Hypothermic Adaptation and Endothelial Protection from Severe Cold Stress

低温适应和内皮保护免受严重冷应激

• 批准号: 8091454
• 负责人: Michael A Zieger
• 金额: $ 16.88万
• 依托单位: INDIANA UNIVERSITY HEALTH, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8091454
• 关键词: Antioxidants; Area; Blood Vessels; Cardiac; Cardiac Surgery procedures; Cell Death; Cell Nucleus; Cell Survival; Cells; Clinical; Congenital Heart Defects; Coronary Artery Bypass; Coronary artery; Cryopreservation; Data; Dependence; Endothelial Cells; Enzymes; Flushing; Funding; Future; Gene Targeting; Genetic Transcription; Glutathione; Goals; Heart; Heart Arrest; Heart Transplantation; Heat shock proteins; Hour; Human; In Vitro; Injury; Kinetics; Knowledge; Lead; Link; Mammalian Cell; Mediating; Mediator of activation protein; Methods; Molecular; Molecular Chaperones; Morbidity - disease rate; Myocardial Ischemia; National Heart, Lung, and Blood Institute; Nuclear; Operative Surgical Procedures; Organ; Organ Preservation; Organ Transplantation; Organism; Outcome; Oxidative Stress; Pathology; Pathway interactions; Patients; Phenotype; Proteins; Proteome; Proteomics; RNA Interference; Reperfusion Injury; Reperfusion Therapy; Research; Resistance; Response Elements; Rewarming; Risk; Role; Signal Transduction; Stress; Stroke; Techniques; Temperature; Testing; Thapsigargin; Therapeutic; Tissues; Transplant Recipients; Traumatic Brain Injury; Vascular Endothelium; Work; base; cell injury; cold temperature; endoplasmic reticulum stress; exposed human population; heart preservation; high risk; implantation; improved; induced hypothermia; innovation; mortality; natural hypothermia; neurological recovery; novel; preconditioning; public health relevance; response; stress protein; tool; trait; transcription factor; working group

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.

描述（由申请人提供）：目前用于保存心脏以供移植的技术仍然不足，并且血管内皮损伤显著导致心脏受体的发病率和死亡率。内皮细胞在心脏保存期间处于危险之中，因为它们在血管冲洗和冷藏期间直接暴露于快速冷却和接近-00 ° C的温度，并且在再灌注期间暴露于快速复温和复氧，这些条件产生氧化损伤。长期目标是了解心功能减退介导的心脏组织损伤的分子基础，并利用这些知识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）在体外适应长期中度（250 ℃）低温具有高水平的细胞谷胱甘肽，广泛修饰的蛋白质组和增加的抵抗冷诱导的氧化应激和细胞死亡在00 ℃，温度是高度破坏性的。本R21应用的目的是确定内皮冷适应如何导致对00 C损伤的增强保护。中心假设是轻度-中度低温诱导非致死性内质网（ER）应激，随后的反应激活了具有细胞保护作用的转录途径。拟议研究的基本原理是，定义冷适应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。将通过追求以下两个具体目标来检验中心假设：1）确定轻度-中度低温对ER应激信号传导的影响;和2）确定轻度-中度低温对Nrf 2/ARE途径的影响。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，然后使用这些知识、UPR通路诱导剂和UPR介导剂的RNAi抑制来调节抗氧化蛋白和伴侣蛋白的表达、谷胱甘肽合成以及随后的细胞在0 ℃下的存活。在第二个目标中，温度、ER应激的诱导/抑制、氧化应激的促进/减少和Nrf 2/Keap 1的RNAi抑制将用于调节Nrf 2/ARE途径的活化，从而改变抗氧化蛋白的表达和谷胱甘肽合成以及随后在0 ℃下的细胞存活。这项研究是创新的，因为它专注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，则不会考虑这些机制。 公共卫生关系：拟议的研究是在一个重要的研究领域最近推荐的NHLBI工作组在心脏外科研究的未来方向。这项拟议中的研究将建立一种保护心脏的全新策略，即利用细胞对寒冷的固有适应性所涉及的分子途径。改进的心脏保存方法不仅有利于心脏移植受者，而且有利于需要外科手术的患者，如冠状动脉旁路移植术或先天性心脏缺陷的矫正。