Intranasal Delivery of RGD-Containing Osteopontin Heptamer Peptide Confers Neuroprotection in the Ischemic Brain and Augments Microglia M2 Polarization.

Dashdulam Davaanyam,Il-Doo Kim,Ja-Kyeong Lee

doi:10.3390/ijms22189999

Dashdulam Davaanyam, Il-Doo Kim + Show 1 more

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https://doi.org/10.3390/ijms22189999

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Journal: International Journal of Molecular Sciences	Publication Date: Sep 16, 2021
Citations: 13	License type: CC BY 4.0

Affiliation: Inha University

Abstract

Osteopontin (OPN), a phosphorylated glycoprotein, is induced in response to tissue damage and inflammation in various organs, including the brain. In our previous studies, we reported the robust neuroprotective effects of the icosamer OPN peptide OPNpt20, containing arginine-glycine-aspartic acid (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs, in an animal model of transient focal ischemia and demonstrated that its anti-inflammatory, pro-angiogenic, and phagocytosis inducing functions are responsible for the neuroprotective effects. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motifs (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S), and their neuroprotective efficacy was examined in a rat middle cerebral artery occlusion (MCAO) model. Intranasal administration of all four peptides significantly reduced infarct volume; OPNpt7R (VPNGRGD), the 7-amino-acid peptide containing an RGD motif, was determined to be the most potent, with efficacy comparable to that of OPNpt20. Additionally, sensory–motor functional deficits of OPNpt7R-administered MCAO animals were significantly improved, as indicated by the modified neurological severity scores and rotarod test. Notably, the expression of M1 markers was suppressed, whereas that of M2 markers (Arginase 1, CD206, and VEGF) was significantly enhanced in OPNpt7R-treated primary microglia cultures. Inflammation resolution by OPNpt7R was further confirmed in MCAO animals, in which upregulation of anti-inflammatory cytokines (Arg1, IL-10, IL-4, and CD36) and enhanced efferocytosis were detected. Moreover, studies using three mutant peptides (OPNpt7R-RAA or OPNpt7R-RAD, where RGD was replaced with RAA or RAD, respectively, and OPNpt7R-sc containing scrambled sequences) revealed that the RGD motif plays a vital role in conferring neuroprotection. In conclusion, the RGD-containing OPN heptamer OPNpt7R exhibits neuroprotective effects in the post-ischemic brain by suppressing M1 markers and augmenting M2 polarization of microglia and the RGD motif plays a critical role in these activities.

Highlights

Osteopontin (OPN) is a multifunctional glycoprotein that is highly phosphorylated and expressed in numerous cell types
We revealed that the OPN heptamer peptide (OPNpt7R) containing the RGD motif confers a robust neuroprotective effect in the post-ischemic brain and that it exhibits anti-inflammatory effects, suppresses the expression of M1 markers, and induces M2 polarization of microglia
Together with our previous report that demonstrated the enhancement of phagocytic efficacy of microglia by OPNpt7R [21], our present results indicate that augmentation of microglia M2 polarization may represent, at least in part, an important molecular mechanism underlying the neuroprotective effects of OPNpt7R in the post-ischemic brain

Summary

Introduction

Osteopontin (OPN) is a multifunctional glycoprotein that is highly phosphorylated and expressed in numerous cell types. OPN plays important roles in various cell types by interacting with multiple receptors, including integrins and CD44 variants [1]. We demonstrated a robust neuroprotective effect of recombinant OPN in a rat MCAO model and enhanced efficiency by encapsulating the recombinant OPN in biodegradable gelatin microspheres [11]. Administration of OPN peptides that are 20 or even 10 amino acids in length and contain RGD and SLAYGLR motifs can significantly reduce infarct volume in MCAO animal models [12], and a similar 15 amino acid peptide exerts neuroprotective effects in the rat substantia nigra following toxic insult [2]. We reported that an OPN icosamer peptide (OPNpt20) containing RGD and SLAYGLR motifs exerted anti-inflammatory effects in the post-ischemic brain [13,14]. We demonstrated that the same peptide confers pro-angiogenic effects in the post-ischemic brain [15]

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