LRRC8 complex regulation of endothelial function

LRRC8 复合物调节内皮功能

• 批准号: 10638931
• 负责人: Abhinav Diwan
• 金额: $ 57.39万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638931
• 关键词: Angiotensin II; Anions; Biochemical; Biology; Blood Pressure; Blood Vessels; Blood flow; Cell membrane; Cell physiology; Chemicals; Chronic; Complex; Data; Diabetes Mellitus; Diffuse; Disease; Endothelial Cells; Endothelium; Exhibits; FRAP1 gene; Health; Heart Diseases; Human; Human Genome; Hydrostatic Pressure; Hypertension; Image; Impairment; In Vitro; Infusion procedures; Knockout Mice; Knowledge; Laboratories; Leucine-Rich Repeat; Lysosomes; Mechanics; Metabolic syndrome; Mission; Modeling; Molecular; Molecular Biology; Mus; Mutation; National Heart, Lung, and Blood Institute; Non-Insulin-Dependent Diabetes Mellitus; PIK3CG gene; Pathway interactions; Prevention; Proteins; Proto-Oncogene Proteins c-akt; Reagent; Regulation; Reporting; Research; Retina; Signal Transduction; Societies; Stretching; Testing; Therapeutic; Treatment Efficacy; Umbilical vein; Vascular Diseases; Vascular Endothelium-Dependent Relaxation; angiogenesis; detection of nutrient; diabetic; experimental study; fluid flow; genome wide association study; improved; in vivo; innovation; novel; patch clamp; pharmacologic; phenome; pre-clinical; response; small molecule; vascular endothelial dysfunction

Project Summary The endothelium responds to a multitude of chemical and mechanical factors in regulating vascular tone, angiogenesis, blood pressure and blood flow. The endothelial volume regulatory anion channel (VRAC) has been proposed to be mechano-sensitive, to activate in response to fluid flow/hydrostatic pressure and putatively regulate vascular reactivity and angiogenesis. We recently reported that the Leucine Rich Repeat Containing Protein 8a, LRRC8a (LRRC8A) is a required component of the heterohexameric complex that forms VRAC in human umbilical vein ECs (HUVECs). Endothelial LRRC8A regulates AKT-eNOS and mTOR signaling under basal conditions, and with stretch and shear-flow stimulation and is required for EC alignment to laminar shear flow. Endothelium-restricted LRRC8A KO (LRRC8A KO) mice have impaired endothelium-dependent vascular relaxation, develop hypertension in response to chronic angiotensin II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of Type 2 diabetes (T2D). These data demonstrate that LRRC8a regulates AKT-eNOS, and mTOR signaling in endothelium and is required for maintaining vascular function. There remains a knowledge gap in (a) the molecular identity of specific LRRC8 heteromers that form VRAC in endothelium, (b) the molecular mechanisms that connect the endothelial LRRC8 complex to AKT-eNOS and mTOR signaling, (c) the therapeutic potential of small molecules targeting the LRRC8 complex needs to be evaluated, leading to a novel class of compounds to improve vascular function and hypertension in metabolic syndrome. We have biochemical, patch-clamp and imaging evidence that LRRC8 channel complexes are expressed and functional in lysosomes (Lyso-LRRC8) and have identified a critical channel pore mutation (R103E) that specifically disrupts LRRC8 channel activity. Given that lysosomes are signaling hubs that integrate nutrient sensing and AKT-mTOR signaling, we hypothesize LRRC8A/C channels co-regulate plasma membrane PI3K-AKT signaling and lysosome centered mTOR signaling in endothelium, and that small molecule LRRC8 complex modulators can restore dysfunctional endothelial LRRC8A/C in diabetes associated vascular disease and hypertension. To test the above hypotheses, we propose three specific AIMs that develop endothelial LRRC8 biology from molecular signaling mechanisms to proof of concept in vivo therapeutic: AIM#1: Delineate the mechanisms of plasma membrane versus lysosomal LRRC8 signaling to AKT- mTOR signaling in endothelium. AIM#2: Examine LRRC8 molecular contributions to EC function in vitro, ex vivo and in vivo AIM#3: Examine the therapeutic efficacy of small molecule LRRC8 modulators to improve vascular function and blood pressure in diabetes associated hypertension models

项目摘要 内皮对调节血管张力的多种化学和机械因素反应， 血管生成，血压和血液流动。内皮体积调节阴离子通道（VRAC）具有 被提议具有机械敏感性，以响应流体流量/静水压力，并在 调节血管反应性和血管生成。我们最近报道说，含亮氨酸富含的重复 蛋白质8a，lrrc8a（LRRC8A）是杂异的综合体的必需组成部分，该组合形成VRAC 人脐静脉EC（HUVECS）。内皮LRC8A调节Akt-enos和MTOR信号传导 基础条件，并具有拉伸和剪切流刺激，并且需要EC对齐层状剪切 流动。内皮限制的LRRC8A KO（LRRC8A KO）小鼠损害了内皮依赖性血管 放松，响应于慢性血管紧张素II输注并表现出视网膜血液受损的高血压 在2型糖尿病（T2D）的情况下，以弥漫性和局灶性血管的流动缩小。这些数据 证明LRRC8A调节Akt-enos和内皮中的MTOR信号传导，是必需的 维持血管功能。 （a）特定的分子身份仍然存在知识差距 在内皮中形成VRAC的LRRC8异构体，（b）连接的分子机制 内皮LRRC8复合物至Akt-enos和MTOR信号传导，（c）小的治疗潜力 需要评估靶向LRRC8复合物的分子，从而导致一类新的化合物 改善代谢综合征中的血管功能和高血压。 我们有生化，斑块钳和成像证据，表明LRRC8通道复合物被表达，并且 在溶酶体（lyso-lrrc8）中的功能，并确定了一个关键的通道孔突变（R103E） 特别破坏LRRC8通道活动。鉴于溶酶体是整合养分的信号枢纽 传感和AKT-MTOR信号传导，我们假设LRRC8A/C通道共同调节质膜 pi3k-akt信号传导和溶酶体中心的MTOR信号传导，该小分子小分子 LRRC8复合调节剂可以在相关的糖尿病中恢复功能失调的内皮LRRC8A/C 血管疾病和高血压。为了检验上述假设，我们提出了三个特定目标， 从分子信号传导机制到体内治疗概念证明：开发内皮LRC8生物学： AIM＃1：描述质膜与溶酶体LRC8信号的机理至Akt- 内皮中的MTOR信号传导。 AIM＃2：检查LRRC8分子对EC功能的贡献，体内和体内 目标＃3：检查小分子LRRC8调节剂的治疗功效以改善血管 与糖尿病相关的高血压模型的功能和血压