Nck adaptor proteins in atherogenic endothelial activation

项目摘要 动脉粥样硬化易发部位血流动力学切应力的改变促进内皮活化， 以核因子-κ B B（NF-κ B B）驱动的细胞粘附分子的表达为特征， 归航除了流动模式之外，促炎细胞因子（例如IL-1 β）促进NF-κ B依赖性炎症反应。 内皮激活虽然最近使用IL-1 β拮抗剂的CANTOS试验强调了 除了单独的降脂治疗外，我们对内皮细胞如何作用的理解， 活化对这种效果的贡献仍然有限。在上一个资助期间，我的实验室确定了 信号适配器Nck 1作为致动脉粥样硬化内皮激活的关键调节因子。内皮细胞缺乏 在体外和体内均缺乏流动诱导NF-κ B B活化和促炎基因表达的Nck 1中， 结构域交换实验和点突变分析表明，Nck 1 SH 2结构域和Nck 1 SH 2结构域的关键作用。 第一Nck 1 SH 3结构域介导这些作用。为了了解Nck 1如何介导动脉粥样硬化内皮细胞 激活后，我们将Nck 1的促炎功能分离到Nck 1的SH 2结构域和第一个SH 3结构域。 此外，我们证明了Nck 1和IL-1 β信号传导之间的相互作用， 显示干扰的血流激活IL-1 β受体（IL 1 R1）信号伴侣IRAK-1， 在体外和体内的方式。IRAK-1耗竭抑制了对血流紊乱的促炎反应，但 受扰流刺激IRAK-1信号传导的机制仍然未知。 Nck家族的信号转导衔接子（Nck 1和Nck 2）在血管发育过程中起着冗余的作用， 视网膜新生血管形成，只有当两种亚型都被抑制时才观察到缺陷。与我们的细胞一致 培养研究中，整体Nck 1敲除小鼠显示动脉粥样硬化减少，其特征在于减少 巨噬细胞和平滑肌掺入，而内皮Nck 2敲除则不然。虽然骨髓 嵌合体表明非造血Nck 1介导了这种反应，关于嵌合体的相对作用的问题， 内皮和平滑肌Nck 1在这种反应中持续存在。最近的一项GWAS分析将Nck 1与冠状动脉疾病联系起来， 动脉疾病，强调其对人类疾病的重要性。因此，我们假设Nck 1衔接子 介导IL-1相关信号复合物的形成以诱导致动脉粥样硬化内皮活化，和 选择性抑制Nck 1或其关键结构域将有效抑制致动脉粥样硬化的内皮活化 而不影响缺血性血管重塑。我们将确定Nck 1调节的机制， 干扰流诱导的IRAK-1激活（Aim 1），表征Nck 1和IL-1相关 在体内干扰血流诱导的内皮细胞活化中的信号传导（目的2），并评估选择性Nck 1 抑制不同地影响动脉粥样硬化和缺血性血管生成（Aim 3）。成功完成 这些目标将确定Nck 1介导致动脉粥样硬化内皮激活的新机制， 将Nck 1表征为限制动脉粥样硬化的治疗靶点。

PROJECT SUMMARY Alterations in hemodynamic shear stress at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-B (NF-B)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition to flow patterns, proinflammatory cytokines (e.g. IL-1) promote NF-B-dependent endothelial activation. While the recent CANTOS trial using an IL-1 antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited. During the previous funding period, my laboratory identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack flow-induced NF-B activation and proinflammatory gene expression both in vitro and in vivo, and domain swap experiments and point mutation analysis suggest a critical role for the Nck1 SH2 domain and the first Nck1 SH3 domain in mediating these effects. To understand how Nck1 mediates atherogenic endothelial activation, we isolated Nck1’s proinflammatory functions to the Nck1 SH2 domain and fist SH3 domain. Additionally, we demonstrated an interaction between Nck1 and IL-1 signaling in response to disturbed flow, showing that disturbed flow activates the IL-1 receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both in vitro and in vivo. IRAK-1 depletion inhibited the proinflammatory response to disturbed flow, but the mechanism by which disturbed flow stimulates IRAK-1 signaling remains unknown. The Nck family of signaling adaptors (Nck1 and Nck2) play redundant roles during vascular development and retinal neovascularization, with defects only observed when both isoforms are inhibited. Consistent with our cell culture studies, global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation, whereas endothelial Nck2 knockouts do not. While bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response, questions concerning the relative roles of endothelial and smooth muscle Nck1 in this response persist. A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, we hypothesize that Nck1 adaptors mediate the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherogenic endothelial activation without affecting ischemic vascular remodeling. We will determine the mechanisms by which Nck1 regulates disturbed flow-induced IRAK-1 activation (Aim 1), characterize the interplay between Nck1 and IL-1-associated signaling in disturbed flow-induced endothelial activation in vivo (Aim 2), and assess whether selective Nck1 inhibition differentially affects atherosclerosis and ischemic angiogenesis (Aim 3). Successful completion of these aims will identify new mechanisms by which Nck1 mediates atherogenic endothelial activation and will characterize Nck1 as a therapeutic target to limit atherosclerosis.