Smooth muscle LRRC8A knockout reduces superoxide influx, inflammation, senescence and atherosclerosis

BackgroundLeucine Rich Repeat Containing 8A (LRRC8A) anion channels (VRACs) associate with NADPH oxidase 1 (Nox1) and support extracellular superoxide (O2{middle dot}-) production, inflammation, and contractility in vascular smooth muscle cells (VSMCs). We proposed previously that VRACs also support influx of O2{middle dot}- to localize cytoplasmic redox signals. MethodsWe assayed O2{middle dot}- influx, assessed changes in mRNA expression (RNAseq), and tested multiple phenotypes of cultured LRRC8A knockout (KO) VSMCs. Aortic atherosclerotic burden and inflammation, and mesenteric vascular reactivity were compared between wild type (WT) apolipoprotein E null (ApoE-/-) mice and VSMC-specific LRRC8A KO, ApoE-/- mice. ResultsKO cells were less permeable to extracellular O2{middle dot}-, produced less mitochondrial O2{middle dot}-, and experienced less oxidant stress (GSH/GSSG, lipid peroxidation, Nrf2 activity) than WT. RNAseq and reporter assays demonstrated reduced pro-inflammatory (NF-{kappa}B and Hif1) transcription. KO cells also under-expressed multiple senescence markers and had longer telomeres. Inflammation causes a metabolic shift from oxidative phosphorylation (OCR) to glycolysis (ECAR). Both pathways were less active in KO cells, as was expression of glycolytic enzymes. Mitochondrial membrane potential was lower in KO cells, but ADP/ATP and NADP+/NADPH were unaltered, suggesting lower energy demand. Consistent with this, proliferation and migration were both reduced in KO cells. Protein-protein interaction analysis of RNAseq data (PPI Hub) identified Epidermal Growth Factor Receptor (EGFR) signaling. Resting phosphorylation of EGFR (pY1068) and AKT (pS473 and pT308) were all reduced in KO cells, as was and EGF-induced pY1068 and pS308. Following 15 weeks of exposure to a high fat diet (42%) VSMC-specific LRRC8A-/- and 8A+/-, ApoE-/- mice had reduced atherosclerotic lesion area, aortic senescence ({beta}-Gal), inflammation (ICAM, VCAM) and proliferation marker (PCNA) expression compared to WT, ApoE-/- controls. Mesenteric artery vasomotor function was also preserved, and the abundance of MYPT1 and CPI17 was lower in KO vessels. Uptake of oxidized LDL (OxLDL) was significantly reduced in KO VSMCs and impaired by VRAC block in WT cells. ConclusionsLoss of LRRC8A reduced O2{middle dot}- influx, oxidative stress, inflammation and senescence, lowers energy demand, and in the setting of hypercholesterolemia impaired uptake of oxidized LDL and abrogated atherosclerosis. VSMC VRACs may be novel targets of vascular anti-inflammatory therapy. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIChronically inflamed vascular smooth muscle cells (VSMCs) are critical drivers of atherosclerotic plaque development. C_LIO_LIIn VSMCs, Nox1-derived superoxide drives redox signaling that promotes inflammation, phenotypic switching, and senescence. C_LIO_LILRRC8A family volume-regulated anion channels (VRACs) physically associate with Nox1 and support TNF-induced ROS production, receptor endocytosis, NF-{kappa}B activation, and proliferation. C_LIO_LIHypercholesterolemia causes VSMC inflammation via oxidized LDL (OxLDL) activation of scavenger receptors (e.g., LOX-1, CD36) that stimulate oxidant production and EGFR/PI3K-Akt-NF-{kappa}B signaling. C_LI What New Information Does This Article Contribute?O_LILRRC8A channels support influx of extracellular superoxide which impacts cytosolic redox status, metabolism and Nrf2, HIF-1, and NF-{kappa}B activity. C_LIO_LILoss of LRRC8A reprograms VSMCs into a low-oxidative-stress, low-energy-demand state, characterized by diminished proliferation and migration, attenuated senescence, and preservation of telomere length. C_LIO_LILoss of LRRC8A inhibits EGF-stimulated EGFR and Akt phosphorylation and uncouples oxidant-dependent growth factor signaling from downstream inflammatory and metabolic remodeling. C_LIO_LIVSMC-specific LRRC8A knockout or heterozygosity in hypercholesterolemic ApoE-/- mice reduces aortic inflammation and senescence, reduces atherosclerotic lesion burden, and preserves mesenteric artery vasomotor function. C_LIO_LILRRC8A/VRAC activity is necessary for uptake of oxidized but not native LDL in VSMCs. C_LIO_LILRRC8A is a novel regulator of scavenger receptor-dependent lipoprotein handling and a potential therapeutic target to uncouple oxidant signaling from VSMC lipid overload and inflammation in the vessel wall. C_LI