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 a (TNFa). Pathology may result from excess activation, loss of spatiotemporal constraints, or
dysregulation of the feedback mechanisms that control these signals. AngII and TNFa 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-¿B-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.