CFTR limits F-actin formation and promotes morphological alignment with flow in human lung microvascular endothelial cells.

Adam J Causer,Zoe L Saynor,Janis K Shute,Stephen J Bailey,Emily Klein Rot,Maha Khalaf,Anthony I Shepherd,Kimberly Brand,James Smith,Michael H Cummings

doi:10.14814/phy2.15128

Abstract

Micro‐ and macrovascular endothelial dysfunction in response to shear stress has been observed in cystic fibrosis (CF), and has been associated with inflammation and oxidative stress. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) regulates endothelial actin cytoskeleton dynamics and cellular alignment in response to flow. Human lung microvascular endothelial cells (HLMVEC) were cultured with either the CFTR inhibitor GlyH‐101 (20 µM) or CFTRinh‐172 (20 µM), tumor necrosis factor (TNF)‐α (10 ng/ml) or a vehicle control (0.1% dimethyl sulfoxide) during 24 and 48 h of exposure to shear stress (11.1 dynes/cm2) or under static control conditions. Cellular morphology and filamentous actin (F‐actin) were assessed using immunocytochemistry. [Nitrite] and endothelin‐1 ([ET‐1]) were determined in cell culture supernatant by ozone‐based chemiluminescence and ELISA, respectively. Treatment of HLMVECs with both CFTR inhibitors prevented alignment of HLMVEC in the direction of flow after 24 and 48 h of shear stress, compared to vehicle control (both p < 0.05). Treatment with TNF‐α significantly increased total F‐actin after 24 h versus control (p < 0.05), an effect that was independent of shear stress. GlyH‐101 significantly increased F‐actin after 24 h of shear stress versus control (p < 0.05), with a significant (p < 0.05) reduction in cortical F‐actin under both static and flow conditions. Shear stress decreased [ET‐1] after 24 h (p < 0.05) and increased [nitrite] after 48 h (p < 0.05), but neither [nitrite] nor [ET‐1] was affected by GlyH‐101 (p > 0.05). CFTR appears to limit cytosolic actin polymerization, while maintaining a cortical rim actin distribution that is important for maintaining barrier integrity and promoting alignment with flow, without effects on endothelial nitrite or ET‐1 production.

Highlights

Cystic fibrosis (CF) is a multi-organ, life-shortening autosomal recessive disease that affects 90–100,000 people worldwide (Bell et al, 2020)
Expression of CFTR has been reported in human lung microvascular endothelial cells (HLMVEC; Khalaf et al, 2020; Tousson et al, 1998), human umbilical vein endothelial cells (HUVEC; Declercq et al, 2021; Tousson et al, 1998) and human pulmonary artery endothelial cells (HPAEC; Plebani et al, 2017; Totani et al, 2017)
This is the first study to investigate the role of functional CFTR in the regulation of the actin cytoskeleton and cellular alignment of endothelial cells in response to flow

Summary

Introduction

Cystic fibrosis (CF) is a multi-organ, life-shortening autosomal recessive disease that affects 90–100,000 people worldwide (Bell et al, 2020). In CF, defective flow- mediated dilation (FMD) of the brachial artery suggests NO bioavailability is reduced in response to shear stress (Poore et al, 2013), and improved by agents that increase NO availability (Rodriguez-Miguelez et al, 2018). This notion is further supported by the observation that acute supplementation with 20 mg/kg of oral tetrahydrobiopterin (an essential cofactor for eNOS activity) significantly improved FMD, through reduced superoxide production and increased NO, which is indicative of improved eNOS coupling in people with CF (Jeong et al, 2018)

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