Anion channel regulation of vascular superoxide signaling in hypertension

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.