Angiotensin II downregulates vascular endothelial cell hydrogen sulfide production by enhancing cystathionine γ-lyase degradation through ROS-activated ubiquitination pathway

Highlights

• AngII downregulates endogenous EC H₂S/CSE pathway.

• ROS mediates AngII-induced CSE degradation via K48-linked ployubiquitination.

Abstract

The interactions between vasoactive peptides and gasotransmitters have attracted considerable attention from scientists. However, the impact of angiotensin II (AngII) on the endogenous hydrogen sulfide/cystathionine γ-lyase (H₂S/CSE) pathway in vascular endothelial cells remains unclear. In this study, we found, for the first time, that AngII downregulated the endogenous H₂S/CSE pathway in a time-dependent manner. Mechanistically, AngII accelerated the degradation of the CSE protein and shortened its half-life in endothelial cells. AngII significantly induced Lys48 (K48)-linked CSE ubiquitination and subsequent CSE degradation but did not affect Lys63 (K63)-linked CSE ubiquitination in vascular endothelial cells. Treatment with the proteasome inhibitor MG132 and mutation of Lys48 to Arg in ubiquitin successfully blunted the inhibitory effects of AngII on the endogenous H₂S/CSE pathway in vascular endothelial cells. Furthermore, we found that superoxide anion levels were significantly increased in AngII-treated endothelial cells compared with controls and that the ROS scavenger N-acetyl-l-cysteine (NAC) significantly abolished CSE ubiquitination. Taken together, our data suggested that AngII inhibited endogenous H₂S generation through ubiquitination-mediated CSE degradation via the ROS pathway in vascular endothelial cells.

Introduction

Vascular endothelial cells that form the continuous cellular linings of blood vessels are important elements of critical regulatory nodes in the cardiovascular homeostatic network. Normal vascular endothelial function depends on the balance between vascular injury factors and vascular protective factors [1]. Endothelial dysfunction is regarded as an initial step in the development of hypertension, pulmonary hypertension and aneurysms [[2], [3], [4]].

Angiotensin II (AngII) is a critical factor responsible for vascular endothelial injury. A previous study showed that AngII caused vascular endothelial dysfunction by upregulating the NAD(P)H oxidase 2 expression [5]. In addition, AngII activated endoplasmic reticulum stress in vascular endothelial cells, thereby promoting endothelial dysfunction [6]. However, the molecular mechanisms by which AngII injures the vascular endothelium have not yet been fully elucidated.

Endogenous hydrogen sulfide (H₂S) is recognized as a gasotransmitter in the cardiovascular system [7]. It is produced endogenously through the catalytic reactions by cystathionine γ-lyase (CSE) in the mammalian sulfur-containing amino acid metabolic pathway in cardiovascular system [8] and plays an important protective role in the regulation of vascular endothelial cell functions [9,10]. H₂S has been found to protect against endothelial injury by inhibiting endoplasmic reticulum stress [6], activating NFE2-related factor 2 [11], and decreasing the inflammatory response [12] in vascular endothelial cells. In addition, deficiency of the endogenous H₂S/CSE pathway has been found to be associated with vascular injury in animal models of pulmonary hypertension, arterial hypertension and atherosclerosis [[13], [14], [15]].

In the present study, we aimed to explore the effect of AngII on the endogenous H₂S/CSE pathway in the vascular endothelial cell line (EA.hy926). Furthermore, we demonstrated the posttranslational regulatory mechanism by which Ang II inhibited endogenous H₂S production by targeting on CSE degradation.