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.

Integrating single-nucleus RNA sequencing and spatial transcriptomics to elucidate a specialized subpopulation of astrocytes, microglia and vascular cells in brains of mouse model of lipopolysaccharide-induced sepsis-associated encephalopathy.

BACKGROUND: Understanding the mechanism behind sepsis-associated encephalopathy (SAE) remains a formidable task. This study endeavors to shed light on the complex cellular and molecular alterations that occur in the brains of a mouse model with SAE, ultimately unraveling the underlying mechanisms of this condition. METHODS: We established a murine model using intraperitoneal injection of lipopolysaccharide (LPS) in wild type and Anxa1-/- mice and collected brain tissues for analysis at 0-hour, 12-hour, 24-hour, and 72-hour post-injection. Utilizing advanced techniques such as single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq, we conducted a comprehensive characterization of the cellular responses and molecular patterns within the brain. RESULTS: Our study uncovered notable temporal differences in the response to LPS challenge between Anxa1-/- (annexin A1 knockout) and wild type mice, specifically at the 12-hour and 24-hour time points following injection. We observed a significant increase in the proportion of Astro-2 and Micro-2 cells in these mice. These cells exhibited a colocalization pattern with the vascular subtype Vas-1, forming a distinct region known as V1A2M2, where Astro-2 and Micro-2 cells surrounded Vas-1. Moreover, through further analysis, we discovered significant upregulation of ligands and receptors such as Timp1-Cd63, Timp1-Itgb1, Timp1-Lrp1, as well as Ccl2-Ackr1 and Cxcl2-Ackr1 within this region. In addition, we observed a notable increase in the expression of Cd14-Itgb1, Cd14-Tlr2, and Cd14-C3ar1 in regions enriched with Micro-2 cells. Additionally, Cxcl10-Sdc4 showed broad upregulation in brain regions containing both Micro-2 and Astro-2 cells. Notably, upon LPS challenge, there was an observed increase in Anxa1 expression in the mouse brain. Furthermore, our study revealed a noteworthy increase in mortality rates following Anxa1 knockdown. However, we did not observe substantial differences in the types, numbers, or distribution of other brain cells between Anxa1-/- and wildtype mice over time. Nevertheless, when comparing the 24-hour post LPS injection time point, we observed a significant decrease in the proportion and distribution of Micro-2 and Astro-2 cells in the vicinity of blood vessels in Anxa1-/- mice. Additionally, we noted reduced expression levels of several ligand-receptor pairs including Cd14-Tlr2, Cd14-C3ar1, Cd14-Itgb1, Cxcl10-Sdc4, Ccl2-Ackr1, and Cxcl2-Ackr1. CONCLUSIONS: By combining snRNA-seq and Stereo-seq techniques, our study successfully identified a distinctive cellular colocalization, referred to as a special pathological niche, comprising Astro-2, Micro-2, and Vas-1 cells. Furthermore, we observed an upregulation of ligand-receptor pairs within this niche. These findings suggest a potential association between this cellular arrangement and the underlying mechanisms contributing to SAE or the increased mortality observed in Anxa1 knockdown mice.

ANXA1 is identified as a key gene associated with high risk and T cell infiltration in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease, with unclear pathogenesis. Although immune disorders, especially T cell infiltration, are thought to play a vital role in PSC, the specific pathogenesis mechanisms remain incompletely understood. This study evaluated the potential key gene associated with the PSC pathogenesis and analyzed the associations of the key gene with prognosis and immune cell infiltration by combining bioinformatics analysis and experimental verification. METHODS: Transcriptome data of PSC and normal human liver tissues (GSE159676) were obtained from the gene expression omnibus database. Differentially expressed genes (DEGs) were identified, and differences in biological states were analyzed. A protein-protein interaction (PPI) network was constructed. Hub genes were identified, and their expression was verified using transcriptome data of mice fed 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and Mdr2-/- mice (GSE179993, GSE80776), as well as by immunohistochemistry staining on clinical samples. The correlations between the key gene and other factors were evaluated by Pearson's correlation coefficient. Immune cell infiltration into human liver (GSE159676) was analyzed by xCell and verified by immunofluorescence staining on PSC liver samples. RESULTS: Of the 185 DEGs identified, 113 were upregulated and 72 were downregulated genes in PSC. Genes associated with immune cell infiltration and fibrosis were significantly enriched in PSC. PPI network showed close interactions among DEGs. A module strongly associated with immune infiltration was identified, with annexin A1 (ANXA1) being the core gene. High expression of ANXA1 in PSC was confirmed in two public datasets and by immunohistochemistry staining on clinical samples. High ANXA1 expression was strongly associated with high-risk score for PSC. Also, ANXA1 expression was positively associated with chemokines and chemokine receptors and with the infiltration of immune cells, especially T cells, into liver with PSC. Immune infiltration, fibrosis, and cancer-related processes were markedly enriched in PSC with high expression of ANXA1. CONCLUSION: ANXA1 is a key gene associated with high risk and infiltration of immune cells, especially T cells, in PSC. These findings provide new insight into the key biomarker of PSC and suggest that targeting ANXA1 may be a valuable strategy for the treatment of PSC.

Neuroprotective effects of annexin A1 tripeptide in rats with sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is characterized by high incidence and mortality rates, with limited treatment options available. The underlying mechanisms and pathogenesis of SAE remain unclear. Annexin A1 (ANXA1), a membrane-associated protein, is involved in various in vivo pathophysiological processes. This study aimed to explore the neuroprotective effects and mechanisms of a novel bioactive ANXA1 tripeptide (ANXA1sp) in SAE. Forty Sprague-Dawley rats were randomly divided into four groups (n = 10 each): control, SAE (intraperitoneal injection of lipopolysaccharide), vehicle (SAE + normal saline), and ANXA1sp (SAE + ANXA1sp) groups. Changes in serum inflammatory factors (interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), hippocampal reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) levels were measured. The Morris water maze and Y maze tests were used to assess learning and memory capabilities in the rats. Further, changes in peroxisome proliferator-activated receptor-gamma (PPAR-γ) and apoptosis-related protein expression were detected using western blot. The IL-6, TNF-α, and ROS levels were significantly increased in the SAE group compared with the levels in the control group. Intraperitoneal administration of ANXA1sp led to a significant decrease in the IL-6, TNF-α, and ROS levels (p < 0.05). Compared with the SAE group, the ANXA1sp group exhibited reduced escape latency on day 5, a significant increase in the number of platform crossings and the percent spontaneous alternation, and significantly higher hippocampal MMP and ATP levels (p < 0.05). Meanwhile, the expression level of PPAR-γ protein in the ANXA1sp group was significantly increased compared with that in the other groups (p < 0.05). The expressions of apoptosis-related proteins (nuclear factor-kappa B [NF-κB], Bax, and Caspase-3) in the SAE and vehicle groups were significantly increased, with a noticeable decrease in Bcl-2 expression, compared with that noted in the control group. Moreover, the expressions of NF-κB, Bax, and Caspase-3 were significantly decreased in the ANXA1sp group, and the expression of Bcl-2 was markedly increased (p < 0.05). ANXA1sp can effectively reverse cognitive impairment in rats with SAE. The neuroprotective effect of ANXA1sp may be attributed to the activation of the PPAR-γ pathway, resulting in reduced neuroinflammatory response and inhibition of apoptosis.

Annexin-A1 tripeptide enhances functional recovery and mitigates brain damage in traumatic brain injury by inhibiting neuroinflammation and preventing ANXA1 nuclear translocation in mice.

This study explores the role and mechanism of Annexin-A1 Tripeptide (ANXA1sp) in mitigating neuronal damage and promoting functional recovery in a mouse model of traumatic brain injury (TBI). Our goal is to identify ANXA1sp as a potential therapeutic drug candidate for TBI treatment. Adult male C57BL/6J mice were subjected to controlled cortical impact (CCI) to simulate TBI, supplemented by an in vitro model of glutamate-induced TBI in HT22 cells.  We assessed neurological deficits using the Modified Neurological Severity Score (mNSS), tested sensorimotor functions with beam balance and rotarod tests, and evaluated cognitive performance via the Morris water maze. Neuronal damage was quantified using Nissl and TUNEL staining, while microglial activation and inflammatory responses were measured through immunostaining, quantitative PCR (qPCR), Western blotting, and ELISA. Additionally, we evaluated cell viability in response to glutamate toxicity using the Cell Counting Kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release. Intraperitoneal administration of ANXA1sp significantly enhanced neurological outcomes, markedly reducing sensorimotor and cognitive impairments caused by TBI. This treatment resulted in a significant reduction in lesion volume and decreased neuronal cell death in the ipsilateral cortex. Moreover, ANXA1sp effectively diminished microglial activation around the brain lesion and decreased the levels of pro-inflammatory markers such as IL-6, IL-1ß, TNF-α, and TGF-ß in the cortex, indicating a significant reduction in neuroinflammation post-TBI. ANXA1sp also offered protection against neuronal cell death induced by glutamate toxicity, primarily by inhibiting the nuclear translocation of ANXA1, highlighting its potential as a neuroprotective strategy in TBI management. Administration of ANXA1sp significantly reduced neuroinflammation and neuronal cell death, primarily by blocking the nuclear translocation of ANXA1. This treatment substantially reduced brain damage and improved neurological functional recovery after TBI. Consequently, ANXA1sp stands out as a promising neuroprotective agent for TBI therapy.

MiR-374a/b-5p Suppresses Cell Growth in Papillary Thyroid Carcinoma Through Blocking Exosomal ANXA1-Induced Macrophage M2 Polarization.

Papillary thyroid carcinoma (PTC), comprising 85% of all thyroid cancers, is an epithelial malignancy. The potential for malignant transformation in normal cells by thyroid cancer cells via exosomal Annexin A1 (ANXA1) delivery is investigated in this study. Our aim is to determine the impact of PTC cells on macrophage polarization through exosomal ANXA1 secretion and its implications for tumor progression. Exosomes in PTC cells were examined using transmission electron microscopy, exosome labeling, and nanoparticle tracking analysis. Real-time quantitative polymerase chain reaction was employed to quantify gene expression levels. Protein levels were determined through Western blot analysis. The interplay between genes was assessed using luciferase reporter and RNA pull-down assays. Functional experiments were conducted to investigate PTC cell proliferation and apoptosis. Our findings reveal that ANXA1 promotes PTC cell proliferation and inhibits apoptosis. Exosomes derived from PTC cells were found to promote macrophage M2 polarization. ANXA1 stimulates M2 polarization through the activation of the PI3K/AKT pathway. MicroRNA-374a-5p (miR-374a-5p) and microRNA-374b-5p (miR-374b-5p) were identified as inhibitors of ANXA1 expression and PI3K/AKT pathway activity, thereby inhibiting macrophage M2 polarization. Furthermore, miR-374a-5p and miR-374b-5p were observed to suppress PTC cell proliferation through their regulatory action on ANXA1. Our study suggests that miR-374a/b-5p inhibits PTC cell growth by blocking the macrophage M2 polarization induced by exosomal ANXA1.

The time-dependent expression of FPR2 and ANXA1 in murine deep vein thrombosis model and its relation to thrombus age.

Thrombus age determination in fatal venous thromboembolism cases is an important task for forensic pathologists. In this study, we investigated the time-dependent expressions of formyl peptide receptor 2 (FPR2) and Annexin A1 (ANXA1) in a stasis-induced deep vein thrombosis (DVT) murine model, with the aim of obtaining useful information for thrombus age timing. A total of 75 ICR mice were randomly classified into thrombosis group and control group. In thrombosis group, a DVT model was established by ligating the inferior vena cava (IVC) of mice, and thrombosed IVCs were harvested at 1, 3, 5, 7, 10, 14, and 21 days after modeling. In control group, IVCs without thrombosis were taken as control samples. The expressions of FPR2 and ANXA1 during thrombosis were detected using immunohistochemistry and double immunofluorescence staining. Their protein and mRNA levels in the samples were determined by Western blotting and quantitative real-time PCR. The results reveal that FPR2 was predominantly expressed by intrathrombotic neutrophils and macrophages. ANXA1 expression in the thrombi was mainly distributed in neutrophils, endothelial cells of neovessels, and fibroblastic cells. After thrombosis, the expressions of FPR2 and ANXA1 were time-dependently up-regulated. The percentage of FPR2-positive cells and the level of FPR2 protein significantly elevated at 1, 3, 5 and 7 days after IVC ligation as compared to those at 10, 14 and 21 days after ligation (p < 0.05). Moreover, the mRNA level of FPR2 were significantly higher at 5 days than that at the other post-ligation intervals (p < 0.05). Besides, the levels of ANXA1 mRNA and protein peaked at 10 and 14 days after ligation, respectively. A significant increase in the mRNA level of ANXA1 was found at 10 and 14 days as compared with that at the other post-ligation intervals (p < 0.01). Our findings suggest that FPR2 and ANXA1 are promising as useful markers for age estimation of venous thrombi.

FMDV 3A Antagonizes the Effect of ANXA1 to Positively Modulate Viral Replication.

The RIG-I-like receptor signaling pathway is crucial for producing type I interferon (IFN-I) against RNA viruses. The present study observed that viral infection increased annexin-A1 (ANXA1) expression, and ANXA1 then promoted RNA virus-induced IFN-I production. Compared to ANXA1 wild-type cells, ANXA1-/- knockout cells showed IFN-ß production decreasing after viral stimulation. RNA virus stimulation induced ANXA1 to regulate IFN-ß production through the TBK1-IRF3 axis but not through the NF-κB axis. ANXA1 also interacted with JAK1 and STAT1 to increase signal transduction induced by IFN-ß or IFN-γ. We assessed the effect of ANXA1 on the replication of foot-and-mouth disease virus (FMDV) and found that ANXA1 inhibits FMDV replication dependent on IFN-I production. FMDV 3A plays critical roles in viral replication and host range. The results showed that FMDV 3A interacts with ANXA1 to inhibit its ability to promote IFN-ß production. We also demonstrated that FMDV 3A inhibits the formation of ANXA1-TBK1 complex. These results indicate that ANXA1 positively regulates RNA virus-stimulated IFN-ß production and FMDV 3A antagonizes ANXA1-promoted IFN-ß production to modulate viral replication. IMPORTANCE FMDV is a pathogen that causes one of the world's most destructive and highly contagious animal diseases. The FMDV 3A protein plays a critical role in viral replication and host range. Although 3A is one of the viral proteins that influences FMDV virulence, its underlying mechanisms remain unclear. ANXA1 is involved in immune activation against pathogens. The present study demonstrated that FMDV increases ANXA1 expression, while ANXA1 inhibits FMDV replication. The results also showed that ANXA1 promotes RNA virus-induced IFN-I production through the IRF3 axis at VISA and TBK1 levels. ANXA1 was also found to interact with JAK1 and STAT1 to strengthen signal transduction induced by IFN-ß and IFN-γ. 3A interacted with ANXA1 to inhibit ANXA1-TBK1 complex formation, thereby antagonizing the inhibitory effect of ANXA1 on FMDV replication. This study helps to elucidate the mechanism underlying the effect of the 3A protein on FMDV replication.

Endothelial CYP2J2 overexpression restores the BRB via METTL3-mediated ANXA1 upregulation.

Blood-retinal barrier (BRB) breakdown is responsible for multiple ocular diseases, such as diabetic retinopathy, age-related macular degeneration, and retinal vascular occlusive diseases. Increased vascular permeability contributes to vasogenic edema and tissue damage, with consequent adverse effects on vision. Herein, we found that endothelial CYP2J2 overexpression maintained BRB integrity after ischemia-reperfusion injury and consequently protected against retinal ganglion cell loss. Oxidative stress repressed endothelial ANXA1 expression in vivo and in vitro. CYP2J2 upregulated methyltransferase-like 3 (METTL3) expression and hence promoted ANXA1 translation via ANXA1 m6 A modification in endothelium under oxidative stress. CYP2J2 maintained the distribution of endothelial tight junctions and adherens junctions in an ANXA1-dependent manner. Endothelial ANXA1 plays an indispensable role in vascular homeostasis and stabilization during development. Endothelial ANXA1 deletion disrupted retinal vascular perfusion as well as BRB integrity. CYP2J2 metabolites restored BRB integrity in the presence of ANXA1. Our findings identified the CYP2J2-METTL3-ANXA1 pathway as a potential therapeutic target for relieving BRB impairments.

Annexin A1 (Ac2-26)-dependent Fpr2 receptor alleviates sepsis-induced acute kidney injury by inhibiting inflammation and apoptosis in vivo and in vitro.

OBJECTIVES: Excessive inflammatory responses and apoptosis are critical pathologies that contribute to sepsis-induced acute kidney injury (SI-AKI). Annexin A1 (ANXA1), a member of the calcium-dependent phospholipid-binding protein family, protects against SI-AKI through its anti-inflammatory and antiapoptotic effects, but the underlying mechanisms are still largely unknown. METHODS: In vivo, SI-AKI mouse models were established via caecal ligation and puncture (CLP) and were then treated with the Ac2-26 peptide of ANXA1 (ANXA1 (Ac2-26)), WRW4 (Fpr2 antagonist) or both. In vitro, HK-2 cells were induced by lipopolysaccharide (LPS) and then treated with ANXA1 (Ac2-26), Fpr2-siRNA or both. RESULTS: In the present study, we found that the expression levels of ANXA1 were decreased, and the expression levels of TNF-α, IL-1ß, IL-6, cleaved caspase-3, cleaved caspase-8 and Bax were significantly increased, accompanied by marked kidney tissue apoptosis in vivo. Moreover, we observed that ANXA1 (Ac2-26) significantly reduced the levels of TNF-α, IL-1ß and IL-6 and cleaved caspase-3, cleaved caspase-8, FADD and Bax and inhibited apoptosis in kidney tissue and HK-2 cells, accompanied by pathological damage to kidney tissue. Seven-day survival, kidney function and cell viability were significantly improved in vivo and in vitro, respectively. Furthermore, the administration of ANXA1 (Ac2-26) inhibited the CLP- or LPS-induced phosphorylation of PI3K and AKT and downregulated the level of NF-κB in vivo and in vitro. Moreover, our data demonstrate that blocking the Fpr2 receptor by the administration of WRW4 or Fpr2-siRNA reversed the abovementioned regulatory role of ANXA1, accompanied by enhanced phosphorylation of PI3K and AKT and upregulation of the level of NF-κB in vivo and in vitro. CONCLUSIONS: Taken together, this study provides evidence that the protective effect of ANXA1 (Ac2-26) on SI-AKI largely depends on the negative regulation of inflammation and apoptosis via the Fpr2 receptor.

Deletion of Annexin A1 in Mice Upregulates the Expression of Its Receptor, Fpr2/3, and Reactivity to the AnxA1 Mimetic Peptide in Platelets.

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.

Identification and prognostic analysis of biomarkers to predict the progression of pancreatic cancer patients.

BACKGROUND: Pancreatic cancer (PC) is a malignancy with a poor prognosis and high mortality. Surgical resection is the only "curative" treatment. However, only a minority of patients with PC can obtain surgery. Improving the overall survival (OS) rate of patients with PC is still a major challenge. Molecular biomarkers are a significant approach for diagnostic and predictive use in PCs. Several prediction models have been developed for patients newly diagnosed with PC that is operable or patients with advanced and metastatic PC; however, these models require further validation. Therefore, precise biomarkers are urgently required to increase the efficiency of predicting a disease-free survival (DFS), OS, and sensitivity to immunotherapy in PC patients and to improve the prognosis of PC. METHODS: In the present study, we first evaluated the highly and selectively expressed targets in PC, using the GeoMxTM Digital Spatial Profiler (DSP) and then, we analyzed the roles of these targets in PCs using TCGA database. RESULTS: LAMB3, FN1, KRT17, KRT19, and ANXA1 were defined as the top five upregulated targets in PC compared with paracancer. The TCGA database results confirmed the expression pattern of LAMB3, FN1, KRT17, KRT19, and ANXA1 in PCs. Significantly, LAMB3, FN1, KRT19, and ANXA1 but not KRT17 can be considered as biomarkers for survival analysis, univariate and multivariate Cox proportional hazards model, and risk model analysis. Furthermore, in combination, LAMB3, FN1, KRT19, and ANXA1 predict the DFS and, in combination, LAMB3, KRT19, and ANXA1 predict the OS. Immunotherapy is significant for PCs that are inoperable. The immune checkpoint blockade (ICB) analysis indicated that higher expressions of FN1 or ANXA1 are correlated with lower ICB response. In contrast, there are no significant differences in the ICB response between high and low expression of LAMB3 and KRT19. CONCLUSIONS: In conclusion, LAMB3, FN1, KRT19, and ANXA1 are good predictors of PC prognosis. Furthermore, FN1 and ANXA1 can be predictors of immunotherapy in PCs.

Annexin A1 restores cerebrovascular integrity concomitant with reduced amyloid-ß and tau pathology.

Alzheimer's disease, characterized by brain deposits of amyloid-ß plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood-brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer's disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood-brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-ß levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood-brain barrier function and decreasing amyloid-ß and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood-brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-ß load, due to increased clearance and degradation of amyloid-ß by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood-brain barrier breakdown early in Alzheimer's disease can be restored by hrANXA1 as a potential therapeutic approach.

Four Chemotherapeutic Compounds That Limit Blood-Brain-Barrier Invasion by Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii, an intracellular protozoan parasite, exists in the host brain as cysts, which can result in Toxoplasmic Encephalitis (TE) and neurological diseases. However, few studies have been conducted on TE, particularly on how to prevent it. Previous proteomics studies have showed that the expression of C3 in rat brains was up-regulated after T. gondii infection. METHODS: In this study, we used T. gondii to infect mice and bEnd 3 cells to confirm the relation between T. gondii and the expression of C3. BEnd3 cells membrane proteins which directly interacted with C3a were screened by pull down. Finally, animal behavior experiments were conducted to compare the differences in the inhibitory ability of TE by four chemotherapeutic compounds (SB290157, CVF, NSC23766, and Anxa1). RESULTS: All chemotherapeutic compounds in this study can inhibit TE and cognitive behavior in the host. However, Anxa 1 is the most suitable material to inhibit mice TE. CONCLUSION: T. gondii infection promotes TE by promoting host C3 production. Anxa1 was selected as the most appropriate material to prevent TE among four chemotherapeutic compounds closely related to C3.

ANXA1 directs Schwann cells proliferation and migration to accelerate nerve regeneration through the FPR2/AMPK pathway.

Many factors are involved in the process of nerve regeneration. Understanding the mechanisms regarding how these factors promote an efficient remyelination is crucial to deciphering the molecular and cellular processes required to promote nerve repair. Schwann cells (SCs) play a central role in the process of peripheral nerve repair/regeneration. Using a model of facial nerve crush injury and repair, we identified Annexin A1 (ANXA1) as the extracellular trigger of SC proliferation and migration. ANXA1 activated formyl peptide receptor 2 (FPR2) receptors and the downstream adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling cascade, leading to SC proliferation and migration in vitro. SCs lacking FPR2 or AMPK displayed a defect in proliferation and migration. After facial nerve injury (FNI), ANXA1 promoted the proliferation of SCs and nerve regeneration in vivo. Collectively, these data identified the ANXA1/FPR2/AMPK axis as an important pathway in SC proliferation and migration. ANXA1-induced remyelination and SC proliferation promotes FNI regeneration.

Maintenance of cellular annexin A1 level is essential for PI3K/AKT/mTOR-mediated proliferation of pancreatic beta cells.

Annexin A1 (AnxA1, also known as lipocortin-1), is a calcium-dependent phospholipid binding protein with diverse functions. Previous studies have indicated that AnxA1 is associated with age-related ß-cell dysfunction and aging, which lead to decreased ß-cell proliferation capacity. However, it has been uncertain whether AnxA1 affects the proliferation of pancreatic beta (ß) cells. In the present study, we reduced AnxA1 expression in the MIN6 islet ß-cell line using small interfering RNA (AnxA1-siRNA), then measured cell cycle distribution and cellular proliferation. We also measured the expression levels of cell cycle-related proteins such as cyclin D1, cyclin E, and cyclin-dependent kinase 2 (CDK2) by Western blot analysis. We investigated the phosphatidylinositol 3-kinase (PI3K)/ serine/threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway to explore the potential mechanism underlying the observed effects. Knockdown of AnxA1 expression using siRNA reduced the rates of MIN6 cell proliferation. The proportions of cells in S and G2/M phases also decreased upon inhibition of AnxA1. Moreover, AnxA1 protein expression in MIN6 cells was positively related to the protein levels of cyclin D1, cyclin E, and CDK2. Activation of the PI3K/Akt/mTOR signaling pathway by AnxA1 may be involved in the signaling cascade to regulate cell proliferation. This study identified a positive correlation between AnxA1 protein and pancreatic ß-cell proliferation. AnxA1 protein expression might affect the proliferation of MIN6 cells via regulation of cyclin D1, cyclin E, and CDK2 proteins, as well as the PI3K/Akt/mTOR signaling pathway.

ANXA1-GSK3ß interaction and its involvement in NSCLC metastasis.

Although initially discovered and extensively studied for its role in inflammation, Annexin A1 (ANXA1) has been reported to be closely related to cancer in recent years, and its role in cancer is specific to tumor types and tissues. In the present study, we identified ANXA1 as an interaction partner of glycogen synthase kinase 3 beta (GSK3ß), a multi-functional serine/threonine kinase tightly associated with cell fate determination and cancer, and assessed the functional significance of GSK3ß-ANXA1 interaction in the metastasis of non-small cell lung cancer (NSCLC). We confirmed the interaction between GSK3ß and ANXA1 in vitro and in H1299 and A549 cells by Glutathione-S-transferase (GST) pull-down assay and co-immunoprecipitation. We found that ANXA1 negatively regulated the phosphorylation of GSK3ß and inhibited the epithelial-mesenchymal transformation (EMT) process and migration and invasion of NSCLC cells. By functional rescue assay, we confirmed that ANXA1 inhibited EMT through the regulation of GSK3ß activity and thereby inhibited the migration and invasion of NSCLC cells. Our study sheds light on the function of ANXA1 and GSK3ß and provides new elements for the understanding of NSCLC pathogenesis.

Induction of cell death and modulation of Annexin A1 by phytoestrogens in human leukemic cell lines.

BACKGROUND: Phytoestrogens are polyphenolic plant compounds which are structurally similar to the endogenous mammalian estrogen, 17ß-estradiol. Annexin A1 (ANXA1) is an endogenous protein which inhibits cyclo-oxygenase 2 (COX-2) and phospholipase A2, signal transduction, DNA replication, cell transformation, and mediation of apoptosis. OBJECTIVE: This study aimed to determine the effects of selected phytoestrogens on annexin A1 (ANXA1) expression, mode of cell death and cell cycle arrest in different human leukemic cell lines. METHODS: Cells viability were examined by MTT assay and ANXA1 quantification via Enzyme-linked Immunosorbent Assay. Cell cycle and apoptosis were examined by flow cytometer and phagocytosis effect was evaluated using haematoxylin-eosin staining. RESULTS: Coumestrol significantly (p < 0.05) reduced the total level of ANXA1 in both K562 and U937 cells and genistein significantly (p < 0.05) reduced it in K562, Jurkat and U937 cells, meanwhile estradiol and daidzein induced similar reduction in U937 and Jurkat cells. Coumestrol and daidzein induced apoptosis in K562 and Jurkat cells, while genistein and estradiol induced apoptosis in all tested cells. Coumestrol and estradiol induced cell cycle arrest at G2/M phase in K562 and Jurkat cells with an addition of U937 cells for estradiol. Genistein induced cell cycle arrest at S phase for both K562 and Jurkat cells. However, daidzein induced cell cycle arrest at G0/G1 phase in K562, and G2/M phase of Jurkat cells. Coumestrol, genistein and estradiol induced phagocytosis in all tested cells but daidzein induced significant (p < 0.05) phagocytosis in K562 and Jurkat cells only. CONCLUSION: The selected phytoestrogens induced cell cycle arrest, apoptosis and phagocytosis and at the same time they reduced ANXA1 level in the tested cells. The IC50 value of phytoestrogens was undetectable at the concentrations tested, their ability to induce leukemic cells death may be related with their ability to reduce the levels of ANXA1. These findings can be used as a new approach in cancer treatment particularly in leukemia.

The crosstalk of ABCA1 and ANXA1: a potential mechanism for protection against atherosclerosis.

Atherosclerosis, characterized by the formation of fat-laden plaques, is a chronic inflammatory disease. ABCA1 promotes cholesterol efflux, reduces cellular cholesterol accumulation, and regulates anti-inflammatory activities in an apoA-I- or ANXA1-dependent manner. The latter activity occurs by mediating the efflux of ANXA1, which plays a critical role in anti-inflammatory effects, cholesterol transport, exosome and microparticle secretion, and apoptotic cell clearance. ApoA-I increases ANXA1 expression via the ERK, p38MAPK, AKT, and PKC pathways. ApoA-I regulates the signaling pathways by binding to ABCA1, suggesting that apoA-I increases ANXA1 expression by binding to ABCA1. Furthermore, ANXA1 may increase ABCA1 expression. ANXA1 increases PPARγ expression by modulating STAT6 phosphorylation. PPARγ also increases ANXA1 expression by binding to the promoter of ANXA1. Therefore, ABCA1, PPARγ, and ANXA1 may form a feedback loop and regulate each other. Interestingly, the ANXA1 needs to be externalized to the cell membrane or secreted into the extracellular fluids to exert its anti-inflammatory properties. ABCA1 transports ANXA1 from the cytoplasm to the cell membrane by regulating lipidization and serine phosphorylation, thereby mediating ANXA1 efflux, likely by promoting microparticle and exosome release. The direct role of ABCA1 expression and ANXA1 release in atherosclerosis has been unclear. In this review, we focus on the role of ANXA1 in atheroprogression and its novel interaction with ABCA1, which may be useful for providing basic knowledge for the development of novel therapeutic targets for atherosclerosis and cardiovascular disease.

Reduced expression of annexin A1 promotes gemcitabine and 5-fluorouracil drug resistance of human pancreatic cancer.

Intrinsic chemoresistance is the main reason for the failure of human pancreatic ductal adenocarcinoma (PDAC) therapy. To identify the candidate protein, we compared the protein expression profiling of PDAC cells and its distinct surviving cells following primary treatment with gemcitabine (GEM) and 5-fluorouracil (5-FU) by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry or mass spectrometry. A total of 20 differentially expressed proteins were identified, and annexin A1 (ANXA1) was analyzed for further validation. The functional validation showed that the downregulation of ANXA1 contributes to GEM and 5-FU resistance in PDAC cells through protein kinase C/c-Jun N-terminal kinase/P-glycoprotein signaling pathway. Our findings provide a platform for the further elucidation of the underlying mechanisms of PDAC intrinsic chemoresistance and demonstrated that ANXA1 may be a valid marker for anticancer drug development.