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Transcription factor TFEB is thought to control cellular functions-including in the vascular bed-primarily via regulation of lysosomal biogenesis and autophagic flux. Here, we report that TFEB also orchestrates a non-canonical program that controls the cell cycle/VEGFR2 pathway in the developing vasculature. In endothelial cells, TFEB depletion halts proliferation at the G1-S transition by inhibiting the CDK4/Rb pathway. TFEB-deficient cells attempt to compensate for this limitation by increasing VEGFR2 levels at the plasma membrane via microRNA-mediated mechanisms and controlled membrane trafficking. TFEB stimulates expression of the miR-15a/16-1 cluster, which limits VEGFR2 transcript stability and negatively modulates expression of MYO1C, a regulator of VEGFR2 trafficking to the cell surface. Altered levels of miR-15a/16-1 and MYO1C in TFEB-depleted cells cause increased expression of plasma membrane VEGFR2, but in a manner associated with low signaling strength. An endothelium-specific Tfeb-knockout mouse model displays defects in fetal and newborn mouse vasculature caused by reduced endothelial proliferation and by anomalous function of the VEGFR2 pathway. These previously unrecognized functions of TFEB expand its role beyond regulation of the autophagic pathway in the vascular system.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Proliferação de Células , Embrião de Mamíferos/citologia , Endotélio Vascular/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/fisiologia , Endotélio Vascular/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Over sixty years, laser technologies have undergone a technological revolution and become one of the main tools in biomedicine, particularly in neuroscience, neurodegenerative diseases and brain tumors. Glioblastoma is the most lethal form of brain cancer, with very limited treatment options and a poor prognosis. In this study on rats, we demonstrate that glioblastoma (GBM) growth can be suppressed by photosensitizer-free laser treatment (PS-free-LT) using a quantum-dot-based 1267 nm laser diode. This wavelength, highly absorbed by oxygen, is capable of turning triplet oxygen to singlet form. Applying 1267 nm laser irradiation for a 4 week course with a total dose of 12.7 kJ/cm2 firmly suppresses GBM growth and increases survival rate from 34% to 64%, presumably via LT-activated apoptosis, inhibition of the proliferation of tumor cells, a reduction in intracranial pressure and stimulation of the lymphatic drainage and clearing functions. PS-free-LT is a promising breakthrough technology in non- or minimally invasive therapy for superficial GBMs in infants as well as in adult patients with high photosensitivity or an allergic reaction to PSs.
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Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFß1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFß1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFß1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFß1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFß1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.
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Células Endoteliais , Fator 2 de Crescimento de Fibroblastos , Fator de Crescimento Transformador beta1/fisiologia , Animais , Proteína Morfogenética Óssea 4 , Diferenciação Celular , Células-Tronco Embrionárias , Fator 2 de Crescimento de Fibroblastos/farmacologia , Camundongos , Proteínas Proto-Oncogênicas , Células Estromais , Fator de Crescimento Transformador beta1/genética , Proteínas WntRESUMO
In the last several years, accumulating evidence indicates that noncoding RNAs, especially long-noncoding RNAs (lncRNAs) and microRNAs, play essential roles in regulating angiogenesis. However, the contribution of lncRNA-mediated competing-endogenous RNA (ceRNA) activity in the control of capillary sprouting from the pre-existing ones has not been described so far. Here, by exploiting the transcriptomic profile of VEGF-A-activated endothelial cells in a consolidate three-dimensional culture system, we identified a list of lncRNAs whose expression was modified during the sprouting process. By crossing the lncRNAs with a higher expression level and the highest fold change value between unstimulated and VEGF-A-stimulated endothelial cells, we identified the unknown LINC02802 as the best candidate to take part in sprouting regulation. LINC02802 was upregulated after VEGF-A stimulation and its knockdown resulted in a significant reduction in sprouting activity. Mechanistically, we demonstrated that LINC02802 acts as a ceRNA in the post-transcriptional regulation of Mastermind-like-3 (MAML3) gene expression through a competitive binding with miR-486-5p. Taken together, these results suggest that LINC02802 plays a critical role in preventing the miR-486-5p anti-angiogenic effect and that this inhibitory effect results from the reduction in MAML3 expression.
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Células Endoteliais da Veia Umbilical Humana/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , RNA Longo não Codificante/metabolismo , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.
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Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Técnicas Imunoenzimáticas , Camundongos , Chaperonas Moleculares , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.
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Glioma , Gliose , Proteínas de Membrana , Humanos , Proteínas de Membrana/metabolismo , Glioma/metabolismo , Glioma/patologia , Gliose/metabolismo , Gliose/patologia , Animais , Receptores de PeptídeosRESUMO
OBJECTIVE: Liver X receptors (LXRα, LXRß) are master regulators of cholesterol homeostasis. In the endothelium, perturbations of cell cholesterol have an impact on fundamental processes. We, therefore, assessed the effects of LXR activation on endothelial functions related to angiogenesis in vitro and in vivo. METHODS AND RESULTS: LXR agonists (T0901317, GW3965) blunted migration, tubulogenesis, and proliferation of human umbilical vein endothelial cells. By affecting endothelial cholesterol homeostasis, LXR activation impaired the compartmentation of vascular endothelial growth factor receptor-2 in lipid rafts/caveolae and led to defective phosphorylation and downstream signaling of vascular endothelial growth factor receptor-2 upon vascular endothelial growth factor-A stimulation. Consistently, the antiangiogenic actions of LXR agonists could be prevented by coadministration of exogenous cholesterol. LXR agonists reduced endothelial sprouting from wild-type but not from LXRα(-/-)/LXRß(-/-) knockout aortas and blunted the vascularization of implanted angioreactors in vivo. Furthermore, T0901317 reduced the growth of Lewis lung carcinoma grafts in mice by impairing angiogenesis. CONCLUSIONS: Pharmacological activation of endothelial LXRs reduces angiogenesis by restraining cholesterol-dependent vascular endothelial growth factor receptor-2 compartmentation and signaling. Thus, administration of LXR agonists could exert therapeutic effects in pathological conditions characterized by uncontrolled angiogenesis.
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Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colesterol/metabolismo , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Nucleares Órfãos/deficiência , Fosforilação , Interferência de RNA , Sulfonamidas/farmacologia , Fatores de Tempo , Transfecção , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) are each leading causes of mortality and morbidity worldwide, and present additional treatment considerations when they are comorbid (TBI+HS) as a result of competing pathophysiological responses. The current study rigorously quantified injury biomechanics with high precision sensors and examined whether blood-based surrogate markers were altered in general trauma as well as post-neurotrauma. Eighty-nine sexually mature male and female Yucatan swine were subjected to a closed-head TBI+HS (40% of circulating blood volume; n = 68), HS only (n = 9), or sham trauma (n = 12). Markers of systemic (e.g., glucose, lactate) and neural functioning were obtained at baseline, and at 35 and 295 min post-trauma. Opposite and approximately twofold differences existed for both magnitude (device > head) and duration (head > device) of quantified injury biomechanics. Circulating levels of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and ubiquitin C-terminal hydrolase L1 (UCH-L1) demonstrated differential sensitivity for both general trauma (HS) and neurotrauma (TBI+HS) relative to shams in a temporally dynamic fashion. GFAP and NfL were both strongly associated with changes in systemic markers during general trauma and exhibited consistent time-dependent changes in individual sham animals. Finally, circulating GFAP was associated with histopathological markers of diffuse axonal injury and blood-brain barrier breach, as well as variations in device kinematics following TBI+HS. Current findings therefore highlight the need to directly quantify injury biomechanics with head mounted sensors and suggest that GFAP, NfL, and UCH-L1 are sensitive to multiple forms of trauma rather than having a single pathological indication (e.g., GFAP = astrogliosis).
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Lesões Encefálicas Traumáticas , Choque Hemorrágico , Masculino , Feminino , Suínos , Animais , Fenômenos Biomecânicos , Biomarcadores , Modelos Animais , Proteína Glial Fibrilar Ácida , Ubiquitina TiolesteraseRESUMO
The connections existing between vessels and nerves go beyond the structural architecture of vascular and nervous systems to comprise cell fate determination. The analysis of functional/molecular links that interconnect endothelial and neural commitments requires a model in which the two differentiation programs take place at the same time in an artificial controllable environment. To this regard, this work presents an in vitro model to differentiate embryonic stem (ES) cells simultaneously into mature neurons and endothelial cells. Murine ES cells are differentiated within an artificial environment composed of PA6 stromal cells and a serum-free medium. Upon these basal culture conditions ES cells preferentially differentiate into neurons. The addition of basic fibroblast growth factor (FGF2) to the medium allows the simultaneous maturation of neurons and endothelial cells, whereas bone morphogenetic protein (BMP)4 drives endothelial differentiation to the disadvantage of neural commitment. The responsiveness of the system to exogenous cytokines was confirmed by genes expression analysis that revealed a significant up-regulation of endothelial genes in presence of FGF2 and a massive down-regulation of the neural markers in response to BMP4. Furthermore, the role played by single genes in determining endothelial and neural fate can be easily explored by knocking down the expression of the target gene with lentiviruses carrying the corresponding shRNA sequence. The possibility to address the neural and the endothelial fate separately or simultaneously by exogenous stimuli combined with an efficient gene silencing strategy make this model an optimal tool to identify environmental signals and genes pathways involved in both endothelial and neural specification.
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Células-Tronco Embrionárias/citologia , Endotélio/citologia , Endotélio/crescimento & desenvolvimento , Neurônios/citologia , Animais , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Técnicas de Cultura de Células , Diferenciação Celular , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Camundongos , Células Estromais/citologiaRESUMO
BACKGROUND AIMS: Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. METHODS: The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34(+) cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34(+) cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. RESULTS: Gene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14(+) subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. CONCLUSIONS: Our data suggest that combining basal and expanded BM-derived CD34(+) cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.
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Vasos Coronários/cirurgia , Ligadura , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/terapia , Animais , Antígenos CD/metabolismo , Antígenos CD34/biossíntese , Medula Óssea/patologia , Caderinas/metabolismo , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Endotélio/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos NOD , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Recuperação de Função FisiológicaRESUMO
[This corrects the article DOI: 10.1016/j.csbj.2020.11.048.].
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Glioblastomas (GBM) are the most aggressive tumors originating in the brain. Histopathologic features include circuitous, disorganized, and highly permeable blood vessels with intermittent blood flow. These features contribute to the inability to direct therapeutic agents to tumor cells. Known targets for anti-angiogenic therapies provide minimal or no effect in overall survival of 12-15 months following diagnosis. Identification of novel targets therefore remains an important goal for effective treatment of highly vascularized tumors such as GBM. We previously demonstrated in zebrafish that a balanced level of expression of the transmembrane protein TMEM230/C20ORF30 was required to maintain normal blood vessel structural integrity and promote proper vessel network formation. To investigate whether TMEM230 has a role in the pathogenesis of GBM, we analyzed its prognostic value in patient tumor gene expression datasets and performed cell functional analysis. TMEM230 was found necessary for growth of U87-MG cells, a model of human GBM. Downregulation of TMEM230 resulted in loss of U87 migration, substratum adhesion, and re-passaging capacity. Conditioned media from U87 expressing endogenous TMEM230 induced sprouting and tubule-like structure formation of HUVECs. Moreover, TMEM230 promoted vascular mimicry-like behavior of U87 cells. Gene expression analysis of 702 patients identified that TMEM230 expression levels distinguished high from low grade gliomas. Transcriptomic analysis of patients with gliomas revealed molecular pathways consistent with properties observed in U87 cell assays. Within low grade gliomas, elevated TMEM230 expression levels correlated with reduced overall survival independent from tumor subtype. Highest level of TMEM230 correlated with glioblastoma and ATP-dependent microtubule kinesin motor activity, providing a direction for future therapeutic intervention. Our studies support that TMEM230 has both glial tumor and endothelial cell intracellular and extracellular functions. Elevated levels of TMEM230 promote glial tumor cell migration, extracellular scaffold remodeling, and hypervascularization and abnormal formation of blood vessels. Downregulation of TMEM230 expression may inhibit both low grade glioma and glioblastoma tumor progression and promote normalization of abnormally formed blood vessels. TMEM230 therefore is both a promising anticancer and antiangiogenic therapeutic target for inhibiting GBM tumor cells and tumor-driven angiogenesis.
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Embryonic stem (ES) cells are pluripotent cells capable of differentiating in all the cell types present in a living organism. They derive from the inner cell mass of blastocysts of different species including humans. Given their unlimited potential, ES cells represent an invaluable resource of different cell types for transplantation and tissue engineering applications. However, in order to accomplish these therapeutic purposes, efficient and controlled in vitro systems of directing ES cell differentiation are mandatory. ES cell differentiation is strongly influenced by physical, chemical and cellular signals provided by the local microenvironment. Understanding the relationships occurring between differentiating cells and surrounding environment is pivotal for a successful ES cells-based therapy. This review describes three different methods of in vitro differentiation of ES cells by outlining the environmental elements required for endothelial fate specification. For each system, the efficiency of endothelial differentiation, the accessibility and the advantages are discussed. The main conclusion that arises from this analysis is that the knowledge of the role played by microenvironment in cell fate determination is essential to control and take advantage of ES cells potential.
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Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Células Endoteliais/fisiologia , Nicho de Células-Tronco , Animais , Comunicação Celular , Linhagem da Célula , Meios de Cultura/metabolismo , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Neovascularização FisiológicaRESUMO
The observation that the architecture of the cardiovascular and nervous systems is drawn by common guidance cues and the closeness between neural progenitors and endothelial cells in the vascular niche strongly suggests the existence of links between endothelial and neural cell fates. We identified an embryonic stem cell-derived discrete, nonclonal cell population expressing the two vascular endothelial growth factor receptors neuropilin-1 (Nrp1) and Flk1 that differentiates in vitro toward endothelial or neural phenotypes depending on microenvironmental cues. When microinjected in the chick embryo, Nrp1(+) cells integrate within the host, developing vessels and brain, and acquire endothelial and neural markers, respectively. These results show that precursors of endothelial cells and precursors of neural cells arise from the same pool of differentiating embryonic stem cells and share the expression of Nrp1 and Flk1. These data reinforce the parallelism between vascular and nervous system at the level of cell fate and commitment and open new perspective in regenerative medicine of neurovascular diseases.
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Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Neuropilina-1/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Embrião de Galinha , Fibroblastos/metabolismo , Camundongos , Neuropilina-1/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Interferon-α (IFN-α) comprises a family of 13 cytokines involved in the modulation of antiviral, immune, and anticancer responses by orchestrating a complex transcriptional network. The activation of IFN-α signaling pathway in endothelial cells results in decreased proliferation and migration, ultimately leading to suppression of angiogenesis. In this study, we knocked-down the expression of seven established or candidate modulators of IFN-α response in endothelial cells to reconstruct a gene regulatory network and to investigate the antiangiogenic activity of IFN-α. This genetic perturbation approach, along with the analysis of interferon-induced gene expression dynamics, highlighted a complex and highly interconnected network, in which the angiostatic chemokine C-X-C Motif Chemokine Ligand 10 (CXCL10) was a central node targeted by multiple modulators. IFN-α-induced secretion of CXCL10 protein by endothelial cells was blunted by the silencing of Signal Transducer and Activator of Transcription 1 (STAT1) and of Interferon Regulatory Factor 1 (IRF1) and it was exacerbated by the silencing of Ubiquitin Specific Peptidase 18 (USP18). In vitro sprouting assay, which mimics in vivo angiogenesis, confirmed STAT1 as a positive modulator and USP18 as a negative modulator of IFN-α-mediated sprouting suppression. Our data reveal an unprecedented physiological regulation of angiogenesis in endothelial cells through a tonic IFN-α signaling, whose enhancement could represent a viable strategy to suppress tumor neoangiogenesis.
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Angiogenesis requires the temporal coordination of the proliferation and the migration of endothelial cells. Here, we investigated the regulatory role of microRNAs (miRNAs) in harmonizing angiogenesis processes in a three-dimensional in vitro model. We described a microRNA network which contributes to the observed down- and upregulation of proliferative and migratory genes, respectively. Global analysis of miRNA-target gene interactions identified two sub-network modules, the first organized in upregulated miRNAs connected with downregulated target genes and the second with opposite features. miR-424-5p and miR-29a-3p were selected for the network validation. Gain- and loss-of-function approaches targeting these microRNAs impaired angiogenesis, suggesting that these modules are instrumental to the temporal coordination of endothelial migration and proliferation. Interestingly, miR-29a-3p and its targets belong to a selective biomarker that is able to identify colorectal cancer patients who are responding to anti-angiogenic treatments. Our results provide a view of higher-order interactions in angiogenesis that has potential to provide diagnostic and therapeutic insights.
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Células Endoteliais , MicroRNAs , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Biomarcadores Tumorais , Células Cultivadas , Neoplasias Colorretais/tratamento farmacológico , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , FenótipoRESUMO
Many of the existing three-dimensional (3D) cancer models in vitro fail to represent the entire complex tumor microenvironment composed of cells and extra cellular matrix (ECM) and do not allow a reliable study of the tumoral features and progression. In this paper we reported a strategy to produce 3D in vitro microtissues of pancreatic ductal adenocarcinoma (PDAC) for studying the desmoplastic reaction activated by the stroma-cancer crosstalk. Human PDAC microtissues were obtained by co-culturing pancreatic cancer cells (PT45) and normal or cancer-associated fibroblasts within biodegradable microcarriers in a spinner flask bioreactor. Morphological and histological analyses highlighted that the presence of fibroblasts resulted in the deposition of a stromal matrix rich in collagen leading to the formation of tumor microtissues composed of a heterotypic cell population embedded in their own ECM. We analyzed the modulation of expression of ECM genes and proteins and found that when fibroblasts were co-cultured with PT45, they acquired a myofibroblast phenotype and expressed the desmoplastic reaction markers. This PDAC microtissue, closely recapitulating key PDAC microenvironment characteristics, provides a valuable tool to elucidate the complex stroma-cancer interrelationship and could be used in a future perspective as a testing platform for anticancer drugs in tissue-on-chip technology. STATEMENT OF SIGNIFICANCE: Tumor microenvironment is extremely complex and its organization is due to the interaction between different kind of cells and the extracellular matrix. Tissue engineering could give the answer to the increasing need of 3D culture model that better recapitulate the tumor features at cellular and extracellular level. We aimed in this work at developing a microtissue tumor model by mean of seeding together cancer cells and fibroblasts on gelatin microsphere in order to monitor the crosstalk between the two cell populations and the endogenous extracellular matrix deposition. Results are of particular interest because of the need of heterotypic cancer model that can replicate the complexity of the tumor microenvironment and could be used as drug screening platform.
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Bioengenharia/métodos , Neoplasias Pancreáticas/patologia , Biomarcadores Tumorais/metabolismo , Ciclo Celular/genética , Regulação para Baixo/genética , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Metaloproteinases da Matriz/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/genética , Reprodutibilidade dos Testes , Transdução de Sinais/genética , SoftwareRESUMO
Neuronal pentraxins (NPTX) and their corresponding receptors (NPTXR) have been studied as synapse-associated proteins in the nervous system, but their role in cancer is largely unknown. By applying a multidisciplinary, high-throughput proteomic approach, we have recently identified a peptide ligand motif for targeted drug delivery to neuroblastoma. Here, we report the sequence similarity between this peptide and a conserved portion of the pentraxin domain that is involved in the homo- and hetero-oligomerization of NPTX2 and NPTXR. We show that, in comparison with normal tissues, NPTX2 and NPTXR are overexpressed in vivo in mouse models, as well as in human Schwannian stroma-poor, stage IV neuroblastoma. Both proteins are concentrated in the vicinity of tumor blood vessels, with NPTXR also present on neuroblastic tumor cells. In vivo targeting of NPTX2 and NPTXR with the selected peptide or with specific antibodies reduces tumor burden in orthotopic mouse models of human neuroblastoma. In vitro interference with this ligand/receptor system inhibits the organization of neuroblastoma cells in tumor-like masses in close contact with vascular cells, as well as their adhesion to normal microenvironment-derived cells, suggesting a role in the cross-talk between tumor and normal cells in the early steps of neuroblastoma development. Finally, we show that NPTX2 is a marker of poor prognosis for neuroblastoma patients.
Assuntos
Proteína C-Reativa/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/farmacologia , Proteína C-Reativa/química , Proteína C-Reativa/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Ligantes , Camundongos , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/mortalidade , Peptídeos/química , Peptídeos/farmacologia , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIMS: Liver X receptors alpha and beta (LXRalpha, LXRbeta) are key regulators of cholesterol homeostasis. The effects of LXR ligands on endothelial cells are largely unknown. While oxysterol LXR agonists can increase the endothelial-leukocyte interaction, synthetic LXR agonists are anti-atherogenic and anti-inflammatory. Mechanistic differences may underlie such findings. METHODS AND RESULTS: LXRalpha and LXRbeta were found to be expressed in human endothelial cells. While synthetic LXR agonists could blunt the LPS-induced up-regulation of adhesion molecules (ICAM-1, VCAM-1, E-Selectin), 22-hydroxycholesterol and 24,25-epoxycholesterol enhanced such response. Microarray profiling further showed that the endothelial gene expression fingerprints of 22-hydroxycholesterol and T0901317 largely differed and unexpectedly shared only a restricted number of genes. Indeed, 22-hydroxycholesterol down-regulated eNOS and up-regulated a vast cohort of inflammatory mediators such as adhesion molecules, cytokines, enzymes and transcription factors. Other LXR-activating oxysterols such as 24,25-epoxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol could also stimulate the endothelial expression of inflammatory markers, although significant differences were observed. These effects persisted in LXR-silenced cells, confirming the mechanistic dissociation of oxysterol and LXR pathways. Furthermore, the oxysterol-induced expression of inflammatory markers was not secondary to cell apoptosis and may relate to oxidative stress. CONCLUSIONS: LXR-activating oxysterols comprehensively activate the expression of endothelial inflammation markers independently from LXRs. At proper dosage, synthetic LXR agonists are safe on endothelial cells and may even transrepress inflammatory reactions.