LRRC8 complex regulation of endothelial function

项目摘要 血管内皮细胞对调节血管张力的多种化学和机械因素做出反应， 血管生成、血压和血流量。血管内皮细胞容量调节阴离子通道(Vrac)具有 被提议是机械敏感的，响应流体流动/流体静压而激活，并被认为是 调节血管反应性和血管生成。我们最近报道，富含亮氨酸的重复序列含有 蛋白质8a，LRRC8a(LRRC8A)是形成vrac的异六聚体复合体的必需成分 人脐静脉内皮细胞(HUVEC)。内皮细胞LRRC8A调节AKT-eNOS和mTOR信号转导 基本条件，并具有拉伸和剪切流动刺激，是EC对准层流剪切所必需的 流。内皮限制性LRRC8A KO(LRRC8A KO)小鼠血管内皮依赖性受损 放松，因慢性血管紧张素II注射而发展为高血压，并表现出视网膜血液受损 2型糖尿病(T2D)患者血流弥漫性和局灶性血管狭窄。这些数据 证明LRRC8a调节血管内皮细胞内AKT-eNOS和mTOR信号，并且是 维持血管功能。在(A)特定的分子同一性方面仍然存在知识差距 在内皮细胞中形成vrac的LRRC8异构体，(B)连接血管内皮的分子机制 内皮细胞LRRC8复合体对AKT-eNOS和mTOR信号转导，(C)Small的治疗潜力 靶向LRRC8复合体的分子需要评估，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LRRC8通道复合体表达并 在溶酶体中具有功能(lyso-LRRC8)，并发现了一个关键的通道孔突变(R103E)，该突变 具体扰乱LRRC8通道活动。鉴于溶酶体是整合营养物质的信号中枢 传感和AKT-mTOR信号，我们假设LRRC8A/C通道共同调节质膜 PI3K-AKT信号和溶酶体介导的内皮细胞mTOR信号，以及该小分子 LRRC8复合调节剂对糖尿病相关血管内皮细胞功能紊乱的修复作用 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目标 发展内皮细胞LRRC8生物学从分子信号机制到体内治疗的概念验证： 目的#1：阐明质膜与溶酶体LRRC8信号转导AKT-1的机制 内皮细胞中的mTOR信号转导。 目的#2：检测LRRC8分子在体外、体外和体内对EC功能的贡献 目的#3：检测小分子LRRC8调节剂改善血管的疗效 糖尿病合并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