Histone deacetylase 11 (HDAC11), a sole member of the class IV HDAC subfamily, participates in various cardiovascular diseases. Recent evidence showed that pyroptosis was a form of inflammatory programmed cell death and is critical for atherosclerosis (AS). However, little is known about the effect of HDAC11 on endothelial cell pyroptosis in AS. Thus, this study aims to investigate the role of HDAC11 in vascular endothelial cell pyroptosis and its molecular mechanism. Firstly, we found that HDAC11 expression was up-regulated and pyroptosis occurred in the aorta of ApoE−/− mice fed with a high-fat diet (HFD) for 8 or 12 weeks. Then, in vitro study found the treatment of human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor-α (TNF-α) resulted in pyroptosis, as evidenced by activation of caspase-1 and caspase-3 activation, cleavage of downstream gasdermin D (GSDMD) and gasdermin E (GSDME/DFNA5), the release of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and IL-18, as well as elevation of LDH activity and increase of propidium iodide (PI)-positive cells. Besides, TNF-α increased HDAC11 expression and induced pyroptosis via TNFR1 in HUVECs. HDAC11 knockdown mitigated pyroptosis by suppressing both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways in TNF-α-induced HUVECs. Moreover, GSDME knockdown by siRNA significantly decreased pyroptosis and inflammatory response, while treatment with disulfiram or necrosulfonamide (NSA) further augmented the inhibitory effects of GSDME siRNA on pyroptosis and inflammatory response. Further studies found HDAC11 formed a complex with ERG and decreased the acetylation levels of ERG. More importantly, ERG knockdown augmented vascular endothelial cell pyroptosis in TNF-α-induced HUVECs. Taken together, our study suggests that HDAC11 might promote both NLRP3/caspase-1/GSDMD and caspase-3/GSDME pathways leading to pyroptosis via regulation of ERG acetylation in HUVECs. Modulation of HDAC11 may serve as a potential target for therapeutic strategies of AS.

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Hydroxytyrosol acetate (HT-AC), a natural polyphenolic compound in olive oil, exerts an anti-inflammatory effect in cardiovascular diseases (CVDs). Pyroptosis is a newly discovered form of programmed inflammatory cell death and is suggested to be involved in the atherosclerosis (AS) process. However, the effect of HT-AC on vascular endothelial cell pyroptosis remains unknown. Thus, we aimed to investigate the effect of HT-AC on vascular endothelial cell pyroptosis in AS and related signaling pathways. In vivo studies showed that HT-AC alleviated the formation of atherosclerotic lesions and inhibited pyroptosis in the aortic intima of ApoE−/− mice fed a high-fat diet (HFD) for 12 weeks. In vitro, we found that HT-AC treatment of human umbilical vein endothelial cells (HUVECs) alleviated tumor necrosis factor-alpha (TNF-α)-induced pyroptosis by decreasing the number of PI positive cells, decreasing the enhanced protein expressions of activated caspase-1 and gasdermin D (GSDMD), as well as by decreasing the release of pro-inflammatory interleukin (IL)-1β and IL-6. Besides, HT-AC down-regulated HDAC11 expression in the aortic intima of HFD-fed ApoE−/− mice and TNF-α-stimulated HUVECs. To determine the underlying mechanism of action, molecular docking and drug affinity responsive target stability (DARTS) were utilized to identify whether HDAC11 protein is a target of HT-AC. The molecular docking result showed good compatibility between HT-AC and HDAC11. DARTS study's result showed that HDAC11 protein may be a target of HT-AC. Further study demonstrated that knockdown of HDAC11 augmented the inhibition of HT-AC on pyroptosis in TNF-α-stimulated HUVECs. These findings indicate that HT-AC might prevent vascular endothelial pyroptosis through down-regulation of HDAC11 related signaling pathway in AS.

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Endothelial cell (EC) dysfunction is an important predictor of and contributor to the pathobiology of cardiovascular diseases. However, most in vitro studies are performed using monolayer cultures of ECs on 2D tissue polystyrene plates (TCPs), which cannot reflect the physiological characteristics of cells in vivo. Here, we used 2D TCPs and a 3D culture model to investigate the effects of dimensionality and cardiovascular risk factors in regulating endothelial dysfunction. Cell morphology, oxidative stress, inflammatory cytokines and endothelial function were investigated in human umbilical vein endothelial cells (HUVECs) cultured in 2D/3D. The differentially expressed genes in 2D/3D-cultured HUVECs were analysed using Enrichr, Cytoscape and STRING services. Finally, we validated the proteins of interest and confirmed their relevance to TNF-α and the culture microenvironment. Compared with 2D TCPs, 3D culture increased TNF-α-stimulated oxidative stress and the inflammatory response and changed the mediators secreted by ECs. In addition, the functional characteristics, important pathways and key proteins were determined by bioinformatics analysis. Furthermore, we found that some key proteins, notably ACE, CD40, Sirt1 and Sirt6, represent a critical link between endothelial dysfunction and dimensionality, and these proteins were screened by bioinformatics analysis and verified by western blotting. Our observations provide insight into the interdependence between endothelial dysfunction and the complex microenvironment, which enhances our understanding of endothelial biology or provides a therapeutic strategy for cardiovascular-related diseases.