Anion channel regulation of vascular superoxide signaling in hypertension

项目总结 高血压(HTN)是一种慢性炎症性疾病，是缺血性心脏的主要危险因素 疾病和中风是全球两大主要死亡原因。HTN与血管“氧化”有关 压力“，然而抗氧化剂疗法并未被证明有效。这可能是因为活性氧(ROS) 还通过血管紧张素II(AngII)和肿瘤坏死等分子参与正常的生理信号 因子α(肿瘤坏死因子α)。病理可能是由于过度激活、时空约束的丧失或 控制这些信号的反馈机制的失调。血管紧张素Ⅱ和肿瘤坏死因子α均激活NADPH 氧化酶1(Nox1)，产生细胞外超氧化物(O2-·)。通过一种未知的机制，这会生成一个 细胞内信号，并破坏这一信号可保护血管炎症和血管紧张素转换酶诱导的HTN。 我们先前发现Nox1在物理上与容量调节的阴离子通道(VRAC)相关 由富含亮氨酸重复序列的8个家族蛋白编码。LRRC8A与四个 相关异构体(LRRC8B-E)，以产生具有独特属性的通道。Nox1生产ROS需要 功能VRAC，可能用于变化补偿，以及由Nox1创建的氧化环境 监管VRAC。因此，VRAC和Nox1在功能上是相互依赖的。我们现在提供新的证据 O2-·也通过这些紧密联系的阴离子通道进入细胞。这可能允许对O2-·进行严格的监管。 传递到细胞质，提供氧化还原信号的空间控制，从而限制非靶标氧化。 缺乏LRRC8A的小鼠血管仅在血管平滑肌细胞(VSMCs)中显示正常 收缩但血管扩张增强，这些小鼠可免受血管紧张素Ⅱ诱导的HTN的影响。我们假设 LRRC8阴离子通道通过调节Nox1活性和O2-·进入VSMC控制细胞质 促进炎症和损害血管扩张的氧化还原信号通路。目标1将决定如何 LRRC8通道促进O2-·内流到VSMC，并决定其如何受局部氧化还原条件的调节。 我们将使用我们开发的两种新的O2-·通量分析结合膜片钳记录来实现 这些目标。Aim#2将确定LRRC8A通道和O2-·如何通过 两个细胞质靶标：1)RhoA，一种控制血管运动功能的小GTP酶；2)TRIM21，E3 泛素连接酶，我们通过质谱仪鉴定为LRRC8A的一个新的结合伙伴。TRIM21 调节依赖于NF-κB的炎症和依赖于NRF2的抗氧化反应。目标#3将 明确特定的LRRC8亚型在血管紧张素Ⅱ诱导的小鼠高血压中的作用。血压 记录、血管反应性和分子生物学研究将定义LRRC8通道亚型， 控制HTN中的Nox1和血管功能。相关性：炎症、氧化应激和 心血管疾病是明确的，但缺乏控制氧化剂依赖信号转导的方法。这个项目 将确定适用于HTN和血管炎症治疗的新治疗策略。

PROJECT SUMMARY Hypertension (HTN) is a chronic inflammatory disease and is a primary risk factor for ischemic heart disease and stroke, the two leading causes of death worldwide. HTN is associated with vascular “oxidative stress”, yet antioxidant therapy has not proven effective. This may be because reactive oxygen species (ROS) also participate in normal physiological signaling by molecules like Angiotensin II (AngII) and tumor necrosis factor α (TNFα). Pathology may result from excess activation, loss of spatiotemporal constraints, or dysregulation of the feedback mechanisms that control these signals. AngII and TNFα both activate NADPH Oxidase 1 (Nox1), producing extracellular superoxide (O2-•). By an unknown mechanism, this generates an intracellular signal, and disruption of this protects against vascular inflammation and AngII-induced HTN. We previously found that Nox1 physically associates with Volume-Regulated Anion Channels (VRACs) that are encoded by Leucine-Rich Repeat-Containing 8 family proteins. LRRC8A associates with one of four related isoforms (LRRC8B-E) to produce channels with unique properties. ROS production by Nox1 requires functional VRACs, potentially for change compensation, and the oxidized environment created by Nox1 regulates VRACs. Thus, VRACs and Nox1 are functionally interdependent. We now provide new evidence that O2-• also enters cells via these closely associated anion channels. This may allow tight regulation of O2-• delivery to the cytoplasm, providing spatial control of redox signaling which limits off-target oxidation. Blood vessels from mice lacking LRRC8A only in vascular smooth muscle cells (VSMCs) exhibit normal contractility but enhanced vasodilation and these mice are protected from AngII-induced HTN. We hypothesize that by regulating Nox1 activity and O2-• entry into VSMCs, LRRC8 anion channels control cytoplasmic redox signaling pathways that promote inflammation and impair vasodilation. Aim #1 will determine how LRRC8 channels facilitate O2-• influx into VSMCs and determine how this is regulated by local redox conditions. We will use two novel O2-• flux assays that we have developed combined with patch-clamp recording to achieve these goals. Aim #2 will determine how LRRC8A channels and O2-• modulate inflammation and contractility via two cytoplasmic targets: 1) RhoA, a small GTPase that controls vasomotor function, and 2) TRIM21, an E3 ubiquitin ligase that we identified by mass spectrometry as a novel binding partner of LRRC8A. TRIM21 modulates both NF-κB-dependent inflammation and the Nrf2-dependent antioxidant response. Aim #3 will define the contribution of specific LRRC8 isoforms to AngII-induced hypertension in mice. Blood pressure recording, vascular reactivity and molecular biologic studies will define the LRRC8 channel subtype that controls Nox1 and vascular function in HTN . Relevance: Links between inflammation, oxidative stress and cardiovascular disease are clear, but methods to control oxidant-dependent signaling are lacking. This project will identify novel therapeutic strategies that are applicable to the treatment of HTN and vascular inflammation.