Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-ß1: Induction of Cystathionine γ-Lyase and Cystathionine ß-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways.

Transforming growth factor-ß1 (TGF-ß1) 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-ß1. Bovine aortic endothelial cells in a culture system were treated with TGF-ß1 to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-ß1, induction of RSS-producing enzymes by TGF-ß1, and intracellular signal pathways that mediate this induction. The results suggest that TGF-ß1 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 ß-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-ß1 regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-ß1, may modulate the regulation activity in vascular endothelial cells.

Transcriptional Induction of Cystathionine γ-Lyase, a Reactive Sulfur-Producing Enzyme, by Copper Diethyldithiocarbamate in Cultured Vascular Endothelial Cells.

As toxic substances can enter the circulating blood and cross endothelial monolayers to reach parenchymal cells in organs, vascular endothelial cells are an important target compartment for such substances. Reactive sulfur species protect cells against oxidative stress and toxic substances, including heavy metals. Reactive sulfur species are produced by enzymes, such as cystathionine γ-lyase (CSE), cystathionine ß-synthase, 3-mercaptopyruvate sulfurtransferase, and cysteinyl-tRNA synthetase. However, little is known about the regulatory mechanisms underlying the expression of these enzymes in vascular endothelial cells. Bio-organometallics is a research field that analyzes biological systems using organic-inorganic hybrid molecules (organometallic compounds and metal coordinating compounds) as molecular probes. In the present study, we analyzed intracellular signaling pathways that mediate the expression of reactive sulfur species-producing enzymes in cultured bovine aortic endothelial cells, using copper diethyldithiocarbamate (Cu10). Cu10 selectively upregulated CSE gene expression in vascular endothelial cells independent of cell density. This transcriptional induction of endothelial CSE required both the diethyldithiocarbamate scaffold and the coordinated copper ion. Additionally, the present study revealed that ERK1/2, p38 MAPK, and hypoxia-inducible factor (HIF)-1α/HIF-1ß pathways mediate transcriptional induction of endothelial CSE by Cu10. The transcription factors NF-κB, Sp1, and ATF4 were suggested to act in constitutive CSE expression, although the possibility that they are involved in the CSE induction by Cu10 cannot be excluded. The present study used a copper complex as a molecular probe to reveal that the transcription of CSE is regulated by multiple pathways in vascular endothelial cells, including ERK1/2, p38 MAPK, and HIF-1α/HIF-1ß. Bio-organometallics appears to be an effective strategy for analyzing the functions of intracellular signaling pathways in vascular endothelial cells.

Sodium trisulfide, a sulfane sulfur donor, stimulates bovine aortic endothelial cell proliferation in culture.

Reactive sulfur species (RSS) include biological persulfide molecules that protect cells against oxidative stress and heavy metal toxicity. Vascular endothelial cells regulate blood coagulation and fibrinolytic activity, and prevent vascular disorders such as atherosclerosis. We hypothesized that RSS protect vascular endothelial cells not only from nonspecific cell damage but also from specific functional damage through regulation of specific cell functions. In the present study, cultured bovine aortic endothelial cells were treated with sodium trisulfide, a sulfane sulfur donor, and both [3H]thymidine incorporation and effects on cell cycle were analyzed. These results suggest that RSS stimulate vascular endothelial cell proliferation. RSS may reduce the functional cytotoxicity of antiproliferative agents.