RESUMEN
BACKGROUND: During muscle regeneration, the chemokine CXCL12 (SDF-1) and the synthesis of some specific heparan sulfates (HS) have been shown to be critical. CXCL12 activity has been shown to be heavily influenced by its binding to extracellular glycosaminoglycans (GAG) by modulating its presentation to its receptors and by generating haptotactic gradients. Although CXCL12 has been implicated in several phases of tissue repair, the influence of GAG binding under HS influencing conditions such as acute tissue destruction remains understudied. METHODS: To investigate the role of the CXCL12/HS proteoglycan interactions in the pathophysiology of muscle regeneration, we performed two models of muscle injuries (notexin and freeze injury) in mutant CXCL12Gagtm/Gagtm mice, where the CXCL12 gene having been selectively mutated in critical binding sites of CXCL12 to interact with HS. Histological, cytometric, functional transcriptomic, and ultrastructure analysis focusing on the satellite cell behavior and the vessels were conducted on muscles before and after injuries. Unless specified, statistical analysis was performed with the Mann-Whitney test. RESULTS: We showed that despite normal histology of the resting muscle and normal muscle stem cell behavior in the mutant mice, endothelial cells displayed an increase in the angiogenic response in resting muscle despite the downregulated transcriptomic changes induced by the CXCL12 mutation. The regenerative capacity of the CXCL12-mutated mice was only delayed after a notexin injury, but a severe damage by freeze injury revealed a persistent defect in the muscle regeneration of CXCL12 mutant mice associated with vascular defect and fibroadipose deposition with persistent immune cell infiltration. CONCLUSION: The present study shows that CXCL12 is crucial for proper muscle regeneration. We highlight that this homing molecule could play an important role in drastic muscle injuries and that the regeneration defect could be due to an impairment of angiogenesis, associated with a long-lasting fibro-adipogenic scar.
Asunto(s)
Quimiocina CXCL12/genética , Quimiocina CXCL12/fisiología , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/fisiopatología , Neovascularización Fisiológica/genética , Regeneración/genética , Regeneración/fisiología , Animales , Venenos Elapídicos/toxicidad , Células Endoteliales/patología , Células Endoteliales/fisiología , Congelación/efectos adversos , Perfilación de la Expresión Génica , Proteoglicanos de Heparán Sulfato/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Músculo Esquelético/lesiones , Regeneración/efectos de los fármacos , Células Satélite del Músculo Esquelético/patología , Células Satélite del Músculo Esquelético/fisiologíaRESUMEN
The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the (epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.
Asunto(s)
Neoplasias Cerebelosas/patología , Meduloblastoma/patología , Receptor ErbB-4/metabolismo , Familia-src Quinasas/metabolismo , Adolescente , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Neoplasias Cerebelosas/genética , Cerebelo/patología , Niño , Preescolar , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Masculino , Meduloblastoma/genética , Ratones , Ratones Transgénicos , Fosforilación , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal , Familia-src Quinasas/genéticaRESUMEN
Numerous molecular alterations have been described in supratentorial high-grade gliomas (1p19q co-deletion, IDH1/2, histone H3, hTERT promotor mutations, loss of ATRX) which have led to a new histomolecular classification of diffuse gliomas. We aimed at describing these alterations in a series of 19 adults with pure cerebellar high-grade gliomas. Systematic immunohistochemical analyses, including that of IDH1R132H, ATRX, p53, PTEN, EGFR, p16, FGFR3, BRAFV600E, mismatch repair proteins, H3K27me3, H3K36me3, and H3K27M; molecular analyses of IDH1/2, hTERT, BRAF, H3F3A, and HIST1H3B mutation hotspots; and EGFR, PTEN FISH were retrospectively performed in a multicentric study. We histopathologically identified 14 glioblastomas, 4 grade III astrocytomas and 1 gliosarcoma. Two cases showed a H3F3A K27M mutation. Only one case harbored a classical profile of glioblastoma with hTERT mutation, EGFR gain and 10q loss. The most frequent alteration was the absence of p16 immunoexpression. We report a histomolecular analysis of pure cerebellar high grade gliomas. The histomolecular profile appears to be different from that of supratentorial gliomas, with no IDH1/2 gene mutations and only 1 case with a classic profile of de novo glioblastoma. In 2 cases, we identified H3F3A K27M mutation, classically described in pediatric midline gliomas.â©.
Asunto(s)
Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Glioma/genética , Glioma/patología , Histonas/genética , Neoplasias Supratentoriales/genética , Neoplasias Supratentoriales/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mutación/genética , Proteínas de Neoplasias/genética , Estudios Retrospectivos , Adulto JovenRESUMEN
Ganglioglioma (GG) is a grade I tumor characterized by alterations in the MAPK pathway, including BRAF V600E mutation. Recently, diffuse midline glioma with an H3 K27M mutation was added to the WHO 2016 classification as a new grade IV entity. As co-occurrence of H3 K27M and BRAF V600E mutations has been reported in midline tumors and anaplastic GG, we searched for BRAF V600E and H3 K27M mutations in a series of 54 paediatric midline grade I GG (midline GG) to determine the frequency of double mutations and its relevance for prognosis. Twenty-seven patients (50%) possessed the BRAF V600E mutation. The frequency of the co-occurrence of H3F3A/BRAF mutations at diagnosis was 9.3%. No H3 K27M mutation was detected in the absence of the BRAF V600E mutation. Double-immunostaining revealed that BRAF V600E and H3 K27M mutant proteins were present in both the glial and neuronal components. Immunopositivity for the BRAF V600E mutant protein correlated with BRAF mutation status as detected by massARRAY or digital droplet PCR. The median follow-up of patients with double mutation was 4 years. One patient died of progressive disease 8 years after diagnosis, whereas the four other patients were all alive with stable disease at the last clinical follow-up (at 9 months, 1 year and 7 years) without adjuvant therapy. We demonstrate in this first series of midline GGs that the H3 K27M mutation can occur in association with the BRAF V600E mutation in grade I glioneuronal tumors. Despite the presence of H3 K27M mutations, these cases should not be graded and treated as grade IV tumors because they have a better spontaneous outcome than classic diffuse midline H3 K27M-mutant glioma. These data suggest that H3 K27M cannot be considered a specific hallmark of grade IV diffuse gliomas and highlight the importance of integrated histomolecular diagnosis in paediatric brain tumors.
Asunto(s)
Neoplasias Encefálicas/genética , Ganglioglioma/genética , Histonas/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias de la Médula Espinal/genética , Adolescente , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Niño , Preescolar , Femenino , Estudios de Seguimiento , Ganglioglioma/diagnóstico por imagen , Ganglioglioma/patología , Ganglioglioma/terapia , Humanos , Inmunohistoquímica , Masculino , Clasificación del Tumor , Neoplasias de la Médula Espinal/diagnóstico por imagen , Neoplasias de la Médula Espinal/patología , Neoplasias de la Médula Espinal/terapia , Resultado del TratamientoRESUMEN
Clear cell meningioma (CCM) is a rare grade II histopathological subtype that usually occurs in young patients and displays high recurrence rate. Germline SMARCE1 mutations have been described in hereditary forms of this disease and more recently in small syndromic and sporadic CCM series. The diagnostic value of SMARCE1 in distinguishing between CCM and other meningioma variants has not been yet established. The aim of our study was to investigate the status of SMARCE1 in a series of CCMs and its morphological mimickers. We compared the performance of an anti-SMARCE1 antibody and the molecular analysis of the SMARCE1 gene in a retrospective multicenter series of CCMs. All CCMs lossed SMARCE1 immunoexpression. Bi-allelic inactivating events were found by NGS-based sequencing in all of these cases, except for one, which was incompletely explored, but had a wild-type sequence. We then validated the anti-SMARCE1 antibody specificity by analyzing additional 305 pediatric and adult meningiomas of various subtypes and 15 non-meningioma clear cell tumors by SMARCE1 immunohistochemistry. A nuclear immunostaining was preserved in all other meningioma variants, as well as non-meningioma clear cell tumors. In conclusion, our series showed, for the first time, that SMARCE1 immunostaining is a highly sensitive biomarker for CCM, useful as a routine diagnostic biomarker.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias Meníngeas , Meningioma , Adolescente , Adulto , Anciano , Anticuerpos/administración & dosificación , Biomarcadores de Tumor/metabolismo , Niño , Proteínas Cromosómicas no Histona/inmunología , Proteínas de Unión al ADN/inmunología , Femenino , Genotipo , Humanos , Inmunohistoquímica , Masculino , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/metabolismo , Meningioma/diagnóstico , Meningioma/genética , Meningioma/metabolismo , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
UNLABELLED: Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH), to initiate growth, protect liver cells, and sustain remnant liver functions. Extracellular adenosine triphosphate rises in blood and bile after PH and contributes to liver regeneration, although purinergic receptors and mechanisms remain to be precisely explored. In this work we analyzed during regeneration after PH the involvement of P2X4 purinergic receptors, highly expressed in the liver. P2X4 receptor expression in the liver, liver histology, hepatocyte proliferation, plasma bile acid concentration, bile flow and composition, and lysosome distribution in hepatocytes were studied in wild-type and P2X4 knockout (KO) mice, before and after PH. P2X4 receptors were expressed in hepatocytes and Kupffer cells; in hepatocytes, P2X4 was concentrated in subcanalicular areas closely costained with lysosomal markers. After PH, delayed regeneration, hepatocyte necrosis, and cholestasis were observed in P2X4-KO mice. In P2X4-KO mice, post-PH biliary adaptation was impaired with a smaller increase in bile flow and HCO3 (-) biliary output, as well as altered biliary composition with reduced adenosine triphosphate and lysosomal enzyme release. In line with these data, lysosome distribution and biogenesis were altered in P2X4-KO compared with wild-type mice. CONCLUSION: During liver regeneration after PH, P2X4 contributes to the complex control of biliary homeostasis through mechanisms involving pericanalicular lysosomes, with a resulting impact on hepatocyte protection and proliferation. (Hepatology 2016;64:941-953).
Asunto(s)
Sistema Biliar/fisiología , Regeneración Hepática , Hígado/metabolismo , Lisosomas/fisiología , Receptores Purinérgicos P2X4/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Ácidos y Sales Biliares/sangre , Proliferación Celular , Células Cultivadas , Hepatectomía , Hepatocitos/fisiología , Homeostasis , Hígado/ultraestructura , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: A longstanding goal in regenerative medicine is to reconstitute functional tissues or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised. METHODS: We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®. RESULTS: We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models. CONCLUSIONS: Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.
Asunto(s)
Modelos Animales , Músculo Esquelético/fisiología , Regeneración , Animales , Compuestos de Bario/toxicidad , Cloruros/toxicidad , Proteínas Cardiotóxicas de Elápidos/toxicidad , Lesión por Frío/patología , Lesión por Frío/fisiopatología , Citocinas/fisiología , Venenos Elapídicos/toxicidad , Fibrosis , Congelación/efectos adversos , Proteínas Fluorescentes Verdes/análisis , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Desarrollo de Músculos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Mioblastos/fisiología , Necrosis , Neovascularización Fisiológica , Regeneración/inmunología , Regeneración/fisiología , Células Satélite del Músculo Esquelético/fisiología , Células Madre/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/análisisRESUMEN
UNLABELLED: Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH) to initiate growth, protect liver cells, and sustain functions of the remnant liver. Bile acids (BAs), whose levels rise in the blood early after PH, stimulate both hepatocyte proliferation and protection, in part through their binding to the nuclear farnesoid X receptor (FXR). However, the effect of the BA receptor, TGR5 (G-protein-coupled BA receptor 1) after PH remains to be studied. Liver histology, hepatocyte proliferation, BA concentrations (plasma, bile, liver, urine, and feces), bile flow and composition, and cytokine production were studied in wild-type (WT) and TGR5 KO (knockout) mice before and after PH. BA composition (plasma, bile, liver, urine, and feces) was more hydrophobic in TGR5 KO than in WT mice. After PH, severe hepatocyte necrosis, prolonged cholestasis, exacerbated inflammatory response, and delayed regeneration were observed in TGR5 KO mice. Although hepatocyte adaptive response to post-PH BA overload was similar in WT and TGR5 KO mice, kidney and biliary adaptive responses were strongly impaired in TGR5 KO mice. Cholestyramine treatment, as well as Kupffer cell depletion, significantly improved the post-PH TGR5 KO mice phenotype. After bile duct ligation or upon a cholic acid-enriched diet, TGR5 KO mice exhibited more severe liver injury than WT as well as impaired BA elimination in urine. CONCLUSION: TGR5 is crucial for liver protection against BA overload after PH, primarily through the control of bile hydrophobicity and cytokine secretion. In the absence of TGR5, intrahepatic stasis of abnormally hydrophobic bile and excessive inflammation, in association with impaired bile flow adaptation and deficient urinary BA efflux, lead to BA overload-induced liver injury and delayed regeneration.