Dysregulation of brain iron levels causes functional disturbances and damages neurons. Hepcidin (a peptide hormone) plays a principal role in regulating intracellular iron levels by modulating ferroportin (FPN, the only known iron exporter) through triggering its internalization and lysosomal degradation. We observed a significant and rapid iron surge in the cortices of ischemic hemispheres at 3 h after cerebral ischemia (middle cerebral artery occlusion, MCAO) that was maintained until 4 d post-MCAO. We showed upregulation of hepcidin expression in the brain as early as 3 h post-MCAO, mainly in astrocytes, and significant hepcidin accumulation in serum from 6 h post-MCAO, and these inductions were maintained for 1 day and 7 days, respectively. High mobility group box 1 (HMGB1), a prototypic danger-associated molecular pattern, accumulates markedly after transient MCAO and plays critical roles in damage aggravation via its proinflammatory effects. Here, we demonstrated that treatment with recombinant HMGB1 stimulated astrocytes to induce hepcidin expression in a TLR4- and CXCR4-dependent manner. Furthermore, hepcidin-mediated intracellular iron accumulation in neurons was confirmed by an experiment using N-methyl-D-aspartate (NMDA)-conditioned medium-treated primary astrocytes and fresh primary cortical neurons treated with hepcidin-containing astrocyte-conditioned medium. Moreover, HMGB1-mediated local hepcidin upregulation and subsequent local iron surge were found to cause ferroptosis in the postischemic brain, which was suppressed by the functional blocking of HMGB1 using intranasally administered HMGB1 A box or anti-HMGB1 antibody. These findings show that HMGB1 serves as a ferroptosis inducer by upregulating hepcidin in astrocytes and thus aggravates acute damage in the postischemic brain.

In ischemic stroke, neutrophils infiltrate damaged brain tissue immediately following the ischemic insult and aggravate inflammation via various mechanisms which include neutrophil extracellular traps (NETs) formation. In the present study, we showed that adenosine triphosphate (ATP), a DAMP molecule, accumulates in the brain and induces NETosis in brain parenchyma and in circulating neutrophils (PMNs) isolated from a murine model of stroke induced by middle cerebral artery occlusion (MCAO). Expression of peptidylarginine deiminase-4 (PAD4), which induces citrullination of histones H3 (CitH3) and initiates NETosis, was significantly enhanced in brain parenchyma and blood PMNs following MCAO. ATP or BzATP (a prototypic P2X7R agonist) significantly enhanced the inductions of PAD4 and CitH3 in a P2X7R-dependent manner and intracellular Ca²⁺ influx, PKCα activation, and NADPH oxidase-dependent reactive oxygen species (ROS) production play critical roles in this ATP-P2X7R-mediated NETosis. In our MCAO animal model, NETosis was markedly suppressed by treatment with apyrase, an enzyme hydrolyzing ATP, but enhanced by co-treatment of BzATP, confirming ATP-P2X7R-mediated NETosis. Since ATP not only induced NETosis but was also extruded after NETosis, our results indicate that ATP accumulated in the ischemic brain induces NETosis, mediating a cross-talk linking NETosis with neuronal damage that might aggravate inflammation and brain damage.

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.

Following cerebral ischemia, neutrophil extracellular traps (NETs) contribute significantly to brain damage by exacerbating delayed immune cell infiltration and vascular injury. They are detected both in brain tissue and within blood vessels. Danger-associated molecular pattern (DAMP) molecules have been implicated in inducing NETosis after cerebral ischemia. This study investigated the role of High mobility group box 1 (HMGB1), a prototype DAMP molecule, in NETosis induction following photothrombotic stroke (PTS), with a particular focus on neutrophil-platelet interactions. In PTS, thrombi consist primarily of aggregated platelets and neutrophils, lacking significant fibrin content. Triphenyltetrazolium chloride (TTC) staining revealed rapid but progressive expansion of the infarct area in the PTS model, commencing within 1 h and continuing until 24 h. Concomitant with this, peripheral neutrophils isolated following PTS exhibited progressive NETosis, particularly intravascular NETosis. This was evidenced by significant increase in citrullinated histone H3 (CitH3), a marker of NETosis, as early as 1 h post-PTS. Furthermore, serum levels of free DNA gradually and significantly increased, further supporting the induction of NETosis following PTS. Intranasal administration of BBCA, a peptidylarginine deiminase (PAD) inhibitor, effectively suppressed the induction of intravascular NETosis. Importantly, BBCA administration, both 30 min before and 4 h after PTS surgery, significantly reduced infarct volumes at 24 h and improved neurological outcomes. These findings underscore the crucial role of NETosis in both the initiation and progression of ischemic brain damage in this model. Following PTS, HMGB1 rapidly accumulated in serum, detectable as early as 1 h. Immunofluorescence staining revealed initial localization of HMGB1 in neurons, followed by its accumulation within activated neutrophils and platelets within blood vessels. Functional inhibition of HMGB1 by intranasal administration of an HMGB1 A box 4 h post-PTS significantly suppressed NETosis induction, reduced infarct volume, and improved neurological deficits, confirming the pivotal role of HMGB1 in NETosis induction. Notably, we observed a rapid platelet activation and concomitant HMGB1 induction within activated platelets after PTS. Co-culture experiments using naïve PMNs-platelets isolated following PTS demonstrate that extracellular HMGB1, particularly one derived from platelets, plays a critical role in activating neutrophils and inducing intravascular NETosis via a TLR4-dependent manner. Collectively, these findings highlight the critical role of NETosis not only in the initial stages of thrombus formation but also in the subsequent progression of ischemic brain damage in the PTS animal model. HMGB1, particularly platelet-derived HMGB1, emerges as a key mediator to this process. Therefore, targeting NETosis through modulation of HMGB1 presents a promising multipotent therapeutic strategy for mitigating ischemic brain damage.

Peptidylarginine deiminase 4 (PAD4) is an enzyme that modifies proteins by converting positively charged arginine residues to neutral citrulline residues. This process, termed citrullination, has been known to trigger NETosis, a neutrophil cell death pathway involving the release of neutrophil extracellular traps (NETs). Abnormal PAD4 activity and protein citrullination have been linked to various diseases, including those affecting the central nervous system. Herein we investigated the profile of PAD4 expression in an animal model of stroke induced by middle cerebral artery occlusion (MCAO). PAD4 levels were significantly elevated in the ischemic core and penumbra of the affected hemisphere at 3–6 and 6–48 h post-MCAO, respectively. Notably, NETosis induction, indicated by the upregulation of CitH3 (citrullinated histone H3, a NETosis marker), was observed between 48 and 96 h post-MCAO, peaking at 96 h. While PAD4 was present in most brain cell types of sham controls, strong PAD4 induction was primarily observed in neurons during the peak PAD4 induction period (12–24 h post-MCAO). Importantly, intranasal administration of the PAD4 inhibitor BB-Cl-amidine (BBCA) significantly reduced infarct volume and improved neurological and functional outcomes at 24 h post-MCAO, demonstrating a strong protective effect of PAD4 inhibition in ischemic stroke. Staining with an antibody that recognizing citrullinated proteins (F95) revealed an accumulation of these proteins, especially degenerating neurons, however, BBCA treatment significantly suppressed this accumulation in dying neurons. These findings indicate that PAD4-mediated protein citrullination in neurons plays a critical role in promoting ischemic brain damage. Furthermore, delayed administration of BBCA (at 48/72 h post-MCAO) suppresses the NETosis induction observed at 96 h post-MCAO, potentially ameliorating repair processes such as blood vessel regeneration. Collectively, these findings suggest a complex role of PAD4 in cerebral ischemia, with neuroprotective effects (NETosis-independent function) during the acute to subacute period and NETosis-suppressive effects at later time points.

Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In our previous paper, we have shown that Ninj1 plays an important role in the infiltration of neutrophils in the postischemic brain and that the dodecamer peptide harboring the Ninj1 N-terminal adhesion motif (N-NAM, Pro²⁶-Asn³⁷) inhibits infiltration of neutrophils in the postischemic brain and confers robust neuroprotective and anti-inflammatory effects. In the present study, we examinedt the pro-angiogenic effect of N-NAM using human umbilical vein endothelial cells (HUVECs) and rat MCAO (middle cerebral artery occlusion) model of stroke. We found that N-NAM promotes proliferation, migration, and tube formation of HUVECs and demonstrate that the suppression of endogenous Ninj1 is responsible for the N-NAM-mediated pro-angiogenic effects. Importantly, a pull-down assay revealed a direct binding between exogenously delivered N-NAM and endogenous Ninj1 and it is N-terminal adhesion motif dependent. In addition, N-NAM activated the Ang1-Tie2 and AKT signaling pathways in HUVECs, and blocking those signaling pathways with specific inhibitors suppressed N-NAM-induced tube formation, indicating critical roles of those signaling pathways in N-NAM-induced angiogenesis. Moreover, in a rat MCAO model, intranasal administration of N-NAM beginning 4 days post-MCAO (1.5 µg daily for 3 days) augmented angiogenesis in the penumbra of the ipsilateral hemisphere of the brain and significantly enhanced total vessel lengths, vessel densities, and pro-angiogenic marker expression. These results demonstrate that the 12-amino acid Ninj1 peptide, which contains the N-terminal adhesion motif of Ninj1, confers pro-angiogenic effects and suggest that those effects might contribute to its neuroprotective effects in the postischemic brain.