Abstract We0008: Endothelial LRRC8C associates with LRRC8A and LRRC8B to regulate vascular reactivity and blood pressure

Vascular tone is impacted by the endothelium's ability to detect both mechanical and chemical stimulation. The Leucine-Rich Repeat-Containing protein 8a, (LRRC8A), was previously identified as a required component of the mechanoresponsive endothelial LRRC8 complex regulating AKT-endothelial nitric oxide synthase (eNOS) signaling and vascular function. While LRRC8A is broadly expressed, LRRC8B, C, D and E have tissue-restricted expression. Here, we identified 2 single nucleotide polymorphisms (SNPs) in Lrrc8c that are highly associated with diastolic and systolic blood pressure in human Genome-Wide Association Studies (GWAS) and Phenome Wide Association Studies (PheWAS), implicating LRRC8C as a required LRRC8 heteromer that regulates endothelial function. While LRRC8A/B/C/D are expressed in endothelium, co-immunoprecipitation experiments from lung endothelium using LRRC8A-3xFlag knock-in mice and endothelium-specific LRRC8A-3xFlag overexpression mice revealed the endothelial LRRC8 complex to be composed largely of LRRC8A/B/C heteromers. LRRC8A/B/C knock-out studies in mice and knock-down studies in human umbilical vein endothelial cells (HUVECs) show co-dependent expression of LRRC8A/B/C proteins, but not LRRC8D. Functionally, LRRC8A and LRRC8C depletion reduces endothelial LRRC8 currents, reduces AKT-eNOS signaling, increases myogenic tone, impairs eNOS dependent vasodilation, and exacerbates angiotensin-induced hypertension. These data identify LRRC8A, LRRC8B and LRRC8C as components of the endothelial LRRC8 complex and reveal LRRC8C as having a non-redundant role in regulating endothelial AKT-eNOS, vascular relaxation and susceptibility to hypertension.