RESUMO
The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Humanos , Animais , Camundongos , Clostridioides difficile/genética , Via de Sinalização Hippo , Toxinas Bacterianas/metabolismo , Células HeLa , Clostridioides , RNA Mensageiro/metabolismo , Proteínas de Membrana/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismoRESUMO
Spinal cord injury (SCI) stimulates reactive astrogliosis and the infiltration of macrophages, which interact with each other at the injured area. We previously found Photobiomodulation (PBM) significantly decreases the number of M1 macrophages at the injured area of SCI. But the exact nature of the astrocyte response following PBM and relationship with the macrophage have not been explored in detail. In this study, a BALB/c mice model with standardized bilateral spinal cord compression and a macrophage-astrocyte co-culture model were applied to study effects of PBM on astrocytes. Results showed that PBM inhibit the expression of the astrocyte markers glial fibrillary acidic protein (GFAP) and the secretion of chondroitin sulfate proteoglycans (CSPG) in the para-epicenter area, decrease the number of M1 macrophage in vivo. The in vitro experiments indicated M1 macrophages promote the cell viability of astrocytes and the expression of CSPG. However, PBM significantly inhibited the expression of GFAP, decreased activation of astrocyte, and downregulated the expression of CSPG by regulating M1 macrophages. These results demonstrate that PBM may regulate the interaction between macrophages and astrocytes after spinal cord injury, which inhibited the formation of glial scar.
Assuntos
Astrócitos/efeitos da radiação , Polaridade Celular/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Macrófagos/efeitos da radiação , Animais , Astrócitos/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Meios de Cultivo Condicionados/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Atividade Motora/efeitos dos fármacos , Atividade Motora/efeitos da radiação , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Recuperação de Função Fisiológica/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/radioterapiaRESUMO
Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumour. Patients with GBM respond poorly to chemotherapy and have poor survival outcomes. Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), has been shown to contribute to critical processes, such as cell survival, proliferation, and chemotherapy resistance, during glioma progression. In this study, we found that furanodienone (FUR), a diene-type sesquiterpene isolated from the rhizomes of Rhizoma curcumae, exhibited a potential cytotoxic effect on temozolomide (TMZ)-resistant GBM cells in vitro by inhibiting CSPG4 and related signalling pathways. Studies investigating the mechanism demonstrated that FUR suppressed CSPG4-Akt-ERK signalling, inflammatory responses, and cytokine levels but activated caspase-dependent pathways and mitochondrial dysfunction. Furthermore, an immunofluorescence assay and a dual-luciferase reporter assay revealed that inhibition of EGR1-mediated transcription might have contributed to the FUR-dependent blockade of CSPG4 signalling and glioma cell survival. These results established a link between FUR-induced CSPG4 inhibition and the suppression of EGR1-dependent transcription. Attenuation of ERK1/2 and cytokine signalling might have generated the EGR1-dependent negative feedback loop of the CSPG4 pathway during FUR-induced apoptosis. These findings suggested that FUR could be a therapeutic candidate for the treatment of malignant glioma via targeting CSPG4 signalling.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Furanos/farmacologia , Glioblastoma/tratamento farmacológico , Sesquiterpenos/farmacologia , Temozolomida/uso terapêutico , Adulto , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Furanos/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sesquiterpenos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Temozolomida/administração & dosagem , Transcrição Gênica/efeitos dos fármacosRESUMO
Chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR T cells) show modest therapeutic efficacy in solid tumors. The desmoplastic structure of the tumor and the immunosuppressive tumor microenvironment usually account for the reduced efficacy of CAR T cells in solid tumors. Mild hyperthermia of the tumor reduces its compact structure and interstitial fluid pressure, increases blood perfusion, releases antigens, and promotes the recruitment of endogenous immune cells. Therefore, the combination of mild hyperthermia with the adoptive transfer of CAR T cells can potentially increase the therapeutic index of these cells in solid tumors. It is found that the chondroitin sulfate proteoglycan-4 (CSPG4)-specific CAR T cells infused in Nod scid gamma mice engrafted with the human melanoma WM115 cell line have superior antitumor activity after photothermal ablation of the tumor. The findings suggest that photothermal therapy facilitates the accumulation and effector function of CAR T cells within solid tumors.
Assuntos
Antígenos/metabolismo , Hipertermia Induzida , Imunoterapia Adotiva/métodos , Fototerapia/métodos , Proteoglicanas/metabolismo , Linfócitos T/metabolismo , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Terapia Combinada , Feminino , Xenoenxertos , Humanos , Verde de Indocianina/química , Melanoma/patologia , Melanoma/terapia , Proteínas de Membrana/metabolismo , Camundongos SCID , Nanopartículas/química , Fármacos Fotossensibilizantes/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Linfócitos T/transplanteRESUMO
BACKGROUND/AIMS: Low-level laser therapy (LLLT) leads to complex photochemical responses during the healing process of spinal cord injury (SCI). Confocal Raman Microspectral Imaging (in combination with multivariate analysis) was adopted to illustrate the underlying biochemical mechanisms of LLLT treatment on a SCI rat model. METHODS: Using transversal tissue sections, the Raman spectra can identify areas neighboring the injury site, glial scar, cavity, and unharmed white matter, as well as their correlated cellular alterations, such as demyelination and up-regulation of chondroitin sulfate proteoglycans (CSPGs). Multivariate data analysis methods are used to depict the underlying therapeutic effects by highlighting the detailed content and distribution variations of the biochemical constituents. RESULTS: It is confirmed that photon-tissue interactions might lead to a decay of the inhibitory response to remyelination by suppressing CSPG expression, as also morphologically demonstrated by reduced glial scar and cavity areas. An inter-group comparison semi-quantitatively confirms changes in lipids, phosphatidic acid, CSPGs, and cholesterol during SCI and its LLLT treatment, paving the way for in vitro and in vivo understanding of the biochemical changes accompanying pathobiological SCI events. CONCLUSION: The achieved results in this work not only have once again proved the well-known cellular mechanisms of SCI, but further illustrate the underlying biochemical variability during LLLT treatment, which provide a sound basis for developing real-time Raman methodologies to monitor the efficacy of the SCI LLLT treatment.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Lasers Semicondutores/uso terapêutico , Terapia com Luz de Baixa Intensidade , Traumatismos da Medula Espinal/radioterapia , Animais , Colesterol/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Metabolismo dos Lipídeos , Microscopia Confocal , Ácidos Fosfatídicos/metabolismo , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Análise Espectral Raman , Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
BACKGROUND: Traumatic spinal cord injury (SCI) results in upregulation of chondroitin sulfate proteoglycans (CSPGs) by reactive glia that impedes repair and regeneration in the spinal cord. Degradation of CSPGs is known to be beneficial in promoting endogenous repair mechanisms including axonal sprouting/regeneration, oligodendrocyte replacement, and remyelination, and is associated with improvements in functional outcomes after SCI. Recent evidence suggests that CSPGs may regulate secondary injury mechanisms by modulating neuroinflammation after SCI. To date, the role of CSPGs in SCI neuroinflammation remains largely unexplored. The recent discovery of CSPG-specific receptors, leukocyte common antigen-related (LAR) and protein tyrosine phosphatase-sigma (PTPσ), allows unraveling the cellular and molecular mechanisms of CSPGs in SCI. In the present study, we have employed parallel in vivo and in vitro approaches to dissect the role of CSPGs and their receptors LAR and PTPσ in modulating the inflammatory processes in the acute and subacute phases of SCI. METHODS: In a clinically relevant model of compressive SCI in female Sprague Dawley rats, we targeted LAR and PTPσ by two intracellular functionally blocking peptides, termed ILP and ISP, respectively. We delivered ILP and ISP treatment intrathecally to the injured spinal cord in a sustainable manner by osmotic mini-pumps for various time-points post-SCI. We employed flow cytometry, Western blotting, and immunohistochemistry in rat SCI, as well as complementary in vitro studies in primary microglia cultures to address our questions. RESULTS: We provide novel evidence that signifies a key immunomodulatory role for LAR and PTPσ receptors in SCI. We show that blocking LAR and PTPσ reduces the population of classically activated M1 microglia/macrophages, while promoting alternatively activated M2 microglia/macrophages and T regulatory cells. This shift was associated with a remarkable elevation in pro-regenerative immune mediators, interleukin-10 (IL-10), and Arginase-1. Our parallel in vitro studies in microglia identified that while CSPGs do not induce an M1 phenotype per se, they promote a pro-inflammatory phenotype. Interestingly, inhibiting LAR and PTPσ in M1 and M2 microglia positively modulates their inflammatory response in the presence of CSPGs, and harnesses their ability for phagocytosis and mobilization. Interestingly, our findings indicate that CSPGs regulate microglia, at least in part, through the activation of the Rho/ROCK pathway downstream of LAR and PTPσ. CONCLUSIONS: We have unveiled a novel role for LAR and PTPσ in regulating neuroinflammation in traumatic SCI. Our findings provide new insights into the mechanisms by which manipulation of CSPG signaling can promote recovery from SCI. More importantly, this work introduces the potential of ILP/ISP as a viable strategy for modulating the immune response following SCI and other neuroinflammatory conditions of the central nervous system.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Traumatismos da Medula Espinal/complicações , Animais , Animais Recém-Nascidos , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Citocinas/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/fisiologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Peroxidase/metabolismo , Fagocitose/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Oligodendroglial cell death and demyelination are hallmarks of neurotrauma and multiple sclerosis that cause axonal damage and functional impairments. Remyelination remains a challenge as the ability of endogenous precursor cells for oligodendrocyte replacement is hindered in the unfavorable milieu of demyelinating conditions. Here, in a rat model of lysolecithin lysophosphatidyl-choline (LPC)-induced focal demyelination, we report that Neuregulin-1 (Nrg-1), an important factor for oligodendrocytes and myelination, is dysregulated in demyelinating lesions and its bio-availability can promote oligodendrogenesis and remyelination. We delivered recombinant human Nrg-1ß1 (rhNrg-1ß1) intraspinally in the vicinity of LPC demyelinating lesion in a sustained manner using poly lactic-co-glycolic acid microcarriers. Availability of Nrg-1 promoted generation and maturation of new oligodendrocytes, and accelerated endogenous remyelination by both oligodendrocyte and Schwann cell populations in demyelinating foci. Importantly, Nrg-1 enhanced myelin thickness in newly remyelinated spinal cord axons. Our complementary in vitro studies also provided direct evidence that Nrg-1 significantly promotes maturation of new oligodendrocytes and facilitates their transition to a myelinating phenotype. Nrg-1 therapy remarkably attenuated the upregulated expression chondroitin sulfate proteoglycans (CSPGs) specific glycosaminoglycans in the extracellular matrix of demyelinating foci and promoted interleukin-10 (IL-10) production by immune cells. CSPGs and IL-10 are known to negatively and positively regulate remyelination, respectively. We found that Nrg-1 effects are mediated through ErbB2 and ErbB4 receptor activation. Our work provides novel evidence that dysregulated levels of Nrg-1 in demyelinating lesions of the spinal cord pose a challenge to endogenous remyelination, and appear to be an underlying cause of myelin thinning in newly remyelinated axons.
Assuntos
Doenças Desmielinizantes/terapia , Imunomodulação , Neuregulina-1/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Remielinização/fisiologia , Medula Espinal/imunologia , Animais , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Portadores de Fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Feminino , Gânglios Espinais/imunologia , Gânglios Espinais/patologia , Humanos , Ácido Láctico , Masculino , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/patologia , Oligodendroglia/imunologia , Oligodendroglia/patologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Medula Espinal/patologia , Doenças da Medula Espinal/imunologia , Doenças da Medula Espinal/patologia , Doenças da Medula Espinal/terapiaRESUMO
After spinal cord injury (SCI) there are many recoveries inhibiting factors such as chondroitin sulfate proteoglycan (CSPG) and inflammation. The present study investigated the combinational effect of low level laser therapy (LLLT) as anti-inflammatory agent and Chondroitinase ABC (ChABC) enzyme as CSPG digesting factor on spinal cord after injury. This study performed on 44 male Wistar rats, spinal cord injury induced by a clip compression injury. Animals received two-weeks treatment of 660nm low level laser (LLL) and intraspinal injection of 1µg ChABC. Functional recovery, cavity size, myelination, axonal projections around the cavity, fibroblast invasion and expression of glycogen synthase kinase-3ß (GSk 3ß), CSPG and aquaporin 4 (AQP4) expression were evaluated. In statistical evaluation p<0.05 considered significant. Result showed the combination of LLLT and ChABC have more effect on reduction of cavity size, improvement of myelination and number of axons around the cavity and decreasing the expression of GSK3ß, CSPG and AQP4 expression compared to LLLT and ChABC alone. In the laser and laser+enzyme groups AQP4 expression decreased significantly after SCI. Functional recovery, improved in LLLT and ChABC treated animals, but higher recovery belonged to the combination therapy group. The current study showed combination therapy by LLLT and ChABC is more efficient than a single therapy with each of them.
Assuntos
Condroitina ABC Liase/uso terapêutico , Terapia com Luz de Baixa Intensidade , Regeneração Nervosa , Traumatismos da Medula Espinal/terapia , Animais , Anti-Inflamatórios/uso terapêutico , Aquaporina 4/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Inflamação/complicações , Inflamação/terapia , Masculino , Bainha de Mielina/patologia , Ratos Wistar , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Dysregulated human monocytes/macrophages can synthesize and secrete matrix metalloproteinases (MMPs), which play important roles in the progression of sepsis. In this study, we investigated the effects and mechanism of a novel histone deacetylase (HDAC8) inhibitor, (E)-N-hydroxy-4-methoxy-2-(biphenyl-4-yl)cinnamide (WK2-16), on MMP-9 production and activation in stimulated human monocytic THP-1 cells. Our results demonstrated that the acetylation level of structural maintenance of chromosomes 3 (SMC3) was up-regulated by WK2-16 in THP-1 cells. Consistently, an in vitro enzyme study demonstrated that WK2-16 selectively inhibited HDAC8 activity. Moreover, the WK2-16 concentration dependently suppressed MMP-9-mediated gelatinolysis induced by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). Additionally, WK2-16 significantly inhibited both MMP-9 protein and mRNA expression without cellular toxicity. Nevertheless, WK2-16 suppressed the extracellular levels of interleukin (IL)-6 from LPS-stimulated THP-1 cells. For the signaling studies, WK2-16 had no effect on LPS/TLR4 downstream signaling pathways, such as the NF-κB and ERK/JNK/P38 MAPK pathways. On the other hand, WK2-16 enhanced the recruitment of acetylated Yin Yang 1 (YY1) with HDAC1. Finally, in vivo studies indicated that WK2-16 could reduce the serum levels of TNF-α and IL-6 in endotoxemic mice. These results suggested that HDAC8 inhibition might provide a novel therapeutic strategy of hypercytokinemia in sepsis.
Assuntos
Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/metabolismo , Lipopolissacarídeos/farmacologia , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Acetilação , Animais , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Ciclo-Oxigenase 2/efeitos dos fármacos , Regulação para Baixo , Endotoxemia , Histona Desacetilase 1/efeitos dos fármacos , Humanos , Interleucina-6 , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Sepse/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Transcrição YY1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix (ECM) in the brain. In adult mammals, CSPGs form the specialized ECM structure perineuronal nets (PNNs) that surround somata and dendrites of certain types of neurons. PNNs restrict synaptic plasticity and regulate the closure of critical periods. Although previous studies have examined the starting period of PNN formation, focusing on primary sensory cortices, there are no systematic studies at the whole brain level. Here, we examined the starting period of PNN formation in male mice ranging in age from postnatal day 3 to week 11, mainly focusing on several cortical areas, limbic structures, hypothalamus, and brain stem, using lectin histochemistry with Wisteria floribunda agglutinin (WFA). Results showed that early PNN formation was observed in several reticular formations of the brain stem related to the cranial nerves and primary somatosensory cortices. In the limbic system, PNN formation in the hippocampus started earlier than that of the amygdala. Furthermore, in the medial amygdaloid nucleus and some hypothalamic regions, WFA labeling did not show typical PNN-like forms. The present study suggests spatiotemporal differences at the beginning of PNN formation and a structural variety of CSPG-contained ECM in the brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Animais , Tronco Encefálico/crescimento & desenvolvimento , Tronco Encefálico/metabolismo , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Sistema Límbico/crescimento & desenvolvimento , Sistema Límbico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
The formation of glial scar restricts axon regeneration after spinal cord injury (SCI) in adult mammalian. Chondroitin sulfate proteoglycans (CSPGs) are mostly secreted by reactive astrocytes, which form dense scar tissues after SCI. Chondroitinase ABC (ChABC), which can digest CSPGs, is a promising therapeutic strategy for SCI. However, to date ChABC has exhibited only limited success in the treatment of chronic SCI. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. We targeted glial scar in injured spinal cord by sustained infusion of ChABC and antisense vimentin cDNA. Using anterograde tracing, BBB scoring and hind limb placing response, we found that this combined treatment promoted axon regeneration and functional recovery after SCI in rats. Our results indicate that axon regeneration may be promoted by modified physical and biochemical characteristics of intra- and extracellular architecture in glial scar tissues. Theses findings could potentially help us to understand better the composition of glial scar in central nervous system injury.
Assuntos
Axônios/efeitos dos fármacos , Condroitina ABC Liase/farmacologia , DNA Antissenso/farmacologia , DNA Complementar/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Vimentina/genética , Animais , Axônios/fisiologia , Condroitina ABC Liase/uso terapêutico , Proteoglicanas de Sulfatos de Condroitina/metabolismo , DNA Antissenso/uso terapêutico , DNA Complementar/uso terapêutico , Quimioterapia Combinada , Feminino , Atividade Motora/efeitos dos fármacos , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/fisiopatologia , Tratos Piramidais/ultraestrutura , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
The rapid decline of injury-induced neuronal circuit remodelling after birth is paralleled by the accumulation of chondroitin sulphate proteoglycans (CSPGs) in the extracellular matrix, culminating with the appearance of perineuronal nets (PNNs) around parvalbumin-expressing GABAergic interneurons. We used a spinal cord injury (SCI) model to study the interplay between integrity of PNN CSPGs in the sensorimotor cortex, anatomical remodelling of the corticospinal tract (CST) and motor recovery in adult mice. We showed that thoracic SCI resulted in an atrophy of GABAergic interneurons in the axotomized hindlimb cortex, as well as in a more widespread downregulation of parvalbumin expression. In parallel, spontaneous changes in the integrity of CSPG glycosaminoglycan (GAG) chains associated with PNNs occurred at the boundary between motor forelimb and sensorimotor hindlimb cortex, a region previously showed to undergo reorganization after thoracic SCI. Surprisingly, full digestion of CSPG GAG chains by intracortical chondroitinase ABC injection resulted in an aggravation of motor deficits and reduced sprouting of the axotomized CST above the lesion. Altogether, our data show that changes in the expression pattern of GABAergic markers and PNNs occur in regions of the sensorimotor cortex undergoing spontaneous reorganization after SCI, but suggest that these changes have to be tightly controlled to be of functional benefit.
Assuntos
Membro Posterior/inervação , Córtex Motor/fisiopatologia , Rede Nervosa/fisiopatologia , Plasticidade Neuronal , Tratos Piramidais/fisiopatologia , Córtex Sensório-Motor/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Biomarcadores/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Modelos Animais de Doenças , Feminino , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora , Córtex Motor/metabolismo , Rede Nervosa/metabolismo , Tratos Piramidais/metabolismo , Recuperação de Função Fisiológica , Córtex Sensório-Motor/metabolismo , Traumatismos da Medula Espinal/metabolismo , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismoRESUMO
Chondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of neural regeneration in the peripheral nervous system. Following nerve injury, inhibitory CSPGs accumulate within the endoneurium and Schwann cell basal lamina of the distal nerve stump. The utilization of chondroitinase ABC (chABC) has led to a marked increase in the ability of injured axons to regenerate across gaps through the CSPG-laden extracellular matrix. Experimental models have repeatedly shown chABC to be capable of degrading the CSPGs that hinder neurite outgrowth. In this article, the characterization of CSPGs, their upregulation following peripheral nerve injury, and potential mechanisms behind their growth and inhibition are described. To date, the literature supports that the adjunct use of chABC may be beneficial to peripheral nerve repair in digesting inhibitory CSPGs. chABC has also shown some indication of synergism with other therapies, such as stem cell transplantation. Evidence supporting the use of chondroitinase as a treatment modality in nerve repair, either alone or in combination with other agents, is reviewed within. Finally, several shortcomings of chABC are addressed, notably its thermal stability and physiologic longevity - both hindering its widespread clinical adoption. Future studies are warranted in order to optimize the therapeutic benefits of the chondroitinase enzyme.
Assuntos
Adjuvantes Imunológicos/farmacologia , Condroitina ABC Liase/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/terapia , Animais , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Humanos , Imunomodulação , Traumatismos dos Nervos Periféricos/fisiopatologiaRESUMO
Stroke is the dominant cause of sensorimotor disability that primarily affects the elderly. We now show that neuroplasticity and functional recovery after stroke is constrained by inhibitory chondroitin sulphates. In two blinded, randomized preclinical trials, degradation of chondroitin sulphate using chondroitinase ABC reactivated neuroplasticity and promoted sensorimotor recovery after stroke in elderly rats. Three days after stroke, chondroitinase ABC was microinjected into the cervical spinal cord to induce localized plasticity of forelimb sensorimotor spinal circuitry. Chondroitinase ABC effectively removed chondroitin sulphate from the extracellular matrix and perineuronal nets. Three different tests of sensorimotor function showed that chondroitinase ABC promoted recovery of forelimb function. Anterograde and retrograde tracing showed that chondroitinase ABC also induced sprouting of the contralesional corticospinal tract in the aged treated hemicord. Chondroitinase ABC did not neuroprotect the peri-infarct region. We show for the first time delayed chondroitinase ABC treatment promotes neuroanatomical and functional recovery after focal ischaemic stroke in an elderly nervous system.
Assuntos
Envelhecimento , Condroitina ABC Liase/administração & dosagem , Transtornos Neurológicos da Marcha/tratamento farmacológico , Transtornos Neurológicos da Marcha/etiologia , Plasticidade Neuronal/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral , Estimulação Acústica/efeitos adversos , Amidinas , Análise de Variância , Animais , Biotina/análogos & derivados , Biotina/metabolismo , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/etiologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Dextranos/metabolismo , Modelos Animais de Doenças , Método Duplo-Cego , Feminino , Membro Anterior/fisiopatologia , Lateralidade Funcional/efeitos dos fármacos , Injeções Espinhais/métodos , Masculino , Transtornos dos Movimentos/etiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Lectinas de Plantas , Desempenho Psicomotor/efeitos dos fármacos , Tratos Piramidais/patologia , Ratos , Ratos Long-Evans , Receptores de N-Acetilglucosamina , Transtornos de Sensação/etiologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia , Fatores de TempoRESUMO
OBJECTIVE: Maintenance of chondrocyte phenotype is a major issue in prevention of degeneration and repair of articular cartilage. Although the critical pathways in chondrocyte maturation and homeostasis have been revealed, the in-depth understanding is deficient and novel modifying components and interaction partners are still likely to be discovered. Our focus in this study was to characterize a novel cartilage specific gene that was identified in mouse limb cartilage during embryonic development. METHODS: Open access bioinformatics tools and databases were used to characterize the gene, predicted protein and orthologs in vertebrate species. Immunohistochemistry and mRNA expression methodology were used to study tissue specific expression. Fracture callus and limb bud micromass culture were utilized to study the effects of BMP-2 during experimental chondrogenesis. Fusion protein with C-terminal HA-tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER signal peptide, lumenal/extracellular domain with several threonines/serines prone to O-N-acetylgalactosamine modification, and a cytoplasmic tail with a Yin-Yang site prone to phosphorylation or O-N-acetylglucosamine modification. It is highly conserved in mammals with orthologs in all vertebrate subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis models demonstrated similar expression profiles with Sox9, Acan and Col2a1 and up-regulation by BMP-2. Based on its cartilage specific expression, the molecule was named Snorc, (Small NOvel Rich in Cartilage). CONCLUSION: A novel cartilage specific molecule was identified which marks the differentiating chondrocytes and adult articular chondrocytes with possible functions associated with development and maintenance of chondrocyte phenotype.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Cartilagem Articular/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Idoso , Animais , Cartilagem Articular/embriologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno Tipo II/metabolismo , Membro Posterior/embriologia , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
BACKGROUND: Soft-laser therapy has been used to treat rheumatic diseases for decades. The major effects of laser treatment may be dependent not on thermal mechanisms but rather on cellular, photochemical mechanisms. However, the exact cellular and molecular mechanisms of action have not been elucidated. OBJECTIVE: The aim of this study was to investigate the ex vivo effects of low-level laser treatment (with physical parameters similar to those applied previously) on protein expression in the synovial membrane in rheumatoid arthritis (RA). DESIGN: Synovial tissues were laser irradiated, and protein expression was analyzed. METHODS: Synovial membrane samples obtained from 5 people who had RA and were undergoing knee surgery were irradiated with a near-infrared diode laser at a dose of 25 J/cm(2) (a dose used in clinical practice). Untreated synovial membrane samples obtained from the same people served as controls. Synovial protein expression was assessed with 2-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. RESULTS: The expression of 12 proteins after laser irradiation was different from that in untreated controls. Laser treatment resulted in the decreased expression of α-enolase in 2 samples and of vimentin and precursors of haptoglobin and complement component 3 in 4 samples. The expression of other proteins, including 70-kDa heat shock protein, 96-kDa heat shock protein, lumican, osteoglycin, and ferritin, increased after laser therapy. LIMITATIONS: The relatively small sample size was a limitation of the study. CONCLUSIONS: Laser irradiation (with physical parameters similar to those used previously) resulted in decreases in both α-enolase and vimentin expression in the synovial membrane in RA. Both proteins have been considered to be important autoantigens that are readily citrullinated and drive autoimmunity in RA. Other proteins that are expressed differently also may be implicated in the pathogenesis of RA. Our results raise the possibility that low-level laser treatment of joints affected with RA may be effective, at least in part, by suppressing the expression of autoantigens. Further studies are needed.
Assuntos
Artrite Reumatoide/metabolismo , Artrite Reumatoide/cirurgia , Autoantígenos/metabolismo , Terapia com Luz de Baixa Intensidade/métodos , Fosfopiruvato Hidratase/metabolismo , Membrana Sinovial/metabolismo , Vimentina/metabolismo , Adulto , Idoso , Análise de Variância , Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Estudos de Casos e Controles , Proteoglicanas de Sulfatos de Condroitina/imunologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Ferritinas/imunologia , Ferritinas/metabolismo , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/metabolismo , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sulfato de Queratano/imunologia , Sulfato de Queratano/metabolismo , Lumicana , Espectrometria de Massas , Pessoa de Meia-Idade , Fosfopiruvato Hidratase/imunologia , Membrana Sinovial/imunologia , Vimentina/imunologiaRESUMO
Traumatic brain injury (TBI) results in enduring functional deficits. Strategies aimed at promoting plasticity within the injured brain may aid in enhancing functional outcome. We have previously shown that spontaneous pericontusional axon sprouting occurs within 7-14 days after controlled cortical impact injury in the adult rat, but ultimately fails due to an increasingly growth-inhibitory environment. We therefore sought to determine whether acute infusion of chondroitinase ABC into the site of the cortical contusion, to further reduce pericontusional growth-inhibitory chondroitin sulfate proteoglycans (CSPGs), would enhance and prolong the sprouting response. We also wanted to determine if chondroitinase-enhanced sprouting would ameliorate the behavioral deficits in forelimb function that occur in this model. Acute chondroitinase infusion decreased intact CSPGs and significantly increased pericontusional cortical grey and white matter growth-associated protein 43 (GAP43)-positive axon sprouting at 7 days post-injury. A return of intact CSPGs at later time points likely contributed to the absence of persistently increased levels of axon sprouting by 14-21 days post-injury. There was no overall benefit on forelimb function during the time of maximal sprouting or at any subsequent times in three of four behavioral outcome measures. However, there was a chondroitinase-induced improvement in recovery from unskilled limb use deficits on the staircase forelimb reaching test toward sham-injured values at 28 days, which was not achieved by the vehicle-treated rats, indicating that there is some minor functional benefit of the increased sprouting induced by chondroitinase treatment. The current results, together with data from spinal cord injury models after chondroitinase intervention, suggest that a combinatorial approach with the addition of neurotrophins and rehabilitation would result in more robust axon sprouting and consequently improve behavioral outcome.
Assuntos
Axônios/efeitos dos fármacos , Comportamento Animal/fisiologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Condroitina ABC Liase/farmacologia , Animais , Atrofia , Lesões Encefálicas/psicologia , Contagem de Células , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteína GAP-43/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Córtex Motor/lesões , Córtex Motor/patologia , Procedimentos Neurocirúrgicos , Desempenho Psicomotor/fisiologia , Ratos , Ratos Sprague-Dawley , Córtex Somatossensorial/lesões , Córtex Somatossensorial/patologia , Caminhada/fisiologiaRESUMO
The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans approximately 4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5'-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5'-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/deficiência , Proteoglicanas de Sulfatos de Condroitina/genética , Técnicas de Silenciamento de Genes , Sulfato de Queratano/deficiência , Sulfato de Queratano/genética , Esclera/anatomia & histologia , Esclera/metabolismo , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Bovinos , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno/metabolismo , Sequência Conservada , Substância Própria/metabolismo , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Humanos , Sulfato de Queratano/química , Sulfato de Queratano/metabolismo , Larva/anatomia & histologia , Larva/efeitos dos fármacos , Lumicana , Camundongos , Dados de Sequência Molecular , Antagonistas Muscarínicos/farmacologia , Miopia/tratamento farmacológico , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/genética , Filogenia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Esclera/patologia , Esclera/ultraestrutura , Alinhamento de Sequência , Peixe-Zebra/embriologiaRESUMO
Clinical symptoms in MOG-induced EAE mice significantly exacerbated following chondroitin sulfate A (CS-A) injection, whereas administration of a degraded product, CSPG-DS, caused dramatic inhibition of EAE development. Also, administration of CSPG-DS but not CS-A, after the onset of clinical symptoms of EAE, was able to suppress the disease. Further studies demonstrated that CS-A up-regulated STAT4 expression and thus, induced IFN-gamma production and Th1 CD4 T cell differentiation. CS-A also up-regulated STAT3 and IL-23 expression and thus increased IL-17 producing T cells. CSPG-DS treatment both in vivo and in vitro decreased TNFalpha production from splenocytes. In vitro and in vivo studies indicated that CSPG-DS treatment in EAE mice significantly blocked migration of lymphocytes, whereas CS-A treatment increased lymphocyte infiltration in the brain.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anti-Inflamatórios não Esteroides/administração & dosagem , Proteoglicanas de Sulfatos de Condroitina/administração & dosagem , Dissacarídeos/administração & dosagem , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Sequência de Aminoácidos , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Dissacarídeos/metabolismo , Dissacarídeos/fisiologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Feminino , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/toxicidade , Toxoides/antagonistas & inibidores , Toxoides/toxicidadeRESUMO
The hypothalamo-neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS.