AbstractAtherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Herein, using a fluorometric imaging plate reader-supported Ca²⁺ influx assay, we report semi high-throughput screening-based identification of ginkgetin, a biflavone, as a novel inhibitor of transient receptor potential vanilloid 4 (TRPV4)-dependent proatherogenic and inflammatory processes in macrophages. We found that ginkgetin (1) blocks TRPV4-elicited Ca²⁺ influx into macrophages, (2) inhibits oxidized low-density lipoprotein (oxLDL)-induced foam cell formation by suppressing the uptake but not the binding of oxLDL in macrophages, and (3) attenuates oxLDL-induced phosphorylation of JNK2, expression of TRPV4 proteins, and induction of inflammatory mRNAs. Considered all together, the results of this study show that ginkgetin inhibits proatherogenic/inflammatory macrophage function in a TRPV4-dependent manner, thus strengthening the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.