Control of Endothelial Mechanotransduction by the Mitochondrial Ca2+ Uniporter: Implications for Atherosclerosis

线粒体 Ca2 单向转运蛋白控制内皮机械转导：对动脉粥样硬化的影响

• 批准号: 10197208
• 负责人: Barbara Rita Alevriadou
• 金额: $ 44.51万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197208
• 关键词: ATP Synthesis Pathway; Ablation; Agreement; Animals; Aorta; ApoE knockout mouse; Apolipoprotein E; Apoptosis; Arterial Fatty Streak; Arteries; Atherosclerosis; Autophagocytosis; Beds; Biochemical; Bioenergetics; Biomedical Engineering; CREB1 gene; CRISPR/Cas technology; Calcium; Cardiovascular Diseases; Carotid Arteries; Cause of Death; Cell Death; Cell physiology; Cells; Collaborations; Complex; Consumption; Data; Dependence; Developed Countries; Development; Diabetes Mellitus; Disease; Endothelial Cells; Endothelium; Event; Exhibits; Exposure to; Functional disorder; Future; Gene Proteins; Genes; Genetic Transcription; Goals; Human; Hyperlipidemia; Hypertension; In Vitro; Inflammation; Inner mitochondrial membrane; Ion Channel; Knock-in; Lead; Ligation; Liquid substance; Measures; Mediating; Membrane Potentials; Mitochondria; Modeling; Modification; Molecular; Mus; Nature; Nitric Oxide; Operative Surgical Procedures; Oxidants; Oxidation-Reduction; Oxidative Stress; Permeability; Phenotype; Physiological; Prevention strategy; Production; Reactive Oxygen Species; Regulation; Respiration; Risk Factors; Role; Seminal; Signal Pathway; Signal Transduction; Small Interfering RNA; Smoking; Stress; Structure; Systole; Testing; Transgenic Mice; Transgenic Organisms; VDAC1 gene; Vascular Endothelial Cell; Work; atheroprotective; base; conditional knockout; design; experience; gain of function; hemodynamics; in vivo; insight; knock-down; mechanotransduction; monolayer; mutant; new therapeutic target; novel; novel therapeutic intervention; overexpression; protein expression; sensor; shear stress; small molecule; spatiotemporal; targeted delivery; therapeutic target; transcription factor; uptake; vascular bed

PROJECT SUMMARY / ABSTRACT It is well known that local hemodynamic forces/stresses modulate the phenotype of vascular endothelial cells (ECs), and this phenotypic modulation contributes to the focal nature of atherosclerotic disease. ECs in athero- prone arterial regions, which are exposed to oscillatory shear stress (OS), experience higher levels of reactive oxygen species (ROS) and exhibit inflammation and increased sensitization to apoptosis compared to ECs in atheroprotective regions, which are exposed to pulsatile shear stress (PS). Our group has made seminal discov- eries on the structure/function of the Mitochondrial Calcium (Ca2+) Uniporter (MCU) complex, an inner mitochon- drial membrane channel responsible for mitochondrial Ca2+ ([Ca2+]m) uptake. It consists of a pore-forming protein, also called MCU, and auxiliary subunits. We showed that EC MCU expression is regulated by the redox-sensitive transcription factor CREB. MCU is activated following oxidative modification by mitochondrial ROS (mROS), and persistent activation results in [Ca2+]m overload and cell death. We recently showed that MCU knockdown inhibits the intracellular Ca2+ ([Ca2+]i) oscillations in cultured ECs exposed to steady laminar shear stress suggesting that the MCU activity ([Ca2+]m uptake) is critical for shear-induced [Ca2+]i signaling and EC function. Since MCU expression/activity are redox regulated and OS-exposed ECs encounter oxidative stress, we hy- pothesized that EC MCU expression/activity will be enhanced in atheroprone (OS) regions compared to athero- protective (PS) ones, and the increased EC [Ca2+]m uptake in OS-exposed regions will be responsible for mito- chondrial and cell dysfunction, and for initiation of atherosclerosis. Preliminary data showed significantly higher EC MCU protein expression and activity in OS-exposed, compared to PS-exposed, regions in mouse aortas. We propose to: (a) Determine the differential effects of PS vs. OS on EC MCU gene/protein expression and activity in vivo and in vitro. To establish the causative role of OS in EC MCU expression in vivo, a mouse partial carotid artery ligation model will be employed. (b) Define the signaling events upstream and downstream of MCU in cultured ECs, from different species/vascular beds, exposed to PS vs. OS. The effects of either MCU knock- down, overexpression, or persistent activation on the basal and PS/OS-induced [Ca2+]m, [Ca2+]i, mROS, cytosolic ROS, mitochondrial (bioenergetics, respiration) and EC function will be determined. (c) Assess whether targeting the EC MCU will confer protection from atherosclerotic disease. The MCU role in OS-induced EC inflammation will be examined in EC-specific MCU conditional knockout (MCU∆EC), CRISPR/Cas9 knock-in (gain-of-function mutant), and control mice. To assess the MCU as a therapeutic target, double transgenic MCU∆EC+ApoE-/- mice will be generated and the role of MCU ablation in post-ligation atherosclerosis will be examined in ApoE-/- mice. Completion of this collaborative project will provide new insights into the MCU-mediated Ca2+ signaling and its role in EC mechanotransduction and atherosclerotic disease development. Furthermore, the discovery of novel molecular mechanisms of atherosclerosis will undoubtedly lead to the design of new therapeutic interventions.

项目摘要/摘要 众所周知，局部血流动力/应力调节血管内皮细胞的表型。 (ECS)，这种表型调节有助于动脉粥样硬化性疾病的局灶性。动脉粥样硬化中的ECS- 易受振荡剪切力(OS)影响的动脉区域，会经历更高水平的反应 与内皮细胞相比，氧物种(ROS)和表现出炎症和对细胞凋亡的增敏作用 动脉粥样硬化保护区域，暴露在脉动剪应力(PS)下。我们的团队已经发现了种子- 线粒体钙(Ca~(2+))单一转运体(MCU)复合体结构/功能的研究 线粒体Ca~(2+)([Ca~(2+)]m)摄取的干膜通道。它由一种成孔蛋白组成， 也称为MCU，和辅助子单元。我们发现EC MCU的表达受氧化还原敏感性的调节 转录因子CREB。MCU在线粒体ROS(MROS)氧化修饰后被激活，并且 持续的激活会导致[Ca~(2+)]m超载和细胞死亡。我们最近发现，MCU基因敲除会抑制 恒定层流切应力作用下培养内皮细胞内钙([Ca~(2+)]i)振荡 MCU活性([Ca~(2+)]_m摄取)对切变诱导的[Ca~(2+)]_i信号转导和EC功能至关重要。 由于MCU的表达/活性受氧化还原调节，而暴露于OS的内皮细胞遇到氧化应激，我们认为- 与动脉粥样硬化相比，动脉粥样硬化(OS)区EC MCU的表达/活性将增强。 保护性(PS)蛋白和OS暴露区域内EC[Ca~(2+)]m摄取的增加将导致丝裂原反应。 软骨和细胞功能障碍，以及动脉粥样硬化的启动。初步数据显示， 与PS暴露相比，OS暴露的小鼠主动脉中EC MCU蛋白的表达和活性。 我们建议：(A)确定PS和OS对EC MCU基因/蛋白表达的不同影响和 体内和体外活性。为了确定OS在体内EC MCU表达中的作用，建立了一种小鼠部分 将采用颈动脉结扎模型。(B)定义MCU上下游的信令事件 在来自不同物种/血管床的培养内皮细胞中，暴露于PS和OS。无论是MCU敲门的效果- 下调、过表达或持续激活基础和PS/OS诱导的胞浆[Ca~(2+)]_m、[Ca~(2+)]_i、MRO、 将测定ROS、线粒体(生物能量学、呼吸作用)和EC功能。(C)评估目标是否 EC MCU将提供对动脉粥样硬化性疾病的保护。MCU在OS诱导的EC炎症中的作用 将在EC特定的MCU条件敲除(MCU∆EC)、CRISPR/CAS9敲入(功能增益)中进行检查 突变)，并控制小鼠。以∆EC+载脂蛋白E-/-双转基因小鼠为靶点 将在载脂蛋白E-/-小鼠身上检测MCU消融在结扎后动脉粥样硬化中的作用。 这一合作项目的完成将为研究MCU介导的钙信号转导及其 在EC机械转导和动脉粥样硬化性疾病发展中的作用。此外，小说的发现 动脉粥样硬化的分子机制无疑将导致新的治疗干预措施的设计。