The SWELL1-LRRC8 complex regulates endothelial AKT-eNOS signaling and vascular function.

Ahmad F Alghanem,Urooj Fatima,Oluwaseun Adeola,Ashutosh Kumar,Macaulay Elliot-Hudson,Javier Abello,Litao Xie,Chau My Ta,Rachel A Minerath,Rajan Sah,Susheel K Gunasekar,Robert F Mullins,Amber N Stratman,Chad E Grueter,Chen Kang,Megan Riker,Joshua M Maurer

doi:10.7554/elife.61313

Abstract

The endothelium responds to numerous chemical and mechanical factors in regulating vascular tone, blood pressure, and blood flow. The endothelial volume-regulated anion channel (VRAC) has been proposed to be mechanosensitive and thereby sense fluid flow and hydrostatic pressure to regulate vascular function. Here, we show that the leucine-rich repeat-containing protein 8a, LRRC8A (SWELL1), is required for VRAC in human umbilical vein endothelial cells (HUVECs). Endothelial LRRC8A regulates AKT-endothelial nitric oxide synthase (eNOS) signaling under basal, stretch, and shear-flow stimulation, forms a GRB2-Cav1-eNOS signaling complex, and is required for endothelial cell alignment to laminar shear flow. Endothelium-restricted Lrrc8a KO mice develop hypertension in response to chronic angiotensin-II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of type 2 diabetes (T2D). These data demonstrate that LRRC8A regulates AKT-eNOS in endothelium and is required for maintaining vascular function, particularly in the setting of T2D.

Highlights

46 47 The endothelium integrates mechanical and chemical stimuli to regulate vascular tone, angiogenesis, blood flow and blood pressure[1]
LRRC8A and eNOS co-localized in plasma membrane and peri-nuclear intracellular domains (Figure 3C, inset), consistent with the IP data revealing a LRRC8A-GRB2-Cav-eNOS interaction (Figure 3A&B), and with previously described intracellular eNOS localization[48]. 157 Given that endothelial cells respond to stretch stimuli to regulate vascular tone via activation of eNOS[49], we examined the LRRC8A-dependence of stretch-induced AKT, ERK1/2 and eNOS signaling in human umbilical vein endothelial cells (HUVECs) (Figure 4), similar to several previous studies[50,51,52,53]
276 277 Our findings demonstrate that the LRRC8 heterohexamer functionally encodes endothelial 278 volume regulatory anion channel (VRAC), whereby LRRC8A associates with GRB2 and Cav1 and positively regulates PI3K-AKT279 eNOS and ERK1/2 signaling

Summary

Introduction

46 47 The endothelium integrates mechanical and chemical stimuli to regulate vascular tone, angiogenesis, blood flow and blood pressure[1]. Endothelial cells express a variety of mechanosensitive and mechanoresponsive ion channels that regulate vascular function[2], including TRPV4(3-6) and Piezo1(7-9). The volume-regulated anion current (VRAC) is prominent in endothelium, has been proposed to be mechano-responsive[10], to activate in response to fluid flow/hydrostatic pressure[11] and putatively regulate vascular reactivity. For ~11 years, LRRC8A was conceived of as a membrane protein that regulates PI3K-AKT mediated lymphocyte function[12, 13]. Historically, LRRC8A was first described as a membrane protein that signaled via protein-protein interactions and later found to form an ion channel signaling complex. Historically, LRRC8A was first described as a membrane protein that signaled via protein-protein interactions and later found to form an ion channel signaling complex. 69

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Results

Conclusion