Supplemental Review Material to Sleep Restriction Exacerbates Atherogenesis via Monocyte-Derived CCL15/CCR1 Signaling v1

Sleep Restriction (SR) is a prevalent condition linked to heightened risk of atherosclerotic cardiovascular disease (ASCVD), yet the molecular mechanisms and biomarkers linking sleep deficiency to cardiovascular risk remain poorly defined. Here we identified the monocyte-derived CCL15/CCR1 axis as a critical mediator of SR-induced atherogenesis through cross-species translational approaches. In a murine SRmodel, we observed a markedexpansion of Gr1⁺CD115⁺ monocytes, paralleled by increased CD11b⁺CD14⁺ monocytes in sleep-deprived humans. SRexacerbated atherosclerotic plaque burden in Ldlr⁻/⁻ mice fed a high-fat diet, with greater aortic plaque area versus controls. Integrated single-cell transcriptomics and proteomics identified CCL9 (murine) and its human homolog CCL15 as top upregulated SF-associated factors. Clinically, plasma CCL15 levels correlated with carotid plaque vulnerability in patients with sleep deficiency(R = 0.6102, P = 0.0015). Congruently, recombinant adeno-associated virus (AAV)-Lyz2-shRNA interference and bone marrow transplantation from SR-exposed donors to irradiated Ldlr⁻/⁻ mice confirmed myeloid CCL9-dependent plaque progression. Mechanistically, CCL15 impaired oxidized LDL uptake in human macrophagesand enhanced apoptosis via CCR1, while these effects could be partially reversed by CCR1 antagonist BX471. In vivo, BX471 treatment reduced SR-driven plaque burden in mice, although BX471 did not reduce the leukocyte number. Collectively, these findings establish CCR1 antagonism as a therapeutic strategy to mitigate SR-related ASCVD by targeting theCCL15/CCR1 signaling.