Knockout of LRRC8A Anion Channels in Vascular Smooth Muscle Cells Impairs NF-κB and Hif1α-mediated Signaling

Abstract

Vascular smooth muscle cell (VSMC) inflammation associated with the development of vascular disease, including hypertension. We have demonstrated that knockout (KO) or inhibition of LRRC8A anion channels in VSMCs attenuates reactive oxygen species (ROS) production by NADPH oxidase 1, impairs TNFα-induced inflammation, and promotes vascular relaxation in murine mesenteric arteries. To explore the mechanism of these effects we performed RNA sequencing (RNAseq) on LRRC8A wild-type (WT) and knockout (KO) VSMCs (N = 3 pairs) under control conditions and 6hrs following TNFα treatment (10ng/ml). The data were normalized by Median ratio (DESeq2) and differential expression was determined by DESeq2 method using Partek software and pathway enrichment analysis was done by KEGG pathway. Differential expression of genes of interest was confirmed by quantitative PCR (qPCR) in both cultured cells and in whole aortic tissues (endothelium-denuded, N = 3) from LRRC8A WT and KO mice. HIF1α transcriptional activity was quantified by luciferase reporter assays in cells exposed to hypoxia (1% oxygen) or cobalt chloride (300μM) for 6 hrs. RNAseq demonstrated broad impairment of pro-inflammatory signaling in KO cells. This was consistent with previous observations of impaired nuclear factor kappa B (NF-κB) reporter activation by TNFα in the absence of LRRC8A. Compared to WT VSMCs, resting LRRC8A KO VSMCs exhibited statistically significant downregulation of 33 NF-κB target genes including the IL-6 (Il6) and IL-1β (Il1r1) receptors. TNFα treatment increased the expression of 37 NF-κB target genes in WT cells. Most of these genes (34 genes) were not significantly activated by TNFα in KO cells, while 3 genes (chemokine (C-C motif) ligand 20; CCL20, prostaglandin E synthase; PTGES and vascular endothelial growth factor C; VEGFC) were significantly downregulated. Pathway analysis data revealed that Hif1α signaling appear in the top downregulated pathway and it was impaired as 14 Hif1α target genes were downregulated in unstimulated KO VSMCs. In addition, expression of Rps4i, a long non-coding mRNA associated with inhibition of HIF1α signaling was significantly upregulated. Luciferase reporter assays following exposure to hypoxia or cobalt chloride confirmed impaired Hif1α activation in LRRC8A KO cells. Important target genes for HIF1α, including HK2, LDHB, ACE, BMP4 and PFKP, were less activated, while RPS4i remained upregulated compared to WT cells. These findings demonstrate disruption of both NF-κB and HIF1α signaling in VSMCs lacking LRRC8A. This anion channel represents a novel target for anti-inflammatory therapy in vascular disease. This work was supported by DK132948 and HL160975. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.