Abstract Body: Atherosclerosis, a major cause of cardiovascular death, is driven by lipid-induced phenotypic changes in macrophages originating from the bone marrow. This study aims to explore the role of circadian clock genes Period circadian regulator 1 (Per1), Per2 and Per3 triple (Per 1, 2 and 3) genes and Cryptochrome 1 (Cry1) and Cry2 (Cry2) double genes in mediating bone marrow macrophage phenotypic changes and underlying molecular mechanisms, and the regulation of Per1 to 3 and Cry1 to 2 in atherosclerosis.

We investigated the role of Per and Cry in regulating macrophage cholesterol handling and foam-cell formation.

The effects of circadian clock genes Per and Cry on reactive oxygen species, lipid peroxidation, mitochondrial activity, membrane potential, lipid accumulation, oxLDL uptake, and cholesterol efflux were investigated ex vivo. The Per and Cry mechanisms were verified by J774A.1 cells.

Compared with control group mice, Per 1 to 3 triple knockout (TKO) and Cry 1 and 2 double knockout (DKO) mice showed significantly increased ROS production and lipid peroxidation in bone marrow-derived macrophages (BMDMs). These effects were further exacerbated by high-fat/cholesterol diet feeding, with the highest oxidative burden observed in high-fat/cholesterol diet fed Per TKO or Cry DKO macrophages. Per TKO and Cry DKO BMDMs exhibited marked neutral lipid accumulation. At the same time, Per TKO and Cry DKO BMDMs showed pronounced mitochondrial membrane depolarization, an effect amplified by obesogenic diets. Per TKO and Cry DKO significantly increased oxidized LDL uptake in BMDMs. The effect of Per and Cry on oxLDL uptake may be related to their inhibition of oxLDL uptake-associated genes CD36 and Lox-1 protein expressions. Further, Per TKO and Cry DKO macrophages exhibited reduced cholesterol efflux and decreased cholesterol-related transporters Abca1 and Abcg1 mRNA and protein levels. Moreover, Per TKO and Cry DKO significantly increased lysosomal markers LAMP1 and LAMP2 of BMDMs to control cholesterol handling in the lysosome. The mechanism of the study showed that KD of Per 1 to 3 or Cry 1 and 2, increased Cd36 expression and decreased Abca1 and Abcg1 expression, and lamp1 and lamp2 expression in vitro.

Disruption of circadian genes Per and Cry increases oxidative stress, mitochondrial and lysosomal dysfunction, impairs cholesterol efflux, and promotes lipid accumulation in macrophages - key drivers of foam-cell formation and atherosclerosis.