The Sirtuin 5 Inhibitor MC3482 Ameliorates Microglia­induced Neuroinflammation Following Ischaemic Stroke by Upregulating the Succinylation Level of Annexin-A1.

In our previous study, we concluded that sirtuin 5 (SIRT5) was highly expressed in microglia following ischaemic stroke, which induced excessive neuroinflammation and neuronal injury. Therefore, SIRT5-targeting interventions should reduce neuroinflammation and protect against ischaemic brain injury. Here, we showed that treatment with a specific SIRT5 inhibitor, MC3482, alleviated microglia-induced neuroinflammation and improved long-term neurological function in a mouse model of stroke. The mice were administrated with either vehicle or 2 mg/kg MC3482 daily for 7 days via lateral ventricular injection following the onset of middle cerebral artery occlusion. The outcome was assessed by a panel of tests, including a neurological outcome score, declarative memory, sensorimotor tests, anxiety-like behavior and a series of inflammatory factors. We observed a significant reduction of infarct size and inflammatory factors, and the improvement of long-term neurological function in the early stages during ischaemic stroke when the mice were treated with MC3482. Mechanistically, the administration of MC3482 suppressed the desuccinylation of annexin-A1, thereby promoting its membrane recruitment and extracellular secretion, which in turn alleviated neuroinflammation during ischaemic stroke. Based on our findings, MC3482 offers promise as an anti-ischaemic stroke treatment that targets directly the disease's underlying factors.

Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

Acute lung injury (ALI) is characterized by respiratory failure resulting from the disruption of the epithelial and endothelial barriers as well as immune system. In this study, we evaluated the therapeutic potential of airway epithelial cell-derived extracellular vesicles (EVs) in maintaining lung homeostasis. We isolated human bronchial epithelial cell-derived EVs (HBEC-EVs), which endogenously express various immune-related surface markers and investigated their immunomodulatory potential in ALI. In ALI cellular models, HBEC-EVs demonstrated immunosuppressive effects by reducing the secretion of proinflammatory cytokines in both THP-1 macrophages and HBECs. Mechanistically, these effects were partially ascribed to nine of the top 10 miRNAs enriched in HBEC-EVs, governing toll-like receptor-NF-κB signaling pathways. Proteomic analysis revealed the presence of proteins in HBEC-EVs involved in WNT and NF-κB signaling pathways, pivotal in inflammation regulation. ANXA1, a constituent of HBEC-EVs, interacts with formyl peptide receptor (FPR)2, eliciting anti-inflammatory responses by suppressing NF-κB signaling in inflamed epithelium, including type II alveolar epithelial cells. In a mouse model of ALI, intratracheal administration of HBEC-EVs reduced lung injury, inflammatory cell infiltration, and cytokine levels. Collectively, these findings suggest the therapeutic potential of HBEC-EVs, through their miRNAs and ANXA1 cargo, in mitigating lung injury and inflammation in ALI patients.

The Ability of Clinically Relevant Chemotherapeutics to Induce Immunogenic Cell Death in Squamous Cell Carcinoma.

BACKGROUND: Immunogenic cell death (ICD) is a crucial mechanism for triggering the adaptive immune response in cancer patients. Damage-associated molecular patterns (DAMPs) are critical factors in the detection of ICD. Chemotherapeutic drugs can cause ICD and the release of DAMPs. The aim of this study was to assess the potential for paclitaxel and platinum-based chemotherapy regimens to induce ICD in squamous cell carcinoma (SCC) cell lines. In addition, we examined the immunostimulatory effects of clinically relevant chemotherapeutic regimens utilized in the treatment of SCC. METHODS: We screened for differentially expressed ICD markers in the supernatants of three SCC cell lines following treatment with various chemotherapeutic agents. The ICD markers included Adenosine Triphosphate (ATP), Calreticulin (CRT), Annexin A1 (ANXA 1), High Mobility Group Protein B1 (HMGB1), and Heat Shock Protein 70 (HSP70). A vaccination assay was also employed in C57BL/6J mice to validate our in vitro findings. Lastly, the levels of CRT and HMGB1 were evaluated in Serum samples from SCC patients. RESULTS: Addition of the chemotherapy drugs cisplatin (DDP), carboplatin (CBP), nedaplatin (NDP), oxaliplatin (OXA) and docetaxel (DOC) increased the release of ICD markers in two of the SCC cell lines. Furthermore, mice that received vaccinations with cervical cancer cells treated with DDP, CBP, NDP, OXA, or DOC remained tumor-free. Although CBP induced the release of ICD-associated molecules in vitro, it did not prevent tumor growth at the vaccination site in 40% of mice. In addition, both in vitro and in vivo results showed that paclitaxel (TAX) and LBP did not induce ICD in SCC cells. CONCLUSION: The present findings suggest that chemotherapeutic agents can induce an adjuvant effect leading to the extracellular release of DAMPs. Of the agents tested here, DDP, CBP, NDP, OXA and DOC had the ability to act as inducers of ICD.

Annexin A1-Loaded Alginate Hydrogel Promotes Cardiac Repair via Modulation of Macrophage Phenotypes after Myocardial Infarction.

Myocardial infarction (MI) is associated with inflammatory reaction, which is a pivotal component in MI pathogenesis. Moreover, excessive inflammation post-MI can lead to cardiac dysfunction and adverse remodeling, emphasizing the critical need for an effective inflammation-regulating treatment for cardiac repair. Macrophage polarization is crucial in the inflammation process, indicating its potential as an adjunct therapy for MI. In this study, we developed an injectable alginate hydrogel loaded with annexin A1 (AnxA1, an endogenous anti-inflammatory and pro-resolving mediator) for MI treatment. In vitro results showed that the composite hydrogel had good biocompatibility and consistently released AnxA1 for several days. Additionally, this hydrogel led to a reduced number of pro-inflammatory macrophages and an increased proportion of pro-healing macrophages via the adenosine monophosphate (AMP)-activated protein kinase (AMPK)-mammalian target of the rapamycin (mTOR) axis. Furthermore, the intramyocardial injection of this composite hydrogel into a mouse MI model effectively modulated macrophage transition to pro-healing phenotypes. This transition mitigated early inflammatory responses and cardiac fibrosis, promoted angiogenesis, and improved cardiac function. Therefore, our study findings suggest that combining biomaterials and endogenous proteins for MI treatment is a promising approach for limiting adverse cardiac remodeling, preventing cardiac damage, and preserving the function of infarcted hearts.

[Banxia Xiexin Decoction inhibiting colitis-associated colorectal cancer infected with Fusobacterium nucleatum by regulating Wnt/ß-catenin pathway].

This paper investigates the intervention effect and mechanism of Banxia Xiexin Decoction(BXD) on colitis-associated colorectal cancer(CAC) infected with Fusobacterium nucleatum(Fn). C57BL/6 mice were randomly divided into a control group, Fn group, CAC group [azoxymethane(AOM)/dextran sulfate sodium salt(DSS)](AOM/DSS), model group, and BXD group. Except for the control and AOM/DSS groups, the mice in the other groups were orally administered with Fn suspension twice a week. The AOM/DSS group, model group, and BXD group were also injected with a single dose of 10 mg·kg~(-1) AOM combined with three cycles of 2.5% DSS taken intragastrically. The BXD group received oral administration of BXD starting from the second cycle until the end of the experiment. The general condition and weight changes of the mice were monitored during the experiment, and the disease activity index(DAI) was calculated. At the end of the experiment, the colon length and weight of the mice in each group were compared. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-2, IL-4, and IL-6 inflammatory factors in the serum. Immunohistochemistry(IHC) was used to detect the expression of Ki67, E-cadherin, and ß-catenin in the colon tissue. Western blot was used to detect the protein content of Wnt3a, ß-catenin, E-cadherin, annexin A1, cyclin D1, and glycogen synthase kinase-3ß(GSK-3ß) in the colon tissue. The results showed that compared with the control group, the Fn group had no significant lesions. The mice in the AOM/DSS group and model group had decreased body weight, increased DAI scores, significantly increased colon weight, and significantly shortened colon length, with more significant lesions in the model group. At the same time, the colon histology of the model group showed more severe adenomas, inflammatory infiltration, and cellular dysplasia. The levels of IL-4 and IL-6 in the serum were significantly increased, while the IL-2 content was significantly decreased. The IHC results showed low expression of E-cadherin and high expression of Ki67 and ß-catenin in the model group, with a decreased protein content of E-cadherin and GSK-3ß and an increased protein content of Wnt3a, ß-catenin, annexin A1, and cyclin D1. After intervention with BXD, the body weight of the mice increased; the DAI score decreased; the colon length increased, and the tumor decreased. The histopathology showed reduced tumor proliferation and reduced inflammatory infiltration. The levels of IL-6 and IL-4 in the serum were significantly decreased, while the IL-2 content was increased. Meanwhile, the expression of E-cadherin was upregulated, and that of Ki67 and ß-catenin was downregulated. The protein content of E-cadherin and GSK-3ß increased, while that of Wnt3a, ß-catenin, annexin A1, and cyclin D1 decreased. In conclusion, BXD can inhibit CAC infected with Fn, and its potential mechanism may be related to the inhibition of Fn binding to E-cadherin, the decrease in annexin A1 protein level, and the regulation of the Wnt/ß-catenin pathway.

An overview of the regulatory role of annexin A1 in the tumor microenvironment and its prospective clinical application (Review).

Although annexin A1 (ANXA1), a 37 kDa phospholipid­binding anti­inflammatory protein expressed in various tissues and cell types, has been investigated extensively for its regulatory role in cancer biology, studies have mainly focused on its intracellular role. However, cancer cells and stromal cells expressing ANXA1 have the ability to transmit signals within the tumor microenvironment (TME) through autocrine, juxtacrine, or paracrine signaling. This bidirectional crosstalk between cancer cells and their environment is also crucial for cancer progression, contributing to uncontrolled tumor proliferation, invasion, metastasis and resistance to therapy. The present review explored the important role of ANXA1 in regulating the cell­specific crosstalk between various compartments of the TME and analyzed the guiding significance of the crosstalk effects in promotion or suppressing cancer progression in the development of cancer treatments. The literature shows that ANXA1 is critical for the regulation of the TME, indicating that ANXA1 signaling between cancer cells and the TME is a potential therapeutic target for the development of novel therapeutic approaches for impeding cancer development.

Annexin A1 conveys neuroprotective function via inhibiting oxidative stress in diffuse axonal injury of rats.

Diffuse axonal injury (DAI) is a critical pathological facet of traumatic brain injury (TBI). Oxidative stress plays a significant role in the progress of DAI. Annexin A1 (AnxA1) has been demonstrated to benefit from recovery of neurofunctional outcomes after TBI. However, whether AnxA1 exhibits neuronal protective function by modulating oxidative stress in DAI remains unknown. Expression of AnxA1 was evaluated via real-time PCR and western blotting in rat brainstem after DAI. The neurological effect of AnxA1 following DAI through quantification of modified neurologic severity score (mNSS) was compared between wild-type and AnxA1-knockout rats. Brain edema and neuronal apoptosis, as well as expression of oxidative factors and inflammatory cytokines, were analyzed between wild-type and AnxA1 deficiency rats after DAI. Furthermore, mNSS, oxidative and inflammatory cytokines were assayed after timely administration of recombinant AnxA1 for DAI rats. In the brainstem of DAI, the expression of AnxA1 remarkably increased. Ablation of AnxA1 increased the mNSS score and brain water content of rats after DAI. Neuron apoptosis in the brainstem after DAI was exaggerated by AnxA1 deficiency. In addition, AnxA1 deficiency significantly upregulated the level of oxidative and inflammatory factors in the brainstem of DAI rats. Moreover, mNSS decreased by AnxA1 treatment in rats following DAI. Expression of oxidative and inflammatory molecules in rat brainstem subjected to DAI inhibited by AnxA1 administration. AnxA1 exhibited neuronal protective function in the progression of DAI mainly dependent on suppressing oxidative stress and inflammation.

The role of ANXA1 in the tumor microenvironment.

Annexin A1 (ANXA1) is widely expressed in a variety of body tissues and cells and is also involved in tumor development through multiple pathways. The invasion, metastasis, and immune escape of tumor cells depend on the interaction between tumor cells and their surrounding environment. Research shows that ANXA1 can act on a variety of cells in the tumor microenvironment (TME), and subsequently affect the proliferation, invasion and metastasis of tumors. This article describes the role of ANXA1 in the various components of the tumor microenvironment and its mechanism of action, as well as the existing clinical treatment measures related to ANXA1. These findings provide insight for the further design of strategies targeting ANXA1 for the diagnosis and treatment of malignant tumors.

Annexin A1 improves immune responses and control of tissue parasitism during Leishmania amazonensis infection in BALB/c mice.

Leishmaniases, a group of diseases caused by the species of the protozoan parasite Leishmania, remains a significant public health concern worldwide. Host immune responses play a crucial role in the outcome of Leishmania infections, and several mediators that regulate inflammatory responses are potential targets for therapeutic approaches. Annexin A1 (AnxA1), an endogenous protein endowed with anti-inflammatory and pro-resolving properties, has emerged as a potential player. We have shown that during L. braziliensis infection, deficiency of AnxA1 exacerbates inflammatory responses but does not affect parasite burden. Here, we have investigated the role of AnxA1 in L. amazonensis infection, given the non-healing and progressive lesions characteristic of this infectious model. Infection of AnxA1 KO BALB/c mice resulted in increased lesion size and tissue damage associated with higher parasite burdens and enhanced inflammatory response. Notably, therapeutic application of the AnxA1 peptidomimetic Ac2-26 improves control of parasite replication and increases IL-10 production in vivo and in vitro, in both WT and AnxA1 KO mice. Conversely, administration of WRW4, an inhibitor of FPR2/3, resulted in larger lesions and decreased production of IL-10, suggesting that the effects of AnxA1 during L. amazonensis infection are associated with the engagement of these receptors. Our study illuminates the role of AnxA1 in L. amazonensis infection, demonstrating its impact on the susceptibility phenotype of BALB/c mice. Furthermore, our results indicate that targeting the AnxA1 pathway by using the Ac2-26 peptide could represent a promising alternative for new treatments for leishmaniasis.

The pro-resolving mediator, annexin A1 regulates blood pressure, and age-associated changes in cardiovascular function and remodeling.

Aging is associated with chronic, low-level inflammation which may contribute to cardiovascular pathologies such as hypertension and atherosclerosis. This chronic inflammation may be opposed by endogenous mechanisms to limit inflammation, for example, by the actions of annexin A1 (ANXA1), an endogenous glucocorticoid-regulated protein that has anti-inflammatory and pro-resolving activity. We hypothesized the pro-resolving mediator ANXA1 protects against age-induced changes in blood pressure (BP), cardiovascular structure and function, and cardiac senescence. BP was measured monthly in conscious mature (4-month) and middle-aged (12-month) ANXA1-deficient (ANXA1-/- ) and wild-type C57BL/6 mice. Body composition was measured using EchoMRI, and both cardiac and vascular function using ultrasound imaging. Cardiac hypertrophy, fibrosis and senescence, vascular fibrosis, elastin, and calcification were assessed histologically. Gene expression relevant to structural remodeling, inflammation, and cardiomyocyte senescence were also quantified. In C57BL/6 mice, progression from 4 to 12 months of age did not affect the majority of cardiovascular parameters measured, with the exception of mild cardiac hypertrophy, vascular calcium, and collagen deposition. Interestingly, ANXA1-/- mice exhibited higher BP, regardless of age. Additionally, age progression had a marked impact in ANXA1-/- mice, with markedly augmented vascular remodeling, impaired vascular distensibility, and body composition. Consistent with vascular dysfunction, cardiac dysfunction, and hypertrophy were also evident, together with markers of senescence and inflammation. These findings suggest that endogenous ANXA1 plays a critical role in regulating BP, cardiovascular function, and remodeling and delays cardiac senescence. Our findings support the development of novel ANXA1-based therapies to prevent age-related cardiovascular pathologies.

Loss of Endothelial Annexin A1 Aggravates Inflammation-Induched Vascular Aging.

Chronic inflammation is increasingly considered as the most important component of vascular aging, contributing to the progression of age-related cardiovascular diseases. To delay the process of vascular aging, anti-inflammation may be an effective measure. The anti-inflammatory factor annexin A1 (ANXA1) is shown to participate in several age-related diseases; however, its function during vascular aging remains unclear. Here, an ANXA1 knockout (ANXA1-/-) and an endothelial cell-specific ANXA1 deletion mouse (ANXA1△EC) model are used to investigate the role of ANXA1 in vascular aging. ANXA1 depletion exacerbates vascular remodeling and dysfunction while upregulates age- and inflammation-related protein expression. Conversely, Ac2-26 (a mimetic peptide of ANXA1) supplementation reverses this phenomenon. Furthermore, long-term tumor necrosis factor-alpha (TNF-α) induction of human umbilical vein endothelial cells (HUVECs) increases cell senescence. Finally, the senescence-associated secretory phenotype and senescence-related protein expression, rates of senescence-ß-galactosidase positivity, cell cycle arrest, cell migration, and tube formation ability are observed in both ANXA1-knockdown HUVECs and overexpressed ANXA1-TNF-α induced senescent HUVECs. They also explore the impact of formyl peptide receptor 2 (a receptor of ANXA1) in an ANXA1 overexpression inflammatory model. These data provide compelling evidence that age-related inflammation in arteries contributes to senescent endothelial cells that promote vascular aging.

A therapeutic antibody targeting annexin-A1 inhibits cancer cell growth in vitro and in vivo.

In this study we conducted the first investigation to assess the efficacy of a novel therapeutic antibody developed to target annexin-A1 (ANXA1). ANXA1 is an immunomodulatory protein which has been shown to be overexpressed in, and promote the development and progression of, several cancer types. In particular, high ANXA1 expression levels correlate with poorer overall survival in pancreatic and triple-negative breast cancers, two cancers with considerable unmet clinical need. MDX-124 is a humanised IgG1 monoclonal antibody which specifically binds to ANXA1 disrupting its interaction with formyl peptide receptors 1 and 2 (FPR1/2). Here we show that MDX-124 significantly reduced proliferation (p < 0.013) in a dose-dependent manner across a panel of human cancer cell lines expressing ANXA1. The anti-proliferative effect of MDX-124 is instigated by arresting cell cycle progression with cancer cells accumulating in the G1 phase of the cell cycle. Furthermore, MDX-124 significantly inhibited tumour growth in both the 4T1-luc triple-negative breast and Pan02 pancreatic cancer syngeneic mouse models (p < 0.0001). These findings suggest ANXA1-targeted therapy is a viable and innovative approach to treat tumours which overexpress ANXA1.

Glioma-derived ANXA1 suppresses the immune response to TLR3 ligands by promoting an anti-inflammatory tumor microenvironment.

A highly immunosuppressive tumor microenvironment (TME) and the presence of the blood‒brain barrier are the two major obstacles to eliciting an effective immune response in patients with high-grade glioma (HGG). Here, we tried to enhance the local innate immune response in relapsed HGG by intracranially injecting poly(I:C) to establish a robust antitumor immune response in this registered clinical trial (NCT03392545). During the follow-up, 12/27 (44.4%) patients who achieved tumor control concomitant with survival benefit were regarded as responders in our study. We found that the T-cell receptor (TCR) repertoire in the TME was reshaped after poly(I:C) treatment. Based on the RNA-seq analysis of tumor samples, the expression of annexin A1 (ANXA1) was significantly upregulated in the tumor cells of nonresponders, which was further validated at the protein level. In vitro and in vivo experiments showed that ANXA1 could induce the production of M2-like macrophages and microglia via its surface receptor formyl peptide receptor 1 (FPR1) to establish a Treg cell-driven immunosuppressive TME and suppress the antitumor immune response facilitated by poly(I:C). The ANXA1/FPR1 signaling axis can inhibit the innate immune response of glioma patients by promoting an anti-inflammatory and Treg-driven TME. Moreover, ANXA1 could serve as a reliable predictor of response to poly(I:C), with a notable predictive accuracy rate of 92.3%. In light of these notable findings, this study unveils a new perspective of immunotherapy for gliomas.

The interplay between associated proteins, redox state and Ca2+ in the intraluminal ER compartment regulates the IP3 receptor.

There have been in the last three decades repeated publications indicating that the inositol 1,4,5-trisphosphate receptor (IP3R) is regulated not only by cytosolic Ca2+ but also by intraluminal Ca2+. Although most studies indicated that a decreasing intraluminal Ca2+ level led to an inhibition of the IP3R, a number of publications reported exactly the opposite effect, i.e. an inhibition of the IP3R by high intraluminal Ca2+ levels. Although intraluminal Ca2+-binding sites on the IP3Rs were reported, a regulatory role for them was not demonstrated. It is also well known that the IP3R is regulated by a vast array of associated proteins, but only relatively recently proteins were identified that can be linked to the regulation of the IP3R by intraluminal Ca2+. The first to be reported was annexin A1 that is proposed to associate with the second intraluminal loop of the IP3R at high intraluminal Ca2+ levels and to inhibit the IP3R. More recently, ERdj5/PDIA19 reductase was described to reduce an intraluminal disulfide bridge of IP3R1 only at low intraluminal Ca2+ levels and thereby to inhibit the IP3R. Annexin A1 and ERdj5/PDIA19 can therefore explain most of the experimental results on the regulation of the IP3R by intraluminal Ca2+. Further studies are needed to provide a fuller understanding of the regulation of the IP3R from the intraluminal side. These findings underscore the importance of the state of the endoplasmic reticulum in the control of IP3R activity.

Modeling the Annexin A1-S100A11 heterotetramer: a molecular dynamics investigation of structure and correlated motion.

Annexin A1 (A1) has been shown to form a tetrameric complex (A1t) with S100A11 which is implicated in calcium homeostasis and EGFR pathways. In this work, a full-length model of the A1t was generated for the first time. Multiple molecular dynamics simulations were performed on the complete A1t model for several hundred nanoseconds each to assess the structure and dynamics of A1t. These simulations yielded three structures for the A1 N-terminus (ND) which were identified via principal component analysis. The orientations and interactions of the first 11 A1-ND residues for all three structures were conserved, and their binding modes were strikingly similar to those of the Annexin A2 N-terminus in the Annexin A2-p11 tetramer. In this study, we provided detailed atomistic information for the A1t. Strong interactions were identified within the A1t between the A1-ND and both S100A11 monomers. Residues M3, V4, S5, E6, L8, K9, W12, E15, and E18 of A1 were the strongest interactions between A1 and the S100A11 dimer. The different conformations of the A1t were attributed to the interaction between W12 of the A1-ND with M63 of S100A11 which caused a kink in the A1-ND. Cross-correlation analysis revealed strong correlated motion throughout the A1t. Strong positive correlation was observed between the ND and S100A11 in all simulations regardless of conformation. This work suggests that the stable binding of the first 11 residues of A1-ND to S100A11 is potentially a theme for Annexin-S100 complexes and that the flexibility of the A1-ND allows for multiple conformations of the A1t.Communicated by Ramaswamy H. Sarma.

Annexin A1 may contribute to the morphological changes in podocytes by mediating endocytic vesicle fusion and transport via promotion of SNARE assembly in idiopathic membranous nephropathy.

BACKGROUND: Annexin A1 is a membrane-associated calcium-binding protein that participates in the progression of many diseases by facilitating vesicle aggregation. It has been documented that reducing vesicle formation alleviates podocyte injury and albuminuria in idiopathic membranous nephropathy (IMN). However, the role of Annexin A1 (ANXA1) in IMN is unknown. METHODS: Electron microscopy was used to observe the numbers of vesicles in podocytes. The expression of ANXA1 in IMN was investigated by bioinformatics analysis. We validated the hub genes with the Nephroseq V5 online tool and microarray data from the GEO. Immunohistochemical staining and qPCR were performed to measure gene and protein expression. RESULTS: The numbers of vesicles in IMN podocytes were significantly increased. Bioinformatics analysis showed that ANXA1, one of the differentially expressed genes, was upregulated in glomeruli from IMN patients. In the validation database and dataset, we confirmed that ANXA1 expression was upregulated in the glomeruli of IMN patients. We revealed that the increased expression of ANXA1 was negatively correlated with the glomerular filtration rate (GFR) and proteinuria. Moreover, ANXA1 was enriched in the biological process of vesicle fusion, in which the expression of SNAREs and the SNARE complex was increased. Finally, the expression of ANXA1 and genes related to SNAREs and the SNARE complex was upregulated in glomeruli from IMN patients according to immunohistochemical staining and qPCR. CONCLUSION: We conclude that ANXA1 may mediate endocytic vesicle fusion and transport by promoting SNARE assembly, contributing to the morphological changes in podocytes and massive proteinuria in IMN.

The expression of annexin-A1 in esophageal squamous cell carcinoma and its association with the biological behavior of the primary human esophageal squamous carcinoma cell line.

This study aimed to observe the differential expression of Annexin-A1 in esophageal squamous cell carcinoma (ESCC) and explored the effect of small interfering ribonucleic acid (RNAi)-Annexin-A1 on the biological behavior of CE81T-0 cells. An immunohistochemical approach was used to detect the expression of Annexin-A1 in 86 pairs of ESCC samples. Quantitative reverse transcription polymerase chain reaction was used to detect the expression of Annexin-A1 in CE81T-0 and CE81T-4 cells, and the expression of Annexin-A1 in CE81T-0 cells was knocked out by RNAi. A methyl-thiazolyl-tetrazolium assay was used to observe the effect of Annexin-A1 on cell proliferation, and flow cytometry was conducted to analyze its effect on cell cycles and apoptosis. A scratch assay and a Transwell chamber were used to detect changes in cell migration and invasion. From the results, compared with the Annexin-A1 expression rate of 59.3% in para-carcinoma tissues, the expression of Annexin-A1 in cancer was reduced to only 32.6% in ESCC cells. Annexin-A1 was strongly expressed in highly differentiated ESCC cells without lymphatic metastasis and highly expressed in the CE81T-0 cell group with low metastasis. Annexin-A1 gene silencing promoted cell proliferation and inhibited apoptosis, blocked cells in the S-phase, and increased cell migration, leading to an increase in the number of invaded cells. Above all, Annexin-A1 could reflect the differentiation degree and lymph node metastasis of ESCC cells to some extent and was involved in the invasion, metastasis, proliferation, and other biological behaviors of ESCC cells, indicating an experimental basis for Annexin-A1 as a molecular marker in the early diagnosis of ESCC and the prediction of cell metastasis, invasion, and differentiation degree.

The Annexin-A1 mimetic RTP-026 promotes acute cardioprotection through modulation of immune cell activation.

AIMS: The cardio-protective and immuno-regulatory properties of RTP-026, a synthetic peptide that spans the Annexin-A1 (AnxA1) N-terminal region, were tested in rat acute myocardial infarction. METHODS AND RESULTS: In vitro, selective activation of formyl-peptide receptor type 2 (FPR2) by RTP-026 occurred with apparent EC50 in the 10-30 nM range. With human primary cells, RTP-026 counteracted extension of neutrophil life-span and augmented phagocytosis of fluorescent E.coli by blood myeloid cells. An in vivo model of rat acute infarction was used to quantify tissue injury and phenotype immune cells in myocardium and blood. The rat left anterior descending coronary artery was occluded and then reopened for 2-hour or 24-hour reperfusion. For the 2-hour reperfusion protocol, RTP-026 (25-500 µg/kg; given i.v. at the start of reperfusion) significantly reduced infarct size by ∼50 %, with maximal efficacy at 50 µg/kg. Analyses of cardiac immune cells showed that RTP-026 reduced neutrophil and classical monocyte recruitment to the damaged heart. In the blood, RTP-026 (50 µg/kg) attenuated activation of neutrophils and monocytes monitored through CD62L and CD54 expression. Modulation of vascular inflammation by RTP-026 was demonstrated by reduction in plasma levels of mediators like TNF-α, IL-1ß, KC, PGE2 and PGF2α⊡ For the 24-hour reperfusion protocol, RTP-026 (30 µg/kg given i.v. at 0, 3 and 6 h reperfusion) reduced necrotic myocardium by ∼40 %. CONCLUSIONS: RTP-026 modulate immune cell responses and decreases infarct size of the heart in preclinical settings. Tempering over-exuberant immune cell activation by RTP-026 is a suitable approach to translate the biology of AnxA1 for therapeutic purposes.

Tat-NTS peptide protects neurons against cerebral ischemia-reperfusion injury via ANXA1 SUMOylation in microglia.

Rationale: Recent studies indicate that microglial activation and the resulting inflammatory response could be potential targets of adjuvant therapy for ischemic stroke. Many studies have emphasized a well-established function of Annexin-A1 (ANXA1) in the immune system, including the regulation of microglial activation. Nevertheless, few therapeutic interventions targeting ANXA1 in microglia for ischemic stroke have been conducted. In the present study, Tat-NTS, a small peptide developed to prevent ANXA1 from entering the nucleus, was utilized. We discovered the underlying mechanism that Tat-NTS peptide targets microglial ANXA1 to protect against ischemic brain injury. Methods: Preclinical studies of ischemic stroke were performed using an oxygen-glucose deprivation and reperfusion (OGD/R) cell model in vitro and the middle cerebral artery occlusion (MCAO) animal model of ischemic stroke in vivo. Confocal imaging and 3D reconstruction analyses for detecting the protein expression and subcellular localization of microglia in vivo. Co-immunoprecipitation (Co-IP), immunoblotting, ELISA, quantitative real-time PCR (qRT-PCR), Luciferase reporter assay for determining the precise molecular mechanism. Measurement on the cytotoxicity of Tat-NTS peptide for microglia was assessed by CCK-8 and LDH assay. TUNEL staining was used to detect the microglia conditioned medium-mediated neuronal apoptosis. Adeno-associated viruses (AAVs) were injected into the cerebral cortex, striatum and hippocampal CA1 region of adult male Cx3cr1-Cre mice, to further verify the neurofunctional outcome and mechanism of Tat-NTS peptide by TTC staining, the modified Neurological Severity Score (mNSS) test, the open field test (OFT), the novel object recognition task (NORT), the Morris water maze (MWM) test, the long-term potentiation (LTP) and the Transmission electron microscopy (TEM). Results: It was observed that administration of Tat-NTS led to a shift of subcellular localization of ANXA1 in microglia from the nucleus to the cytoplasm in response to ischemic injury. Notably, this shift was accompanied by an increase in ANXA1 SUMOylation in microglia and a transformation of microglia towards an anti-inflammatory phenotype. We confirmed that Tat-NTS-induced ANXA1 SUMOylation in microglia mediated IKKα degradation via NBR1-dependent selective autophagy, then blocking the activation of the NF-κB pathway. As a result, the expression and release of the pro-inflammatory factors IL-1ß and TNF-α were reduced in both in vitro and in vivo experiments. Furthermore, we found that Tat-NTS peptide's protective effect on microglia relieved ischemic neuron apoptosis. Finally, we demonstrated that Tat-NTS peptide administration, through induction of ANXA1 SUMOylation in microglia, reduced infarct volume, improved neurological function and facilitated behavioral recovery in MCAO mice. Conclusions: Our study provides evidence for a novel mechanism of Tat-NTS peptide in regulating microglial ANXA1 function and its substantial neuroprotective effect on neurons with ischemic injuries. These findings suggest that Tat-NTS peptides have a high potential for clinical application and may be a promising therapeutic candidate for treating cerebral ischemia.

Use of the selected metal-dependent enzymes for exploring applicability of human annexin A1 as a purification tag.

Several fusion tags have been developed for non-chromatographic fusion protein purification. Previously, we identified that human annexin A1 as a novel N-terminal purification tag was used for purifying the fusion proteins produced in Escherichia coli through precipitation in 10 mM Ca2+ buffer, and redissolution of the precipitate in 15 mM EDTA buffer. In this work, we selected four metal-dependent enzymes including E. coli 5-aminolevulinate dehydratase, yeast 3-hydroxyanthranilate 3,4-dioxygenase, maize serine racemase and copper amine oxidase for investigating the annexin A1 tag applicability. Fusion of the His6-tag or the enzyme changed the behavior of precipitation-redissolution. The relatively high recovery yields of three tagged enzymes with the improved purities were obtained through two rounds of purification, whereas low recovery yield of the annexin A1 tagged maize amine oxidase was prepared. The added EDTA displayed different abilities to redissolve the fusion proteins precipitates in two precipitation-redissolution cycles. It inactivated three enzymes and obviously inhibited the activity of the fused maize serine racemase. Based on current findings, we believe that four enzymes could be applied for evaluating applicability of the proteins or peptides as affinity tags for chromatographic purification in a calcium dependent manner.