CIRP Promotes Redox-Inflammatory Endothelial Injury in High-Fat Diet-Fed ApoE-/- Mice and HUVECs: Involvement of TLR4/SIRT6 Signaling.

Cold-inducible RNA-binding protein (CIRP/CIRBP) is recognized as an extracellular damage-associated molecular pattern. However, its relationship to endothelial redox-inflammatory injury in atherosclerosis remains poorly characterized. Here, we explored the associations among CIRP, endothelial dysfunction, and TLR4-/SIRT6-related changes. In high-fat diet (HFD)-fed ApoE-/- mice, circulating CIRP was elevated and positively correlated with atherosclerotic plaque burden. This increase coincided with systemic redox imbalance, impaired NO/eNOS activity, and vascular inflammation. In HUVECs, CIRP exposure reduced cell viability and impaired NO/eNOS function. These effects were accompanied by increased ROS accumulation and MDA content, together with reduced GSH-Px activity. CIRP also increased inflammatory cytokine production, NF-κB p65 phosphorylation, and THP-1 adhesion. At the molecular level, CIRP reduced SIRT6 expression. Overexpression of SIRT6 attenuated CIRP-induced endothelial injury, oxidative stress, and NO/eNOS dysfunction. CIRP also increased TLR4 expression. Accordingly, pharmacological TLR4 inhibition with TAK-242 attenuated CIRP-associated endothelial injury and partially restored SIRT6 expression and protein stability. Conversely, SIRT6 silencing weakened the protective effects of TAK-242. In addition, CIRP exposure was accompanied by increased overall eNOS acetylation. This increase was attenuated by TAK-242 and further enhanced by SIRT6 silencing. Exploratory cross-context transcriptomic analysis identified overlapping inflammatory and oxidative stress-related signatures. Representative antioxidant-related changes were further examined in CIRP-treated HUVECs through assessment of GPX4 and CAT mRNA expression and CAT activity. Finally, in a preliminary clinical cohort, serum CIRP levels were higher in patients with coronary heart disease and were positively associated with Gensini score, including selected exploratory multivariable models. Overall, these findings provide preliminary evidence that CIRP is associated with redox-inflammatory endothelial injury. The results are also consistent with the possible involvement of TLR4-/SIRT6-related signaling. These observations should be interpreted cautiously because of the relatively high CIRP concentration used in vitro, the cross-context transcriptomic comparison, and the small clinical cohort.