RESUMO
BACKGROUND: The thymus is responsible for thymocyte differentiation into immunocompetent T lymphocytes. Different cell types in the thymic microenvironment actively cooperate in this process, interacting with the developing thymocytes through soluble factors, extracellular matrix (ECM) molecules, and receptors. In addition, this microenvironment can be influenced by several factors, such as semaphorin-3A (Sema3A), which is a multifunctional protein involved in cell migration. We evaluated the Sema3A effects on the cellular parameters and functional features of thymic stromal cells. METHODS: Thymic stromal cells were obtained by enzymatic digestion of the murine thymus. These cells were treated with Sema3A and evaluated as follows: cell morphology by scanning electron microscope, F-actin cytoskeleton and deposition of ECM molecules by fluorescence microscopy, and adhesion assays with freshly obtained thymocytes. RESULTS: The obtained thymic stroma was composed of 67% of thymic epithelial cells (TECs), and 90% of the TECs were positive for the Sema3A receptor neuropilin-1. These cells secreted CXCL12, IL-7 and extended thymocyte survival. Sema3A changed the morphology of thymic stromal cells and promoted F-actin reorganization. In addition, the fibronectin fibers were reoriented, and the laminin production was increased in Sema3A-treated thymic stromal cells. In the adhesion assays, there was an increase in the number of adhered thymocytes when thymic stromal cells were pretreated with Sema3A. CONCLUSION: Our data strongly suggest the active participation of Sema3A in thymic physiology, highlighting its role as an immunomodulatory molecule. This may provide important knowledge for understanding the interactions of thymic cells.
Assuntos
Semaforina-3A , Timócitos , Animais , Movimento Celular , Células Epiteliais , Camundongos , Células EstromaisRESUMO
Sympathetic nerves innervate most organs and regulate organ blood flow. Specifically, in the uterus, estradiol (E2) elicits rapid degeneration of sympathetic axons and stimulates the growth of blood vessels. Both physiological remodeling processes, critical for reproduction, have been extensively studied but as independent events and are still not fully understood. Here, we examine the neuropilin-1 (NRP1), a shared receptor for axon guidance and angiogenic factors. Systemic estradiol or vehicle were chronically injected to prepubertal rats and uterine and sympathetic chain sections immunostained for NRP1. Uterine semaphorin-3A mRNA was evaluated by in situ hybridization. Control sympathetic uterine-projecting neurons (1-month-old) expressed NRP1 in their somas but not in their intrauterine terminal axons. Estradiol did not affect NRP1 in the distal ganglia. However, at the entrance of the organ, some sympathetic NRP1-positive nerves were recognized. Vascular NRP1 was confined to intrauterine small-diameter vessels in both hormonal conditions. Although the overall pattern of NRP1-IR was not affected by E2 treatment, a subpopulation of infiltrated eosinophil leukocytes showed immunoreactivity for NRP1. Sema3A transcripts were detected in this cellular type as well. No NRP1-immunoreactive axons nor infiltrated eosinophils were visualized in other estrogenized pelvic organs. Together, these data suggest the involvement of NRP1/Sema3A signaling in the selective E2-induced uterine neurovascular remodeling. Our data support a model whereby NRP1 could coordinate E2-induced uterine neurovascular remodeling, acting as a positive regulator of growth when expressed in vessels and as a negative regulator of growth when expressed on axons.
Assuntos
Plasticidade Neuronal , Neuropilina-1/fisiologia , Semaforina-3A/fisiologia , Sistema Nervoso Simpático , Útero , Remodelação Vascular , Animais , Estradiol/farmacologia , Feminino , Ratos , Ratos Wistar , Útero/irrigação sanguínea , Útero/inervaçãoRESUMO
BACKGROUND: Class 3 semaphorins are soluble proteins involved in cell adhesion and migration. Semaphorin-3A (Sema3A) was initially shown to be involved in neuronal guidance, and it has also been reported to be associated with immune disorders. Both Sema3A and its receptors are expressed by most immune cells, including monocytes, macrophages, and lymphocytes, and these proteins regulate cell function. Here, we studied the correlation between Sema3A-induced changes in biophysical parameters of thymocytes, and the subsequent repercussions on cell function. METHODS: Thymocytes from mice were treated in vitro with Sema3A for 30min. Scanning electron microscopy was performed to assess cell morphology. Atomic force microscopy was performed to further evaluate cell morphology, membrane roughness, and elasticity. Flow cytometry and/or fluorescence microscopy were performed to assess the F-actin cytoskeleton and ROCK2. Cell adhesion to a bovine serum albumin substrate and transwell migration assays were used to assess cell migration. RESULTS: Sema3A induced filopodia formation in thymocytes, increased membrane stiffness and roughness, and caused a cortical distribution of the cytoskeleton without changes in F-actin levels. Sema3A-treated thymocytes showed reduced substrate adhesion and migratory ability, without changes in cell viability. In addition, Sema3A was able to down-regulate ROCK2. CONCLUSIONS: Sema3A promotes cytoskeletal rearrangement, leading to membrane modifications, including increased stiffness and roughness. This effect in turn affects the adhesion and migration of thymocytes, possibly due to a reduction in ROCK2 expression. GENERAL SIGNIFICANCE: Sema3A treatment impairs thymocyte migration due to biomechanical alterations in cell membranes.
Assuntos
Fenômenos Biomecânicos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Semaforina-3A/farmacologia , Timócitos/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Timócitos/metabolismo , Timócitos/ultraestrutura , Quinases Associadas a rho/metabolismoRESUMO
Contrast-induced acute kidney injury (CI-AKI) is a serious complication of diagnostic coronary angiograph and percutaneous coronary intervention (PCI). However, the exact pathophysiological mechanisms underlying CI-AKI development are largely unknown. The present study examined whether urinary semaphorin 3A levels predict the development of CI-AKI in patients undergoing PCI. This study enrolled 168 patients with stable angina undergoing elective PCI. Serial urine samples, obtained at baseline and 2, 6, 12, 24, 36, and 48 h post-PCI were analyzed by semaphorin 3A and neutrophil gelatinase-associated lipocalin (NGAL) ELISA kit. AKI was defined as an increase in serum creatinine beyond 50% according to the RIFLE classification system. Receiver operator characteristic (ROC) curve analyses identified optimal semaphorin 3A and NGAL values for diagnosing CI-AKI. CI-AKI occurred in 20 of 168 patients. There were no significant differences in the baseline clinical characteristics and angiographic findings between non-AKI patients group and AKI patients group. Both urinary semaphorin 3A and NGAL levels significantly increased at 2 and 6 h post-PCI. ROC analysis showed that the cut-off value of 389.5 pg/mg semaphorin 3A at 2 h post-PCI corresponds to 94% sensitivity and 75% specificity and the cut-off value of 94.4 ng/mg NGAL at 2 h post-PCI corresponds to 74% sensitivity and 82% specificity. Logistic regression showed that semaphorin 3A levels at 2 and 6 h post-PCI were the significant predictors of AKI in our cohort. Urinary semaphorin 3A may be a promising early biomarker for predicting CI-AKI in patients undergoing PCI.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Meios de Contraste/efeitos adversos , Semaforina-3A/urina , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/urina , Intervenção Coronária Percutânea/efeitos adversos , Biomarcadores/urina , Valor Preditivo dos Testes , Curva ROC , Injúria Renal Aguda/diagnósticoRESUMO
UNLABELLED: Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. SIGNIFICANCE STATEMENT: Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel therapeutic modality for promoting nerve repair and preventing axonal loss.
Assuntos
Actinas/metabolismo , Axônios/fisiologia , Endocitose/fisiologia , Galectina 1/metabolismo , Peróxido de Hidrogênio/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Galectina 1/genética , Galectina 1/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , Ratos , Semaforina-3A/farmacologia , Transdução de Sinais , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
When spinal cord injury (SCI) occurs, a great number of inhibitors of axonal regeneration consecutively invade the injured site. The first protein to reach the lesion is known as semaphorin 3A (Sema3A), which serves as a powerful inhibitor of axonal regeneration. Mechanistically binding of Sem3A to the neuronal receptor complex neuropilin-1 (NRP-1) / PlexinA4 prevents axonal regeneration. In this special article we review the effects of galectin-1 (Gal-1), an endogenous glycan-binding protein, abundantly present at inflammation and injury sites. Notably, Gal1 adheres selectively to the NRP-1/PlexinA4 receptor complex in injured neurons through glycan-dependent mechanisms, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. While both the monomeric and dimeric forms of Gal-1 contribute to "switch-off" classically-activated microglia, only dimeric Gal-1 binds to the NRP-1/PlexinA4 receptor complex and promotes axonal regeneration. Thus, dimeric Gal-1 promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A adhering to the NRP-1/PlexinA4 complex, supporting the use of dimeric Gal-1 for the treatment of SCI patients.
Assuntos
Axônios/fisiologia , Galectina 1/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Humanos , Camundongos , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforina-3A/fisiologiaRESUMO
Al producirse una lesión de médula espinal (LME), un sinnúmero de proteínas inhibidoras de la regeneración axonal ocupan el sitio de lesión en forma secuencial. La primer proteína en llegar al mismo se conoce como semaforina 3A (Sema3A), siendo además una de las más potentes por su acción de inhibir la regeneración axonal. A nivel mecanístico la unión de esta proteína al complejo-receptor neuronal neuropilin-1 (NRP-1)/PlexinA4 evita que se produzca regeneración axonal. En este trabajo de revisión se discutirá la acción de galectin-1 (Gal-1), una proteína endógena de unión a glicanos, que selectivamente se une al complejo-receptor NRP-1/PlexinA4 de las neuronas lesionadas a través de un mecanismo dependiente de interacciones lectina-glicano, interrumpiendo la señalización generada por Sema3A y permitiendo de esta manera la regeneración axonal y recuperación locomotora luego de producirse la LME. Mientras ambas formas de Gal-1 (monomérica y dimérica) contribuyen a la inactivación de la microglia, solo la forma dimérica de Gal-1 es capaz de unirse al complejo-receptor NRP-1/PlexinA4 y promover regeneración axonal. Por lo tanto, Gal-1 dimérica produce recuperación de las lesiones espinales interfiriendo en la señalización de Sema3A a través de la unión al complejo-receptor NRP-1/PlexinA4, sugiriendo el uso de esta lectina en su forma dimérica para el tratamiento de pacientes con LME.
When spinal cord injury (SCI) occurs, a great number of inhibitors of axonal regeneration consecutively invade the injured site. The first protein to reach the lesion is known as semaphorin 3A (Sema3A), which serves as a powerful inhibitor of axonal regeneration. Mechanistically binding of Sem3A to the neuronal receptor complex neuropilin-1 (NRP-1) / PlexinA4 prevents axonal regeneration. In this special article we review the effects of galectin-1 (Gal-1), an endogenous glycan-binding protein, abundantly present at inflammation and injury sites. Notably, Gal1 adheres selectively to the NRP-1/PlexinA4 receptor complex in injured neurons through glycan-dependent mechanisms, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. While both the monomeric and dimeric forms of Gal-1 contribute to ’switch-off’ classically-activated microglia, only dimeric Gal-1 binds to the NRP-1/PlexinA4 receptor complex and promotes axonal regeneration. Thus, dimeric Gal-1 promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A adhering to the NRP-1/PlexinA4 complex, supporting the use of dimeric Gal-1 for the treatment of SCI patients.
Assuntos
Animais , Humanos , Camundongos , Axônios/fisiologia , Galectina 1/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/metabolismo , Receptores de Superfície Celular/metabolismo , /fisiologiaRESUMO
Al producirse una lesión de médula espinal (LME), un sinnúmero de proteínas inhibidoras de la regeneración axonal ocupan el sitio de lesión en forma secuencial. La primer proteína en llegar al mismo se conoce como semaforina 3A (Sema3A), siendo además una de las más potentes por su acción de inhibir la regeneración axonal. A nivel mecanístico la unión de esta proteína al complejo-receptor neuronal neuropilin-1 (NRP-1)/PlexinA4 evita que se produzca regeneración axonal. En este trabajo de revisión se discutirá la acción de galectin-1 (Gal-1), una proteína endógena de unión a glicanos, que selectivamente se une al complejo-receptor NRP-1/PlexinA4 de las neuronas lesionadas a través de un mecanismo dependiente de interacciones lectina-glicano, interrumpiendo la señalización generada por Sema3A y permitiendo de esta manera la regeneración axonal y recuperación locomotora luego de producirse la LME. Mientras ambas formas de Gal-1 (monomérica y dimérica) contribuyen a la inactivación de la microglia, solo la forma dimérica de Gal-1 es capaz de unirse al complejo-receptor NRP-1/PlexinA4 y promover regeneración axonal. Por lo tanto, Gal-1 dimérica produce recuperación de las lesiones espinales interfiriendo en la señalización de Sema3A a través de la unión al complejo-receptor NRP-1/PlexinA4, sugiriendo el uso de esta lectina en su forma dimérica para el tratamiento de pacientes con LME.(AU)
When spinal cord injury (SCI) occurs, a great number of inhibitors of axonal regeneration consecutively invade the injured site. The first protein to reach the lesion is known as semaphorin 3A (Sema3A), which serves as a powerful inhibitor of axonal regeneration. Mechanistically binding of Sem3A to the neuronal receptor complex neuropilin-1 (NRP-1) / PlexinA4 prevents axonal regeneration. In this special article we review the effects of galectin-1 (Gal-1), an endogenous glycan-binding protein, abundantly present at inflammation and injury sites. Notably, Gal1 adheres selectively to the NRP-1/PlexinA4 receptor complex in injured neurons through glycan-dependent mechanisms, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. While both the monomeric and dimeric forms of Gal-1 contribute to ’switch-off’ classically-activated microglia, only dimeric Gal-1 binds to the NRP-1/PlexinA4 receptor complex and promotes axonal regeneration. Thus, dimeric Gal-1 promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A adhering to the NRP-1/PlexinA4 complex, supporting the use of dimeric Gal-1 for the treatment of SCI patients.(AU)