Osteopontin as a candidate of therapeutic application for the acute brain injury

Abstract Acute brain injury is the leading cause of human death and disability worldwide, which includes intracerebral haemorrhage, subarachnoid haemorrhage, cerebral ischaemia, traumatic brain injury and hypoxia‐ischaemia brain injury. Currently, clinical treatments for neurological dysfunction of acute brain injury have not been satisfactory. Osteopontin (OPN) is a complex adhesion protein and cytokine that interacts with multiple receptors including integrins and CD44 variants, exhibiting mostly neuroprotective roles and showing therapeutic potential for acute brain injury. OPN‐induced tissue remodelling and functional repair mainly rely on its positive roles in the coordination of pro‐inflammatory and anti‐inflammatory responses, blood‐brain barrier maintenance and anti‐apoptotic actions, as well as other mechanisms such as affecting the chemotaxis and proliferation of nerve cells. The blood OPN strongly parallel with the OPN induced in the brain and can be used as a novel biomarker of the susceptibility, severity and outcome of acute brain injury. In the present review, we summarized the molecular signalling mechanisms of OPN as well as its overall role in different kinds of acute brain injury.

rupture of cerebral arteries. [6][7][8] The latter further consists of subarachnoid haemorrhage (SAH) and intracerebral haemorrhage (ICH) and accounts for approximately 10%-20% of strokes yet has higher mortality vs the former. [9][10][11][12][13] TBI refers to sudden damage caused by mechanical force, occurring in traffic accidents, blast, wars, violence, terrorism, falls and sporting activity. 14 TBI is currently the major source of fatality in young adults, with an annual global economic loss of approximately US$ 400 billion. 1,2,[14][15][16] Hypoxic-ischaemic brain injury is another frequent, fatal and crippling neurologic disease, particularly perinatal hypoxia-ischaemia remains the dominating cause of acute brain injury in the neonate. [17][18][19] These acute brain injuries impose a heavy socio-economic burden, whereas effective therapies are still scarce. Notably, acute neurologic disorders share many common features and processes within the pathophysiology. 20 Although pathogenic mechanisms involved in acute brain injury have been studied extensively, which include cellular apoptosis, neuroinflammation, blood-brain barrier (BBB) disruption, 21 the prognosis of patients remains poor under current therapeutic strategies. 1 New treatments targeting acute brain injury are urgently needed.
Osteopontin (OPN), a highly phosphorylated glycoprotein, is a complex adhesion protein and cytokine that interacts with multiple receptors including integrins and CD44 variants. 22 OPN has been found in various tissues, including the brain, and plays an important role in cellular processes such as adhesion, motility and survival. 23 Altered expression patterns of OPN have been observed in pathological conditions such as multiple sclerosis, atherosclerosis, myocardial infarction and cancers. 24,25 Under normal conditions, OPN expression is weak in the brain, while under pathological conditions including Alzheimer's disease, Parkinson's disease, TBI, stroke and hypoxia-ischaemia brain injury, it is significantly increased in macrophages/microglia and astrocytes and exerts neuroprotective effects. [26][27][28][29][30] In this review, we will highlight the molecular signalling pathways involved in neuroprotective part of OPN as well as its value as a potential therapeutic target, biomarker and predictor; we will also discuss the potential reason why exogenous OPN is not effective in some experimental models and put forth the limitations of current OPN research.

| G ENER AL FE ATURE S OF OPN
Osteopontin is a highly phosphorylated extracellular matrix glycoprotein that is rich in aspartic acid and has acidic characteristics consisting of approximately 314 amino acids with a molecular weight ranging between 44 and 75 kD. 31,32 OPN is initially found in osteoblasts and is later independently identified as secreted phosphoprotein 1 associated with neoplastic transformation and early T lymphocyte activation 1. [33][34][35] The multiplicity of functions ascribed to OPN may reflect the presence of various isoforms, post-translational modifications, proteolytic processing, and diversity of cell types that OPN can interact with. 32,36 OPN gene is located in the small integrin-binding ligand, N-linked glycoproteins (SIBLING) cluster on chromosome 4 (4q13) in the human genome and on mouse chromosome 5. 37 The gene contains seven exons, six of which are translated in the full-length isoform OPN-a. 38 Alternative translation and splicing result in two splice variants with deletion of exon 5 (OPN-b) or deletion of exon 4 (OPN-c), which correlates with cancer progression and poor prognosis. 38,39 Besides important isoforms, OPN is subject to significant post-translational modifications, including phosphorylation, sulfation, glycosylation and transglutamination, and regulation of these modifications represents the potential to control OPN function. 32,36 When cleaved by thrombin, OPN transforms into two types, the N-terminal fragment (trOPN-N) and C-terminal fragment (trO-PN-C). 40 TrOPN-N contains several highly conserved cell adhesive motifs including an Arg-Gly-Asp (RGD) sequence which binds to integrins such as αvβ1, αvβ3, αvβ5, α8β1 and α5β1, as well as a cryptic Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR)-containing domain that is only exposed after thrombin cleavage and binds to α4β1, α9β1 and α4β7 integrins, 22 whereas trOPN-C binds to CD44 variants. 41 Moreover, mouse OPN cleaved by matrix metalloproteinase (MMP) 3/7 produces LRSKSRSFQVSDEQY, a novel α9β1 integrin-binding motif in the C-terminal fragment. 41 In the human bone marrow, trO-PN-N constitutes the predominant form and acts as a chemotactic factor promoting hematopoietic progenitor cell homing as well as T cell-derived IFN-γ secretion through its binding to α9β1 and α4β1 integrins. 42 In T cells, trOPN-N upregulates IL-17, whereas OPN-C induces IL-10 downregulation by selectively interacting with CD44 isoforms. 43 In carotid specimens, inflammation severity is only associated with trOPN-N expression, not with full-length OPN or trO-PN-C. 44 Thus, different terminal fragments may perform different functions.

| THE ROLE S OF OS TEOP ONTIN IN ACUTE B R AIN INJ URIE S AND OTHER DISE A SE S
Osteopontin can be secreted by multiple tissues as well as body fluids and serves as a regulator in various biological mechanisms including osteoclast function, wound healing, cell migration, immune response, insulin resistance and cellular processes. 24,45,46 Expression levels of OPN vary in different cell types; however, the mechanism underlying the release of OPN is not yet fully understood. 23 In the central nervous system, OPN expression is weak under normal conditions and can be distinctly upregulated in response to either injuries or inflammation. 47 Accumulating evidence has demonstrated that OPN plays a significant role in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis, 26,27,48 as well as acute brain injury including TBI, stroke, and hypoxia-ischaemia brain injury. [49][50][51][52] Different acute brain injury models share many common features and processes within the pathophysiology. To date, most studies suggest a neuroprotective role of OPN in these neurological diseases. 49,[53][54][55][56][57][58][59][60][61] Furthermore, numerous studies have demonstrated that intracerebroventricular injection or intranasal administration of exogenous OPN possesses neuroprotective roles and improves neurological outcome following ischaemic stroke and haemorrhagic stroke [53][54][55][56][57][58] (Table 1) showed exogenous administration of OPN treatment following TBI did not alter lesion characteristics. Moreover, inconsistent therapeutic effects were found in the experimental neonatal hypoxia-ischaemia stroke treated with exogenous OPN. 60,61,63,64 The aforementioned experimental hypoxia-ischaemia strokes were performed on neonatal mice or neonatal rats. 60,61,63,64 Accordingly, an age-dependent manner of the neuroprotective effects of exogenous OPN has been introduced, in which OPN potentiates injury in the neonatal brain while resisting injury in the adult brain due to the different integrin subunit expression levels and distribution at different neuronal development stages 64 Additionally, the above models of the neuroprotective group 60,61 and the non-neuroprotective group 63,64 were established on rat pups and mice pups, respectively, and consistently, the animal heterogeneity of OPN efficacy has been proposed. 62 Jullienne et al 62 were the first to test the function of exogenous OPN after experimental TBI on adult male SD rats (similar ages to the models in cited experimental stroke researches); although several potential beneficial alterations were observed, exogenous OPN did not substantially improve lesion characteristics. It is worthy of note that the protective effects of OPN may be overridden by the hyperacute cerebral injury but sufficient to attenuate delayed thalamic neurodegeneration. 65 Indeed, the regulation and function of OPN may be specific to each pathophysiological condition and may be context-dependent, spatiotemporal-dependent or cell-dependent, which perhaps explain the conflicting results regarding the efficacy of exogenous OPN in distinct disorders. 25,27,[62][63][64][65][66][67][68][69] Notably, OPN is proposed to play a dichotomous role in the neuroinflammation 70,71 (see Section 4.1 for details) and can propel the progress of various autoimmune diseases such as multiple sclerosis. 25,67 Thus, the ability to maintain the balance of anti-inflammatory and pro-inflammatory responses and to coordinate these signals with other inputs received by the cell (eg T cell-independent neurodegeneration in ischaemic brain injury vs T cell-mediated aggressive signals in multiple sclerosis contribute to the altered efficacy of OPN 65 ) may define the ultimate role of OPN.
Crucially, as mentioned above, different isoforms, different terminal fragments, various post-translational modifications of OPN may yield different impact on acute brain injury. 32,43,44

| THE C AND IDATE MECHANIS MS OF THE NEUROPROTEC TIVE EFFEC TS OF OS TEOP ONTIN IN ACUTE B R AIN INJ URY
The pleiotropic effects of OPN in acute brain injury are reflected in its multifunctional regulation of various physiological processes, such as inflammation, apoptosis, BBB reconstruction and neurogenesis, and some of the mechanisms are interrelated and overlapping.
In the present section, we will summarize current research findings concerning the OPN signalling in acute brain injury and integrate knowledge about its underlying mechanisms of neuroprotection and therapeutic potential. Some potential mechanisms for its beneficial effects are summarized in Figure 1, and promoting these signalling pathways may elicit a neuroprotective role of OPN.

| OPN and neuroinflammation
Inflammation plays a crucial role in the pathogenesis of acute brain injury. 9,16,72,73 Potentially exacerbating secondary brain injury via inflammatory cascade in the acute stage, whereas beneficially promoting tissue remodelling and functional repair, inflammatory response induced by acute brain injury is suggested to be a double-edged sword. 74 By early inhibition of inflammatory cascade, the coordination of pro-inflammatory and anti-inflammatory responses leads to the alleviation of the brain injury and better patient outcome. 75 Interestingly, OPN is also indicated to exert dual roles in neuroinflammation. 70 Many studies have highlighted the pro-inflammatory role of OPN in the pathogenesis of various autoimmune diseases, such as multiple sclerosis. 25,65,67,76 During acute brain injury, although some researchers have reported that OPN exacerbates cerebral injury via neuroinflammation, 71 OPN is also considered a promising target for anti-inflammation. 58 An important role of OPN in the inflammatory response is to trigger various leucocytes to cause a functional response and induce cytokine secretion, thereby forming an entire immune response. 78 Activated microglia/macrophages and astrocytes are the main cellular sources of OPN induction in the central nervous system. 27 Kang et al 79 showed that microglia/macrophages and astrocytes induced OPN upregulation at different stages after brain injury. And then, the expression of OPN inversely recruited, activated and polarized additional microglial/macrophages and astrocytes in the lesional and perilesional area, which was based on the interaction of OPN with αvβ3 integrin and/or interaction with CD44. [79][80][81][82][83] These receptors can also be upregulated after brain injury. 52 The activated macrophages/microglia and astrocytes, as well as the induced OPN and its receptors, contribute to the succedent cytokine secretion, removal of the necrotic tissue, extracellular matrix formation, tissue remodelling, angiogenesis and gliosis. 52,80,84,85 Inducible nitric oxide synthase (iNOS) exerts vital roles in inflammation response and is identified to be excitotoxic and neurotoxic. 77 Moreover, iNOS-derived nitric oxide is known to activate MMP-9, which is involved in neuroinflammation, cell death and the BBB disruption. [86][87][88] Previous studies have demonstrated that OPN is involved in iNOS pathway. 50,89,90 Induced OPN provides a dose-dependent pattern suppressing iNOS expression after acute brain in-

| OPN and apoptosis
Apoptosis is a highly complex energy-dependent programmed cell death process closely in correlation with neuron development and homeostasis under normal physiological conditions. 94,95 Nevertheless, apoptosis also participates in the process of neurological disorders under pathological conditions and is considered as a key player for the progression and prognosis of patients. [96][97][98] Previous studies have suggested that OPN ameliorates apoptosis and promotes cell survival during brain injury through several molecular signalling pathways, which may suggest a potential therapeutic strategy for treating acute brain injury. 61 Among all the proteins involved in the initiation and execution of apoptosis, the caspases stand out as being crucial mediators of this process. [103][104][105] Furthermore, of all the caspases, caspase-3 is probably the best understood and required for a large portion of distinct apoptotic processes and cell deaths. 104  The PI3K/Akt signalling pathway is involved in numerous cellular processes, including apoptosis, BBB disruption, neurogenesis and angiogenesis. 60 is considered to be a common downstream protein, and the regulation of phosphorylated Akt results in the suppression of caspase-3 and apoptosis. 61,112 In addition, phosphorylated Akt induces the phosphorylation and subsequent inactivation of GSK-3β, which is also pro-apoptotic and has been found to aggravate brain injury in experimental ICH, ischaemic stroke and traumatic brain injury. [113][114][115] Additionally, inhibitors of FAK and PI3K administrated to the rat models could abolish the protective effects of OPN. 55,99 Thus, the PI3K/Akt pathway may play a critical role in the OPN-induced anti-apoptotic actions.
Besides, some proteins involved in the process of apoptosis after acute brain injury such as FAK, Bcl-2, PI3K and Akt may overlap with autophagic pathways. 116,117 Emerging studies suggest that OPN enhances autophagy and reduces apoptosis after experimental SAH through FAK signalling, resulting in the attenuation of early brain injury and improvement of long-term outcome. 51

| OPN and BBB disruption
The BBB is built up by specialized monolayer endothelial cells which are connected by tight junctions without fenestrations. 118 The endothelial barrier is also supplemented with a large number of pericytes, which share the common basal membrane with the endothelial cells. 118 Besides, the abluminal surface of the microvascular basement membranes is covered by astrocytic perivascular end-feet. 119 This major barrier plays a significant role in maintaining brain haemostasis and protecting the brain from disease and injury.
Dysfunction of the BBB has been described as one of the independent risk factors for poor prognosis after acute brain injury, which may amplify inflammation and lead to further parenchyma damage and oedema by allowing more blood-borne cells and substances to flow into the brain parenchyma. [120][121][122][123] Previous studies have F I G U R E 1 Some neuroprotective signalling pathways induced by osteopontin following acute brain injury. OPN exerts multiple roles in acute brain injury via interaction with integrins and CD44, which is reflected in its multifunctional regulation of various physiological processes, such as inflammation, apoptosis and BBB reconstruction. The mechanisms in these processes are interrelated and overlapping. AK2/STAT1, Janus kinase/signal transducers and activators of transcription 1; Ang-1, angiopoietin-1; BBB, blood-brain barrier; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; GSK-3β, glycogen synthase kinase 3 beta; IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKP-1, MAPK phosphatase-1; MMP, matrix metalloproteinase; NFκB, factor-κ-gene binding; P-gp, P-glucoprotein; Rac-1, Ras-related C3 botulinum toxin substrate 1; ROS, reactive oxygen species; TIMP-1, tissue Inhibitor of MMP-1; VEGF, vascular endothelial growth factor demonstrated that OPN is significantly induced and locates at reactive capillary endothelial cells and astrocytes during the recovery of BBB function, [124][125][126] suggesting an important role for OPN in BBB reconstruction. And still, OPN exerts effects of BBB maintenance via interaction with integrins and CD44 receptors. 127 As mentioned above, MMP-9 is involved in aggravation of BBB disruption in addition to facilitating the inflammatory cascade and cell death. [86][87][88] The balanced interaction between MMP-9 and its corresponding inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), determines the severity of BBB disruption. 128 Induced by acute brain injury, oxidative stress and IL-1β then activate NF-κB, which directly upregulates MMP-9 and inhibits TIMP-1 levels in the brain. 56,129 There is plenty of evidence that MMP-9 plays a role in degrading the extracellular matrix of cerebral microvessel basal lamina including laminin, fibronectin, collagen IV and zona occludens-1 (ZO-1) and causing BBB disruption. 130-132 ZO-1 belongs to endothelial tight junction-related proteins, loss of which will increase BBB permeability. 133 The inhibition of IL-1β/NFκB pathway and reduction of oxidative stress have been widely reported by exogenous OPN administration, through which OPN downregulates MMP-9 expression and upregulates TIMP-1 to protect cerebral microvessel basal lamina and subsequently maintain BBB integrity. 56,89,92,93 Furthermore, the FAK/PI3K signalling pathway is also involved in BBB dysfunction following experimental brain injury. 60 It has been demonstrated that exogenous OPN promotes the phosphorylation of FAK and subsequently activates the PI3K, 60,99,111 leading to the induction of Ras-related C3 botulinum toxin substrate 1 (Rac-1) expression and the preservation of BBB integrity. 60,134 Interestingly, endogenous OPN induced after acute brain injury may ameliorate BBB disruption through different mechanisms.

| Additional aspects of OPN
Previous studies also revealed that OPN exerted positive roles in the proliferation, survival and differentiation of various cells, including cerebrovascular smooth muscle cells, [138][139][140] neural progenitor cells 119,141 and neural stem cells. 142 Moreover, adhesive and chemotactic properties of OPN exert function in the lateral migration of neuroblasts from the subventricular zone to the injured region following focal cerebral ischaemia and ICH. 143,144 OPN also reportedly enhances the sensitivity of adult corticospinal neurons to insulin-like growth factor 1, 145 ameliorates cerebral vasospasm 109,[146][147][148] and stabilizes smooth muscle cell phenotype 57 following acute brain injury.

| OS TEOP ONTIN A S A B I OMARK ER OF ACUTE B R AIN INJ URY
Considering the upregulation and multifarious effects of OPN following acute brain injury, it may reflect the occurrence and severity of neurological damage. 149 And the OPN induced in the brain strongly parallels increased OPN protein in the blood, which may owe to the leakage of OPN produced in the brain or at the interface between brain and blood into the circulation. [150][151][152] Thus, the plasma/serum OPN can be used as a novel biomarker of the susceptibility, severity and outcome of acute brain injury. 149

| CON CLUS I ON S AND FUTURE PROS PEC TS
In the present review, we mainly focused on the roles and therapeutic potential of OPN in acute brain injury including ICH, SAH, cerebral ischaemia, TBI and hypoxia-ischaemia brain injury. OPN plays a bidirectional role in neuroinflammation. Following acute brain injury, blasts. Collectively, OPN is a pleiotropic extracellular matrix glycoprotein that is mainly believed to be neuroprotective during acute brain injury. These beneficial effects of OPN are induced through its interaction with integrins and CD44. Therefore, OPN represents a potential therapeutic target for acute brain injury. Furthermore, the blood OPN strongly parallels the OPN induced in the brain and can be used as a novel biomarker of the susceptibility, severity and outcome of acute brain injury.
Notably, although endogenous OPN is believed to be neuroprotective, studies regarding TBI and neonatal hypoxia-ischaemia brain injury suggest that exogenous OPN treatment may be ineffective or even harmful. Thus far, the reason for this discrepancy remains unclear. The efficacy of OPN may depend on the region, injury pattern and stage of brain lesions, and the age and species of victims.
Additional experiments based on these variables need to be con-

This work was supported by grants from China Postdoctoral
Science Foundation (2017M612010) and National Natural Science Foundation of China (81701144).

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no competing interests. Zhang: Conceptualization (equal); Writing-review & editing (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
No data, models or code were generated or used during the study.