The EZH2 Inhibitor GSK126 Alleviates Thromboinflammation in Deep Vein Thrombosis by Suppressing TLR4 Signaling via H3K27me3 Modulation.

Background: Deep vein thrombosis (DVT) is characterized by abnormal clot formation, often accompanied by endothelial dysfunction and inflammation. Among various inflammatory mediators, extracellular histone H3-acting as a damage-associated molecular pattern (DAMP)-has been implicated in DVT pathogenesis by activating Toll-like receptor 4 (TLR4). Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, regulates gene expression via H3K27me3. Because TLR4 transcription may be epigenetically modulated, this study aimed to evaluate whether GSK126, an EZH2 inhibitor, mitigates DVT by modulating H3K27me3 and suppressing TLR4 signaling.

Methods: To evaluate whether GSK126 attenuates histone H3-exacerbated thromboinflammation in vivo, we employed a stenosis-induced DVT mouse model combined with exogenous histone H3 injection. Tlr4-deficient (Tlr4 ^-/^-) mice were used to assess the role of TLR4 signaling in thrombus formation and inflammation. GSK126 was administered intraperitoneally, and thrombus burden along with inflammatory gene expression were quantified. In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated with lipopolysaccharide (LPS) and treated with GSK126, either alone or in combination with the TLR4-specific inhibitor TAK-242. TLR4 mRNA and protein levels, as well as downstream inflammatory signaling, were analyzed using qPCR and Western blotting.

Results: GSK126 significantly reduced thrombus burden, TLR4 expression, and inflammatory mediators in vivo. In endothelial cells, GSK126 decreased TLR4 and phosphorylated IκBα levels, which was consistently accompanied by reduced H3K27me3 levels. Co-treatment with TAK-242 enhanced these effects. These findings suggest that GSK126 alleviates TLR4-mediated inflammation, likely through its modulation of histone methylation, specifically H3K27me3.

Conclusion: Our results support a role for TLR4 signaling in DVT pathogenesis and suggest that EZH2 inhibition with GSK126 may represent a novel therapeutic approach to thromboinflammation by modulating H3K27me3 and suppressing TLR4-driven inflammatory pathways.