Targeting GALNT4 suppresses atherosclerosis via TNFR1/NF-κB mediated attenuation of endothelial inflammation.

Endothelial inflammation is a critical driver of atherosclerosis (AS). Although N-acetylgalactosaminyltransferase 4 (GALNT4) has been genetically linked to coronary artery disease, its specific function and mechanism within endothelial cells (ECs) during AS progression remain poorly understood, presenting a significant knowledge gap. This study aimed to elucidate the precise role of GALNT4 in endothelial inflammation and AS development. GALNT4 expression was assessed in human and mouse atherosclerotic plaques. ApoE-/- mice fed on a high-fat diet (HFD) received AAV-shRNA-mediated GALNT4 knockdown to evaluate atherosclerotic lesions and vascular inflammation. In vitro, gain and loss-of-function studies were conducted in TNF-α-treated human umbilical vein endothelial cells (HUVECs) to investigate monocyte adhesion and NF-κB activation. Mechanistic insights were gained through lectin blot, co-immunoprecipitation, and chromatin immunoprecipitation (ChIP) assays. Results showed that GALNT4 expression was significantly upregulated in human and mouse atherosclerotic plaques. In ApoE-/- mice, GALNT4 knockdown markedly attenuated atherosclerotic lesion area (45.1 % reduction) and reduced ICAM-1/VCAM-1 expression without altering plasma lipids. In HUVECs, TNF-α induced GALNT4 expression in a dose and time-dependent manner. Functionally, GALNT4 knockdown suppressed TNF-α-induced monocyte adhesion, while its overexpression exacerbated these effects. Mechanistically, GALNT4 knockdown reduced O-GalNAcylation of TNFR1, diminished TNF-α-triggered phosphorylation and nuclear translocation of NF-κB p65, and inhibited NF-κB binding to the ICAM-1 and VCAM-1 promoters. Our findings demonstrate that GALNT4, upregulated in AS, promotes endothelial inflammation by enhancing the O-GalNAcylation of TNFR1 and subsequent activation of the NF-κB pathway. Targeting GALNT4 represents a promising therapeutic strategy for attenuating AS.