Determining the Involvement of PERK in TMAO Induced Atherosclerosis

摘要： 世界卫生组织报告说，心血管疾病（CVD）是全球主要的死亡原因。 我们的长期目标是通过推进CVD的临床治疗和管理来改善公共卫生。 氧化三甲胺（TMAO）是一种可高度预测动脉粥样硬化的膳食代谢产物 CVD死亡率。在多项独立研究中，它在CVD患者中显著升高， 膳食补充剂在动物模型中引起动脉粥样硬化。但是利用氧化三甲胺途径的努力 然而，TMAO的治疗作用受到怀疑，因为TMAO作用的明确机制仍然难以捉摸。 因此，明确TMAO诱导动脉粥样硬化的机制对于产生 对于那些死于CVD的人来说，治疗方案的改变是有意义的。最近，我们的实验室报告了第一个也是唯一一个 已知的TMAO受体; TMAO特异性结合并激活应激信号激酶PKR样 内质网激酶（PERK）。然而，两个核心问题仍然没有得到解决：1）如何 TMAO激活PERK驱动疾病，和2）PERK活性需要TMAO引起动脉粥样硬化？ 本建议提出了一个研究策略来回答上述问题。我们报告的初步数据表明， TMAO引起小鼠主动脉中PERK磷酸化（pPERK），并且慢性TMAO喂养增加了主动脉 以PERK依赖的方式炎症。我们还表明，使用PERK抑制剂可以防止TMAO诱导的 CVD危险因素、血糖和血浆胆固醇增加。此外，我们确认TMAO激活 pPERK，而且它诱导了一种独特的构象状态，不同于经典描述的PERK激活 由未折叠的蛋白质组成。因此，我们的中心假设是TMAO通过一种独特的 激活PERK轴。在目标1中，我们提出定义TMAO对PERK信号复合物的影响 在脉管系统中。在目标2中，我们将测试PERK在TMAO引起动脉粥样硬化的能力中的需求， ApoE-/-小鼠。所描述的实验将确定TMAO如何引起 他们将研究应激反应蛋白PERK的激活如何诱导细胞凋亡， 功能障碍拟议的培训计划包括分子和生理工具的培训，以评估代谢 对CVD的贡献，演示，交流和出版机会，以及 成长为一名富有成效和合作精神的独立调查员。此外，体制环境 在哈佛T. H. Chan公共卫生学院和波士顿儿童医院确保了 丰富的资源和设备，核心设施和辉煌的头脑，将不断支持不断增长的 项目的需要。我们相信，所附的目标将询问PERK在调解良好- 描述了TMAO诱导的CVD的影响，他们将打开一扇已经存在的治疗选择的闸门。 预防过早的心血管死亡。

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.