Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-β1: Induction of Cystathionine γ-Lyase and Cystathionine β-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways.

Tsuyoshi Nakano,Yasushi Hara,Yasuhiro Shinkai,Musubu Takahashi,Takato Hara,Toshiyuki Kaji,Chika Yamamoto,Ryoko Takasawa,Tomoya Fujie,Yoshito Kumagai

doi:10.3390/ijms222111762

Abstract

Transforming growth factor-β1 (TGF-β1) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-β1. Bovine aortic endothelial cells in a culture system were treated with TGF-β1 to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β1, induction of RSS-producing enzymes by TGF-β1, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β1 increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine β-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-β1 regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-β1, may modulate the regulation activity in vascular endothelial cells.

Highlights

Vascular endothelial cells cover the vascular lumen in a monolayer and function as a barrier between the blood and the subendothelial matrix
When total sulfane sulfur levels in vascular endothelial cells treated with TGFβ1 were detected using SSP4, intracellular sulfane sulfur levels were increased by transforming growth factor-β1 (TGF-β1) in a concentration-dependent manner after
Low-molecular-mass Reactive sulfur species (RSS) and the corresponding substrates were quantified in vascular endothelial cells treated with TGF-β1 (Figure 1b); cysteine persulfide was not detected

Summary

Introduction

Vascular endothelial cells cover the vascular lumen in a monolayer and function as a barrier between the blood and the subendothelial matrix. Repeated and severe injury of endothelial monolayer initiates the blood coagulation process. Damage to the endothelial monolayer is a causative factor of vascular lesions such as atherosclerosis [6] due to the activated procoagulant state of the blood. As vascular endothelial cells are in direct contact with the blood, their monolayers can be repeatedly or severely injured by toxic environmental and nutritional substances. In the atherosclerotic vascular wall, TGF-β1 is released from vascular smooth muscle cells, macrophages, and T lymphocytes, and as a result, TGF-β1 is present at high levels at damage sites of vascular endothelial cell layers [6]

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