RESUMO
Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.
Assuntos
Moléculas de Adesão Celular/metabolismo , Endotélio/química , Heparitina Sulfato/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Proteômica/métodos , Animais , Sítios de Ligação , Células Cultivadas , Endotélio/citologia , Humanos , Camundongos , Ligação Proteica , Células U937RESUMO
The development of long-lived luminescent nanoparticles for lifetime imaging is of wide interest as luminescence lifetime is environmentally sensitive detection independent of probe concentration. We report novel iridium-coated gold nanoparticles as probes for multiphoton lifetime imaging with characteristic long luminescent lifetimes based on iridium luminescence in the range of hundreds of nanoseconds and a short signal on the scale of picoseconds based on gold allowing multichannel detection. The tailor-made IrC6 complex forms stable, water-soluble gold nanoparticles (AuNPs) of 13, 25, and 100 nm, bearing 1400, 3200, and 22â¯000 IrC6 complexes per AuNP, respectively. The sensitivity of the iridium signal on the environment of the cell is evidenced with an observed variation of lifetimes. Clusters of iridium nanoparticles show lifetimes from 450 to 590 ns while lifetimes of 660 and 740 ns are an average of different points in the cytoplasm and nucleus. Independent luminescence lifetime studies of the nanoparticles in different media and under aggregation conditions postulate that the unusual long lifetimes observed can be attributed to interaction with proteins rather than nanoparticle aggregation. Total internal reflection fluorescence microscopy (TIRF), confocal microscopy studies and 3D luminescence lifetime stacks confirm the presence of bright, nonaggregated nanoparticles inside the cell. Inductively coupled plasma mass spectrometry (ICPMS) analysis further supports the presence of the nanoparticles in cells. The iridium-coated nanoparticles provide new nanoprobes for lifetime detection with dual channel monitoring. The combination of the sensitivity of the iridium signal to the cell environment together with the nanoscaffold to guide delivery offer opportunities for iridium nanoparticles for targeting and tracking in in vivo models.
Assuntos
Irídio/química , Nanopartículas Metálicas/química , Complexos de Coordenação , Ouro/química , Células HeLa , Humanos , Luminescência , Imagem Óptica , TensoativosRESUMO
Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.
Assuntos
Neoplasias Encefálicas/genética , Redes Reguladoras de Genes/genética , Glioma/genética , Invasividade Neoplásica/genética , Proteínas rho de Ligação ao GTP/genética , Apoptose/genética , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica/genética , Glioma/patologia , Células HEK293 , Humanos , Interferência de RNA , RNA Interferente PequenoRESUMO
C-type lectin family 14, member A (CLEC14A), is a single-pass transmembrane glycoprotein that is overexpressed in tumor endothelial cells, and it promotes sprouting angiogenesis and modulates endothelial function via interactions with extracellular matrix proteins. Here, we show that CLEC14A is cleaved by rhomboid-like protein 2 (RHBDL2), one of 3 catalytic mammalian rhomboid-like (RHBDL) proteases, but that it is not cleaved by RHBDL1 or -3. Site-directed mutagenesis identified the precise site at which RHBDL2 cleaves CLEC14A, and targeted, small interfering RNAs that knockdown endogenous CLEC14A and RHBDL2 in human endothelial cells validated the specificity of CLEC14A shedding by RHBDL2. Loss of endogenous cleaved CLEC14A increased endothelial migration 2-fold, whereas that addition of recombinant cleaved CLEC14A inhibited the sprouting of human and murine endothelial cells 3-fold in several in vitro models. We assessed the in vivo role of cleaved CLEC14A in angiogenesis by using the rodent subcutaneous sponge implant model, and we found that CLEC14A protein inhibited vascular density by >50%. Finally, we show that cleaved CLEC14A binds to sprouting endothelial tip cells. Our data show that the ectodomain of CLEC14A regulates sprouting angiogenesis and suggests a role for RHBDL2 in endothelial function.-Noy, P. J., Swain, R. K., Khan, K., Lodhia, P., Bicknell, R. Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).
Assuntos
Moléculas de Adesão Celular/metabolismo , Endopeptidases/metabolismo , Células Endoteliais/fisiologia , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica/fisiologia , Serina Proteases/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Moléculas de Adesão Celular/genética , Movimento Celular/fisiologia , Endopeptidases/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Lectinas Tipo C/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos , Serina Endopeptidases , Serina Proteases/genéticaRESUMO
The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks in a wide spectrum of biological systems.
Assuntos
Redes Reguladoras de Genes , Próstata/citologia , Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Teorema de Bayes , Comunicação Celular , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Biologia Computacional , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Modelos Biológicos , Neoplasias da Próstata/metabolismo , Transdução de Sinais/genéticaRESUMO
RhoJ is a Rho GTPase expressed in endothelial cells and tumour cells, which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; small interfering RNA (siRNA)-mediated knockdown of RhoJ increased focal adhesion disassembly time, whereas expression of an active mutant (daRhoJ) decreased it. Furthermore, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast two-hybrid experiments, and this depended on the Spa homology domain of GIT1. GIT1, GIT2, ß-PIX (also known as ARHGEF7) and RhoJ all colocalised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of both GIT1 and GIT2, or ß-PIX phenocopying RhoJ knockdown. RhoJ-knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give new insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Adesões Focais/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Movimento Celular/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models. APPROACH AND RESULTS: FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL's critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl(+/-) mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish. CONCLUSIONS: FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.
Assuntos
Aorta/metabolismo , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Imunofilinas/metabolismo , Neovascularização Patológica , Proteínas de Ligação a Tacrolimo/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Carcinoma Pulmonar de Lewis/patologia , Hipóxia Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Imunofilinas/genética , Células MCF-7 , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Fisiológica , Fenótipo , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/genética , Fatores de Tempo , Carga Tumoral , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Tumor endothelial specific expression of Robo4 in adults identifies this plasma membrane protein as an anti-cancer target for immunotherapeutic approaches, such as vaccination. In this report, we describe how vaccination against Robo4 inhibits angiogenesis and tumor growth. To break tolerance to the auto-antigen Robo4, mice were immunised with the extracellular domain of mouse Robo4, fused to the Fc domain of human immunoglobulin within an adjuvant. Vaccinated mice show a strong antibody response to Robo4, with no objectively detectable adverse effects on health. Robo4 vaccinated mice showed impaired fibrovascular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The anti-tumor effect of Robo4 vaccination was present in CD8 deficient mice but absent in B cell or IgG1 knockout mice, suggesting antibody dependent cell mediated cytotoxicity as the anti-vascular/anti-tumor mechanism. Finally, we show that an adjuvant free soluble Robo4-carrier conjugate can retard tumor growth in carrier primed mice. These results point to appropriate Robo4 conjugates as potential anti-angiogenic vaccines for cancer patients.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Imunoterapia/métodos , Neoplasias/prevenção & controle , Neovascularização Patológica/prevenção & controle , Proteínas do Tecido Nervoso/imunologia , Receptores Imunológicos/imunologia , Vacinas Sintéticas/farmacologia , Adulto , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Vetores Genéticos/genética , Células HEK293 , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Papaína , Reação em Cadeia da Polimerase , Receptores de Superfície Celular , Células Tumorais Cultivadas , Vacinas Sintéticas/imunologiaRESUMO
UNLABELLED: Hepatitis C virus (HCV) is a major cause of global morbidity, causing chronic liver injury that can progress to cirrhosis and hepatocellular carcinoma. The liver is a large and complex organ containing multiple cell types, including hepatocytes, sinusoidal endothelial cells (LSEC), Kupffer cells, and biliary epithelial cells. Hepatocytes are the major reservoir supporting HCV replication; however, the role of nonparenchymal cells in the viral lifecycle remains largely unexplored. LSEC secrete factors that promote HCV infection and transcript analysis identified bone morphogenetic protein 4 (BMP4) as a candidate endothelial-expressed proviral molecule. Recombinant BMP4 increased HCV replication and neutralization of BMP4 abrogated the proviral activity of LSEC-conditioned media. Importantly, BMP4 expression was negatively regulated by vascular endothelial growth factor A (VEGF-A) by way of a VEGF receptor-2 (VEGFR-2) primed activation of p38 MAPK. Consistent with our in vitro observations, we demonstrate that in normal liver VEGFR-2 is activated and BMP4 expression is suppressed. In contrast, in chronic liver disease including HCV infection where there is marked endothelial cell proliferation, we observed reduced endothelial cell VEGFR-2 activation and a concomitant increase in BMP4 expression. CONCLUSION: These studies identify a role for LSEC and BMP4 in HCV infection and highlight BMP4 as a new therapeutic target for treating individuals with liver disease.
Assuntos
Células Endoteliais/metabolismo , Hepacivirus/fisiologia , Fígado/metabolismo , Comunicação Parácrina/fisiologia , Replicação Viral/fisiologia , Proteína Morfogenética Óssea 4/metabolismo , Proliferação de Células , Células Cultivadas , Células Endoteliais/patologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Técnicas In Vitro , Fígado/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.
Assuntos
Proteínas Aviárias/metabolismo , Embrião de Galinha/metabolismo , Membrana Corioalantoide/metabolismo , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Animais , Biotinilação , Linhagem Celular Tumoral , Membrana Corioalantoide/lesões , Humanos , Intestino Delgado/embriologia , Intestino Delgado/metabolismo , Rim/embriologia , Rim/metabolismo , Fígado/embriologia , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , ProteomaRESUMO
The process of angiogenesis requires endothelial cells (ECs) to undergo profound changes in shape and polarity. Although this must involve remodelling of the EC cytoskeleton, little is known about this process or the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of ECs actively undergoing angiogenic morphogenesis. We found that elongation of ECs during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified formin-like 3 (FMNL3; also known as FRL2) as a crucial regulator of EC elongation during angiogenesis. We showed that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for this alignment during angiogenic morphogenesis. FMNL3 was highly expressed in the ECs of zebrafish during development and embryos that were depleted for FMNL3 showed profound defects in developmental angiogenesis that were rescued by expression of the human gene. We conclude that FMNL3 is a new regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent ECs into their elongated angiogenic forms.
Assuntos
Citoesqueleto/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Membrana/genética , Neovascularização Fisiológica/genética , Proteínas/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Técnicas de Cocultura , Forminas , Células Endoteliais da Veia Umbilical Humana , Humanos , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The behavior of vascular EC is greatly altered in sites of pathological angiogenesis, such as a developing tumor or atherosclerotic plaque. Until recently it was thought that this was largely due to abnormal chemical signaling, i.e., endothelial cell chemo transduction, at these sites. However, we now demonstrate that the shear stress intensity encountered by EC can have a profound impact on their gene expression and behavior. We review the growing body of evidence suggesting that mechanotransduction, too, is a major regulator of pathological angiogenesis. This fits with the evolving story of physiological angiogenesis, where a combination of metabolic and mechanical signaling is emerging as the probable mechanism by which tight feedback regulation of angiogenesis is achieved in vivo.
Assuntos
Endotélio Vascular/fisiologia , Regulação da Expressão Gênica/fisiologia , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Animais , Endotélio Vascular/citologia , Humanos , Resistência ao CisalhamentoRESUMO
It is now accepted that the growth of solid tumours is dependent on their capacity to acquire a blood supply, and much effort has been directed towards the development of agents (known as anti-angiogenics) that disrupt this process. More recently, it has become apparent that targeted destruction of the established tumour vasculature is another avenue for exciting therapeutic opportunities. In this article, we present evidence that vascular targeting is an effective antitumour strategy in animal models, describe strategies for identifying putative tumour vascular targets and discuss future prospects for vascular targeting in the clinic.
Assuntos
Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica , Animais , Vacinas Anticâncer , Modelos Animais de Doenças , Células Endoteliais , Humanos , Ligantes , Neoplasias/fisiopatologiaRESUMO
Platelets have recently been shown to drive liver injury in murine models of viral hepatitis and promote liver regeneration through the release of serotonin. Despite their emerging role in inflammatory liver disease, little is known about the mechanisms by which platelets bind to the hepatic vasculature. Therefore, we referenced public expression data to determine the profile of potential adhesive receptors expressed by hepatic endothelium. We then used a combination of tissue-binding and flow-based endothelial-binding adhesion assays to show that resting platelets bind to human hepatic sinusoidal endothelial cells and that the magnitude of adhesion is greatly enhanced by thrombin-induced platelet activation. Adhesion was mediated by the integrins Gp1b, αIIbßIII, and αvß3, as well as immobilized fibrinogen. Platelet binding to hepatic endothelial cells resulted in NF-κB activation and increased chemokine secretion. The functional relevance of platelet binding was confirmed by experiments that showed markedly increased binding of neutrophils and lymphocytes to hepatic endothelial cells under shear conditions replicating those found in the hepatic sinusoid, which was in part dependent on P-selectin expression. Thus the ability of platelets to activate endothelium and promote leukocyte adhesion may reflect an additional mechanism through which they promote liver injury.
Assuntos
Plaquetas/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Integrinas/fisiologia , Fígado/metabolismo , Infiltração de Neutrófilos/fisiologia , Ativação Plaquetária/fisiologia , Anticorpos Bloqueadores/farmacologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Quimiocina CCL2/biossíntese , Quimiocinas/biossíntese , Quimiocinas/metabolismo , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Humanos , Técnicas In Vitro , Integrina alfaV/biossíntese , Interleucina-8/biossíntese , NF-kappa B/metabolismo , Peptídeos/farmacologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Fator de von Willebrand/biossínteseRESUMO
OBJECTIVE: RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. METHODS AND RESULTS: Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho-myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. CONCLUSIONS: RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers.
Assuntos
Actomiosina/metabolismo , Movimento Celular , Células Endoteliais/enzimologia , Adesões Focais/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Neovascularização Fisiológica , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Forma Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Imunofluorescência , GTP Fosfo-Hidrolases/genética , Humanos , Hibridização In Situ , Camundongos , Cadeias Leves de Miosina/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Fibras de Estresse/metabolismo , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
OBJECTIVE: To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). METHODS AND RESULTS: Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain-containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. CONCLUSIONS: Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell-expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Perfilação da Expressão Gênica , Semaforinas/metabolismo , Animais , Comunicação Autócrina , Proteína C-Reativa/metabolismo , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/embriologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Comunicação Parácrina , Fenótipo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Interferência de RNA , Proteínas Recombinantes/metabolismo , Semaforinas/deficiência , Semaforinas/genética , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to progressive liver disease, frequently culminating in fibrosis and hepatocellular carcinoma. The mechanisms underlying liver injury in chronic hepatitis C are poorly understood. This study evaluated the role of vascular endothelial growth factor (VEGF) in hepatocyte polarity and HCV infection. METHODS: We used polarized hepatoma cell lines and the recently described infectious HCV Japanese fulminant hepatitis (JFH)-1 cell culture system to study the role of VEGF in regulating hepatoma permeability and HCV infection. RESULTS: VEGF negatively regulates hepatocellular tight junction integrity and cell polarity by a novel VEGF receptor 2-dependent pathway. VEGF reduced hepatoma tight junction integrity, induced a re-organization of occludin, and promoted HCV entry. Conversely, inhibition of hepatoma expressed VEGF with the receptor kinase inhibitor sorafenib or with neutralizing anti-VEGF antibodies promoted polarization and inhibited HCV entry, showing an autocrine pathway. HCV infection of primary hepatocytes or hepatoma cell lines promoted VEGF expression and reduced their polarity. Importantly, treatment of HCV-infected cells with VEGF inhibitors restored their ability to polarize, showing a VEGF-dependent pathway. CONCLUSIONS: Hepatic polarity is critical to normal liver physiology. HCV infection promotes VEGF expression that depolarizes hepatoma cells, promoting viral transmission and lymphocyte migration into the parenchyma that may promote hepatocyte injury.
Assuntos
Carcinoma Hepatocelular/virologia , Polaridade Celular , Hepacivirus/patogenicidade , Neoplasias Hepáticas/virologia , Junções Íntimas/virologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Internalização do Vírus , Comunicação Autócrina , Benzenossulfonatos/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Niacinamida/análogos & derivados , Ocludina , Permeabilidade , Compostos de Fenilureia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Sorafenibe , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Regulação para Cima , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. It is characterized by substantial tumor cell invasion and early-stage metastasis. We developed an in vivo model to analyze interactions between cancer and stromal cells during early stages of PDAC. METHODS: Human pancreatic adenocarcinoma cells were grafted onto the chick chorioallantoic membrane (CAM). Human and chicken GeneChips were used simultaneously to study gene regulation during PDAC cell invasion. Bioinformatic analysis was used to identify human orthologs and cell specificity of gene expression. The effects of netrin-1 encoded by NTN1 were investigated in adhesion, invasion, and apoptosis assays. The effects of NTN1 silencing with small interfering RNAs were investigated in PDAC cells in vivo. NTN1 expression was measured in human PDAC samples. RESULTS: PDAC cells rapidly invade the CAM stroma and remodel the CAM vasculature. Around 800 stromal genes were up-regulated by >2-fold; the angiogenesis regulators vascular endothelial growth factor D, thrombospondin 1, and CD151 were among the most highly regulated genes. Silencing of tumor cell NTN1, which is up-regulated 4-fold in the PDAC model, inhibited tumor cell invasion in vivo. Netrin-1 conferred apoptosis resistance to tumor and endothelial cells in vitro, induced their invasion, and provided an adhesive substrate for tumor cells. NTN1 and its gene product are strongly overexpressed in human PDAC samples. CONCLUSIONS: We developed a useful tool to study the invasive mechanisms of early-stage PDAC. Netrin-1 might be an important regulator of pancreatic tumor growth that functions in tumor and endothelial cells.
Assuntos
Adenocarcinoma/patologia , Células Endoteliais/fisiologia , Fatores de Crescimento Neural/fisiologia , Neoplasias Pancreáticas/patologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Apoptose , Antígeno CA-19-9/sangue , Linhagem Celular Tumoral , Embrião de Galinha , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Invasividade Neoplásica , Netrina-1 , Neoplasias Pancreáticas/metabolismoRESUMO
Capillaries have been recognized for over a century as one of the most important components in regulating tissue oxygen transport, and their formation or angiogenesis a pivotal element of tissue remodelling during development and adaptation. Clinical interest stems from observations that both excessive and inadequate vascular growth plays a major role in human diseases, and novel developments in treatments for cancer and eye disease increasingly rely on anti-angiogenic therapies. Although the discovery of VEGF (vascular endothelial growth factor) provided the first clue for specificity of signalling in endothelial cell activation, understanding the integrative response that drives angiogenesis requires a much broader perspective. The Advances in the Cellular and Molecular Biology of Angiogenesis meeting brought together researchers at the forefront of this rapidly moving field to provide an update on current understanding, and the most recent insights into molecular and cellular mechanisms of vascular growth. The plenary lecture highlighted the integrative nature of the angiogenic process, whereas invited contributions from basic and clinician scientists described fundamental mechanisms and disease-associated issues of blood vessel formation, grouped under a number of themes to aid discussion. These articles will appeal to academic, clinical and pharmaceutical scientists interested in the molecular and cellular basis of angiogenesis, their modulation or dysfunction in human diseases, and application of these findings towards translational medicine.
Assuntos
Biologia Celular , Biologia Molecular , Neovascularização Fisiológica , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Humanos , Camundongos , Neovascularização Fisiológica/fisiologia , Organogênese , Transdução de SinaisRESUMO
We have in recent years described several endothelial-specific genes that mediate cell migration. These include Robo4 (roundabout 4), CLEC14A (C-type lectin 14A) and ECSCR (endothelial cell-specific chemotaxis regulator) [formerly known as ECSM2 (endothelial cell-specific molecule 2)]. Loss of laminar shear stress induces Robo4 and CLEC14A expression and an endothelial 'tip cell' phenotype. Low shear stress is found not only at sites of vascular occlusion such as thrombosis and embolism, but also in the poorly structured vessels that populate solid tumours. The latter probably accounts for strong expression of Robo4 and CLEC14A on tumour vessels. The function of Robo4 has, in the past, aroused controversy. However, the recent identification of Unc5B as a Robo4 ligand has increased our understanding and we hypothesize that Robo4 function is context-dependent. ECSCR is another endothelial-specific protein that promotes filopodia formation and migration, but, in this case, expression is independent of shear stress. We discuss recent papers describing ECSCR, including intracellular signalling pathways, and briefly contrast these with signalling by Robo4.