Nck adaptor proteins in atherogenic endothelial activation

项目总结 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激活， 以核因子-B(NF-B)为特征的，介导白细胞的细胞黏附分子的表达 归巢。除血流模式外，促炎症细胞因子(如IL-1)促进NF-B依赖 血管内皮细胞激活。虽然最近使用IL-1拮抗剂的CATOS试验突显了 治疗动脉粥样硬化性炎症不仅仅是降脂治疗，我们对血管内皮细胞如何 激活对这一效果的贡献仍然有限。在上一次资助期间，我的实验室确定了 信号转接子NKK1是动脉粥样硬化血管内皮细胞活化的关键调节因子。内皮细胞缺陷性 在体外和体内缺乏Flow诱导的NF-B激活和促炎基因表达，以及 结构域交换实验和点突变分析表明，NKK1 SH2结构域和 第一个NKK1 SH3结构域参与了这些效应的调节。了解Nock1如何介导致动脉粥样硬化的内皮细胞 激活后，我们分离到了NKK1‘S的促炎功能到NKK1的SH2结构域和第一个SH3结构域。 此外，我们还展示了NKK1和IL-1信号之间的相互作用，以响应干扰的流动， 显示干扰流激活依赖于的IL-1受体(IL-1R1)信号伙伴IRAK-1 在体外和体内的作用方式。IRAK-1的耗竭抑制了对干扰血流的促炎反应，但 干扰流刺激IRAK-1信号转导的机制尚不清楚。 Nck信号适配器家族(Nck 1和Nck 2)在血管发育和修复过程中扮演多余的角色。 视网膜新生血管，只有当两种异构体都被抑制时才能观察到缺陷。与我们的细胞一致 培养研究表明，全球Nock1基因敲除小鼠的动脉粥样硬化减少的特征是减少 巨噬细胞和平滑肌掺入，而内皮细胞基因敲除不能。而骨髓 嵌合体提示非造血细胞的NK1介导了这一反应，关于NK1的相关作用的问题 血管内皮细胞和血管内皮细胞的NK1在这种反应中持续存在。最近的一项GWAS分析将Nock1与冠状动脉联系起来 动脉疾病，强调了它对人类疾病的重要性。因此，我们假设NCK1适配器 介导IL-1相关信号复合体的形成，以诱导致动脉粥样硬化的内皮激活，以及 选择性地抑制Nck 1或其关键结构域将允许有效地抑制致动脉粥样硬化的内皮激活 而不影响缺血血管重塑。我们将确定NCK1调节的机制 干扰流诱导的IRAK-1激活(AIM 1)，表征NKK-1和IL-1相关的相互作用 干扰血流诱导体内内皮细胞激活的信号转导(AIM 2)，并评估选择性NKK1是否 抑制作用不同地影响动脉粥样硬化和缺血血管生成(目标3)。成功完成 这些目的将确定新的机制，其中NKK1介导动脉粥样硬化内皮细胞的激活，并将 将Nock1定性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.