Angiotensin II downregulates vascular endothelial cell hydrogen sulfide production by enhancing cystathionine γ-lyase degradation through ROS-activated ubiquitination pathway
Graphical abstract
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.
Section snippets
Cell culture
The human umbilical vein endothelial cell (HUVEC) line (EA.hy926) was obtained from the China Infrastructure of Cell Line Resources Center, China. To prepare complete culture medium, DMEM-F12 (Gibco, USA) was supplemented with 2 mM l-glutamine (Gibco, USA), 1% streptomycin and penicillin (Gibco, USA) and 10% fetal bovine serum (Gibco, USA). The culture medium for cell synchronization contained DMEM-F12, 2 mM l-glutamine and 1% streptomycin and penicillin. The endothelial cells were cultured in
AngII downregulated the endogenous H2S/CSE pathway in vascular endothelial cells
The results of the in situ fluorescent probe experiment showed that the H2S content in vascular endothelial cells was significantly decrease in the AngII group compared with the control group (P < 0.05, Fig. 1A). Furthermore, vascular endothelial cells were stimulated by AngII for different durations. As presented in Fig. 1B, the H2S content in the endothelial cell culture supernatant was significantly decreased by 3.7%, 21% and 43.3% (P < 0.05) after AngII stimulation for 6 h, 12 h and 24 h,
Discussion
In the present study, we discovered, for the first time, that AngII downregulated the H2S/CSE pathway. CSE is regarded as the key enzyme that catalyzes endogenous H2S production in the cardiovascular system [19]. Notably, treating mesenteric artery endothelial cells with nuclear factor of activated T cells (NFAT) inhibits CSE transcription after exposure to intermittent hypoxia [20]. In cardiomyocytes, specificity protein-1 upregulates CSE expression by downregulating miR-22 [21]. These data
Authors’ contributions
Junbao Du, Hongfang Jin, Yongfen Qi, Jiadong Wang and Chaoshu Tang designed the research. Lu Bai and Yaqian Huang performed the experiments. Lu Bai, Hongfang Jin, Selena Chen and Yaqian Huang analyzed the data. Lu Bai wrote the manuscript. Hongfang Jin, Yaqian Huang, Junbao Du, Chaoshu Tang and Selena Chen revised the manuscript. All authors read and approved the final manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
We thank professor Xijing Tang for providing us the H2S fluorescent probe. Our work was supported by the National Natural Science Foundation of China (81622004 and 81670395), the Beijing Natural Science Foundation (7171010), the Changjiang Scholar Program of the Ministry of Education of China, and the National Youth Top-notch Talent Support Program.
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