The protective effects of annexin A1 against oxidized-LDL-induced monocytes adhesion to endothelial cells: implication in atherosclerosis.

Oxidized low-density lipoprotein (ox-LDL)-associated endothelial dysfunction is a critical factor in the initiation and progression of Atherosclerosis (AS). Annexin A1 is an important member of the annexin family. Despite its wide range of biological functions across various tissues and cells, the role of Annexin A1 in AS remains largely unexplored. In this study, we demonstrate that Annexin A1 treatment effectively reduced the expression of LOX-1 at both the mRNA and protein levels in HUVECs exposed to ox-LDL. Annexin A1 also ameliorated oxidative stress (OS) by decreasing mitochondrial ROS levels and restoring reduced GSH levels. Moreover, Annexin A1 decreased the expression of pro-inflammatory cytokines, including IL-6 and MCP-1. Importantly, Annexin A1 inhibited ox-LDL-induced expressions of the endothelial adhesion molecules, such as E-selectin and VCAM-1 in HUVECs, which leads to reduced attachment of THP-1 monocytes to HUVECs. Mechanically, we found that Annexin A1 reversed the expression of KLF2 against ox-LDL mediated by the PI3K/Akt axis. Notably, the silencing of KLF2 abrogated the protective effects of Annexin A1 on E-selectin and VCAM-1 expression and the attachment of THP-1 monocytes to HUVECs. Our findings suggest that Annexin A1 is a potential therapeutic agent for atherosclerosis, offering a novel approach to mitigate endothelial dysfunction and inflammation.

CAM-A-dependent HBV core aggregation induces apoptosis through ANXA1.

Background & Aims: Chronic HBV infection is the leading cause of liver disease and of hepatocellular carcinoma. The improvement of antiviral therapy remains an unmet medical need. Capsid assembly modulators (CAMs) target the HBV core antigen (HBc) and inhibit HBV replication. Although CAM-A compounds are well-known inducers of aberrant viral capsid aggregates, their mechanisms of action in HBV-hepatocyte interactions are poorly understood. Recently, we demonstrated that CAM-A molecules lead to a sustained reduction of HBsAg in the serum of HBV replicating mice and induce HBc aggregation in the nucleus of HBc-expressing cells leading to cell death. Methods: The mechanism of action by which CAM-A compounds induce cell death was investigated using an HBV infection model, HBc-overexpressing HepG2-NTCP cells, primary human hepatocytes, and HBV replicating HepAD38 cells. Results: We first confirmed the decrease in HBsAg levels associated with CAM-A treatment and the induction of cell toxicity in HBV-infected differentiated HepaRG cells. Next, we showed that CAM-A-mediated nuclear aggregation of HBc was associated with cell death through the activation of apoptosis. Transcriptomic analysis was used to investigate the mechanism of action driving this phenotype. CAM-A-induced HBc nuclear aggregation led to the upregulation of ANXA1 expression, a documented driver of apoptosis. Finally, silencing of ANXA1 expression delayed cell death and apoptosis in CAM-A-treated cells, confirming its direct involvement in CAM-A-induced cell death. Conclusions: Our results unravel a previously undiscovered mechanism of action involving CAM-As and open the door to new therapeutic strategies involving CAM to achieve a functional cure in patients with chronic infections. Impact and implications: Chronic HBV infection is a global health threat. To date, no treatment achieves viral clearance in chronically infected patients. In this study, we characterized a new mechanism of action of an antiviral molecule targeting the assembly of the viral capsid (CAM). The study demonstrated that a CAM subtype, CAM-A-induced formation of aberrant structures from HBV core protein aggregates in the nucleus leading to cell death by ANXA1-driven apoptosis. Thus, CAM-A treatment may lead to the specific elimination of HBV-infected cells by apoptosis, paving the way to novel therapeutic strategies for viral cure.

Identification of ANXA1 as a Novel Upstream Negative Regulator of Notch1 Function in AML.

Abnormal Notch1 expression has an important role in tumorigenesis. However, upstream control mechanisms for Notch1 are still insufficiently understood. Acute myeloid leukemia (AML) is one of the most common and lethal blood malignancies with limited possibilities for treatment. Thus, new therapeutic targets are urgently needed to improve current ineffective therapies. Herein, high Annexin A1 (ANXA1) expression is found correlated with hyperproliferation of AML cells, and then ANXA1 is identified as a novel negative regulator of Notch1 function in AML. Mechanistically, ANXA1 directly bound to the intracellular domain of Notch1 (NICD) to target this tumor suppressor for degradation. Furthermore, NICD executed its tumor suppressive function through activation of the p15 promoter. Thus, ablation of the Notch1-p15-mediated tumor suppression by ANXA1 provided a novel mechanism of AML proliferation. In human AML patients, a mutual exclusive relation is discovered between ANXA1 and Notch1/p15, corroborating mechanistic discovery. On the basis of these results, it is reasonably speculated that targeting ANXA1 would provide an effective approach for treatment of AML. In support of this new therapeutic paradigm, provided proof-of-concept data by antagonizing ANXA1 using NICD inhibitory peptides.

GPCRs overexpression and impaired fMLP-induced functions in neutrophils from chronic kidney disease patients.

Introduction: G-protein coupled receptors (GPCRs) expressed on neutrophils regulate their mobilization from the bone marrow into the blood, their half-live in the circulation, and their pro- and anti-inflammatory activities during inflammation. Chronic kidney disease (CKD) is associated with systemic inflammatory responses, and neutrophilia is a hallmark of CKD onset and progression. Nonetheless, the role of neutrophils in CKD is currently unclear. Methods: Blood and renal tissue were collected from non-dialysis CKD (grade 3 - 5) patients to evaluate GPCR neutrophil expressions and functions in CKD development. Results: CKD patients presented a higher blood neutrophil-to-lymphocyte ratio (NLR), which was inversely correlated with the glomerular filtration rate (eGFR). A higher frequency of neutrophils expressing the senescent GPCR receptor (CXCR4) and activation markers (CD18+CD11b+CD62L+) was detected in CKD patients. Moreover, CKD neutrophils expressed higher amounts of GPCR formyl peptide receptors (FPR) 1 and 2, known as neutrophil pro- and anti-inflammatory receptors, respectively. Cytoskeletal organization, migration, and production of reactive oxygen species (ROS) by CKD neutrophils were impaired in response to the FPR1 agonist (fMLP), despite the higher expression of FPR1. In addition, CKD neutrophils presented enhanced intracellular, but reduced membrane expression of the protein Annexin A1 (AnxA1), and an impaired ability to secrete it into the extracellular compartment. Secreted and phosphorylated AnxA1 is a recognized ligand of FPR2, pivotal in anti-inflammatory and efferocytosis effects. CKD renal tissue presented a low number of neutrophils, which were AnxA1+. Conclusion: Together, these data highlight that CKD neutrophils overexpress GPCRs, which may contribute to an unbalanced aging process in the circulation, migration into inflamed tissues, and efferocytosis.

Annexin A1 protects periodontal ligament cells against lipopolysaccharide-induced inflammatory response and cellular senescence: An implication in periodontitis.

Periodontitis is an inflammatory condition that affects the tooth-supporting structures, triggered by the host's immune response toward the bacterial deposits around the teeth. Annexin A1 (AnxA1), a vital member of the annexin superfamily, is known for its diverse physiological functions, particularly its anti-inflammatory and anti-senescence properties. We hypothesized that AnxA1 has a protective effect against lipopolysaccharide (LPS)-induced inflammatory responses and cellular damage in periodontal ligament cells (PDLCs). In this study, we demonstrate that LPS stimulation significantly reduced telomerase activity in PDLCs, a decline that was dose-dependently reversed by AnxA1. Importantly, AnxA1 protected the cells from LPS-induced cellular senescence and the downregulation of human telomerase reverse transcriptase (hTERT) expression. In line with this, AnxA1 suppressed the LPS-induced expression of p21 and p16 at both the mRNA and protein levels. Furthermore, AnxA1 demonstrated potent anti-inflammatory effects by inhibiting the secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). It also mitigated LPS-induced oxidative stress by reducing the levels of phosphorylated Foxo3a (Ser253) and restored sirtuin 1 (SIRT1) expression. Notably, SIRT1 silencing abolished AnxA1's protective effects on Foxo3a phosphorylation and cellular senescence, suggesting that SIRT1 mediates AnxA1's actions. In conclusion, AnxA1 protected PDLCs against LPS-triggered inflammation and cell senescence by activating SIRT1 signal pathway. These findings indicate that AnxA1 could serve as a promising therapeutic strategy for the treatment of periodontitis.

Mesenchymal stromal cells and their secretory products reduce the inflammatory crosstalk between islets and endothelial cells.

PURPOSE: Preculturing isolated islets with Mesenchymal Stromal Cells (MSCs) improves their functional survival in vitro and subsequent transplantation outcomes in vivo. The MSC secretory product Annexin A1 (ANXA1) is a key modulator of MSC-mediated improvements in islet function. The current study aims to determine the influence of MSCs and defined MSC secretory products, including ANXA1, on the inflammatory crosstalk between isolated islets and Endothelial Cells (ECs), using in vitro models of the clinically-preferred intraportal islet transplantation niche. METHODS: Islets were cultured alone, with MSCs, or with MSC secretory products and exposed to pro-inflammatory cytokines. Islet gene expression of C-C Motif Chemokine Ligand 2 (CCL2), C-X-C Motif Chemokine Ligand (CXCL)-10 (CXCL10) and CXCL1 were assessed by RT-qPCR. EC activation was induced with 100 U/ml TNF for 24 h. Islet-EC co-cultures were used to determine the influence of MSCs, or MSC secretory products on the inflammatory crosstalk between isolated islets and ECs. VCAM-1 and ICAM-1 expression were assessed at the mRNA and protein level in ECs, using RT-qPCR and immunofluorescence. RESULTS: MSCs reduce pro-inflammatory cytokine-induced islet CCL2, CXCL10, and CXCL1 gene expression, which is partially mimicked by ANXA1. MSCs and ANXA1 have a similar capacity to reduce TNF-induced EC activation. Isolated islets exacerbate TNF-induced EC activation. Preculturing islets with MSCs reduces islet-exacerbated EC activation. ANXA1 reduces islet-exacerbated EC activation, when present during the islet preculture and islet-EC co-culture period. CONCLUSION: MSC-derived secretory factors, including ANXA1, may be used in islet transplantation protocols to target donor islet and host EC inflammation at the intraportal niche.

Cancer stem cells release interleukin-33 within large oncosomes to promote immunosuppressive differentiation of macrophage precursors.

In squamous cell carcinoma (SCC), macrophages responding to interleukin (IL)-33 create a TGF-ß-rich stromal niche that maintains cancer stem cells (CSCs), which evade chemotherapy-induced apoptosis in part via activation of the NRF2 antioxidant program. Here, we examined how IL-33 derived from CSCs facilitates the development of an immunosuppressive microenvironment. CSCs with high NRF2 activity redistributed nuclear IL-33 to the cytoplasm and released IL-33 as cargo of large oncosomes (LOs). Mechanistically, NRF2 increased the expression of the lipid scramblase ATG9B, which exposed an "eat me" signal on the LO surface, leading to annexin A1 (ANXA1) loading. These LOs promoted the differentiation of AXNA1 receptor+ myeloid precursors into immunosuppressive macrophages. Blocking ATG9B's scramblase activity or depleting ANXA1 decreased niche macrophages and hindered tumor progression. Thus, IL-33 is released from live CSCs via LOs to promote the differentiation of alternatively activated macrophage, with potential relevance to other settings of inflammation and tissue repair.

Implication of the Annexin 1/FPR axis in leishmanial exosome-mediated Leishmania major skin hyperpathogenesis.

Introduction: Exosomes produced by the protozoan parasite Leishmania (LeishEXO) are well-established drivers of virulence, though mechanisms underlying their exacerbation of experimental leishmaniasis remain elusive. Expression of Annexin A1 (ANXA1), a protein implicated in exosome-mediated pathologies and viral internalization, has been shown to correlate with cutaneous leishmaniasis severity. Given ANXA1's regulation of myeloid cells - the canonical hosts for Leishmania - we studied the potential role of ANXA1 and its receptors FPR1/2 in exerting LeishEXO's effects. Methods: Murine and in vitro ANXA1-/- models were used to study the generation of protective TH1 responses during experimental L. major infection with and without LeishEXO. Recruitment of inflammatory cells was assessed using a peritoneal cell recruitment assay and immunophenotyping, and production of inflammatory mediators was measured using a cytokine and chemokine array. Treatment of experimental models with FPR2 antagonist WRW4 and FPR1/2 agonist WKYMVm was used to delineate the role of the FPR/ANXA1 axis in LeishEXO-mediated hyperpathogenesis. Results: We established that ANXA1 deficiency prohibits LeishEXO-mediated pathogenesis and myeloid cell infection, with minimal alterations to adaptive and innate immune phenotypes. FPR2 blockade with WRW4 similarly inhibited leishmanial hyperpathogenesis, while direct activation of FPRs with WKYMVm enhanced infection and recapitulated the LeishEXO-mediated phenotype. This research describes LeishEXO's utilization of the ANXA1/FPR axis to facilitate parasitic internalization and pathogenesis, which may be leveraged in the development of therapeutics for leishmaniasis.

Cracking the code of Annexin A1-mediated chemoresistance.

The annexin superfamily protein, Annexin A1, initially recognized for its glucocorticoid-induced phospholipase A2-inhibitory activities, has emerged as a crucial player in diverse cellular processes, including cancer. This review explores the multifaceted roles of Anx-A1 in cancer chemoresistance, an area largely unexplored. Anx-A1's involvement in anti-inflammatory processes, its complex phosphorylation patterns, and its context-dependent switch from anti-to pro-inflammatory in cancer highlights its intricate regulatory mechanisms. Recent studies highlight Anx-A1's paradoxical roles in different cancers, exhibiting both up- and down-regulation in a tissue-specific manner, impacting different hallmark features of cancer. Mechanistically, Anx-A1 modulates drug efflux transporters, influences cancer stem cell populations, DNA damages and participates in epithelial-mesenchymal transition. This review aims to explore Anx-A1's role in chemoresistance-associated pathways across various cancers, elucidating its impact on survival signaling cascades including PI3K/AKT, MAPK/ERK, PKC/JNK/P-gp pathways and NFκ-B signalling. This review also reveals the clinical implications of Anx-A1 dysregulation in treatment response, its potential as a prognostic biomarker, and therapeutic targeting strategies, including the promising Anx-A1 N-terminal mimetic peptide Ac2-26. Understanding Anx-A1's intricate involvement in chemoresistance offers exciting prospects for refining cancer therapies and improving treatment outcomes.

Annexin A1 protects epidermal stem cells against ultraviolet-B irradiation-induced mitochondrial dysfunction.

Ultraviolet-B (UV-B) radiation overexposure causes function impairment of epidermal stem cells (ESCs). We explored the mechanism of Annexin A1 (ANXA1) ameliorating UV-B-induced ESC mitochondrial dysfunction/cell injury. ESCs were cultured in vitro and irradiated with different doses of UV-B. Cell viability/ANXA1 protein level were assessed. After oe-ANXA1 transfection, ESCs were treated with oe-ANXA1/UV-B irradiation/CCCP/CCG-1423/3-methyladenine for 12 h. Cell viability/death, and adenosine triphosphate (ATP)/reactive oxygen species (ROS) levels were determined. Mitochondrial membrane potential (MMP) changes/DNA (mtDNA) content/oxygen consumption and RhoA activation were assessed. ROCK1/p-MYPT1/MYPT1/(LC3BII/I)/Beclin-1/p62 protein levels were determined. Mitochondrial morphology was observed. Mito-Tracker Green (MTG) and LC3B levels were determined. UV-B irradiation decreased cell viability/ANXA1 expression in a dose-dependent manner. UV-B-treated ESCs exhibited reduced cell viability/ATP content/MMP level/mitochondrial respiratory control ratio/mtDNA number/RhoA activity/MYPT1 phosphorylation/MTG+LC3B+ cells/(LC3BII/I) and Beclin-1 proteins, increased cell death/ROS/p62/IL-1ß/IL-6/TNF-α expression, contracted mitochondrial, disappeared mitochondrial cristae, and increased vacuolar mitochondria, which were averted by ANXA1 overexpression, suggesting that UV-B induced ESC mitochondrial dysfunction/cell injury/inflammation by repressing mitophagy, but ANXA1 promoted mitophagy by activating the RhoA/ROCK1 pathway, thus repressing UV-B's effects. Mitophagy activation ameliorated UV-B-caused ESC mitochondrial dysfunction/cell injury/inflammation. Mitophagy inhibition partly diminished ANXA1-ameliorated UV-B's effects. Conjointly, ANXA1 promoted mitophagy by activating the RhoA/ROCK1 pathway, thereby improving UV-B-induced ESC mitochondrial dysfunction/cell injury.

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.

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.

Development of Ac2-26 Mesoporous Microparticle System as a Potential Therapeutic Agent for Inflammatory Bowel Diseases.

Introduction: Inflammatory bowel diseases (IBDs) disrupt the intestinal epithelium, leading to severe chronic inflammation. Current therapies cause adverse effects and are expensive, invasive, and ineffective for most patients. Annexin A1 (AnxA1) is a pivotal endogenous anti-inflammatory and tissue repair protein in IBD. Nanostructured compounds loading AnxA1 or its active N-terminal mimetic peptides improve IBD symptomatology. Methods: To further explore their potential as a therapeutic candidate, the AnxA1 N-terminal mimetic peptide Ac2-26 was incorporated into SBA-15 ordered mesoporous silica and covered with EL30D-55 to deliver it by oral treatment into the inflamed gut. Results: The systems SBA-Ac2-26 developed measurements revealed self-assembled rod-shaped particles, likely on the external surface of SBA-15, and 88% of peptide incorporation. SBA-15 carried the peptide Ac2-26 into cultured Raw 264.7 macrophages and Caco-2 epithelial cells. Moreover, oral administration of Eudragit-SBA-15-Ac2-26 (200 µg; once a day; for 4 days) reduced colitis clinical symptoms, inflammation, and improved epithelium recovery in mice under dextran-sodium sulfate-induced colitis. Discussion: The absorption of SBA-15 in gut epithelial cells is typically low; however, the permeable inflamed barrier can enable microparticles to cross, being phagocyted by macrophages. These findings suggest that Ac2-26 is successfully delivered and binds to its receptors in both epithelial and immune cells, aligning with the clinical results. Conclusion: Our findings demonstrate a simple and cost-effective approach to delivering Ac2-26 orally into the inflamed gut, highlighting its potential as non-invasive IBD therapy.

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 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.

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.

ANXA1sp attenuates sepsis-induced myocardial injury by promoting mitochondrial biosynthesis and inhibiting oxidative stress and autophagy via SIRT3 upregulation.

Sepsis-induced myocardial injury is one of the most difficult complications of sepsis in intensive care units. Annexin A1 (ANXA1) short peptide (ANXA1sp) protects organs during the perioperative period. However, the protective effect of ANXA1sp against sepsis-induced myocardial injury remains unclear. We aimed to explore the protective effects and mechanisms of ANXA1sp against sepsis-induced myocardial injury both in vitro and in vivo. Cellular and animal models of myocardial injury in sepsis were established with lipopolysaccharide. The cardiac function of mice was assessed by high-frequency echocardiography. Elisa assay detected changes in inflammatory mediators and markers of myocardial injury. Western blotting detected autophagy and mitochondrial biosynthesis-related proteins. Autophagic flux changes were observed by confocal microscopy, and autophagosomes were evaluated by TEM. ATP, SOD, ROS, and MDA levels were also detected.ANXA1sp pretreatment enhanced the 7-day survival rate, improved cardiac function, and reduced TNF-α, IL-6, IL-1ß, CK-MB, cTnI, and LDH levels. ANXA1sp significantly increased the expression of sirtuin-3 (SIRT3), mitochondrial biosynthesis-related proteins peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM). ANXA1sp increased mitochondrial membrane potential (△Ψm), ATP, and SOD, and decreased ROS, autophagy flux, the production of autophagosomes per unit area, and MDA levels. The protective effect of ANXA1sp decreased significantly after SIRT3 silencing in vitro and in vivo, indicating that the key factor in ANXA1sp's protective role is the upregulation of SIRT3. In summary, ANXA1sp attenuated sepsis-induced myocardial injury by upregulating SIRT3 to promote mitochondrial biosynthesis and inhibit oxidative stress and autophagy.

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.

Analysis of clinical cure outcome, macrophages number, cytokines levels and expression of annexin-A1 in the cutaneous infection in patients with Leishmania braziliensis

ABSTRACT Background: Leishmania braziliensis, a protozoan prevalent in Brazil, is the known causative agent of cutaneous leishmaniasis (CL). The activation of M1 macrophages is a pivotal factor in the host's ability to eliminate the parasite, whereas M2 macrophages may facilitate parasite proliferation. This study analyzed the clinical outcomes of CL and the patients' immunological profiles, focusing on the prevalence of M1 and M2 macrophages, cytokine production, and annexin-A1 (ANXA1) expression in the lesion. Methods: Data were obtained by polymerase chain reaction (PCR) and histopathological, immunofluorescence, and cytokine analyses. Results: Patients with exudative and cellular reaction-type (ECR)-type lesions that healed within 90 days showed a significant increase in M1. Conversely, patients with ECR and exudative and granulomatous reaction (EGR)types, who healed within 180 days, showed an elevated number of M2. Cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α were higher in ECR lesions that resolved within 90 days (P<0.05). In contrast, IL-9 and IL-10 levels significantly increased in both ECR and EGR lesions that healed after 180 days (P<0.001). The production of IL-21, IL-23 and TGF-β was increased in patients with ECR or EGR lesions that healed after 180 days (P<0.05). The expression of ANXA1 was higher in M2 within ECR-type lesions in patients who healed after 180 days (P<0.05). Conclusions: These findings suggest that the infectious microenvironment induced by L. braziliensis affects the differentiation of M1 and M2 macrophages, cytokine release, and ANXA1 expression, thereby influencing the healing capacity of patients. Therefore, histopathological and immunological investigations may improve the selection of CL therapy.

A cell-penetrating peptide exerts therapeutic effects against ischemic stroke by mediating the lysosomal degradation of sirtuin 5.

Stroke is a major public health concern worldwide. The lack of effective therapies heightens the need for new therapeutic agents. Previous study identified sirtuin 5 (SIRT5) as a positive regulator of microglia-induced excessive neuroinflammation following ischemic stroke. Interventions targeting SIRT5 should therefore alleviate neuroinflammation and protect against ischemic stroke. Here, we synthesized a membrane-permeable peptide specifically bound to SIRT5 through a chaperone-mediated autophagy targeting motif (Tat-SIRT5-CTM) and examined its therapeutic effect in vitro and in vivo. First, in primary microglia, Tat-SIRT5-CTM suppressed the binding of SIRT5 with annexin-A1 (ANXA1), leading to SIRT5 degradation and thus inhibition of SIRT5-mediated desuccinylation of ANXA1, followed by increased membrane accumulation and secretion of ANXA1. These changes, in turn, alleviated microglia-induced neuroinflammation. Moreover, following intravenous injection, Tat-SIRT5-CTM could efficiently pass through the blood‒brain barrier. Importantly, systemic administration of Tat-SIRT5-CTM reduced the brain infarct area and neuronal loss, mitigated neurological deficit scores, and improved long-term neurological functions in a mouse model of ischemic stroke. Furthermore, no toxicity was observed when high doses Tat-SIRT5-CTM were injected into nonischemic mice. Collectively, our study reveals the promising efficacy of the peptide-directed lysosomal degradation of SIRT5 and suggests it as an effective therapeutic approach for the treatment of ischemic stroke.