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: 10423402
• 负责人: Barbara Rita Alevriadou
• 金额: $ 7.31万
• 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/10423402
• 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.

项目总结/摘要 众所周知，局部血流动力学力/应力调节血管内皮细胞的表型 (ECs)并且这种表型调节有助于动脉粥样硬化疾病的局灶性。动脉粥样硬化中的内皮细胞- 暴露于振荡剪切应力（OS）的易发动脉区域经历更高水平的反应性损伤。 与内皮细胞相比， 动脉粥样硬化保护区域，其暴露于脉动剪切应力（PS）。我们小组有了重大发现- 线粒体钙（Ca 2+）单向转运体（MCU）复合物的结构/功能的系列研究， 线粒体膜通道负责线粒体Ca ~（2+）（[Ca ~（2+）]m）摄取。它由成孔蛋白组成， 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蛋白的调节， 转录因子CREB。MCU在线粒体ROS（mROS）氧化修饰后被激活， 持续激活导致[Ca 2 +]m过载和细胞死亡。我们最近发现MCU敲低抑制了 稳定层流切应力作用下培养的内皮细胞胞内Ca ~（2+）（[Ca ~（2+）]i）振荡表明， MCU活性（[Ca 2 +]m摄取）对于剪切诱导的[Ca 2 +]i信号传导和EC功能是关键的。 由于MCU的表达/活性是氧化还原调节的，暴露于OS的EC会遇到氧化应激，因此我们可以通过以下方式来调节MCU的表达/活性： 假设EC MCU表达/活性将在动脉粥样硬化酮（OS）区域中增强， 保护性（PS）的，以及OS暴露区域EC [Ca 2 +]m吸收的增加将是线粒体的原因。 血管和细胞功能障碍，并引发动脉粥样硬化。初步数据显示， EC MCU蛋白表达和活性在OS暴露，相比PS暴露，在小鼠睾丸的区域。 我们建议：（a）确定PS与OS对EC MCU基因/蛋白表达的差异效应， 体内和体外活性。为了确定OS在体内EC MCU表达中的致病作用，使用小鼠部分细胞培养技术， 采用颈动脉结扎模型。(b)定义MCU上下游的信令事件 在培养的EC中，来自不同物种/血管床，暴露于PS与OS。 在基础和PS/OS诱导的[Ca 2 +]m、[Ca 2 +]i、mROS、胞质内 将测定ROS、线粒体（生物能量学、呼吸）和EC功能。(c)评估目标是否 EC MCU将提供对动脉粥样硬化疾病保护。MCU在OS诱导的EC炎症中的作用 将在EC特异性MCU条件性敲除（MCU CNOEC）、CRISPR/Cas9敲入（功能获得性） 突变体）和对照小鼠。为了评估MCU作为治疗靶点的作用，双转基因MCU转基因小鼠EHEC +ApoE-/-小鼠 并在ApoE-/-小鼠中检查MCU消融在结扎后动脉粥样硬化中的作用。 该合作项目的完成将为MCU介导的Ca 2+信号转导及其在细胞内的作用提供新的见解。 在EC机械传导和动脉粥样硬化疾病发展中的作用。此外，小说的发现 动脉粥样硬化的分子机制无疑将导致设计新的治疗干预措施。