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B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, with long-term overall survival rates of ~85%. However, B-ALL harboring rearrangements of the MLL gene (also known as KMT2A), referred to as MLLr B-ALL, is common in infants and is associated with poor 5-year survival (<30%), frequent relapses, and refractoriness to glucocorticoids (GCs). GCs are an essential part of the treatment backbone for B-ALL and GC resistance is a major clinical predictor of poor outcome. Elucidating the mechanisms of GC resistance in MLLr B-ALL is, therefore, critical to guide therapeutic strategies that deepen the response after induction therapy. Neuron-glial antigen-2 (NG2) expression is a hallmark of MLLr B-ALL and is minimally expressed in healthy hematopoietic cells. We recently reported that NG2 expression is associated with poor prognosis and that anti-NG2 immunotherapy strongly reduces/delays relapse in MLLr B-ALL xenograft models. Despite its contribution to MLLr B-ALL pathogenesis and its diagnostic utility, the role of NG2 in MLLr-mediated leukemogenesis/chemoresistance remains elusive. Here we show that NG2 is an epigenetically regulated direct target gene of the leukemic MLL-AF4 fusion protein. NG2 negatively regulates the expression of the GC receptor NR3C1 and confers GC resistance to MLLr B-ALL cells in vitro and in vivo. Mechanistically, NG2 interacts with FLT3 to render ligand-independent activation of FLT3 signaling (a hallmark of MLLr B-ALL) and downregulation of NR3C1 via AP-1-mediated trans-repression. Collectively, our study elucidates the role of NG2 in GC resistance in MLLr B-ALL through FLT3/AP-1-mediated downregulation of NR3C1, providing novel therapeutic avenues for MLLr B-ALL.
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In the study of tumorigenesis, the involvement of molecules within the extracellular matrix (ECM) is crucial. ADAMTSs (A Disintegrin and Metalloproteinase with Thrombospondin motifs), a group of secreted proteases known for their role in ECM remodeling, were primarily considered to be extracellular proteases. However, our research specifically detected ADAMTS-1, a member of this family, predominantly within the nucleus of mammary cells. Our main objective was to understand the mechanism of ADAMTS-1 translocation to the nucleus and its functional significance in this cellular compartment. Our investigation uncovered that nuclear ADAMTS-1 was present in cells exhibiting an epithelial phenotype, while cells of mesenchymal origin contained the protease in the cytoplasm. Moreover, disruption of ADAMTS-1 secretion, induced by Monensin treatment, resulted in its accumulation in the cytoplasm. Notably, our research indicated that alterations in the secretory pathways could influence the protease's compartmentalization. Additionally, experiments with conditioned medium from cells containing nuclear ADAMTS-1 demonstrated its internalization into the nucleus by HT-1080 cells and fibroblasts. Furthermore, heightened levels of ADAMTS-1 within the ECM reduced the migratory potential of mesenchymal cells. This highlights the potential significance of nuclear ADAMTS-1 as a critical component within the tumor microenvironment due to its functional activity in this specific cellular compartment.
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Proteína ADAMTS1 , Movimiento Celular , Núcleo Celular , Matriz Extracelular , Trombospondinas , Humanos , Proteína ADAMTS1/genética , Proteína ADAMTS1/metabolismo , Carcinogénesis/metabolismo , Endopeptidasas/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Trombospondinas/metabolismo , Microambiente Tumoral , Núcleo Celular/metabolismoRESUMEN
Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.
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Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Patients with AML harboring a constitutively active internal tandem duplication mutation (ITDMUT) in the FMS-like kinase tyrosine kinase (FLT3) receptor generally have a poor prognosis. Several tyrosine kinase/FLT3 inhibitors have been developed and tested clinically, but very few (midostaurin and gilteritinib) have thus far been FDA/EMA-approved for patients with newly diagnosed or relapse/refractory FLT3-ITDMUT AML. Disappointingly, clinical responses are commonly partial or not durable, highlighting the need for new molecules targeting FLT3-ITDMUT AML. Here, we tested EC-70124, a hybrid indolocarbazole analog from the same chemical space as midostaurin with a potent and selective inhibitory effect on FLT3. In vitro, EC-70124 exerted a robust and specific antileukemia activity against FLT3-ITDMUT AML primary cells and cell lines with respect to cytotoxicity, CFU capacity, apoptosis and cell cycle while sparing healthy hematopoietic (stem/progenitor) cells. We also analyzed its efficacy in vivo as monotherapy using two different xenograft models: an aggressive and systemic model based on MOLM-13 cells and a patient-derived xenograft model. Orally disposable EC-70124 exerted a potent inhibitory effect on the growth of FLT3-ITDMUT AML cells, delaying disease progression and debulking the leukemia. Collectively, our findings show that EC-70124 is a promising and safe agent for the treatment of AML with FLT3-ITDMUT.
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Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Disease heterogeneity is well documented, and patient stratification determines treatment decisions. Patient-derived xenografts (PDXs) from risk-stratified AML are crucial for studying AML biology and testing novel therapeutics. Despite recent advances in PDX modeling of AML, reproducible engraftment of human AML is primarily limited to high-risk (HR) cases, with inconsistent or very protracted engraftment observed for favorable-risk (FR) and intermediate-risk (IR) patients. We used NSGS mice to characterize the engraftment robustness/kinetics of 28 AML patient samples grouped according to molecular/cytogenetic classification and assessed whether the orthotopic coadministration of patient-matched bone marrow mesenchymal stromal cells (BM MSCs) improves AML engraftment. PDX event-free survival correlated well with the predictable prognosis of risk-stratified AML patients. The majority (85-94%) of the mice were engrafted in bone marrow (BM) independently of the risk group, although HR AML patients showed engraftment levels that were significantly superior to those of FR or IR AML patients. Importantly, the engraftment levels observed in NSGS mice by week 6 remained stable over time. Serial transplantation and long-term culture-initiating cell (LTC-IC) assays revealed long-term engraftment limited to HR AML patients, fitter leukemia-initiating cells (LICs) in HR AML samples, and the presence of AML LICs in the CD34- leukemic fraction, regardless of the risk group. Finally, orthotopic coadministration of patient-matched BM MSCs and AML cells was dispensable for BM engraftment levels but favored peripheralization of engrafted AML cells. This comprehensive characterization of human AML engraftment in NSGS mice offers a valuable platform for in vivo testing of targeted therapies in risk-stratified AML patient samples.
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Leucemia Mieloide Aguda , Animales , Antígenos CD34 , Médula Ósea , Humanos , Leucemia Mieloide Aguda/terapia , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
The relationship of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteases with inflammatory processes was anticipated since their discovery. Although knowledge of these extracellular proteases in different contexts continues to grow, many questions remain unanswered. In this review, we summarize the most important studies of ADAMTSs and their substrates in inflammation and in the immune system of non-oncological disorders. In addition, we update the findings on cancer and highlight their emerging role in the tumor immune microenvironment. Although the overall functions of extracellular molecules are known to be modulated by proteolysis, specific activities attributed to intact proteins and cleaved fragments in the context of inflammation are still subject to debate. A better understanding of ADAMTS activities will help to elucidate their contribution to the immune phenotype and to open up new therapeutic and diagnostic possibilities.
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ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn2+/Ca2+, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.
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Proteína ADAMTS1/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína ADAMTS1/deficiencia , Proteína ADAMTS1/genética , Sistemas CRISPR-Cas/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Fosforilación , ARN Guía de Kinetoplastida/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
Sarcomas are mesenchymal cancers with poor prognosis, representing about 20% of all solid malignancies in children, adolescents, and young adults. Radio- and chemoresistance are common features of sarcomas warranting the search for novel prognostic and predictive markers. GARP/LRRC32 is a TGF-ß-activating protein that promotes immune escape and dissemination in various cancers. However, if GARP affects the tumorigenicity and treatment resistance of sarcomas is not known. We show that GARP is expressed by human osteo-, chondro-, and undifferentiated pleomorphic sarcomas and is associated with a significantly worse clinical prognosis. Silencing of GARP in bone sarcoma cell lines blocked their proliferation and induced apoptosis. In contrast, overexpression of GARP promoted their growth in vitro and in vivo and increased their resistance to DNA damage and cell death induced by etoposide, doxorubicin, and irradiation. Our data suggest that GARP could serve as a marker with therapeutic, prognostic, and predictive value in sarcoma. We propose that targeting GARP in bone sarcomas could reduce tumour burden while simultaneously improving the efficacy of chemo- and radiotherapy.
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Neoplasias Óseas/metabolismo , Proteínas de la Membrana/metabolismo , Osteosarcoma/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias Óseas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Niño , Preescolar , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Osteosarcoma/patología , Pronóstico , Adulto JovenRESUMEN
Extracellular matrix remodeling within the tumor microenvironment has been recognized as a relevant dynamic framework during tumor growth. However, research on proteases that trigger this remodeling keeps revealing a wide range of actions including both pro- and anti-tumorigenic. The extracellular protease ADAMTS1 exemplifies this dual role. In this work, we first confirmed a positive correlation of ADAMTS1 with endothelial-like phenotype of human melanoma cells together with the finding of associated signatures, including key genes such as endothelial CDH5. Using a CRISPR-Cas9 approach, we observed that the inhibition of ADAMTS1 in an aggressive uveal melanoma model compromised its endothelial-like properties, and more importantly, caused a robust blockade on the progression of tumor xenografts. Although vasculature emerged affected in ADAMTS1-deficient tumors, the most relevant action implied the downregulation of endothelial CDH5 in tumor cells, in association with stemness markers. Indeed, melanoma sphere assays also revealed a deficient commitment to form spheres in the absence of ADAMTS1, directly correlating with stemness markers and, remarkably, also with CDH5. Finally, taking advantage of advanced bioinformatics tools and available public data of uveal melanomas, we disclosed new prognosis factors, including endothelial elements and ADAMTS proteases. Our findings support the key role of ADAMTS proteases for uveal melanoma development since earlier stages, modulating the complex crosstalk between extracellular matrix and the induction of stemness and endothelial-like features. To our knowledge, this is the first report that supports the development of therapeutic targets on the extracellular matrix to overcome uveal melanoma.
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Gliomas in general and the more advanced glioblastomas (GBM) in particular are the most usual tumors of the central nervous system with poor prognosis. GBM patients develop resistance to distinct therapies, in part due to the existence of tumor cell subpopulations with stem-like properties that participate in trans-differentiation events. Within the complex tumor microenvironment, the involvement of extracellular proteases remains poorly understood. The extracellular protease ADAMTS1 has already been reported to contribute to the plasticity of cancer cells. Accordingly, this basic knowledge and the current availability of massive sequencing data from human gliomas, reinforced the development of this work. We first performed an in silico study of ADAMTS1 and endothelial markers in human gliomas, providing the basis to further assess these molecules in several primary glioblastoma-initiating cells and established GBM cells with the ability to acquire an endothelial-like phenotype. Using a co-culture approach of endothelial and GBM cells, we noticed a relevant function of ADAMTS1 in GBM cells leading the organization of endothelial-like networks and, even more significantly, we found a blockade of the formation of tumor-spheres and a deficient response to hypoxia in the absence of ADAMTS1. Our data support a chief role of this protease modulating the phenotypic plasticity of GBM.
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Proteína ADAMTS1/genética , Plasticidad de la Célula/genética , Glioblastoma/genética , Glioma/genética , Línea Celular Tumoral , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/patología , Glioma/patología , Humanos , Masculino , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Microambiente Tumoral/genéticaRESUMEN
The continuous improvement of gene editing tools has allowed a major revolution in biological sciences. Although a variety of gain and loss-of-function approaches have been widely used for the last decades, some limitations arose from non-specific targeting or lack of complete inhibition of the gene of interest. CRISPR/Cas9 editing technology introduced new and significant advantages because it can directly modify the gene of interest and completely blocks its expression.In the context of cancer studies, the heterogeneity of the tumor microenvironment requires comprehensive approaches to unveil the contribution of multiple genes. For example, a deeper understanding of the biology of proteases such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) will improve our perspective of complex phenomena affected by extracellular matrix remodeling, including embryonic development, angiogenesis, immune infiltration, metastasis, and tumor plasticity. Here, we present a method using CRISPR/Cas9 technology to inhibit the expression of the representative ADAMTS1 in cancer cells. Following the first steps of gene edition, we pursue further selection of silenced cells and provide a detailed description of sequence analysis and validation assays. This method leads to inactivation of ADAMTS1 in cancer cells, providing a relevant biological tool that will allow subsequent in vivo and in vitro ADAMTS1 functional analysis.
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Proteína ADAMTS1/antagonistas & inhibidores , Edición Génica/métodos , Análisis de la Célula Individual/métodos , Proteína ADAMTS1/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Células HEK293 , Humanos , Lentivirus/genética , Análisis de Secuencia de ARNRESUMEN
The original version of this Article contained an error in the spelling of the author Juan Carlos Rodriguez-Manzaneque, which was incorrectly given as J Carlos Rodríguez-Manzaneque. This has now been corrected in both the PDF and HTML versions of the Article.
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Recent advances have emphasized the relevance of studying the extracellular microenvironment given its main contribution to tissue homeostasis and disease. Within this complex scenario, we have studied the extracellular protease ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motif 1), implicated in vascularization and development, with reported anti- and pro-tumorigenic activities. In this work we performed a detailed study of the vasculature and substrates in adult organs of wild type and Adamts1-deficient mice. In addition to the expected alterations of organs like kidney, heart and aorta, we found that the lack of ADAMTS1 differently affects lymphocyte and myeloid populations in the spleen and bone marrow. The study of the substrate versican also revealed its alteration in the absence of the protease. With such premises, we challenged our mice with subcutaneous B16F1 syngeneic tumours and closely evaluated the immune repertoire in the tumours but also in the distant spleen and bone marrow. Our results confirmed a pro-inflammatory landscape in the absence of ADAMTS1, correlating with tumour blockade, supporting its novel role as a modulator of the immune cell response.
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Proteína ADAMTS1/metabolismo , Inflamación/inmunología , Inflamación/patología , Neoplasias/inmunología , Neoplasias/patología , Proteína ADAMTS1/genética , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Proliferación Celular , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Neoplasias/irrigación sanguínea , Neoplasias/genética , Neovascularización Patológica/metabolismo , Especificidad de Órganos , Bazo/metabolismo , Bazo/patología , Especificidad por Sustrato , Versicanos/metabolismoRESUMEN
The relevance of tumor vasculature has been extensively recognized, and it is still the focus of numerous lines of research for basic, translational, and clinical scientists. Indeed, the knowledge of some of its regulatory mechanisms has provoked the generation of ongoing cancer therapies. Within the context of the tumor microenvironment, the information that the analysis of the vasculature provides is very valuable, and it might reveal not just its quality and the response against a specific therapy but also its close relationship with neighboring stromal and tumor players.Studies during last decades already supported the contribution of extracellular proteases in neovascularization events, including ADAMTS. However, deeper analyses are still required to better understand the modulation of their proteolytic activity in the tumor microenvironment. Future studies will clearly benefit from existing and ongoing genetically modified mouse models.Here we emphasize the use of syngeneic models to study the vasculature during tumor progression, supported by their intact immunocompetent capacities and also by the range of possibilities to play with engineered mice and with modified tumor cells. Although various high-tech and sophisticated approaches have already been reported to evaluate tumor neovascularization, here we describe a simple and easily reproduced methodology based in the immunofluorescence detection of vascular-specific molecules. A final in silico analysis guarantees an unbiased quantification of tumor vasculature under different conditions.
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Proteína ADAMTS1/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Neoplasias/patología , Neovascularización Patológica/patología , Proteína ADAMTS1/genética , Animales , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Neoplasias/irrigación sanguínea , Neoplasias/genética , Neovascularización Patológica/genética , Programas Informáticos , Microambiente TumoralRESUMEN
Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-All. However, NG2 expression was highly upregulated in blasts infiltrating extramedullar hematopoietic sites and CNS, and specific blockage of NG2 resulted in almost complete loss of engraftment. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ blasts. This study provides new insights on the biology of NG2 in iMLLr-B-ALL and suggests NG2 as a potential therapeutic target to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.
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The matrix metalloprotease ADAMTS1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats 1) has been involved in tumorigenesis although its contributions appeared ambiguous. To understand the multifaceted actions of this protease, it is still required a deeper knowledge of its implication in heterogeneous tumor-stroma interactions. Using a syngeneic B16F1 melanoma model in wild type and ADAMTS1 knockout mice we found distinct stroma versus tumor functions for this protease. Genetic deletion of ADAMTS1 in the host microenvironment resulted in a drastic decrease of tumor growth and metastasis. However, the downregulation of tumor ADAMTS1 did not uncover relevant effects. Reduced tumors in ADAMTS1 KO mice displayed a paradoxical increase in vascular density and vascular-related genes; a detailed characterization revealed an impaired vasculature, along with a minor infiltration of macrophages. In addition, ex-vivo assays supported a chief role for ADAMTS1 in vascular sprouting, and melanoma xenografts showed a relevant induction of its expression in stroma compartments. These findings provide the first genetic evidence that supports the pro-tumorigenic role of stromal ADAMTS1.
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Proteína ADAMTS1/genética , Melanoma Experimental/patología , Melanoma/patología , Neovascularización Patológica/patología , Neoplasias de la Úvea/patología , Proteína ADAMTS1/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo , Eliminación de Gen , Células HEK293 , Humanos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sprouting angiogenesis, where new blood vessels grow from pre-existing ones, is a complex process where biochemical and mechanical signals regulate endothelial cell proliferation and movement. Therefore, a mathematical description of sprouting angiogenesis has to take into consideration biological signals as well as relevant physical processes, in particular the mechanical interplay between adjacent endothelial cells and the extracellular microenvironment. In this work, we introduce the first phase-field continuous model of sprouting angiogenesis capable of predicting sprout morphology as a function of the elastic properties of the tissues and the traction forces exerted by the cells. The model is very compact, only consisting of three coupled partial differential equations, and has the clear advantage of a reduced number of parameters. This model allows us to describe sprout growth as a function of the cell-cell adhesion forces and the traction force exerted by the sprout tip cell. In the absence of proliferation, we observe that the sprout either achieves a maximum length or, when the traction and adhesion are very large, it breaks. Endothelial cell proliferation alters significantly sprout morphology, and we explore how different types of endothelial cell proliferation regulation are able to determine the shape of the growing sprout. The largest region in parameter space with well formed long and straight sprouts is obtained always when the proliferation is triggered by endothelial cell strain and its rate grows with angiogenic factor concentration. We conclude that in this scenario the tip cell has the role of creating a tension in the cells that follow its lead. On those first stalk cells, this tension produces strain and/or empty spaces, inevitably triggering cell proliferation. The new cells occupy the space behind the tip, the tension decreases, and the process restarts. Our results highlight the ability of mathematical models to suggest relevant hypotheses with respect to the role of forces in sprouting, hence underlining the necessary collaboration between modelling and molecular biology techniques to improve the current state-of-the-art.
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Vasos Sanguíneos/crecimiento & desarrollo , Proliferación Celular/fisiología , Modelos Cardiovasculares , Neovascularización Fisiológica/fisiología , Animales , Biología Computacional , Humanos , Ratones , Neovascularización Patológica/fisiopatología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteases comprise the most recently discovered branch of the extracellular metalloenzymes. Research during the last 15years, uncovered their association with a variety of physiological and pathological processes including blood coagulation, tissue repair, fertility, arthritis and cancer. Importantly, a frequent feature of ADAMTS enzymes relates to their effects on vascular-related phenomena, including angiogenesis. Their specific roles in vascular biology have been clarified by information on their expression profiles and substrate specificity. Through their catalytic activity, ADAMTS proteases modify rather than degrade extracellular proteins. They predominantly target proteoglycans and glycoproteins abundant in the basement membrane, therefore their broad contributions to the vasculature should not come as a surprise. Furthermore, in addition to their proteolytic functions, non-enzymatic roles for ADAMTS have also been identified expanding our understanding on the multiple activities of these enzymes in vascular-related processes.
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Proteínas ADAM/química , Proteínas ADAM/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Animales , Coagulación Sanguínea , Glicoproteínas/metabolismo , Humanos , Estructura Terciaria de Proteína , Proteoglicanos/metabolismo , Especificidad por SustratoRESUMEN
The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH-SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH-SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH-SWCNTs' use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 µg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies.
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Materiales Biocompatibles , Diferenciación Celular/efectos de los fármacos , Nanotubos de Carbono , Osteogénesis/efectos de los fármacos , Aloinjertos , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/toxicidad , Línea Celular , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidad , Osteoblastos/efectos de los fármacos , Ratas , Ratas DesnudasRESUMEN
Expression of IGFBP2 (Insulin-like Growth Factor Binding Protein 2) has been positively correlated with glioma progression. Although the proteolysis of IGFBP2 has been widely recognized, with consequences as a major modulator of IGFII signaling, the relevance of this post-translational modification has not been well studied in tumors. Using an in vivo proteomic approach by Isotope-Coded Protein Label (ICPL), we identified IGFBP2 as a target of the extracellular protease ADAMTS1 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 1). Notably, the proteolytic pattern of IGFBP2 was also detected in human glioma culture cells and, more importantly, in all glioma samples evaluated. In addition, high expression of ADAMTS1 correlates with higher levels of cleaved IGFBP2 in glioblastoma multiforme cases. Using gene expression public databases, we confirmed that IGFBP2 is a poor prognosis marker for gliomas, and we also observed an important contribution of ADAMTS1.Finally, we showed the impact of ADAMTS1 on IGFII-mediated IGF1R phosphorylation and cellular migration. Our results support a functional interaction between IGFBP2 and ADAMTS1 and suggest the need to evaluate post-translational modifications of IGFBP2 in glioma, in order to approach new therapies.