A novel nested gene Aff3ir participates in vascular remodelling by enhancing endothelial cell differentiation in mice

Endothelial integrity in the vasculature is critically maintained by vascular stem/progenitor cells (SPCs) giving rise to endothelial cells (ECs). However, the genes significantly activated during differentiation remain incompletely understood. Based on mouse aorta and vein cDNA library, we unearthed a hitherto unidentified gene nested residing within intron 6 of Aff3, christened as Aff3 intron resident (Aff3ir), upregulated during laminar shear stress-induced ECs differentiation in mouse. Proteomic analysis substantiated the presence of a 45-amino acid(aa) peptide (AFF3IR-ORF1) and 109-aa or 151-aa protein (AFF3IR-ORF2) encoded from two transcript variants. During embryonic development, AFF3IR-ORF1 peaked at E14.5, while AFF3IR-ORF2 displayed a continuous increase until E19.5. In adult mice, AFF3IR-ORF1 was detected in the lung, liver, spleen, and kidney, while AFF3IR-ORF2 was most abundant in the aorta. Furthermore, Western blot and immunofluorescence analyses revealed a specific upregulation of AFF3IR-ORF2, but not AFF3IR-ORF1, three days after femoral artery injury or hindlimb ischemia in vivo. Overexpression of AFF3IR-ORF2 enhanced, while its knockdown attenuated, SPCs differentiation into ECs induced by shear stress or vascular endothelial growth factor in vitro. Notably, the upregulated AFF3IR-ORF2 hindered SPCs proliferation by sequestering minichromosome maintenance complex component 3 in the cytoplasm, thereby shifting the status of SPCs from a pro-proliferation to a pro-differentiation state. In conclusion, our discoveries unveil the novel protein-coding gene Aff3ir as a participant in ECs differentiation, providing fresh insights into the regulation of vascular endothelial integrity.