SIRT6 deficiency in endothelial cells exacerbates oxidative stress by enhancing HIF1α accumulation and H3K9 acetylation at the Ero1α promoter

Zhenyang Guo; Xueting Yu; Shuang Zhao; Xin Zhong; Dong Huang; Runyang Feng; Peng Li; Zheyan Fang; Yiqing Hu; Zhentao Zhang; Mukaddas Abdurahman; Lei Huang; Yun Zhao; Xiangdong Wang; Junbo Ge; Hua Li

doi:10.1002/ctm2.1377

SIRT6 deficiency in endothelial cells exacerbates oxidative stress by enhancing HIF1α accumulation and H3K9 acetylation at the Ero1α promoter

Clin Transl Med. 2023 Aug;13(8):e1377. doi: 10.1002/ctm2.1377.

Authors

Zhenyang Guo¹, Xueting Yu¹, Shuang Zhao², Xin Zhong¹, Dong Huang¹, Runyang Feng¹, Peng Li¹, Zheyan Fang¹, Yiqing Hu¹, Zhentao Zhang¹, Mukaddas Abdurahman¹, Lei Huang³, Yun Zhao^{4

5

6}, Xiangdong Wang⁷, Junbo Ge^{1

8

9

10

11

12}, Hua Li¹

Affiliations

¹ Department of Cardiology, Zhongshan Hospital, Shanghai Institute of Cardiovascular Diseases, Fudan University, Shanghai, China.
² Department of Medical Examination, Shanghai Xuhui District Central Hospital, Shanghai, China.
³ Department of Molecular, Cell and Cancer Biology, Program in Molecular Medicine, University of Massachusetts Medical School, MA, USA.
⁴ School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
⁵ State Key Laboratory of Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Shanghai, China.
⁶ Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China.
⁷ Department of Pulmonary and Critical Care Medicine, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
⁸ Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, China.
⁹ National Clinical Research Center for Interventional Medicine, Shanghai, China.
¹⁰ Shanghai Clinical Research Center for Interventional Medicine, Shanghai, China.
¹¹ Key Laboratory of Viral Heart Diseases, National Health Commission, Shanghai, China.
¹² Key Laboratory of Viral Heart Diseases, Chinese Academy of Medical Sciences, Shanghai, China.

Abstract

Background: SIRT6, an important NAD⁺ -dependent protein, protects endothelial cells from inflammatory and oxidative stress injuries. However, the role of SIRT6 in cardiac microvascular endothelial cells (CMECs) under ischemia-reperfusion injury (IRI) remains unclear.

Methods: The HUVECs model of oxygen-glucose deprivation/reperfusion (OGD/R) was established to simulate the endothelial IRI in vitro. Endoplasmic reticulum oxidase 1 alpha (Ero1α) mRNA and protein levels in SIRT6-overexpressing or SIRT6-knockdown cells were measured by qPCR and Western blotting. The levels of H₂ O₂ and mitochondrial reactive oxygen species (ROS) were detected to evaluate the status of oxidative stress. The effects of SIRT6 deficiency and Ero1α knockdown on cellular endoplasmic reticulum stress (ERS), inflammation, apoptosis and barrier function were detected by a series of molecular biological experiments and functional experiments in vitro. Chromatin immunoprecipitation, Western blotting, qPCR, and site-specific mutation experiments were used to examine the underlying molecular mechanisms. Furthermore, endothelial cell-specific Sirt6 knockout (ecSirt6^-/- ) mice were subjected to cardiac ischemia-reperfusion surgery to investigate the effects of SIRT6 in CMECs in vivo.

Results: The expression of Ero1α was significantly upregulated in SIRT6-knockdown endothelial cells, and high Ero1α expression correlated with the accumulation of H₂ O₂ and mitochondrial ROS. In addition, SIRT6 deficiency increased ERS, inflammation, apoptosis and endothelial permeability, and these effects could be significantly attenuated by Ero1α knockdown. The deacetylase catalytic activity of SIRT6 was important in regulating Ero1α expression and these biological processes. Mechanistically, SIRT6 inhibited the enrichment of HIF1α and p300 at the Ero1α promoter through deacetylating H3K9, thereby antagonizing HIF1α/p300-mediated Ero1α expression. Compared with SIRT6-wild-type (SIRT6-WT) cells, cells expressing the SIRT6-H133Y-mutant and SIRT6-R65A-mutant exhibited increased Ero1α expression. Furthermore, ecSirt6^-/- mice subjected to ischemia-reperfusion surgery exhibited increased Ero1α expression and ERS in CMECs and worsened injuries to microvascular barrier function and cardiac function.

Conclusions: Our results revealed an epigenetic mechanism associated with SIRT6 and Ero1α expression and highlighted the therapeutic potential of targeting the SIRT6-HIF1α/p300-Ero1α axis.

Keywords: SIRT6; endoplasmic reticulum stress; ischemia-reperfusion injury.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Acetylation
Animals
CME-Carbodiimide
Endothelial Cells*
Mice
Oxidative Stress
Reactive Oxygen Species
Sirtuins* / genetics

Substances

Reactive Oxygen Species
CME-Carbodiimide
Sirtuins
Sirt6 protein, mouse