种策略来表征EPSINS促进TNFR-NF-？B信号转导的分子机制。具体地说，我们的数据表明，epins在招募和/或稳定TNFR信号复合体的形成方面发挥了新的关键作用。总之，从这项提案中获得的信息将为以下疾病提供一个新的调节剂和潜在的新的治疗靶点 动脉粥样硬化的发展和进展。此外，它还将导致鉴定和表征内毒素的一种新的功能，而不依赖于它们作为内吞适配器的作用。

DESCRIPTION (provided by applicant): Atherosclerosis, the process of vascular wall thickening and hardening, is a significant contributing factor in the development of cardiovascular disease (the leading cause of morbidity and mortality in U.S.). Therefore, understanding the molecular mechanisms involved in atherosclerotic lesion (atheroma) progression has significant relevance in human disease and therapeutic development. We recently identified a novel role for the endocytic adaptor protein, epsin, as a pro-atherogenic and pro-inflammatory regulator in the endothelium. Specifically, we observed that epsins 1 and 2 are upregulated in the aortic atheroma of western diet fed ApoE-/- mice. To further investigate the role of epsins in atherosclerosis, we created endothelial cell-specific epsin deficient ApoE-/- mice (ApoE-/-/EC-DKO) by crossing Epn1fl/fl, Epn2-/- mice with constitutive VEcad Cre mice then backcrossed and bred them to an ApoE-/- background. When fed western diet, and compared to ApoE-/- /WT mice, we found that ApoE-/-/EC-DKO mice were resistant to diet-induced atherosclerosis. Development of the atheroma is dependent, in part, on endothelial activation, i.e. upregulated expression of adhesion molecules, selectins, chemokines and cytokines, which facilitate the recruitment of immune and inflammatory cells. TNFR-NF-?B signaling and the subsequent downstream transcriptional activation, is a critical cascade necessary for endothelial activation. Therefore, we describe a research strategy to test the central hypothesis that epsins play a critical role in atherosclerosis by promoting TNFR-NF-?B signaling resulting in inflammation and subsequent recruitment of immune/inflammatory cells to the developing atheroma. In support, we report that epsin deficiency impaired TNF¿-induced TNFR-NF-?B signaling in aortic endothelial cells in vitro. Impaired activation of epsin deficient endothelial cells corresponded with decreased macrophage rolling/adhesion in vitro, which is a critical step in macrophage recruitment, infiltration and foam cell formation. Furthermore, we have preliminary data implicating a novel regulatory mechanism for epsins in TNFR signaling complex assembly, independent of its conventional role as an endocytic adaptor. Therefore, we also propose a strategy to characterize the molecular mechanism by which epsins promote TNFR-NF-?B signaling. Specifically, our data suggests epsins play a novel and critical role in recruiting and/or stabilizing TNFR signaling complex formation. In summary, the information gained from this proposal will provide a novel regulator, and potential new therapeutic target, for the development and progression of atherosclerosis. In addition, it will result in the identificatin and characterization of a novel function for epsins, independent of their role as endocytic adaptors.