Intranasal Delivery of RGD Motif-Containing Osteopontin Icosamer Confers Neuroprotection in the Postischemic Brain via alpha(v)beta(3) Integrin Binding

Abstract

Osteopontin (OPN) is a phosphorylated glycoprotein possessing an arginine-glycine-aspartate (RGD)-motif, which binds to several cell surface integrins and mediates a wide range of cellular processes. Inductions of OPN have been reported in the postischemic brain, and the neuroprotective effects of OPN have been demonstrated in animal models of stroke. In the present study, we showed a robust neuroprotective effect of RGD-containing icosamer OPN peptide (OPNpt20) in a rat model of focal cerebral ischemia (middle cerebral artery occlusion, MCAO). Intranasally administered OPNpt20 reduced mean infarct volume by 79.7 % compared to the treatment-na < ve MCAO control animals and markedly ameliorated neurological deficits. In addition, OPNpt20 significantly suppressed the inductions of iNOS and of inflammatory markers in postischemic brains and in primary microglial cultures, demonstrating anti-inflammatory effects. Administration of a mutant peptide, in which RGD was replaced by arginine-alanine-alanine (RAA), failed to suppress infarct volumes in MCAO animals and co-administration of OPNpt20 with anti-alpha(v)beta(3) integrin antibody failed to suppress iNOS induction in primary microglia culture, indicating that the RGD motif in OPNpt20 and endogenous alpha(v)beta(3) integrin play critical roles. Furthermore, pull-down assay revealed a direct binding between OPNpt20 and alpha(v)beta(3) integrin in primary microglia culture. Together, these results indicate that RGD-containing OPN icosamer has therapeutic potential in the postischemic brain and alpha(v)beta(3) integrin-mediated anti-inflammatory effect might be an underlying mechanism.