SIRT6 deficiency in endothelial cells exacerbates oxidative stress by enhancing HIF1 accumulation and H3K9 acetylation at the Ero1 promoter

BackgroundSIRT6, 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. MethodsThe HUVECs model of oxygen-glucose deprivation/reperfusion (OGD/R) was established to simulate the endothelial IRI in vitro. Endoplasmic reticulum oxidase 1 alpha (Ero1 & alpha;) mRNA and protein levels in SIRT6-overexpressing or SIRT6-knockdown cells were measured by qPCR and Western blotting. The levels of H2O2 and mitochondrial reactive oxygen species (ROS) were detected to evaluate the status of oxidative stress. The effects of SIRT6 deficiency and Ero1 & alpha; 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. ResultsThe expression of Ero1 & alpha; was significantly upregulated in SIRT6-knockdown endothelial cells, and high Ero1 & alpha; expression correlated with the accumulation of H2O2 and mitochondrial ROS. In addition, SIRT6 deficiency increased ERS, inflammation, apoptosis and endothelial permeability, and these effects could be significantly attenuated by Ero1 & alpha; knockdown. The deacetylase catalytic activity of SIRT6 was important in regulating Ero1 & alpha; expression and these biological processes. Mechanistically, SIRT6 inhibited the enrichment of HIF1 & alpha; and p300 at the Ero1 & alpha; promoter through deacetylating H3K9, thereby antagonizing HIF1 & alpha;/p300-mediated Ero1 & alpha; expression. Compared with SIRT6-wild-type (SIRT6-WT) cells, cells expressing the SIRT6-H133Y-mutant and SIRT6-R65A-mutant exhibited increased Ero1 & alpha; expression. Furthermore, ecSirt6(-/-) mice subjected to ischemia-reperfusion surgery exhibited increased Ero1 & alpha; expression and ERS in CMECs and worsened injuries to microvascular barrier function and cardiac function. ConclusionsOur results revealed an epigenetic mechanism associated with SIRT6 and Ero1 & alpha; expression and highlighted the therapeutic potential of targeting the SIRT6-HIF1 & alpha;/p300-Ero1 & alpha; axis.