Abstract Body: Introduction: Vascular endothelial cells (ECs) dynamically sense shear stress generated by blood flow. At atheroprone regions, oscillatory shear stress (OS) suppresses EC homeostatic genes and promotes endothelial dysfunction, thereby initiating atherosclerosis. These responses are partly regulated by shear stress-dependent histone modifications. O-linked b-N-acetylglucosamine (O-GlcNAc), catalyzed by O-GlcNAc transferase (OGT), integrates metabolic and mechanical cues to regulate endothelial signaling and gene expression, however, the functional role of histone O-GlcNAc in ECs remains poorly understood.
Hypothesis: Histone O-GlcNAc, a novel histone code, mediates OS-induced EC dysfunction by competing with histone acetylation, reducing chromatin accessibility, and suppressing EC homeostatic genes.
Methods: OGT expression and protein O-GlcNAc were assessed by immunoblotting. ECs were exposed to shear stress with or without OGT knockdown, followed by RNA sequencing. Endothelial-specific OGT knockout mice were generated, and atherosclerosis was induced by AAV8-PCSK9 and a high-fat diet. Single-nucleus RNA sequencing and H3T32A mutant ECs were used to investigate endothelial functional alterations mediated by histone O-GlcNAc.
Results: OS upregulated OGT expression and protein O-GlcNAc in ECs while suppressing eNOS and KLF4. RNA sequencing revealed that OGT knockdown attenuated OS-induced pathways enriched in inflammatory, fibrotic, and endothelial-to-mesenchymal transition and partially restored eNOS and KLF4 expression. EC-specific OGT deletion (EC-OGT KO; 8 males and 7 females mice) reduced atherosclerotic lesion burden in the aortic arch compared with wild-type littermates (6 males and 6 females mice) and altered vascular cell composition, with increased ECs and smooth muscle cells and reduced fibroblasts and macrophages, as revealed by single-nucleus RNA sequencing (n=3 per group). Mechanistically, OS induced O-GlcNAc of histone H3 at threonine 32 in an OGT-dependent manner. Loss of O-GlcNAc function in the H3T32A mutant increased H3K27 acetylation, enhanced chromatin accessibility at the KLF4 and eNOS promoters, and elevated expression of EC homeostatic genes.
Conclusions: OS induces OGT expression and H3T32 O-GlcNAc, promoting chromatin restriction at endothelial homeostatic gene loci and driving endothelial dysfunction. Targeting OGT or H3T32 O-GlcNAc confers atheroprotection, revealing a novel epigenetic mechanism linking disturbed flow to atherogenesis.