Annexin A1 in the nervous and ocular systems

Aijia Wang ,Hong Zhang ,Xing Li ,Yin Zhao,

Abstract The therapeutic potential of Annexin A1,an important member of the Annexin superfamily,has become evident in results of experiments with multiple human systems and animal models.The antiinflammatory and pro-resolving effects of Annexin A1 are characteristic of pathologies involving the nervous system.In this review,we initially describe the expression sites of Annexin A1,then outline the mechanisms by which Annexin A1 maintains the neurological homeostasis through either formyl peptide receptor 2 or other molecular approaches;and,finally,we discuss the neuroregenerative potential qualities of Annexin A1.The eye and the nervous system are anatomically and functionally connected,but the association between visual system pathogenesis,especially in the retina,and Annexin A1 alterations has not been well summarized.Therefore,we explain the beneficial effects of Annexin A1 for ocular diseases,especially for retinal diseases and glaucoma on the basis of published findings,and we explore present and future delivery strategies for Annexin A1 to the retina.

Key Words: Annexin A1 (ANXA1);glaucoma;nervous system;neuroprotection;neuroregeneration;ocular disease;retina

From the Contents

Introduction 591

Search Strategy 591

Expression of Annexin A1 in the Nervous System 591

Cellular Roles of Annexin A1 592

Annexin A1 in Ocular Diseases 594

Ocular Delivery of Annexin A1: Now and Future 594

Introduction

Annexin A1 (ANXA1) is a calcium-dependent binding protein with phospholipase A2 (PLA2) inhibitory activity,featuring a functionally specific N-terminal region and a core region consisting of four repetitive structural domains.After binding to calcium,ANXA1 undergoes conformational rearrangements that expose its N-terminal region to the extracellular environment and initiates various cellular cascades through binding to different ligands or receptors.The anti-inflammatory effects of ANXA1 can be reproduced by its N-terminal derived peptide Ac2-26 in many pathological settings,broadening the therapeutic range for ANXA1.The low-affinity N-formyl peptide receptor 2 (FPR2) recognizes diverse effectors and can help balance inflammatory responses (Tylek et al.,2021).FPR2 is extensively expressed in the brain tissues,spinal cord,neurons,and glial cells (Stama et al.,2017;Ho et al.,2018).ANXA1 and its derived peptide Ac2-26 exert pharmacological effects resulting in neuroinflammatory resolution and possible neural regeneration through classical pathways that include binding to FPR2 or interacting with other molecules (Stama et al.,2017;Ho et al.,2018;Galvao et al.,2020).

The eyes are connected to the central nervous system (CNS);and so are retinal,optic nerve disorders,and common CNS diseases (Marchesi et al.,2021).The ocular distribution and roles of the Annexin family proteins have been reviewed (Andre da Silva et al.,2022).In this review paper,we specifically explore the functioning patterns of ANXA1 on ocular diseases.

We seek to summarize the distribution of ANXA1 in the central and peripheral nervous systems and to provide an overall perspective on the various function of ANXA1,focusing both on the well-studied inflammation regulation effects and on ANXA1’s underlying neuroregenerative ability in response to neurological homeostasis disorders.Moreover,we believe ANXA1 acts as a link between the visual and nervous systems,and clarifying this association will broaden potential applications for ANXA1 regulators.

Search Strategy

For this narrative review,we selected studies on the basis of the following inclusion criteria: studies that discussed the localization and function of ANXA1 in the nervous system and in ocular diseases.English language and full-text articles published between September 1994 and December 2022.We searched the PubMed database to identify relevant publications.The literature search strategy was conducted as follows: We combined each of two synonymous phrases,i.e.,(1) Annexin A1,(2) ANXA1,with each of the following terms: (a) nervous system,(b) visual system,(c) retina,(d) optic nerve,(e) neuroregeneration,(f) ocular disease,(g) ocular delivery e.g.,“(Annexin A1) AND (nervous system)”,viz.(1)+(a);“ (ANXA1) AND (ocular disease)”,viz.(2)+(f),etc.We obtained more than ten queries.In addition,we screened the reference lists of the studies included to identify other potentially useful studies.First,we screened the titles and abstracts;after that,we screened the full texts for keywords,such as “ANXA1”,“nervous system”,or “ocular disease” to find potentially suitable studies.We extracted the data by focusing on information about ANXA1 localization and function mechanisms in the nervous and ocular systems,the roles of ANXA1 in ocular diseases,and ocular therapy delivery strategies.

Expression of Annexin A1 in the Nervous System

CNS

Cerebrovascular cells

The blood-brain barrier (BBB) is a physiological barrier protecting the CNS from harmful blood-borne factors,and it consists mainly of cerebral endothelial cells (ECs),pericytes,and astrocytes (Dias et al.,2022;Toth and Nielsen,2023).Endogenous ANXA1 helps maintain the integrity of the BBB by colocalizing with cortex actin microfilaments at tight junctions between brain microvascular ECs.The loss of the tight and adherens junction molecules,occludin and VE-cadherin,results in an EC polarity disruption as evidenced by confocal microscopy inAnxa1–/–mice (Cristante et al.,2013).In fact,ANXA1 has been detected in ECs within murine embryonic forebrains by immunofluorescence(McArthur et al.,2016) and paraffin section staining (Schittenhelm et al.,2009).

Pericytes and smooth muscle cells,the mural cells in the cerebrovasculature(Solito et al.,2008;Grant et al.,2019),express ANXA1.In addition,pericytes secrete ANXA1 to maintain BBB integrity as observed with trichloroacetic acid precipitation and immunoblotting experiments (Park et al.,2017).Moreover,the vascular smooth muscle of human hippocampi expresses ANXA1 as revealed by moderate immunoreactivity after immunohistochemistry(Eberhard et al.,1994).

Ependymal cells

Ependymal cells are a layer of epithelial cells lining the central canal of the spinal cord and the lumen of the brain ventricles.They provide a supportive interface between the brain and the cerebrospinal fluid.Studies using immunofluorescence and immunohistochemistry have demonstrated that ANXA1 is strongly expressed in the central canal of the spinal cord of adult rats and the ventricular system of humans.Another study confirmed the expression of ANXA1 in ependymal cells within the ventricles of both adult and fetal brains,by immunoblotting and polymerase chain reaction(Schittenhelm et al.,2009).

Neuroglial cells

The structural expression of ANXA1 in microglia has been reported in healthy humans and rodents (McKanna and Zhang,1997;Dreier et al.,1998;Luo et al.,2014).At the transcriptional level,quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) results also have indicated a strong expression of ANXA1 mRNA in Müller cells and microglia of the mouse retina(Grosche et al.,2016).Intriguingly,resting microglia express only minimal levels of ANXA1,whereas activated microglia express high levels of ANXA1 under lipopolysaccharide (LPS) stimulation or neuroinflammatory conditions(McArthur et al.,2010;You et al.,2021).Fluctuations in ANXA1 protein levels are dynamically correlated with non-inflammatory phagocytosis of apoptotic neurons and quelling of the inflammatory activation state of microglia.Moreover,in the absence of microglial stimuli,the cells express low ANXA1 levels.

ANXA1 is not commonly expressed in resting astrocytes (Eberhard et al.,1994;McKanna and Zhang,1997;Dreier et al.,1998).However,Schittenhelm et al.(2009) detected ANXA1 in the ventricles and subventricular astrocytes of the spinal cord using immunohistochemistry in healthy human brain specimens.Wei et al.(2021) also confirmed ANXA1 expression in healthy human astrocytes and brain glial cell lines with immunoblotting.However,this expression was significantly increased in pathological tissues of astrocytomas(Schittenhelm et al.,2009) and cerebral infarcts (Shijo et al.,2019),where reactive astrocytes showed a significant increase in ANXA1 expression at both the protein and mRNA levels.Collectively,ANXA1 can be found in astrocytes undergoing specific maturation stages and depending on the immune status of the CNS.

Importantly,ANXA1 has been identified in retinal Müller cells as a potential neuroprotection contributor.Murine Müller cells express abundant ANXA1 protein;and,qRT-PCR validation experiments have revealed that ANXA1 mRNA is more abundant in Müller cells than in microglia or retinal neurons,suggesting that Müller cells are involved in the regulation of microglia activation and inflammation through the secretion of ANXA1.Further explanations are needed to ascertain the role of ANXA1 in Müller cells.

Neurons

Perplexing controversies have arisen after the publication of studies concerning ANXA1 in brain neurons.Studies have failed to detect ANXA1 both in the human cerebral cortex and in murine cortical neurons (McKanna and Zhang,1997;Luo et al.,2014).However,our team showed that ANXA1 is expressed in both the nucleus and cytoplasm of embryonic cortical neurons of rats using immunoblots (Zhao et al.,2015).In addition,in 2016,Liu et al.detected ANXA1 in organotypic hippocampal slice cultures,particularly in the cornu ammonis and dentate gyrus using immunofluorescence.Thus,ANXA1 has been shown to be primarily concentrated in neurons rather than in microglia.We conclude that the presence of ANXA1 in neurons depends on different circumstances,such as the species and age,the specific cortical regions of specimens,and the types of primary cultured cells (Sun et al.,2012;Luo et al.,2014;Liu et al.,2016).

In retinal neurons,immunofluorescence and immunoblotting results have detected ANXA1 expression in the nucleus and cytoplasm of retinal ganglion cells (RGC-5) using fluorescein isothiocyanate-propidium iodide(FITC-PI) fluorescence microscopy.Despite the abundance of ANXA1 in the retina,retinal neurons contribute quantitatively less to this abundance than neuroglial cells.Gardner et al.(2017) discovered the ganglion cell and nerve fiber layers contain the highest amounts of ANXA1 using immunohistochemistry.Thus,by contrast with the picture of ANXA1 expression in brain neurons,the picture is clear in retinal neurons.

Peripheral nervous system

In the peripheral nervous system (PNS),the dorsal root ganglion (DRG)had long been proposed to express ANXA1,and the finding was partially replicated in 2011 (Pei et al.,2011) by researchers showing that ANXA1 was predominantly distributed in the perinuclear and neural membranes of rat DRG neurons using immunofluorescence.Also,ANXA1 was found to colocalize with the glial fibrillary acidic protein in satellite glial cells.Moreover,a study using multiplex immunoblotting,bioinformatic protein network analysis,and immunolabelling techniques has identified the localization of ANXA1 to the DRG in mice (Pogatzki-Zahn et al.,2021).

In addition,the presence of ANXA1 in other PNS components has also been reviewed.In a facial nerve injury model,Xia et al.(2020) identified ANXA1 expression in Schwann cells (SCs) using qRT-PCR,immunoblots,and immunofluorescence.Researchers used a proteomic analysis to identify ANXA1 as a resident protein in mouse olfactory cilia;immunofluorescence results also showed ANXA1 immune signals in dendritic knobs of olfactory sensory neurons (Kuhlmann et al.,2014);and,immunohistochemistry images of the inner ear of normal and cisplatin-treated rats displayed ANXA1 protein in spiral ganglion neurons.Together,the evidence supports the general presence of ANXA1 in the PNS.

Cellular Roles of Annexin A1

The Annexin A1/FPR2/ALX signaling axis

The formyl peptide receptor (FPR) subfamily of proteins are pattern recognition receptors that belong to the G-protein coupled receptor superfamily;they recognize pathogen-associated molecular patterns (PAMPs)and damage-associated molecular patterns (Qin et al.,2022).In humans,the FPR subfamily contains three members,namely FPR1,FPR2,and FPR3.FPR2 has a binding preference for lipoxin A4,a mediator produced in response to aspirin treatment;therefore,FPR2 is also known as aspirin-triggered lipoxin A4 receptor (ALX) (Qin et al.,2022).The murine Fpr gene family shares general sequence homology and pharmacological characteristics with its human counterparts (Trojan et al.,2020) and is crucial for investigating FPR mechanisms.The FPR2/ALX protein undergoes conformational changes following binding to ANXA1,thereby activating a series of signaling pathways(Bena et al.,2012).The expression of ANXA1 in the nervous system is extensive and complicated;as shown inFigure 1,the ANXA1/FPR2/ALX signaling axis is similarly distributed.Exogeneous ANXA1 or Ac2-26 exert overall anti-inflammatory and neuroprotective effects primarily in microglia,cerebral endothelial cells,and peripheral neurons under pathological conditions.Created using Adobe Illustrator 2022.ALX: Aspirin-triggered LXA4 receptor;AMPK: adenosine-monophosphate activated-protein kinase;ANXA1:Annexin A1;CaM: Ca2+-calmodulin;ERK: extracellular signal-regulated kinase;FPR2:formyl peptide receptor 2;LPS: lipopolysaccharide;MAPK: mitogen-activated protein kinase;mTOR: Mammalian target of rapamycin;NF-κB: nuclear factor-κB;PLA2:phospholipase A2;PLCβ: phospholipase C-beta;RhoA: Ras homolog family member A;TSPO: translocator protein 18-kDa.

Figure 1｜Schematic diagram representing the classical ANXA1 signaling pathway,namely the ANXA1/FPR2/ALX signaling axis in the nervous system.

The ANXA1/FPR2/ALX signaling axis is engaged in multiple CNS activities,in which microglia regulate neuroinflammation.Enhanced interactions between ANXA1 and FPR proteins during ischemic injury attenuate the proinflammatory effects of microglia,and this is achieved through the FPR2/ALX-mediated adenosine-monophosphate activated-protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway after oxygen-glucose deprivation and reoxygenation injury or through the p38 mitogen-activated protein kinase (MAPK)/COX-2 pathway after spontaneous intracerebral hemorrhages.The ANXA1/FPR2/ALX signaling axis has diverse functions,it inhibits the expression of LPS-induced TSPO (18-kDa translocator protein)through the MyD88/nuclear factor-κB (NF-κB) pathway to regulate TNF-α secretion;and,it also promotes the phosphorylation of actin-binding protein α-catenin,by activating the casein kinase II,to modulate microglial migration towards the inflammation site.

Cristante et al.(2013) and Park et al.(2017) revealed that exogenous ANXA1 interacts with FPR2/ALX and thereby enhances the stability of the cytoskeleton and tight junctions between cerebral ECs to stabilize the integrity of the BBB by inhibiting the small GTPase Ras homolog family member A (RhoA) in neurodegenerative diseases.Liu et al.(2021) found that ANXA1 inhibits the RhoA signaling after interacting with FPR2/ALX,resulting in increased BBB permeability in cases of traumatic brain injury.Thus,the evidence indicates that endogenous and exogenous ANXA1 work together to ensure the BBB integrity through direct protein colocalization and indirect regulation of the ANXA1/FPR2/ALX signaling axis under physiological and pathological conditions.

The DRG is responsible for sensory transduction and PNS regulation,and it is associated with the development of neuropathic pain by directly recruiting primary sensory neurons (Guo et al.,2022).Pei et al.(2011) explored ANXA1’s potential anti-nociceptive effects at the DRG level by performing FPR2/ALX experiments in a murine model.Subsequent studies showed that Ac2-26 triggers a phospholipase C-beta-Ca2+-calmodulin (CaM) signal,via FPR2/ALX,that protects the DRG from the deleterious effects of excessive calcium and desensitizes the transient receptor potential vanilloid 1 ion channel.The anti-nociceptive role of Ac2-26 has been shown to exert neuroprotective effects in spiral ganglion neurons via the FPR2/ALX/extracellular signal-regulated kinase(ERK) pathway in an experimental rat model of ototoxicity (Sena et al.,2022).

Non-classical Annexin A1 signaling pathway

The anti-inflammatory and pro-resolving effects of ANXA1 in the innate immune system are mostly the result of interactions with FPR2/ALX.In addition,ANXA1 possesses receptor-independent intracellular functions(Galvao et al.,2020) that remain unclear,but the available evidence has shed light on the role of ANXA1 in the microglial phagocytosis of neurons (Figure 2).

Figure 2｜Schematic diagram representing the non-classical ANXA1 signaling pathways in microglia and cerebral neurons.

The nuclear translocation of ANXA1 has been shown to modulate apoptosis in the CNS.Our team demonstrated that nuclear transporter protein importinβ(Impβ)-dependent ANXA1 nuclear translocation,which can be inhibited by the exogenous S100A11 (Xia et al.,2018) or an artificial Tat-NTS peptide,is involved in neuronal apoptosis following oxygen-glucose deprivation and reoxygenation via the p53/Bid/caspase-3/poly (ADP-ribose) polymerase(PARP) pathway in rat cerebral cortex neurons (Li et al.,2016).This proapoptotic process gets initiated by upstream SUMO-specific proteinase 6(SENP6)-mediated deSUMOylation together with transient receptor potential melastatin 7 (TRPM7) and protein kinase C-dependent phosphorylation of ANXA1 (Zhao et al.,2015;Xia et al.,2021).

In neuroglial cells,ANXA1 can carry out cellular activities without FPR2.In a mechanism similar to that in neurons,phosphorylated ANXA1 undergoes nuclear translocation in BV-2 microglia mediated by the activated protein kinase C after oxygen-glucose deprivation and reoxygenation injury,the translocation induces the secretion of pro-inflammatory cytokines (Zhao et al.,2016).SENP6-mediated deSUMOylation of ANXA1 may facilitate the proinflammatory phenotype polarization of microglia by activating the inhibitor of kappa B kinase α (IKKα)-NF-κB signaling pathway (Li et al.,2021b;Mao et al.,2022).We recently demonstrated that ischemic stroke upregulates Sirtuin 5-mediated desuccinylation of ANXA1,which in turn promotes the deSUMOylation and the nuclear translocation of ANXA1 (Xia et al.,2022).Exogenous recombinant ANXA1 and endogenous cytoplasmic ANXA1 (not the translocated protein) collectively regulate the expression of the peroxisome proliferated-activated receptor γ/CD36 leading to ANXA1-mediated efferocytosis (da Rocha et al.,2019).Ac2-26 can enter microglia without the FPR2/ALX receptor,Ac2-26 is involved in the degradation of IKKβ in lysosomes and consequently disrupts the damage caused by the IKKβ-NF-κB pathway(Liu et al.,2018).In astrocytes,the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is partially involved in the ANXA1 regulation of glioma growth and development (Wei et al.,2021).Ac2-26 inhibits LPS-induced astrocyte activation and exerts an antineuralgic effect by modulating the p38/c-Jun N-terminal kinase (JNK)-MAPK pathway (Luo et al.,2020).ANXA1 is known for its anti-inflammatory effects,but its nuclear translocation in cerebral neurons and microglia contributes to the inflammatory apoptosis of neurons;thus,the protein has opposing dual effects,which possibly explains the functional differences observed between endogenous and exogenous ANXA1 proteins in response to different neuroinflammatory factors.In all,ANXA1 and Ac2-26 can participate in tumorigenesis regulation,neuroprotection,and anti-nociception in microglia and astrocytes via non-FPR2 intracellular pathways.A complete understanding of the non-classical ANXA1 pathway will probably aid researchers uncover regulatory patterns of neurological pathologies and expand potential therapeutic venues.

Annexin A1 and its potential neuroregenerative function

The anti-inflammatory and pro-resolving effects of ANXA1 are best represented by its inhibition of neutrophil migration and promotion of apoptosis (Sheikh and Solito,2018).In addition,the brain neuroprotective effects mediated by ANXA1 or Ac2-26 via the activation of microglia have been described in detail above.ANXA1 suppresses inflammation mediated by both innate and adaptive immunity and is a promising candidate for development of anti-inflammatory agents.However,the numerous proteins interacting with ANXA1 result in a wide variety of biological phenomena.In our study of the potential functions of ANXA1 in the nervous system,we have focused on its ability to repair human tissues in the context of immune responses.ANXA1 polarizes macrophages towards a phenotype conducive to tissue recovery,its repairing and regenerative effects have been observed in skeletal and cardiac muscle (Ferraro et al.,2019;McArthur et al.,2020).Whether ANXA1 presents similar capabilities in the nervous system is still unclear.

Correlation with FPR2/ALX

FPR2,the classical ANXA1 receptor,participates in neurogenesis and neuronal differentiation for neuroregeneration.The FPR-mediated non-peptide agonist FPRa14 induces differentiation of neuroblastoma cells as evidenced byin vitroexperiments,indicating the involvement of FPR1 and FPR2/ALX in the differentiation of murine neuronal cells (Cussell et al.,2019).Formylated peptides,acting as mitochondrial damage-associated molecular patterns facilitate the cytoplasmic outgrowth of SCs mediated by FPR2 to accelerate the repair of injured nerves (Korimova et al.,2018).Moreover,FPR2 immunoreactivity has been detected in SCs adjacent to regenerating axons and their growth cones;and FPR2 is mostly present in axon pre-terminals.FPR2 signaling inhibition experiments confirmed the engagement of FPR2 in neuronal axon and dendrite growth as well as differentiation in processes which could be initiated through the pharmacological effects of lipoxin A4 or Resolvin D1.Thus,the evidence from formylation-related ligands to classical anti-inflammatory and pro-catabolic lipid mediators suggest that these molecules can synergize with FPR2/ALX to boost neurogenesis and neuronal differentiation.

Whether the anti-inflammatory agent ANXA1 can promote neuroregenerative actions through FPR2/ALX is an area of active research.The evidence so far shows the effects of ANXA1 on SCs,which retain the ability to recover and are responsible for removing myelin debris as well as guiding axonal repair after injury,both key procedures in axonal regeneration.Wallerian degeneration,a pathological change marked by degeneration of axons and myelin sheaths distal to the site of injury,occurs rapidly after nerve breakage.In a model of facial nerve injury and repair,facial nerve regeneration was achieved by rat recombinant ANXA1-induced nerve remyelination and SC recruitment,with the ANXA1/FPR2/ALX/AMPK signaling axis serving as an instrumental pathway to promote the proliferation and migration of SCs along with the maintenance of axonal myelin integrity.Therefore,ANXA1 seems to participate in neuroregenerative processes via the FPR2/ALX receptor,and it has potential applications in the field of neuroregeneration.

Independent molecular approaches

ANXA1 has also been implicated in neuroregeneration as an independent mediator.Spinal cord injury (SCI) is a common devastating neurological condition with secondary mechanical and chemical damages,in the acute phase (characterized by Ca2+flux imbalance and inflammation)and the subacute phase (characterized by neuronal apoptosis and axonal degeneration),that pose challenges to effective neuroregenerative treatments (Anjum et al.,2020).The application of Ac2-26 after SCI alleviates neuroinflammation and reverses PLA2-induced neuronal death,while effectively increasing white matter and myelin in the injured epicenter,providing the first animal evidence of the neuroregenerative effects of ANXA1 in SCI (Liu et al.,2007).The results of these animal experiments were backed by a bioinformatic analysis of the gene expression profiles of rats after SCI,where ANXA1 was classified as a differentially-expressed gene associated with the repair and regeneration of damaged tissues after SCI.

The protective effects of ANXA1 on the neural conduction pathways also support the notion that ANXA1 participates in neuroregeneration.Dendritic spines are small protrusions along neuronal dendrites that form the major synaptic structures between neurons.The regulatory balance between the growth and elimination of dendritic spines is fundamental to the plasticity of neural circuits during learning and memorization.After analyzing the transcriptional changes of stimulated EphB2 signaling in hippocampal neurons,researchers knocked down ANXA1 in neurons and verified that the protein participates in their normal synaptic function) (Yuan et al.,2021).In addition,they found evidence for the role of ANXA1 and the small GTPase RhoA in the control of the dendritic spine cytoskeleton structure under regulation by the EphB2 signaling-induced,cAMP-response element-binding protein (Yuan et al.,2021).Zheng et al.(2022) further deepened the insights into this process by experimenting with mouse hippocampal neurons,they generated transient ischemic attacks to induce ANXA1 upregulation and increase CX3CR1 levels on microglial membranes,the resulting strengthened CX3CR1-ANXA1 dimer facilitated the interaction between CX3CR1 and CX3CL1 in neurons,specifically causing defects in postsynaptic components,and eventually leading to a reduced synaptic density between neurons.This suggests that ANXA1 participates in the synaptic pruning of microglia by modulating the membrane distribution of CX3CR1,and implies a strict and precise modulation by ANXA1,which should be a useful target to promote neuroregeneration.

To sum up,ANXA1 exerts repairing and regenerative influences in the nervous system either alone or mediated by FPR2.Moreover,ANXA1 could serve both as an upstream molecule to initially trigger or as a downstream molecule to indirectly modulate this function during neural regeneration.Despite the limited evidence available,ANXA1 has exhibited protective and regenerative effects on structures essential for nerve conduction under both normal and pathological conditions.On the basis of the available evidence,we contend that the anti-inflammatory and neuroregenerative abilities of ANXA1 may offer an effective strategy to achieve neurological homeostasis therapies and further research is warranted.

Annexin A1 in Ocular Diseases

Conjunctiva and cornea

Allergic conjunctivitis (AC) is an autoimmune disease triggered by mast cell and/or T cell-mediated hypersensitivity reactions.ANXA1 may be an important target for its treatment and prevention.In an allergic conjunctivitis model,Gimenes et al.(2015) and Marmorato et al.(2019) found that Ac2-26 inhibits the recruitment and activation of immune cells and decreases the release of cytokines and chemokines after ANXA1-FPR activation through the activation of the AMPK cascade and especially the ERK signaling pathway.In addition,exogenous ANXA1 increases production of cellular mucins and mucus secretion to maintain ocular surface homeostasis by activating ERK 1/2,PLA2,phospholipase C,and PLD signals via FPR2/ALX in rat conjunctival goblet cells.

Corneal scarring is a common pathological outcome of ocular trauma caused by the persistent and excessive secretion of extracellular matrix proteins by myofibroblasts and leading to the disruption of the normal corneal stroma structure.Researchers found that Ac2-26 mediated by FPR2/ALX inhibits the myofibroblast transdifferentiation process stimulated by transforming growth factor beta1 and reduces corneal scarring (Yu et al.,2019).In a study of dry eye,endogenous ANXA1 and Fpr2 were significantly increased in HCLE(telomerase-immortalized human corneal limbal epithelium) cells and HCjE(human conjunctival epithelial) cells after hyperosmotic treatment,whereas the application of Ac2-26 suppressed the NLRP3 inflammasome activation and interleukin-1beta (IL-1β) release,thereby attenuating the keratoconjunctival damage due to elevated tear film osmolarity.Altogether,these findings suggest a significant potential for the use of ANXA1 as a therapeutic agent in ocular surface diseases.

Uvea

Uveitis is a general term for inflammation of the iris,ciliary body,and choroidal tissues which can be divided into two main categories according to their etiology: infectious and non-infectious.Endotoxin-induced uveitis (EIU)and experimental autoimmune uveitis are currently the most common models in use to investigate the pathogenesis of uveitis.The anti-inflammatory and pro-resolving potential of ANXA1 and Ac2-26 in the innate immune system has already been mentioned.Girol et al.(2013) used Ac2-26 in a rodent model of EIU to show the protective effects of ANXA1.Under inflammatory conditions,a serine phosphorylated ANXA1 endogenously reduces the negative effects of COX-2 and also migrates to the membrane surface to inhibit the release of NF-kB-independent inflammatory mediators via ANXA1-FPR2 engagement.Such anti-inflammatory effects in EIU have been confirmed repeatedly (Girol et al.,2021).ANXA1 knockdown in mice exacerbates retinal autoimmune lesions in a murine model of Th17 cell-mediated autoimmune uveitis,and systemic application of human recombinant ANXA1 (hrANXA1) attenuates the progression of experimental autoimmune uveitis by inducing SOCS3/STAT3 signaling within peripheral lymphocytes.Moreover,vitreous injection of full-length hrANXA1 effectively attenuates the retinal damage caused by experimental autoimmune uveitis or FPRs-mediated EIU.Collectively,these results partially show the ocular distribution and specific contributions of ANXA1 to the treatment of uveitis.

Retina and optic nerve

Retinal diseases

The retinal pigment epithelium (RPE) is the outermost layer of the retina,elucidating the impacts of ANXA1 on the RPE structure,function,and physiology is fundamental to interpreting ANXA1’s role in retinal diseases.In vitroexperiments have demonstrated that Ac2-26 attenuates ARPE-19 cell proliferation and increases cell migration under LPS-activated conditions,suggesting that this pro-ablative process is managed by Ac2-26 through NFκB and ubiquitin C signaling pathways.RPE/ARPE-19 cells in parasite-infected mice demonstrate evident vacuolization and ANXA1 protein upregulation,this immune protective effect is probably related to ANXA1-activated parasite phagocytosis processes.

The proximity of the RPE to the choroid predisposes both structures to simultaneous inflammation,which explains why studies on uveal diseases include RPE examinations (Girol et al.,2013;Zhong et al.,2021).Contact between choroidal endothelial cells (CECs) and the RPE induces neovascular age-related macular degeneration,whereas the invasion of CECs triggers choroidal neovascularization.Researchers discovered that ANXA1 protein secreted by ARPE-19 cells in hypoxic conditions suppresses NLRP3 inflammasome-mediated pyroptosis in CECs by stimulating the FPR2/SHP2 axis,and the enhanced ANXA1/FPR2/SHP2/NLRP3 signals diminish choroidal neovascularization in a laser-induced murine model that supports the prospective application of ANXA1 in neovascular age-related macular degeneration (Zhu et al.,2022).From a different perspective,the RPE constitutes the outer barrier of the blood-retinal barrier (BRB)and serves to control fluid and nutrients in the subretinal area.Our latest study revealed that in response to oxidative stress,ECs overexpress CYP2J2 in retinal facilitated ANXA1 m6A methylation modification by activating methyltransferase 3,which sustains vascular endothelial tight junctions and delays BRB damage in an ANXA1-dependent manner (Zhao et al.,2022).In parallel,the ANXA1 knockdown impairs retinal vascular perfusion and BRB integrity in both developmental and adult mice (Zhao et al.,2022).These results indicate that ANXA1 is associated with the protection of the BRB.Considering the structural and functional similarities between the BRB and BBB,whether the BBB anti-inflammatory effects of ANXA1 also apply to the BRB awaits further investigation.

Retinopathy due to ischemia and hypoxia is another important ocular pathology.In an ischemic retinal vein occlusion model of porcine eyes,protein profiling and immunohistochemistry results showed that retinal ischemia leads to an increase in the expression level of ANXA1,which was presumably the inflammation-related protein that contributed the most to ischemic damage other than IL-18 and S100A12 (Cehofski et al.,2018).In oxygeninduced ischemic retinopathy,ANXA1 deficiency increases pro-angiogenic cytokines,inhibits restorative retinal revascularization,and initiates pathological neovascularization,reflecting the protective role of ANXA1 on retinal neurons under ischemic retinopathy (Hui et al.,2022).

Glaucoma

Glaucoma is a neurodegenerative disease responsible for irreversible blindness worldwide.Glaucomatous optic neuropathy,with loss of RGCs and their axons along with characteristic fundal retinopathy,is the shared pathological end result of all types of glaucoma (Stein et al.,2021).In RGCs,the mechanism of ANXA1 nuclear translocation adequately mimics the role of glaucoma-induced apoptosis,in a process similar to that observed in hippocampal neurons.Nuclear translocation of ANXA1 in RGCsin vivoincreases IL-1β expression through regulation and recruitment of transcription factor p65 after ischemia/reperfusion injury,thereby triggering apoptosis in RGCs (Zhao et al.,2017).Overexpression of full-length or a specific ABCA1 fragment inhibits the protein binding of ANXA1 to impβ and keeps ANXA1 at its membrane localization,thereby preventing the apoptosis of neurons caused by ANXA1 nuclear translocation (Luo et al.,2021).

Importantly,researchers revealed in the same glaucoma model that membrane co-localization of ANXA1 with the ATP-binding cassette transporter A1 (ABCA1) mitigates ocular inflammation and showed that ischemia/reperfusion injury-induced upregulation of the TANK-binding kinase 1 (TBK1)leads to ABCA1 ubiquitination,and ABCA1 degradation reduces the secretion of ANXA1 and promotes RGCs apoptosis (Li et al.,2018).Given the intimate involvement of TBK1 with neurodegenerative diseases (McCauley and Baloh,2019),we hypothesize that ANXA1 may be the underlying link between glaucoma and CNS pathology.In a microarray study of rat genomic RNA,researchers found a significant increase in ANXA1 RNA expression in the experimental glaucoma model,but no obvious alteration in the optic nerve transection model,implicating that ANXA1 was differentially and specifically expressed in both optic nerve injury models (Yang et al.,2007).ANXA1 expression and its distinctive apoptosis regulatory role in different RGCs experimental models paint a sophisticated and variable function model for ANXA1 in glaucoma (Yang et al.,2007;Zhao et al.,2017).

Overall,the protective effects of ANXA1 in retinal diseases and glaucoma are extensive and intricate.A multidimensional,well-established neural network is created among the optic nerve,retina,and CNS based on neurovascular units,which are instrumental for preserving the physiological function of normal neurons and restoring damaged ones.Even with the progress on the study of ANXA1’s function in the eye (Figure 3),its mechanisms for maintaining and regenerating the ocular system,particularly the retina and optic nerve,still need to be elucidated.

Figure 3｜The function of ANXA1 in the eye.

Ocular Delivery of Annexin A1: Now and Future

Direct administration of Ac2-26

ANXA1 has been confirmed to achieve pharmacological effects in full-length form,but it also exerts similar effects through its derived peptides.Ac2-26 is one such peptide (formed by the 26 amino acids of the N-terminal ANXA1 domain) commonly used as an alternative to the full-length ANXA1 for its anti-inflammatory properties (Sheikh and Solito,2018).In studies focusing on ocular diseases,direct administration of Ac2-26 to cells or animals has resulted primarily in inhibition of inflammatory processes.Inin vitroassays,co-culture of HCLE,HCjE,ARPE-19,and RGC-5 cells with Ac2-26 under conventional medium environment has led to reduced cellular inflammation and apoptosis (Girol et al.,2013;Cardin et al.,2017;Fernandez-Torres et al.,2022).Ac2-26 has been administered systemically in murine models (intraperitoneal or subcutaneous back-of-the-neck injections) (Girol et al.,2013;Marmorato et al.,2019;Girol et al.,2021) or topically (drops on the corneal surface or in the eyes) (Girol et al.,2013;Yu et al.,2019)with significantly positive therapeutic outcomes for corneal scarring,allergic conjunctivitis,and EIU.Local treatment with Ac2-26 has a more pronounced effect on the eye than systemic treatment,but the beneficial effects of the mimetic peptide are clear in both cases (Girol et al.,2013).The targeted delivery of Ac2-26 to specific ocular tissues or cells could assist researchers investigating the details of ANXA1s’ functions.

Viral delivery vectors

Viral vectors are widely used in ocular gene therapy due to their efficient transduction capabilities.Adenoviruses (Ad) and retroviruses (RV)/lentiviruses(LV) were the earliest vectors.However,the remarkable immunogenicity of adenoviruses and the insertional mutations that can be caused by RV/LV vectors have limited their widespread adoption (Amador et al.,2022).The recombinant adenovirus-associated virus (rAAV) is the most promising viral vector for ocular gene therapeutics.There are 13 different AAV serotypes(AAV1-AAV13),with the capsid serotype and the specific target cell receptors determining the unique tissue tropism of the different AAVs (Srivastava,2016).AAV serotypes 5,6,8,and 9 have been confirmed to transduce the corneal epithelium and/or stroma in the anterior eye segments of humans and mice;and the same AAV vectors transduced targeted genes in the posterior eye segment after subretinal administration (targeting RPEs,photoreceptors) and intravitreal administration (targeting RGCs,inner nuclear layers,and Müller cells).AAV serotypes 1,4,5,7,and 8 are thought to display a natural retinal tropism.As the first serotype used in the human retina,AAV2/2 (both genome and capsids derived from AAV2) has enabled gene delivery to RPE cells by subretinal or intravitreal administration (Surace and Auricchio,2008).Most experimental rAAVs are hybrid vectors generated by combining the AAV2 genome with different capsids,and resulting in exceptional contributions to the treatment of retinal and optic nerve diseases,such as inherited retinal diseases,neovascular age-related macular degeneration,and glaucoma.

We hypothesize that ANXA1 will be very useful for ocular therapeutics with the aid of rAAV vectors.Few cases of severe inflammation or cytotoxicity caused by AAV have been reported,but routine corticosteroid injections remain in use to eliminate and prevent potential AAV immune responses and mild inflammation (Verdera et al.,2020).Just as Hirsch et al.(2017)used AAV-mediated HLA-G to avert the corneal damage caused by vector immunogenicity,we believe that anti-inflammatory ANXA1 may mitigate the negative effects of AAV while fulfilling its ocular treatment purpose.Moreover,the clear tropism of rAAV for retinal cells including RGCs represents a valuable advantage.We are aware that the pre-existing AAV immunity due to natural exposure and its limited transfer capacity cannot be overlooked(Amador et al.,2022),but the engineered capsid replacements or other refined modifications may render rAAVs a valuable tool to advance the study of the neuroregeneration mechanisms or other mechanisms of ANXA1/Ac2-26 in the eye.

Nanomaterial delivery vectors

Non-viral vectors offer a safer delivery system devoid of the immunogenicity and pathogenicity of viral vectors.Compared to conventional viral vectors,non-viral vectors provide a relatively larger cargo-carrying capacity and repeated administration potential,making them a cost-effective engineered vector.The design of non-viral vectors based on material science and nanobiotechnology has been evolving at a high rate,and nanomaterialbased novel routes of drug delivery for ocular therapies are emerging.In addition to carrying traditional drugs for the clinical treatment of ocular diseases,nanomaterials can deliver small molecules and biological agents,such as peptides,proteins,and nucleic acids,to specific ocular tissues (Li et al.,2021a).Confronted with the impediment of the multiple layers of physiological ocular barriers,well-engineered nanomaterial vectors need to overcome the problems of poor permeability,inadequate bioavailability,low targetability,and biosafety during their cargo transfer.Among the ever-changing and numerous nanomaterial types,nanostructured vectors feature prominently in ocular clinical studies.Depending on the nature of the material,nanostructured vectors can be classified as polymer-based organic nanomaterials (nanoparticles (NPs),dendrimers,micelles,nanogels,protein nanoparticles,and drug conjugates),non-polymer-based inorganic nanomaterials (metallic NPs,quantum dots,carbon nanotubes,and silica particles),and lipid-based NPs (liposomes,solid lipid NPs,and exosomes)(Yetisgin et al.,2020;Tang et al.,2022).

Nanotechnology applications have yielded encouraging results in both the anterior and posterior eye segments.Gene delivery requires consideration of the plasmid design,stable expression,and degradation prevention,and it poses stringent nanostructured vector selection requirements (Sahle et al.,2019;Sahu et al.,2021).The nanostructured vectors to target the retina achieving favorable results include polymer-based NPs,micelles,and lipid-based NPs (Li et al.,2021a;Tang et al.,2022);and they provide novel alternatives to explore the mechanisms of ANXA1 neural regeneration in the eye.Notably,different nanomaterials have been used in retinal and optic nerve tissue regeneration studies.Lipoplexes and polyplexes have been successfully used for retinal regeneration-related gene delivery;by simulating the extracellular matrix,Nano scaffolds promote the proliferation and differentiation of cells associated with retinal regeneration,including RPE cells,RGCs,and neural stem cells (Sahle et al.,2019).Gold and silver NPs have enabled the treatment of neovascular retinal diseases;and,nanowires and hybrid nanostructures,together with stem cell transplantation techniques,have boosted retinal regeneration (Sharma et al.,2021).Although the challenges represented by their low delivery and expression efficiencies have slowed the widespread implementation of nanomaterial vectors,we propose that ANXA1 and its derived peptide can be transported by nanomaterial vectors for retinal regeneration studies and the protein may become an efficient and biosafe platform for retinal disease therapy when applied in concert with nanotechnology strategies.

Limitations

This review has several limitations.First,in summarizing the mechanisms by which ANXA1 exerts its protective effects in the central and peripheral nervous system,we focused on the well-studied cell types and failed to mention effects on neurological cells such as astrocytes and peripheral neurons.Second,the direct evidence of the neuroregenerative effects of ANXA1 is scarce,with most results focusing on the peripheral nervous system.Thus,the neuroregenerative effects of ANXA1 in the central nervous system and in the visual system need to be studied in depth.Third,the links established between ANXA1 and the central nervous system are not numerous or direct due to a lack of relevant research.Forth,most results related to ANXA1 and Ac2-26 delivery to the eye have not been validated in clinical trials sometimes due to shortcomings that do not render them suitable for such trials.

Conclusions

Here,we first mention the localization of ANXA1 and explain its antiinflammatory and pro-resolving effects throughout the nervous system.By describing the classical and non-classical ANXA1 pathways,we set the basis for understanding holistic and versatile pharmacological approaches using ANXA1 to promote neurological homeostasis.In light of the repairing and regenerative potential of ANXA1 in human tissues,we address the possibility of ANXA1 challenging neural regeneration and offer affirmative speculations.We discuss the ocular-nervous system connection by comparing the localization of ANXA1 in the eye and the nervous system (Table 1),and we show the ocular ANXA1 targets relevant for ocular diseases.Although research into the mechanisms of the ANXA1 signaling pathway throughout the visual system is scarce,elucidating these would help in establishing a direct “eye-brain” connection.Finally,we believe ANXA1 has a potential role in the treatment of ocular and nervous system pathologies;therefore,we also elaborate on the possible pathogenic mechanisms and prospects for ANXA1 delivery strategies into eye tissues,especially for retinal disorders and glaucoma.Understanding the biological location and function of ANXA1 in the ocular and nervous will be helpful in clarifying the functions of ANXA1,thereby broadening the therapeutic field of neuro-ophthalmology.

Table 1｜Tissues expressing Annexin A1 in the nervous and ocular systems

Author contributions:Writing original draft: AW;review &editing: HZ;supervision,resources,review &editing: XL and YZ.All approved the final version of the manuscript.

Conflicts of interest:The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Data availability statement:Not applicable.

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