Hypothermic Adaptation and Endothelial Protection from Severe Cold Stress

描述（由申请人提供）：目前保存移植心脏的技术仍然不足，血管内皮的损伤是心脏受体发病率和死亡率的重要因素。在心脏保存过程中，内皮细胞处于危险之中，因为它们在血管冲洗和冷藏过程中直接暴露于快速冷却和接近0℃的温度，在再灌注过程中暴露于快速升温和再氧化，这些条件会产生氧化损伤。长期目标是了解低温介导的心脏组织损伤的分子基础，并利用这些知识来开发改进的治疗方法，以在心胸外科手术和移植过程中保护心脏。初步研究表明，人冠状动脉内皮细胞（HCAEC）适应体外长时间中等（250℃）低温，细胞谷胱甘肽水平高，蛋白质组广泛修饰，对低温诱导的氧化应激和细胞死亡的抵抗力增强，温度为00℃，这是高度破坏性的。本R21应用的目的是确定内皮细胞冷适应如何增强对00C损伤的保护。中心假设是，轻度-中度低温诱导非致死内质网应激，随后的反应激活具有细胞保护作用的转录途径。提出的研究的基本原理是，确定冷适应的机制将提供使心脏保存更安全的工具，并将产生更好的临床结果。中心假设将通过以下两个具体目标进行检验：1)确定轻度至中度低温对内质网应激信号的影响；2)确定轻中度低温对Nrf2/ARE通路的影响。第一个目标是确定hcaec中未折叠蛋白反应的温度依赖性，动力学和激活程度，然后利用这些知识，UPR途径诱导剂和RNAi抑制UPR介质来调节抗氧化蛋白和伴侣的表达，谷胱甘肽合成，以及随后在00C下的细胞存活。在第二个目标中，温度、内质网应激的诱导/抑制、氧化应激的促进/减少以及Nrf2/Keap1的RNAi抑制将被用来调节Nrf2/ARE通路的激活，从而改变抗氧化蛋白的表达和谷胱甘肽的合成以及随后在00C下的细胞存活。这项提议的研究具有创新性，因为它侧重于恒温哺乳动物细胞的低温适应机制，而不是使用更传统的方法来进行器官保存研究。这是非常重要的，因为它有助于研究新的保护机制，如果只研究保存器官的病理学，就不会考虑这些机制。

DESCRIPTION (provided by applicant): Current techniques for preserving the heart for transplantation remain inadequate and damage to the vascular endothelium contributes significantly to the morbidity and mortality of heart recipients. Endothelial cells are at risk during heart preservation because they are directly exposed to rapid cooling and near-00C temperatures during vascular flushing and cold storage and to rapid rewarming and re-oxygenation during reperfusion, conditions that generate oxidative injury. The long-term goal is to understand the molecular basis of hypothermia-mediated cardiac tissue damage, and use this knowledge to develop improved therapies for protecting the heart during cardiothoracic surgery and transplantation. Preliminary studies showed that human coronary artery endothelial cells (HCAEC) adapted to prolonged moderate (250C) hypothermia in vitro have high levels of cellular glutathione, an extensively modified proteome and increased resistance to cold-induced oxidative stress and cell death at 00C, a temperature that is highly damaging. The objective of this R21 application is to determine how endothelial cold-adaptation leads to enhanced protection from 00C injury. The central hypothesis is that mild- moderate hypothermia induces a non-lethal endoplasmic reticulum (ER) stress and the ensuing response activates transcriptional pathways that are cytoprotective. The rationale for the proposed studies is that defining the mechanisms of cold-adaptation will provide the tools to make heart preservation safer and will yield better clinical outcomes. The central hypothesis will be tested by pursuing the following two specific aims: 1) Determine the effect of mild-moderate hypothermia on ER stress signaling; and 2) Determine the effect of mild- moderate hypothermia on the Nrf2/ARE pathway. The first aim will determine the temperature dependence, kinetics and extent of activation of the Unfolded Protein Response in HCAECs and then use this knowledge, UPR pathway inducers and RNAi inhibition of UPR mediators to modulate the expression of antioxidant proteins and chaperones, glutathione synthesis, and subsequent cell survival at 00C. In the second aim, temperature, induction/inhibition of ER stress, promotion/reduction of oxidative stress, and RNAi inhibition of Nrf2/Keap1 will be used to modulate the activation of the Nrf2/ARE pathway and thereby vary the expression of antioxidant proteins and glutathione synthesis and subsequent cell survival at 00C. The proposed research is innovative because it focuses on a mechanism of hypothermic adaptation in homeothermic mammalian cells rather than using more conventional approaches to organ preservation research. This is highly significant because it facilitates the study of novel protective mechanisms that would not be considered if one only studied the pathology of preserved organs. PUBLIC HEALTH RELEVANCE: The proposed studies are in an important area of research recently recommended by an NHLBI working group on future directions for research in cardiac surgery. The proposed research will establish a fundamentally new strategy for preserving the heart, which is the exploitation of molecular pathways involved in the inherent adaptability of cells to cold. Improved heart preservation methods will benefit not only heart transplant recipients, but also patients requiring surgical procedures, such as coronary artery bypass grafting or the correction of congenital heart defects.