Determining the Involvement of PERK in TMAO Induced Atherosclerosis

摘要： 世界卫生组织报告称，心血管疾病(CVD)是全球主要死亡原因。 我们的长期目标是通过推进心血管疾病的临床治疗和管理来改善公众健康。 三甲胺N-氧化物(TMAO)是一种饮食衍生代谢物，可高度预测动脉粥样硬化 和心血管疾病死亡率。在多项独立研究中，在患有心血管疾病的人类中，它显著升高，而且它 饮食补充剂会导致动物模型的动脉粥样硬化。但利用TMAO途径的努力 治疗上受到怀疑，因为TMAO的明确作用机制仍然难以捉摸。 因此，明确TMAO诱导动脉粥样硬化的机制具有重要意义。 对那些死于心血管疾病的人来说，有意义的治疗选择发生了变化。最近，我们的实验室报告了第一次也是唯一一次 已知的TMAO受体；TMAO特异性结合并激活应激信号激酶PKR样蛋白 内质网激酶(PERK)。然而，有两个核心问题仍然没有得到解决：1)如何 TMAO-PERK驱动疾病的激活，以及2)PERK活性是否是TMAO引起动脉粥样硬化所必需的？ 本提案提出了一种回答上述问题的研究策略。我们报告的初步数据显示， TMAO引起小鼠主动脉perk磷酸化(PPERK)，长期摄食TMAO增加了小鼠主动脉 依赖于福利的炎症。我们还表明，使用PERK抑制剂可以防止TMAO诱导的 心血管疾病危险因素、血糖和血浆胆固醇的增加。此外，我们确认TMAO激活 PPERK，但它还诱导了一种不同于经典描述的PERK激活的独特构象状态 通过未折叠的蛋白质。因此，我们的中心假设是TMAO通过一种独特的 Perk轴的激活。在目标1中，我们建议定义TMAO对PERK信号复合体的影响 在血管系统中。在目标2中，我们将测试在TMAO导致动脉粥样硬化的能力方面的PERK要求 APOE-/-小鼠。所描述的实验将确定人们长期寻找的TMAO如何导致 他们将研究应激反应蛋白PERK的激活是如何诱导细胞 功能障碍。拟议的培训计划包括分子和生理学工具方面的培训，以评估新陈代谢 对CVD、演示、交流和出版机会的贡献，以及以下方面的总体路线图 成长为富有成效和协作性的独立调查员。此外，制度环境 在哈佛大学公共卫生学院和波士顿儿童医院，都确保了 丰富的资源和设备、核心设施和聪明才智将继续支持不断增长的 项目的需求。我们相信，所附的目标将审问PERK在调解油井- 描述了TMAO诱导的心血管疾病的影响，他们将打开一扇已经铺天盖地的治疗方案 等待预防心血管疾病过早死亡。

ABSTRACT: The World Health Organization reports cardiovascular disease (CVD) as the global leading cause of death. Our long-term goal is to improve public health by advancing clinical therapies and management of CVD. Trimethylamine N-oxide (TMAO) has emerged as a dietary derived metabolite highly predictive of atherosclerosis and CVD mortality. It is robustly elevated in humans with CVD across multiple independent studies, and its dietary supplementation causes atherosclerosis in animal models. But efforts to leverage the TMAO pathway therapeutically are met with skepticism, as a clear mechanism of TMAO action continues to be elusive. Therefore, defining the mechanism of TMAO-induced atherosclerosis is of fundamental importance to produce meaningful change in treatment options for those dying of CVD. Recently, our lab reported the first and only known receptor for TMAO; TMAO specifically binds and activates the stress signaling kinase PKR-like Endoplasmic Reticulum Kinase (PERK). However, two central questions are still unaddressed: 1) how does TMAO-activation of PERK drive disease, and 2) is PERK activity required for TMAO to cause atherosclerosis? This proposal presents a research strategy to answer the above questions. We report preliminary data that acute TMAO causes PERK phosphorylation (pPERK) in mouse aorta, and that chronic TMAO feeding increases aortic inflammation in a PERK dependent manner. We also show use of a PERK inhibitor prevents TMAO-induced increases of CVD risk factors, blood glucose and plasma cholesterol. Further, we confirm that TMAO activates pPERK, but also that it induces a unique conformational state distinct from classically described PERK activation by unfolded proteins. Thus, our central hypothesis is that TMAO drives atherosclerosis via a unique activation of the PERK axis. In Aim 1, we propose to define TMAO’s effects on the PERK signaling complex in the vasculature. In Aim 2, we will test the requirement of PERK in TMAO’s ability to cause atherosclerosis in ApoE-/- mice. The experiments described will identify a long-sought mechanism of how TMAO causes atherosclerosis, and they will investigate how activation of the stress response protein, PERK, induces cellular dysfunction. The proposed training plan covers training in molecular and physiologic tools to assess metabolic contributions to CVD, presentation, communication, and publication opportunities, and an overall roadmap for growing into a productive and collaborative independent investigator. Additionally, the institutional environment at both the Harvard T.H. Chan School of Public Health and Boston Children’s Hospital ensures robust access to abundant resources and equipment, core facilities, and brilliant minds that will continually support the growing needs of the project. We believe the enclosed aims will interrogate the role of PERK in mediating the well- described effect of TMAO-induced CVD, and they will open a flood gate of therapeutic options already lying-in wait to prevent premature cardiovascular deaths.