Intranasal Delivery of RGD Motif-Containing Osteopontin Icosamer Confers Neuroprotection in the Postischemic Brain via αvβ3 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-naïve 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-αvβ3 integrin antibody failed to suppress iNOS induction in primary microglia culture, indicating that the RGD motif in OPNpt20 and endogenous αvβ3 integrin play critical roles. Furthermore, pull-down assay revealed a direct binding between OPNpt20 and αvβ3 integrin in primary microglia culture. Together, these results indicate that RGD-containing OPN icosamer has therapeutic potential in the postischemic brain and αvβ3 integrin-mediated anti-inflammatory effect might be an underlying mechanism.

Osteopontin directly modulates cytokine expression of primary microglia and increases their survival

“…18 molecule consisting of only 20 amino acids (RGD-containing icosamer OPN) displays stronger neuroprotective abilities in vivo compared to full-length OPN, and additionally decreases the induction of iNOS and NO-release in primary microglia in vitro (Jin et al , 2015). In line with these studies, we found OPN to reduce the expression of iNOS and the release of NO from rat microglia stimulated with LPS.…”

“…A pro-survival effect of OPN has been attributed to the phosphatidylinositol 3-kinase (PI3K)/Akt pathway Yang-Yen, 2001, Topkoru et al , 2013), while OPN has been linked to reduce NO-levels through integrin binding and STAT-ubiquitination (Gao, Guo, 2007, Jin, Lee, 2015.…”

LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats

“…Fibronectin and vitronectin induce microglial activation via the integrins α5β1 and αvβ5, respectively ( 26 ). Furthermore, Jin et al found that RGD-containing icosamer OPN peptide (OPNpt20) confers anti-inflammatory effect in primary microglia cultures, which was obtained by αvβ3 integrin mediated suppression of iNOS induction, indicating the critical roles of αvβ3 integrin in mediating the physical and functional cell-cell or cell-matrix interactions ( 27 ).…”

Perinatal Hypoxic-Ischemic Encephalopathy and Neuroprotective Peptide Therapies: A Case for Cationic Arginine-Rich Peptides (CARPs)

“…Mechanisms thought to be associated with OPN-mediated neuroprotection include binding to the cell surface αvβ3 integrin receptor to stimulate pro-survival signaling (i.e., PI3K/Akt pathway), anti-inflammatory effects [126,129,130,131], inhibition of inducible NOS [132,133] STAT1 ubiquitination/degradation and by reducing MMP-9 and NF-κβ activation. Despite OPN’s demonstrated neuroprotective effects, in one study OPN deficient mice demonstrated no change in susceptibility to ischemic brain injury [126].…”