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

Abstract

Background: Leucine Rich Repeat Containing 8A (LRRC8A) anion channels (VRACs) associate with NADPH oxidase 1 (Nox1) and support extracellular superoxide (O2*-) production, inflammation, and contractility in vascular smooth muscle cells (VSMCs). We proposed previously that VRACs also support influx of O2*- to produce localized cytoplasmic signaling. Methods: We assayed O2*- influx, and guided by changes in mRNA expression (RNAseq) 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. Results: KO cells were less permeable to extracellular O2*-, produced less mitochondrial O2*-, 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, suggestive of lower energy demand. Consistent with this, proliferation and migration were both reduced in KO cells. Protein-protein interaction analysis of RNAseq (PPI Hub) identified the Epidermal Growth Factor Receptor (EGFR) signaling pathway. Activating phosphorylation of EGFR (Y1068) as well as downstream AKT (S473 and T308) were reduced. Following 15 weeks of exposure to a high fat diet (42%) VSMC-specific LRRC8A-/- and 8A+/-, ApoE-/- mice had less atherosclerotic lesion area, and reduced aortic senescence ({beta}-Gal), inflammation (ICAM, VCAM) and proliferation marker (PCNA) expression. Mesenteric artery vasomotor function was also preserved compared to WT, ApoE-/- controls, and the abundance of MYPT1 and CPI17 was lower in KO vessels. Uptake of oxidized LDL (OxLDL) was significantly reduced in KO VSMCs and similarly impaired by VRAC block in WT cells. Conclusions: Loss of LRRC8A reduces O2*- influx, oxidative stress, inflammation and senescence, lowers energy demand, and in the setting of hypercholesterolemia impairs uptake of oxidized LDL and abrogates atherosclerosis. VRAC inhibitors warrant further investigation as therapeutic vascular anti-inflammatory agents.