RESUMO
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.
Assuntos
Anexina A1 , Anexina A2 , Sulfonamidas , Anexina A1/química , Anexina A1/metabolismo , Anexina A2/química , Anexina A2/metabolismo , Simulação de Dinâmica Molecular , Proteínas S100/química , Proteínas S100/metabolismoRESUMO
Annexin A1 (AnxA1) is the primary mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca2+] ([Ca2+]i) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12, and Ac9-25. The increase in [Ca2+]i caused by AnxA1 and its N-terminal peptides in goblet cells was measured to determine the formyl peptide receptors used by the compounds and the action of the peptides on histamine stimulation. Changes in [Ca2+]i were determined by using a fluorescent Ca2+ indicator. AnxA1 and its peptides each activated formyl peptide receptors in goblet cells. AnxA1 and Ac2-26 at 10-12 mol/L and Ac2-12 at 10-9 mol/L inhibited the histamine-stimulated increase in [Ca2+]i, as did resolvin D1 and lipoxin A4 at 10-12 mol/L, whereas Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H1 receptor through the p42/p44 mitogen-activated protein kinase/extracellular regulated kinase 1/2, ß-adrenergic receptor kinase, and protein kinase C pathways, whereas Ac2-12 counter-regulated only through ß-adrenergic receptor kinase. In conclusion, current data show that the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca2+]i and counter-regulation of the H1 receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.
Assuntos
Anexina A1 , Ratos , Animais , Anexina A1/farmacologia , Anexina A1/química , Anexina A1/metabolismo , Células Caliciformes/metabolismo , Receptores de Formil Peptídeo/metabolismo , Histamina/farmacologia , Peptídeos/farmacologia , Anti-Inflamatórios/farmacologia , Quinases de Receptores Adrenérgicos beta/metabolismoRESUMO
This study evaluated the antihyperalgesic and anti-inflammatory effects of percutaneous vagus nerve electrical stimulation (pVNS) by comparing the effects of alternating and random frequencies in an animal model of persistent inflammatory hyperalgesia. The model was induced by Freund's complete adjuvant (CFA) intraplantar (i.pl.) injection. Mice were treated with different protocols of time (10, 20, or 30 min), ear laterality (right, left or both), and frequency (alternating or random). Mechanical hyperalgesia was evaluated, and some groups received i.pl. WRW4 (FPR2/ALX antagonist) to determine the involvement. Edema, paw surface temperature, and spontaneous locomotor activity were evaluated. Interleukin-1ß, IL-6, IL-10, and IL4 levels were verified by enzyme-linked immunosorbent assay. AnxA1, FPR2/ALX, neutrophil, M1 and M2 phenotype macrophage, and apoptotic cells markers were identified using western blotting. The antihyperalgesic effect pVNS with alternating and random frequency effect is depending on the type of frequency, time, and ear treated. The pVNS random frequency in the left ear for 10 min had a longer lasting antihyperalgesic effect, superior to classical stimulation using alternating frequency and the FPR2/ALX receptor was involved in this effect. There was a reduction in the levels of pro-inflammatory cytokines and an increase in the immunocontent of AnxA1 and CD86 in mice paw. pVNS with a random frequency in the left ear for 10 min showed to be optimal for inducing an antihyperalgesic effect. Thus, the random frequency was more effective than the alternating frequency. Therefore, pVNS may be an important adjunctive treatment for persistent inflammatory pain.
Assuntos
Anexina A1 , Animais , Camundongos , Anexina A1/química , Anexina A1/genética , Anexina A1/metabolismo , Estimulação Elétrica , Hiperalgesia/complicações , Hiperalgesia/terapia , Hiperalgesia/metabolismo , Inflamação/complicações , Inflamação/metabolismo , Dor , Receptores de Formil Peptídeo , Nervo Vago/metabolismoRESUMO
AIMS: In this study we evaluated the effect of pharmacological treatment with AnxA1-derived peptide Ac2-26 in an experimental model of toxicity induced by cisplatin. MAIN METHODS: Male rats were divided into Sham (control), Cisplatin (received intraperitoneal injections of 10â¯mg/kg/day of cisplatin for 3â¯days) and Ac2-26 (received intraperitoneal injections of 1â¯mg/kg/day of peptide, 15â¯min before cisplatin) groups. KEY FINDINGS: After 6â¯h of the last dose of cisplatin, an acute inflammatory response was observed characterized by a marked increase in the number of neutrophils and GM-CSF, IL-ß, IL-6, IL-10 and TNF-α plasma levels. These findings were associated with increased AnxA1 protein levels in liver and kidneys, as well as positive AnxA1/Fpr2 circulating leukocytes. Treatment with Ac2-26 produced higher levels of GM-CSF, corroborating the high numbers of neutrophils, and the anti-inflammatory cytokine IL-4. Ac2-26 preserved the morphology of liver structures and increased Fpr1 expression, preventing the damage caused by cisplatin. In the kidneys, Ac2-26 caused downregulation of renal Fpr1 and Fpr2 levels and abrogated the increased levels of the CLU and KIM-1 biomarkers of kidney damage induced by cisplatin. However, no effect of peptide treatment was detected in cisplatin-induced kidney morphology injury. SIGNIFICANCE: Despite activation of the anti-inflammatory AnxA1/Fpr axis during cisplatin administration, treatment with Ac2-26 did not efficiently prevent its deleterious effects on the liver and kidneys.
Assuntos
Anexina A1 , Animais , Anexina A1/química , Anexina A1/metabolismo , Anexina A1/farmacologia , Anti-Inflamatórios/farmacologia , Cisplatino/metabolismo , Cisplatino/toxicidade , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Peptídeos/química , RatosRESUMO
Rapid membrane repair is required to ensure cell survival after rupture of the plasma membrane. The annexin family of proteins is involved in plasma membrane repair (PMR) and is activated by the influx of Ca2+ from the extracellular medium at the site of injury. Annexins A1 and A2 (ANXA1 and ANXA2, respectively) are structurally similar and bind to negatively charged phosphatidylserine (PS) to induce membrane cross-linking and to promote fusion, which are both essential processes that occur during membrane repair. The degree of annexin accumulation and the annexin mobility at cross-linked membranes are important aspects of ANXA1 and ANXA2 function in repair. Here, we quantify ANXA1- and ANXA2-induced membrane cross-linking between giant unilamellar vesicles (GUVs). Time-lapse measurements show that ANXA1 and ANXA2 can induce membrane cross-linking on a time scale compatible with PMR. Cross-linked membrane-membrane interfaces between the GUVs persist in time without fusion, and quantification of confocal microscopy images demonstrates that ANXA1, ANXA2, and, to a lesser extent, PS lipids accumulate at the double membrane interface. Fluorescence recovery after photobleaching shows that the annexins are fully immobilized at the double membrane interface, whereas PS lipids display a 75% decrease in mobility. In addition, the complete immobilization of annexins between two membranes indicates a high degree of network formation between annexins, suggesting that membrane cross-linking is mainly driven by protein-protein interactions.
Assuntos
Anexina A1/química , Anexina A2/química , Membrana Celular/química , Proteínas Imobilizadas/química , Microscopia Confocal , Lipossomas Unilamelares/químicaRESUMO
The Coronavirus Diseases-2019 (COVID-19) pandemic leads many researchers around the world to study the SARS-CoV-s2 infection and pathology to find a treatment for it. This generates a massive production of papers including pre-clinical, clinical and revisions but till now no specific treatment were identified. Meanwhile, like other coronavirus infections, COVID-19 leads to the cytokine storm syndrome resulting in hyperinflammation, exacerbated immune response and multiple organ dysfunctions indicating that drugs that modulate this response, as glucocorticoids could be a treatment option. However glucocorticoids have several side effects or usage limitations. In this sense a drug with anti-inflammatory effects and capable to reduce inflammation but with less after-effects could be a powerful tool to combat COVID-19. Thus the Ac2-26 Mimetic Peptide of Annexin A1 emerges as a possible therapy. The peptide has many anti-inflammatory effects described including the reduction of interleukin (IL)-6, one of the main mediators of cytokine storm syndrome. Therefore the hypothesis to use the Ac2-26 peptide to treat severe COVID-19 will be highlighted in this paper.
Assuntos
Anexina A1/química , Tratamento Farmacológico da COVID-19 , COVID-19/imunologia , COVID-19/terapia , Peptídeos/química , Anti-Inflamatórios/química , Citocinas/metabolismo , Humanos , Inflamação , Interleucina-6/metabolismo , Modelos Teóricos , Ligação ProteicaRESUMO
Overexpression of ANXA1 and EphA2 has been linked to various cancers and both proteins have attracted considerable attention for the development of new anticancer drugs. Here we report that ANXA1 competes with Cbl for binding EphA2 and increases its stability by inhibiting Cbl-mediated EphA2 ubiquitination and degradation in nasopharyngeal carcinoma (NPC). Binding of ANXA1 to EphA2 promoted NPC cell growth and metastasis in vitro and in vivo by elevating EphA2 levels and increasing activity of EphA2 oncogenic signaling (pS897-EphA2). Expression of ANXA1 and EphA2 was positively correlated and both were significantly higher in NPC tissues than in the normal nasopharyngeal epithelial tissues. Patients with high expression of both proteins presented poorer disease-free survival and overall survival relative to patients with high expression of one protein alone. Furthermore, amino acid residues 20-30aa and 28-30aa of the ANXA1 N-terminus bound EphA2. An 11 amino acid-long ANXA1-derived peptide (EYVQTVKSSKG) was developed on the basis of this N-terminal region, which disrupted the connection of ANXA1 with EphA2, successfully downregulating EphA2 expression and dramatically suppressing NPC cell oncogenicity in vitro and in mice. These findings suggest that ANXA1 promotes NPC growth and metastasis via binding and stabilization of EphA2 and present a strategy for targeting EphA2 degradation and treating NPC with a peptide. This therapeutic strategy may also be extended to other cancers with high expression of both proteins. SIGNIFICANCE: These findings show that EphA2 is a potential target for NPC therapeutics and an ANXA1-derived peptide suppresses NPC growth and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4386/F1.large.jpg.
Assuntos
Anexina A1/metabolismo , Efrina-A2/metabolismo , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Animais , Anexina A1/química , Anexina A1/genética , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Efrina-A2/química , Efrina-A2/genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/mortalidade , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/mortalidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptor EphA2 , Ubiquitina/metabolismoRESUMO
Mapping the conformational pathway of biomolecules is a great challenge because of the large size and complexity of biomolecules. The nudged elastic band (NEB) method has been applied to study the reaction pathways for both small organic molecules and small peptides of a few amino acids. In this work, for the first time, the NEB method was employed to study the conformational pathways of Annexin A1, a membrane-binding protein of 334 amino acids. The N-terminal domain conformational change from the buried state within the core domain to the exposed state outside the core domain is a vital step for Annexin A1 to interact with membranes or target proteins. In this work, multiple molecular dynamics simulations using the NEB method were performed to simulate the N-terminal domain conformational pathway of Annexin A1. Our results suggested that the N-terminal domain of Annexin A1 is removed from the repeat III of the core domain in a sliding motion. The loop region of repeat III covering the N-terminal helix in the buried state does not lift up for the N-terminal to swing out of the pocket; instead, the N-terminal pulls out from the bottom of the core domain. The N-terminal domain linker region (S27-N42) flexibility is critical for the N-terminal domain conformational changes. Our results also suggested a two-step folding process for the helix D in repeat III, M247-V250 folds first followed by the folding of L251-E254. The results demonstrated that the NEB method could be an effective tool for theoretical studies on conformational pathways of biomolecules.
Assuntos
Anexina A1/química , Simulação de Dinâmica Molecular , Água/química , Humanos , Conformação Proteica em alfa-Hélice , Domínios Proteicos , TermodinâmicaRESUMO
Annexin-A1 (ANXA1), a potent endogenous immunomodulatory protein has been implicated in multiple functions essential in cancer, including cell proliferation, apoptosis, chemosensitivity, metastasis, and invasion. ANXA1 expression is varied depending on tumor type, and there are contradictory reports on its role in the regulation of proliferation and tumor growth. Here, we summarize the differing reports on cell proliferation and metastasis and attempt to discuss the reasons behind these different effects. ANXA1 plays a role as a homeostatic protein that regulates essential transcription factors and miRNAs. A more coherent understanding of ANXA1 in cancer could present a more biologically meaningful and clinically relevant strategy.
Assuntos
Anexina A1/genética , Anexina A1/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Animais , Anexina A1/química , Biomarcadores , Proliferação de Células , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Transdução de SinaisRESUMO
Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. However, drug delivery is limited without the ability of nanocarriers to sense and respond to the diseased microenvironment. In this study, nanomaterials are developed from peptide amphiphiles (PAs) that respond to the increased levels of matrix metalloproteinases 2 and 9 (MMP2/9) or reactive oxygen species (ROS) found within the atherosclerotic niche. A pro-resolving therapeutic, Ac2-26, derived from annexin-A1 protein, is tethered to PAs using peptide linkages that cleave in response to MMP2/9 or ROS. By adjusting the molar ratios and processing conditions, the Ac2-26 PA can be co-assembled with a PA containing an apolipoprotein A1-mimetic peptide to create a targeted, therapeutic nanofiber (ApoA1-Ac226 PA). The ApoA1-Ac2-26 PAs demonstrate release of Ac2-26 within 24 h after treatment with MMP2 or ROS. The niche-responsive ApoA1-Ac2-26 PAs are cytocompatible and reduce macrophage activation from interferon gamma and lipopolysaccharide treatment, evidenced by decreased nitric oxide production. Interestingly, the linkage chemistry of ApoA1-Ac2-26 PAs significantly affects macrophage uptake and retention. Taken together, these findings demonstrate the potential of PAs to serve as an atheroma niche-responsive nanocarrier system to modulate the inflammatory microenvironment, with implications for atherosclerosis treatment.
Assuntos
Anexina A1 , Apolipoproteína A-I , Aterosclerose , Portadores de Fármacos , Imunoterapia , Nanofibras , Peptídeos , Placa Aterosclerótica , Animais , Anexina A1/química , Anexina A1/farmacologia , Apolipoproteína A-I/química , Apolipoproteína A-I/farmacologia , Aterosclerose/imunologia , Aterosclerose/patologia , Aterosclerose/terapia , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/farmacologia , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/farmacologia , Camundongos , Nanofibras/química , Nanofibras/uso terapêutico , Peptídeos/química , Peptídeos/farmacologia , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Placa Aterosclerótica/terapiaRESUMO
Annexin A1 (ANXA1)-formyl peptide receptor (Fpr) system is potent effective mediators in the control of the inflammatory response. In this study, we evaluate the potential involvement of the Fpr family in the protective effect of the mimetic peptide of ANXA1 (ANXA12-26) using an experimental allergic conjunctivitis (AC) model in mice. Ovalbumin (OVA)/Alum-immunized wild-type (WT) and ANXA1-null (ANXA1-/-) Balb/c mice (days 0 and 7) were challenged by eye drops containing OVA on days 14-16, and two groups received ANXA12-26 alone or with Fpr antagonist Boc2 intraperitoneally during challenged days. As expected, plasma IgE anti-OVA levels increased significantly in the OVA-immunized WT and ANXA1-/- mice, supporting the efficacy of AC model. AC increased Fpr1 and Fpr2 levels in the conjunctiva and the lack of endogenous ANXA1 exacerbated Fpr2 expression only. In contrast, administering ANXA12-26 in the WT mice diminished Fpr2 levels in the conjunctiva, and the effect was reverted by Boc2. Ultrastructural analysis showed the co-localization of Fpr2 and ANXA1 in the plasma membrane of mast cells (MCs), eosinophils and neutrophils, supporting this system as being operative in the AC. Boc2 abrogated the ANXA12-26 effect by increasing the MC degranulation and the eosinophil influx in the conjunctiva, and these findings were supported by peroxidase eosinophil, eotaxin and MC protease levels. Additionally, the ANXA12-26-Fpr system in the AC was associated with the activation of ERK and JNK. Collectively, the data provided in vivo supports the anti-allergic effects of the ANXA1-Fpr system and may serve as a therapeutic target in this ocular disorder.
Assuntos
Anexina A1/metabolismo , Conjuntivite Alérgica/metabolismo , Receptores de Formil Peptídeo/metabolismo , Animais , Anexina A1/química , Quimiocinas/metabolismo , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/patologia , Regulação para Baixo/efeitos dos fármacos , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/farmacologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/farmacologiaRESUMO
Tumor-associated macrophages (TAMs) and angiogenesis are increasingly considered as the pivotal factors that affect tumor progress. Herein, we developed the paclitaxel (PTX)-loaded nanoparticles (NP/PTX) and decorated it with an innovative peptide YI (YINP/PTX) for simultaneously targeting delivery of drug to TAMs and angiogenesis. We demonstrated that the modification of YI peptide significantly enhanced the internalization of nanoparticles by cells and accumulation of nanoparticles in tumor tissues, but down regulated the distribution of them in normal tissues especially the liver. We also made a confirmation that the YI peptide decorated nanoparticles had an excellent co-localization with TAMs and angiogenesis in vivo. Finally, in the HT-26 colorectal tumor-bearing mice, a pharmacodynamic evaluation was performed and results showed that the YINP/PTX was more effective than other PTX formulations in anti-tumor growth. These results together suggested that the prepared nanoparticles are promising in targeting delivery of chemotherapeutics to tumor microenvironment for enhancing tumor therapy effect.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Macrófagos/patologia , Nanopartículas/administração & dosagem , Neovascularização Patológica , Paclitaxel/farmacologia , Fragmentos de Peptídeos/química , Animais , Anexina A1/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Humanos , Camundongos , Camundongos Nus , Nanopartículas/química , Paclitaxel/administração & dosagem , Paclitaxel/química , Células Tumorais Cultivadas , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: In recent years, studies have focused on the resolution of inflammation, which can be achieved by endogenous anti-inflammatory agonists such as Annexin A1 (AnxA1). Here, we investigated the effects of mast cells (MCs) on early LPS-induced neutrophil recruitment and the involvement of the AnxA1-formyl peptide receptor 2/ALX (FPR2/ALX or lipoxin A4 receptor) pathway. EXPERIMENTAL APPROACH: Intravital microscopy (IVM) was used to visualize and quantify the effects of LPS (10 µg per mouse i.p.) on murine mesenteric cellular interactions. Furthermore, the role that MCs play in these inflammatory responses was determined in vivo and in vitro, and effects of AnxA1 mimetic peptide Ac2-26 were assessed. KEY RESULTS: LPS increased both neutrophil endothelial cell interactions within the mesenteric microcirculation and MC activation (determined by IVM and ruthenium red dye uptake), which in turn lead to the early stages of neutrophil recruitment. MC recruitment of neutrophils could be blocked by preventing the pro-inflammatory activation (using cromolyn sodium) or enhancing an anti-inflammatory phenotype (using Ac2-26) in MCs. Furthermore, MCs induced neutrophil migration in vitro, and MC stabilization enhanced the release of AnxA1 from neutrophils. Pharmacological approaches (such as the administration of FPR pan-antagonist Boc2, or the FPR2/ALX antagonist WRW4) revealed neutrophil FPR2/ALX to be important in this process. CONCLUSIONS AND IMPLICATIONS: Data presented here provide evidence for a role of MCs, which are ideally positioned in close proximity to the vasculature, to act as sentinel cells in neutrophil extravasation and resolution of inflammation via the AnxA1-FPR2/ALX pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Receptores de Formil Peptídeo/metabolismo , Animais , Anexina A1/química , Anexina A1/farmacologia , Anti-Inflamatórios/química , Cromolina Sódica/química , Cromolina Sódica/farmacologia , Células Endoteliais/efeitos dos fármacos , Microscopia Intravital , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Inflammation in now appreciated to be at the centre of may diseases that affect Western civilization. Current therapeutics for managing these conditions may interfere with the host response leading to immune suppression. We recently developed an annexin (Anx) A1-derived peptide, coined CR-AnxA12-50, which displays potent pro-resolving and tissue protective actions. Herein, we designed a novel peptide using CR-AnxA12-50 as a template that was significantly more resistant to neutrophil-mediated degradation. This peptide, termed CR-AnxA12-48, retained high affinity and specificity to the pro-resolving Lipoxin A4 receptor (ALX) with an IC50 of ~20nM. CR-AnxA12-48 dose dependently (100fM-10nM) promoted the efferocytosis of apoptotic neutrophils, an action that was mediated by the murine orthologue of human ALX. The neutrophil-directed actions were also retained with human primary cells were CR-AnxA12-48 reduced human neutrophil recruitment to activated endothelial cells at concentrations as low as 100 pM. This protective action was mediated by human ALX, since incubation of neutrophils with an anti-ALX antibody reversed this anti-inflammatory actions of CR-AnxA12-48. Administration of this peptide to mice during dermal inflammation led to a significant and dose dependent decrease in neutrophil recruitment. This reduction in neutrophil numbers was more pronounced than that displayed by the parent peptide CR-AnxA12-50. CR-AnxA12-48 was also cardioprotecitve reducing infarct size and systemic chemokine (C-C motif) ligand 5 concentration following ischemia reperfusion injury. These findings identify CR-AnxA12-48 as a new ALX agonist that regulates phagocyte responses and displays tissue-protective actions.
Assuntos
Anexina A1/química , Anti-Inflamatórios/administração & dosagem , Cardiotônicos/administração & dosagem , Inflamação/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Peptídeos/administração & dosagem , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Cardiotônicos/farmacologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Camundongos , Neutrófilos/efeitos dos fármacos , Peptídeos/farmacologia , Fagócitos/efeitos dos fármacosRESUMO
The eye is immunologically privileged when inflammatory responses are suppressed. One component responsible for the suppression of inflammatory responses is the blood retinal barrier, which comprises the retinal pigment epithelium. The destruction of this barrier initiates inflammation, which can affect any part of the eye. Therefore, inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1) protein acts as a modulator of inflammation. In this study we aimed to improve the knowledge of this area by investigating how a peptide of the ANXA1 protein (ANXA1Ac2-26) modulates the morphology, proliferation, migration and expression of genes and proteins in human retinal pigment epithelium cells (ARPE-19). Determining how signaling pathways (NF-κB and UBC) are modulated by the ANXA1Ac2-26 peptide could be important for understanding the inflammatory process. ARPE-19 cells were activated by bacterial lipopolysaccharide endotoxin (LPS) and treated with ANXA1Ac2-26 peptide, in a concentration of 1.7µM and 33.8µM. We observed that the LPS activation diminished the levels of endogenous ANXA1 after 2h and 24h and ANXA1Ac2-26 peptide decreased the proliferation and re-establishes the migration of ARPE-19 cells. After using a hybridization approach, 80 differentially expressed genes were found. Five of these genes were selected (LRAT, CTGF, MAP1B, ALDH1A3 and SETD7) and all were down-regulated after treatment with the peptide. The genes CTGF and LRAT would be considered as potential molecular markers of ophthalmologic inflammation. The expression of pro-inflammatory cytokines was also decreased after the treatment, indicating the efficiency of the anti-inflammatory peptide at high concentrations, since the reduction in the levels of these mediators were observed after the treatment with ANXA1Ac2-26 peptide at 33.8µM. Our results suggest that the retinal pigment epithelial cells are a potential target of the ANXA1 protein and point to possible applications of the ANXA1Ac2-26 peptide as an innovative therapy for the treatment of ocular inflammation.
Assuntos
Anexina A1/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Peptídeos/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Aciltransferases/genética , Aciltransferases/metabolismo , Anexina A1/química , Anexina A1/metabolismo , Anti-Inflamatórios/farmacologia , Western Blotting , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Oftalmopatias/genética , Oftalmopatias/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/prevenção & controle , Lipopolissacarídeos/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Annexin A1 (ANXA1) is a Ca(2+)-regulated phospholipid-binding protein involved in various cell processes. ANXA1 was initially widely studied in inflammation resolution, but its overexpression was later reported in a large number of cancers. Further in-depth investigations have revealed that this protein could have many roles in cancer progression and act at different levels (from cancer initiation to metastasis). This is partly due to the location of ANXA1 in different cell compartments. ANXA1 can be nuclear, cytoplasmic and/or membrane associated. This last location allows ANXA1 to be proteolytically cleaved and/or to become accessible to its cognate partners, the formyl-peptide receptors. Indeed, in some cancers, ANXA1 is found at the cell surface, where it stimulates formyl-peptide receptors to trigger oncogenic pathways. In the present review, we look at the different locations of ANXA1 and their association with the deregulated pathways often observed in cancers. We have specifically detailed the non-classic pathways of ANXA1 externalization, the significance of its cleavage and the role of the ANXA1-formyl-peptide receptor complex in cancer progression.
Assuntos
Anexina A1/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Anexina A1/química , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Ligação Proteica , Conformação Proteica , Proteólise , Receptores de Formil Peptídeo/metabolismo , Relação Estrutura-AtividadeRESUMO
The main function of annexin A1 (ANXA1), a member of the annexin superfamily, is to bind to cellular membranes in a Ca(2+)-dependent manner. In skeletal muscle, ANXA1 is thought to be involved in the repair of damaged membrane tissue and in the migration of muscle cells. We hypothesized that ANXA1 is one of the myokines secreted during muscle contractions to accelerate the repair of cell damage after contraction. Here we performed cell contractions by electric pulse stimulation; the results revealed that a fragmented form of ANXA1 was cleaved by calpain and selectively secreted from skeletal muscle cells by contraction. We therefore realized that muscle-contraction-induced calpain-dependent ANXA1 fragmentation has a wound-healing effect on damaged cells. This suggested that not the intact form but rather fragmented ANXA1 is a contraction-induced myokine.
Assuntos
Anexina A1/metabolismo , Eletricidade , Fibras Musculares Esqueléticas/metabolismo , Animais , Anexina A1/química , Linhagem Celular , Espectrometria de Massas , Camundongos , CicatrizaçãoRESUMO
Annexin A1 (AnxA1) is an endogenous glucocorticoid regulated protein that modulates anti-inflammatory process and its therapeutic potential has recently been recognized in a range of systemic inflammatory disorders. The effect of the N-terminal peptide Ac2-26 of AnxA1 on the toxic activities of Bothrops moojeni crude venom (CV) and its myotoxin II (MjTX-II) were evaluated using a peritonitis rat model. Peritonitis was induced by the intraperitoneal injection of either CV or MjTX-II, a Lys-49 phospholipase A2. Fifteen minutes after the injection, the rats were treated with either Ac2-26 or PBS. Four hours later, the CV and MjTX-II-induced peritonitis were characterized by neutrophilia (in the peritoneal exudate, blood and mesentery) and increased number of mesenteric degranulated mast cells and macrophages. At 24 hours post-injection, the local inflammatory response was attenuated in the CV-induced peritonitis while the MjTX-II group exhibited neutrophilia (peritoneal exudates and blood). Ac2-26 treatment prevented the influx of neutrophils in MjTX-II-induced peritonitis and diminished the proportion of mesenteric degranulated mast cells and macrophages in CV-induced peritonitis. Additionally, CV and MjTX-II promoted increased levels of IL-1ß and IL-6 in the peritoneal exudates which were significantly reduced after Ac2-26 treatment. At 4 and 24 hours, the endogenous expression of AnxA1 was upregulated in the mesenteric neutrophils (CV and MjTX-II groups) and mast cells (CV group). In the kidneys, CV and MjTX-II administrations were associated with an increased number of macrophages and morphological alterations in the juxtamedullary nephrons in proximal and distal tubules. Ac2-26 promoted significant recovery of the juxtamedullary structures, decreased the number of macrophages and diminished the AnxA1 in epithelial cells from distal tubules and renal capsules. Our results show that Ac2-26 treatment significantly attenuates local and systemic inflammatory processes and indicate this peptide as a potential target for the development of new therapeutic strategies for the snakebite envenomation treatment.
Assuntos
Anexina A1/química , Venenos de Crotalídeos/toxicidade , Inflamação/prevenção & controle , Lisina/química , Peptídeos/farmacologia , Fosfolipases A2/toxicidade , Animais , Anexina A1/farmacologia , Bothrops , Modelos Animais de Doenças , Masculino , Mimetismo Molecular , Fosfolipases A2/química , Ratos , Ratos WistarRESUMO
In this paper, for the first time, hydrogels containing Annexin A1 N-terminal derived peptide, Ac2-26, as a novel dressing were successfully developed for dermal wound repair application. High mannuronic (M) content alginate and low molecular weight chitosan have been used as hydrogel carrier. Peptide recovery analyses, FTIR studies and molecular modelling highlighted chemical interactions between peptide and hydrogel polymers. Ac2-26 resulted entrapped into chitosan hydrogel matrix that prevented its release, whereas such interaction in alginate hydrogel slowed down peptide diffusion enabling its sustained release till 72 h. In vivo wound healing studies conducted on mice dorsal wounds indicate that after the 9th day of post wounding Ac2-26/alginate hydrogels could significantly accelerate wound healing, with complete closure of the wound on day 14th. Therefore, these results suggest that the developed of Ac2-26 high M content alginate hydrogel could be a promising wound dressing with potential application in dermal wound healing.
Assuntos
Anexina A1/administração & dosagem , Hidrogéis/administração & dosagem , Peptídeos/administração & dosagem , Cicatrização/efeitos dos fármacos , Administração Tópica , Alginatos/química , Animais , Anexina A1/química , Quitosana/química , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Liberação Controlada de Fármacos , Hidrogéis/química , Injeções , Camundongos Endogâmicos C57BL , Peptídeos/químicaRESUMO
The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1(2-26) (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1(2-26) treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1(2-26) treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.