LRRC8 complex regulation of endothelial function

项目摘要 内皮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。内皮容量调节阴离子通道（VRAC）具有 被提议为机械敏感的，以响应于流体流动/流体静压和压力而激活 调节血管反应性和血管生成。我们最近报道了富含亮氨酸重复序列的 蛋白质8a，LRRC 8a（LRRC 8A）是异源六聚体复合物的必需组分，所述异源六聚体复合物形成VRAC， 人脐静脉内皮细胞（HUVEC）。内皮细胞LRRC 8A调节AKT-eNOS和mTOR信号转导 基本条件，并与拉伸和剪切流刺激，并要求EC对齐层流剪切 流内皮限制性LRRC 8A KO（LRRC 8A KO）小鼠具有受损的内皮依赖性血管内皮细胞。 松弛，对慢性血管紧张素II输注产生高血压，并表现出视网膜血液受损 在2型糖尿病（T2 D）背景下，弥漫性和局灶性血管狭窄的血流。这些数据 表明LRRC 8a调节内皮中的AKT-eNOS和mTOR信号传导，并且是 维持血管功能。在以下方面仍然存在知识差距：（a）特定的 （B）连接内皮细胞中VRAC的LRRC 8异聚体的分子机制， 内皮LRRC 8复合物对AKT-eNOS和mTOR信号传导的治疗潜力，（c）小的 需要对靶向LRRC 8复合物的分子进行评估，从而产生一类新的化合物 改善代谢综合征患者的血管功能和高血压。 我们有生化、膜片钳和成像证据表明LRRC 8通道复合物表达， 在溶酶体中起作用（Lyso-LRRC 8），并已鉴定出一个关键的通道孔突变（R103 E）， 特异性地破坏LRRC 8通道活性。考虑到溶酶体是整合营养物质的信号中枢， 我们假设LRRC 8A/C通道共调节质膜， PI 3 K-AKT信号和溶酶体中心的mTOR信号在内皮中，并且小分子 LRRC 8复合物调节剂可以恢复糖尿病相关性中功能失调的内皮LRRC 8A/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， 开发内皮LRRC 8生物学，从分子信号传导机制到体内治疗的概念验证： 目标#1：阐明质膜与溶酶体LRRC 8信号传导至AKT的机制。 内皮中的mTOR信号传导。 目标#2：检查LRRC 8分子对体外、离体和体内EC功能的贡献 目的#3：检查小分子LRRC 8调节剂改善血管内皮细胞的治疗功效 糖尿病相关高血压模型的功能和血压

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