Recombinant Human Soluble Thrombomodulin Suppresses Monocyte Adhesion by Reducing Lipopolysaccharide-Induced Endothelial Cellular Stiffening.

Takayuki Okamoto,Koji Suzuki,Haruki Usuda,Tetsuro Nikai,Kunihiro Asanuma,Tetsuya Tanaka,Koichiro Wada,Eiji Kawamoto,Motomu Shimaoka

doi:10.3390/cells9081811

Abstract

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells regulate leukocyte adhesion in a substrate- and cellular stiffness-dependent manner. Our result show that LPS-induced cellular stiffening enhances monocytic THP-1 cell line adhesion, whereas rsTM suppresses THP-1 cell adhesion to inflamed endothelial cells by reducing cellular stiffness. Endothelial cells increase cellular stiffness in reaction to inflammation, thereby promoting monocyte adhesion. Treatment of rsTM reduced LPS-induced cellular stiffening and suppressed monocyte adhesion in a cellular stiffness-dependent manner.

Highlights

Endothelial cells are a crucial player in the regulation of inflammation, blood coagulation, leukocyte adhesion, and vascular remodeling in inflamed vessels
We found that LPS-induced endothelial cellular stiffening facilitates monocyte adhesion
We confirmed that recombinant soluble TM (rsTM) attenuates the cellular stiffening of human umbilical vein endothelial cells (HUVECs) from different donors and of endothelial cells from arteries upon LPS stimulation, suggesting that the alteration in cellular stiffness might be a common character of endothelial cells

Summary

Introduction

Endothelial cells are a crucial player in the regulation of inflammation, blood coagulation, leukocyte adhesion, and vascular remodeling in inflamed vessels. Thrombomodulin (TM) is an anticoagulant protein that is predominantly expressed on the surface of endothelial cells [1,2,3]. TM directly binds to thrombin and inhibits thrombin procoagulant activity [1,2]. The thrombin-TM complex alters thrombin substrate specificity and accelerates anticoagulant activated protein C generation [1,2]. Recent preclinical studies have documented the favorable effects of recombinant soluble TM (rsTM) in experimental sepsis, including vascular inflammation and blood coagulation state [4,5,6,7]. In addition to its anticoagulant properties, it is thought that rsTM has anti-inflammatory effects. The C-type lectin-like domain of TM inhibits an alamin high-mobility group box

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