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

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.

The Differences of Metabolites in Different Parts of the Brain Induced by Shuxuetong Injection against Cerebral Ischemia-Reperfusion and Its Corresponding Mechanism

“…During stroke-like episodes in patients with mitochondrial disease, transient changes and fading were observed in brain magnetic resonance imaging (MRI) within 1 week of arginine administration [ 44 ]. Bone bridge protein peptides containing arginine, alanine, and aspartate motifs significantly reduced brain infarct volume in the MCAO model [ 45 ]. Mitaki et al showed that histidine-rich glycoprotein was significantly upregulated in future IS patients, which also provided a possible biomarker for predicting or targeting treatment of IS [ 46 ].…”

Osteopontin/secreted phosphoprotein‐1 harnesses glial‐, immune‐, and neuronal cell ligand‐receptor interactions to sense and regulate acute and chronic neuroinflammation

“…In a rat model of ischemia, intranasal delivery of an OPN/SPP1 seven‐amino acid peptide confers protection against ischemia. The heptamer peptide downregulates the expression of IL‐1β, IL‐6, and TNF‐α 78 through a putative mechanism involving αvβ3 integrin 79 . A very recent elegant study delineated a mechanism between T‐regulatory cells expressing OPN/SPP1 that infiltrate the brain after chronic stroke injury and engage integrin‐β1 expressed specifically on microglia to stimulate the proliferation and differentiation of new oligodendrocytes having the capacity to remyelinate the injured brain 80 .…”

Neuronal Loss after Stroke Due to Microglial Phagocytosis of Stressed Neurons

“…However, ventricular injection of osteopontin reduced the secondary neurodegeneration of the thalamus after cortical stroke [53], consistent with osteopontin being neuroprotective. On the other hand, small peptides containing the RGD integrin-binding motif of osteopontin were protective in transient MCAO models [66,87], suggesting these peptides block integrin receptors that are mediating pathological phagocytosis or neuroinflammation.…”

“…The latter finding suggests that phagocytosis by both microglia and astrocytes can be detrimental in ischemic stroke. The phagocytic vitronectin receptor (α v β 3 ) can be blocked with RGD-containing peptides, and the nasal delivery of these peptides provides substantial protection in rat models of ischemic stroke [66,87]. Hence, more research and clinical trials targeting microglial phagocytosis are likely to be of benefit in stroke medicine.…”