Disruption of the Novel Nested Gene Aff3ir Mediates Disturbed Flow-Induced Atherosclerosis in Mice

Disturbed shear stress-induced endothelial atherogenic responses are pivotal in the initiation and progression of atherosclerosis, contributing to the uneven distribution of atherosclerotic lesions. This study investigates the role of Aff3ir-ORF2, a novel nested gene variant, in disturbed flow-induced endothelial cell activation and atherosclerosis. We demonstrate that disturbed shear stress significantly reduces Aff3ir-ORF2 expression in athero-prone regions. Using three distinct mouse models with manipulated AFF3ir-ORF2 expression, we demonstrate that AFF3ir-ORF2 exerts potent anti-inflammatory and anti-atherosclerotic effects in ApoE ^-/- mice. RNA sequencing revealed that interferon regulatory factor 5 (IRF5), a key regulator of inflammatory processes, mediates inflammatory responses associated with AFF3ir-ORF2 deficiency. AFF3ir-ORF2 interacts with IRF5, promoting its retention in the cytoplasm, thereby inhibiting the IRF5-dependent inflammatory pathways. Notably, IRF5 knockdown in AFF3ir-ORF2 deficient mice almost completely rescues the aggravated atherosclerotic phenotype. Moreover, endothelial-specific AFF3ir-ORF2 supplementation using the CRISPR/Cas9 system significantly ameliorated endothelial activation and atherosclerosis. These findings elucidate a novel role for AFF3ir-ORF2 in mitigating endothelial inflammation and atherosclerosis by acting as an inhibitor of IRF5, highlighting its potential as a valuable therapeutic approach for treating atherosclerosis.