The Increased Endogenous Sulfur Dioxide Acts as a Compensatory Mechanism for the Downregulated Endogenous Hydrogen Sulfide Pathway in the Endothelial Cell Inflammation

Abstract

Endogenous hydrogen sulfide (H₂S) and sulfur dioxide (SO₂) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H₂S on the SO₂ generation in the endothelial cells and explore its significance in the endothelial inflammation <i>in vitro</i> and <i>in vivo</i>. The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for <i>in vitro</i> experiments. A rat model of monocrotaline (MCT)-induced pulmonary vascular inflammation was used for <i>in vivo</i> experiments. We found that endogenous H₂S deficiency caused by cystathionine-γ-lyase (CSE) knockdown increased endogenous SO₂ level in endothelial cells and enhanced the enzymatic activity of AAT, a major SO₂ synthesis enzyme, without affecting the expressions of AAT1 and AAT2. While H₂S donor could reverse the CSE knockdown-induced increase in the endogenous SO₂ level and AAT activity. Moreover, H₂S donor directly inhibited the activity of purified AAT protein, which was reversed by a thiol reductant DTT. Mechanistically, H₂S donor sulfhydrated the purified AAT1/2 protein and rescued the decrease in the sulfhydration of AAT1/2 protein in the CSE knockdown endothelial cells. Furthermore, an AAT inhibitor l-aspartate-β-hydroxamate (HDX), which blocked the upregulation of endogenous SO₂/AAT generation induced by CSE knockdown, aggravated CSE knockdown-activated nuclear factor-κB pathway in the endothelial cells and its downstream inflammatory factors including ICAM-1, TNF-α, and IL-6. In <i>in vivo</i> experiment, H₂S donor restored the deficiency of endogenous H₂S production induced by MCT, and reversed the upregulation of endogenous SO₂/AAT pathway <i>via</i> sulfhydrating AAT1 and AAT2. In accordance with the results of the <i>in vitro</i> experiment, HDX exacerbated the pulmonary vascular inflammation induced by the broken endogenous H₂S production in MCT-treated rat. In conclusion, for the first time, the present study showed that H₂S inhibited endogenous SO₂ generation by inactivating AAT <i>via</i> the sulfhydration of AAT1/2; and the increased endogenous SO₂ generation might play a compensatory role when H₂S/CSE pathway was downregulated, thereby exerting protective effects in endothelial inflammatory responses <i>in vitro</i> and <i>in vivo</i>.