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

项目概要 内皮细胞对调节血管张力的多种化学和机械因素作出反应， 血管生成、血压和血流量。内皮容量调节阴离子通道 (VRAC) 被认为是机械敏感的，可以响应流体流动/静水压力而激活，并且被认为是 调节血管反应性和血管生成。我们最近报道了含有丰富亮氨酸重复序列的 蛋白 8a，LRRC8a (LRRC8A) 是形成 VRAC 的异六聚体复合物的必需成分。 人脐静脉 EC（HUVEC）。内皮 LRRC8A 调节 AKT-eNOS 和 mTOR 信号传导 基础条件，以及拉伸和剪切流刺激，并且是 EC 与层流剪切对齐所必需的 流动。内皮限制性 LRRC8A KO (LRRC8A KO) 小鼠内皮依赖性血管受损 放松，因慢性血管紧张素 II 输注而出现高血压，并表现出视网膜血液受损 在 2 型糖尿病 (T2D) 的情况下，血流会伴有弥漫性和局灶性血管狭窄。这些数据 证明 LRRC8a 调节内皮细胞中的 AKT-eNOS 和 mTOR 信号传导，并且是 维持血管功能。在 (a) 特定物质的分子特性方面仍然存在知识差距 在内皮细胞中形成 VRAC 的 LRRC8 异聚体，(b) 连接 内皮 LRRC8 复合物与 AKT-eNOS 和 mTOR 信号传导，(c) 小分子的治疗潜力 需要评估针对 LRRC8 复合物的分子，从而产生一类新型化合物 改善代谢综合征的血管功能和高血压。 我们有生化、膜片钳和成像证据表明 LRRC8 通道复合物得到表达并且 在溶酶体 (Lyso-LRRC8) 中发挥功能，并鉴定出关键的通道孔突变 (R103E) 特异性破坏 LRRC8 通道活动。鉴于溶酶体是整合营养物质的信号中心 传感和 AKT-mTOR 信号传导，我们假设 LRRC8A/C 通道共同调节质膜 内皮细胞中的 PI3K-AKT 信号传导和以溶酶体为中心的 mTOR 信号传导，以及该小分子 LRRC8 复合物调节剂可以恢复糖尿病相关的功能失调的内皮 LRRC8A/C 血管疾病和高血压。为了检验上述假设，我们提出了三个具体的目标 从分子信号传导机制到体内治疗概念验证，开发内皮 LRRC8 生物学： AIM#1：描述质膜与溶酶体 LRRC8 信号传导至 AKT 的机制 内皮细胞中的 mTOR 信号传导。 AIM#2：在体外、离体和体内检查 LRRC8 分子对 EC 功能的贡献 AIM#3：检验小分子 LRRC8 调节剂改善血管的治疗效果 糖尿病相关高血压模型的功能和血压