Ninjurin 1 dodecamer peptide containing the N-terminal adhesion motif (N-NAM) exerts proangiogenic effects in HUVECs and in the postischemic brain

Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In our previous paper, we have shown that Ninj1 plays an important role in the infiltration of neutrophils in the postischemic brain and that the dodecamer peptide harboring the Ninj1 N-terminal adhesion motif (N-NAM, Pro(26)-Asn(37)) inhibits infiltration of neutrophils in the postischemic brain and confers robust neuroprotective and anti-inflammatory effects. In the present study, we examinedt the pro-angiogenic effect of N-NAM using human umbilical vein endothelial cells (HUVECs) and rat MCAO (middle cerebral artery occlusion) model of stroke. We found that N-NAM promotes proliferation, migration, and tube formation of HUVECs and demonstrate that the suppression of endogenous Ninj1 is responsible for the N-NAM-mediated pro-angiogenic effects. Importantly, a pull-down assay revealed a direct binding between exogenously delivered N-NAM and endogenous Ninj1 and it is N-terminal adhesion motif dependent. In addition, N-NAM activated the Ang1-Tie2 and AKT signaling pathways in HUVECs, and blocking those signaling pathways with specific inhibitors suppressed N-NAM-induced tube formation, indicating critical roles of those signaling pathways in N-NAM-induced angiogenesis. Moreover, in a rat MCAO model, intranasal administration of N-NAM beginning 4 days post-MCAO (1.5 µg daily for 3 days) augmented angiogenesis in the penumbra of the ipsilateral hemisphere of the brain and significantly enhanced total vessel lengths, vessel densities, and pro-angiogenic marker expression. These results demonstrate that the 12-amino acid Ninj1 peptide, which contains the N-terminal adhesion motif of Ninj1, confers pro-angiogenic effects and suggest that those effects might contribute to its neuroprotective effects in the postischemic brain.