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Intranasal Delivery of RGD Motif-Containing Osteopontin Icosamer Confers Neuroprotection in the Postischemic Brain via αvβ3 Integrin Binding

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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.

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Abbreviations

RGD:

Arginine-glycine-aspartate

SLAY:

Serine-leucine-alanine-tyrosine

RAA:

Arginine-alanine-alanine

SLAA:

Serine-leucine-alanine-alanine

OPN:

Osteopontin

OPNpt20:

20 amino acid peptide of osteopontin

OPNpt20-RAA:

Mutant 20 amino acid peptide of osteopontin (in which RGD motif is replaced by RAA)

OPNpt20-SLAA:

Mutant 20 amino acid peptide of osteopontin (in which SLAY motif is replaced by SLAA)

MCAO:

Middle cerebral artery occlusion

mNSS:

Modified neurological severity score

HUVECs:

Human umbilical vein endothelial cells

GMS:

Gelatin microsphere

SEM:

Scanning electron microscope

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Acknowledgments

This work was financially supported by a Medical Research Center Grant 2014R1A5A2009392 (to J.-K.L.) funded by the National Research Foundation (NRF) of Korea.

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    Authors and Affiliations

    1. Department of Anatomy, Inha University School of Medicine, 7-241 Shinheung-dong, Jung-Gu, Inchon, 400-712, Republic of Korea

      Yin-Chuan Jin, Hahnbie Lee, Seung-Woo Kim, Il-Doo Kim, Hye-Kyung Lee & Ja-Kyeong Lee

    2. Medical Research Center, Inha University School of Medicine, Inchon, South Korea

      Hahnbie Lee, Seung-Woo Kim, Il-Doo Kim, Hye-Kyung Lee & Ja-Kyeong Lee

    3. Department of Brain and Cognitive Science, Ewha Womans University, Seoul, South Korea

      Yunjin Lee & Pyung-Lim Han

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    Yin-Chuan Jin and Hahnbie Lee contributed equally to this work.

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    Jin, YC., Lee, H., Kim, SW. et al. Intranasal Delivery of RGD Motif-Containing Osteopontin Icosamer Confers Neuroprotection in the Postischemic Brain via αvβ3 Integrin Binding. Mol Neurobiol 53, 5652–5663 (2016). https://doi.org/10.1007/s12035-015-9480-z

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