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 <i>Aff3ir-ORF2</i>, a novel nested gene variant, in disturbed flow-induced endothelial cell activation and atherosclerosis. We demonstrate that disturbed shear stress significantly reduces <i>Aff3ir-ORF2</i> expression in athero-prone regions. Using three distinct mouse models with manipulated <i>Aff3ir-ORF2</i> expression, we demonstrate that <i>Aff3ir-ORF2</i> exerts potent anti-inflammatory and anti-atherosclerotic effects in <i>Apoe^-/-</i> mice. RNA sequencing revealed that interferon regulatory factor 5 (<i>Irf5</i>), a key regulator of inflammatory processes, mediates inflammatory responses associated with <i>Aff3ir-ORF2</i> deficiency. <i>Aff3ir-ORF2</i> interacts with <i>Irf5</i>, promoting its retention in the cytoplasm, thereby inhibiting the <i>Irf5</i>-dependent inflammatory pathways. Notably, <i>Irf5</i> knockdown in <i>Aff3ir-ORF2</i> deficient mice almost completely rescues the aggravated atherosclerotic phenotype. Moreover, endothelial-specific <i>Aff3ir-ORF2</i> supplementation using the CRISPR/Cas9 system significantly ameliorated endothelial activation and atherosclerosis. These findings elucidate a novel role for <i>Aff3ir-ORF2</i> in mitigating endothelial inflammation and atherosclerosis by acting as an inhibitor of <i>Irf5</i>, highlighting its potential as a valuable therapeutic approach for treating atherosclerosis.