RESUMEN
Heterozygous germline missense variants in the HRAS gene underlie Costello syndrome (CS). The molecular basis for cutaneous manifestations in CS is largely unknown. We used an immortalized human cell line, HaCaT keratinocytes, stably expressing wild-type or CS-associated (p.Gly12Ser) HRAS and defined RIN1 as quantitatively most prominent, high-affinity effector of active HRAS in these cells. As an exchange factor for RAB5 GTPases, RIN1 is involved in endosomal sorting of cell-adhesion integrins. RIN1-dependent RAB5A activation was strongly increased by HRASGly12Ser, and HRAS-RIN1-ABL1/2 signaling was induced in HRASWT- and HRASGly12Ser-expressing cells. Along with that, HRASGly12Ser expression decreased total integrin levels and enriched ß1 integrin in RAB5- and EEA1-positive early endosomes. The intracellular level of active ß1 integrin was increased in HRASGly12Ser HaCaT keratinocytes due to impaired recycling, whereas RIN1 disruption raised ß1 integrin cell surface distribution. HRASGly12Ser induced co-localization of ß1 integrin with SNX17 and RAB7 in early/sorting and late endosomes, respectively. Thus, by retaining ß1 integrin in intracellular endosomal compartments, HRAS-RIN1 signaling affects the subcellular availability of ß1 integrin. This may interfere with integrin-dependent processes as we detected for HRASGly12Ser cells spreading on fibronectin. We conclude that dysregulation of receptor trafficking and integrin-dependent processes such as cell adhesion are relevant in the pathobiology of CS.
Asunto(s)
Síndrome de Costello , Enfermedades de la Piel , Humanos , Integrinas/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Queratinocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
The microenvironment of malignant melanomas defines the properties of tumor blood vessels and regulates infiltration and vascular dissemination of immune and cancer cells, respectively. Previous research in other cancer entities suggested the complement system as an essential part of the tumor microenvironment. Here, we confirm activation of the complement system in samples of melanoma patients and murine melanomas. We identified the tumor endothelium as the starting point of the complement cascade. Generation of complement-derived C5a promoted the recruitment of neutrophils. Upon contact with the vascular endothelium, neutrophils were further activated by complement membrane attack complexes (MACs). MAC-activated neutrophils release neutrophil extracellular traps (NETs). Close to the blood vessel wall, NETs opened the endothelial barrier as indicated by an enhanced vascular leakage. This facilitated the entrance of melanoma cells into the circulation and their systemic spread. Depletion of neutrophils or lack of MAC formation in complement component 6 (C6)-deficient animals protected the vascular endothelium and prevented vascular intravasation of melanoma cells. Our data suggest that inhibition of MAC-mediated neutrophil activation is a potent strategy to abolish hematogenous dissemination in melanoma.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento , Endotelio Vascular , Trampas Extracelulares , Melanoma , Neutrófilos , Microambiente Tumoral , Animales , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento , Endotelio Vascular/fisiopatología , Humanos , Melanoma/irrigación sanguínea , Melanoma/inmunología , Melanoma/patología , Ratones , Neutrófilos/inmunología , PermeabilidadRESUMEN
Cryptococcus neoformans is an opportunistic fungal pathogen that infects â¼280,000 people every year, causing >180,000 deaths. The human immune system recognizes chitin as one of the major cell-wall components of invading fungi, but C. neoformans can circumvent this immunosurveillance mechanism by instead exposing chitosan, the partly or fully deacetylated form of chitin. The natural production of chitosans involves the sequential action of chitin synthases (CHSs) and chitin deacetylases (CDAs). C. neoformans expresses four putative CDAs, three of which have been confirmed as functional enzymes that act on chitin in the cell wall. The fourth (CnCda4/Fpd1) is a secreted enzyme with exceptional specificity for d-glucosamine at its -1 subsite, thus preferring chitosan over chitin as a substrate. We used site-specific mutagenesis to reduce the subsite specificity of CnCda4 by converting an atypical isoleucine residue in a flexible loop region to the bulkier or charged residues tyrosine, histidine, and glutamic acid. We also investigated the effect of CnCda4 deacetylation products on human peripheral blood-derived macrophages, leading to a model explaining the function of CnCda4 during infection. We propose that CnCda4 is used for the further deacetylation of chitosans already exposed on the C. neoformans cell wall (originally produced by CnChs3 and CnCda1 to 3) or released from the cell wall as elicitors by human chitinases, thus making the fungus less susceptible to host immunosurveillance. The absence of CnCda4 during infection could therefore promote the faster recognition and elimination of this pathogen.
Asunto(s)
Amidohidrolasas/metabolismo , Quitosano/metabolismo , Cryptococcus neoformans/enzimología , Proteínas Fúngicas/metabolismo , Amidohidrolasas/genética , Pared Celular/enzimología , Pared Celular/genética , Quitina/química , Quitina/metabolismo , Quitosano/química , Criptococosis/microbiología , Cryptococcus neoformans/química , Cryptococcus neoformans/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Humanos , Especificidad por SustratoRESUMEN
Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC50 values two- or fourfold, respectively. Resistance formation was scarcely related to HSPG deficiency, suggested by enzymatic cleavage of HSPG in control cells. Notably, EXT1kd induced an upregulation of EGFR signaling via JNK and MEK/ERK, and hence blocking these kinases returned resistance to a sensitive level. JNK appeared as a key signal component, also inducing higher migratory activity of EXT1kd cells. Furthermore, EXT1kd upregulated thrombotic properties of MV3 cells, indicated by tissue factor and PAR-1 expression, functionally reflected by a stronger activation of platelet aggregation. EXT1 was confirmed to act as a tumor suppressor, shown here for the first time to affect chemosensitivity of melanoma cells.
Asunto(s)
Antineoplásicos , Melanoma , Humanos , Doxorrubicina , Resistencia a Antineoplásicos/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , MitoxantronaRESUMEN
Brain metastases (BM) represent a growing problem for breast cancer (BC) patients. Recent studies have demonstrated a strong impact of the BC molecular subtype on the incidence of BM development. This study explores the interaction between BC cells of different molecular subtypes and the blood-brain barrier (BBB). We compared the ability of BC cells of different molecular subtypes to overcome several steps (adhesion to the brain endothelium, disruption of the BBB, and invasion through the endothelial layer) during cerebral metastases formation, in vitro as well as in vivo. Further, the impact of these cells on the BBB was deciphered at the molecular level by transcriptome analysis of the triple-negative (TNBC) cells themselves as well as of hBMECs after cocultivation with BC cell secretomes. Compared to luminal BC cells, TNBC cells have a greater ability to influence the BBB in vitro and consequently develop BM in vivo. The brain-seeking subline and parental TNBC cells behaved similarly in terms of adhesion, whereas the first showed a stronger impact on the brain endothelium integrity and increased invasive ability. The comparative transcriptome revealed potential brain-metastatic-specific key regulators involved in the aforementioned processes, e.g., the angiogenesis-related factors TNXIP and CXCL1. In addition, the transcriptomes of the two TNBC cell lines strongly differed in certain angiogenesis-associated factors and in several genes related to cell migration and invasion. Based on the present study, we hypothesize that the tumor cell's ability to disrupt the BBB via angiogenesis activation, together with increased cellular motility, is required for BC cells to overcome the BBB and develop brain metastases.
Asunto(s)
Neoplasias Encefálicas/secundario , Neoplasias de la Mama/patología , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Animales , Barrera Hematoencefálica , Neoplasias Encefálicas/genética , Neoplasias de la Mama/genética , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Ratones , Trasplante de NeoplasiasRESUMEN
Chitinase 3-like 1 (CHI3L1) is an enzymatically inactive mammalian chitinase that is associated with tumor inflammation. Previous research indicated that CHI3L1 is able to interact with different extracellular matrix components, such as heparan sulfate. In the present work, we investigated whether the interaction of CHI3L1 with the extracellular matrix of melanoma cells can trigger an inflammatory activation of endothelial cells. The analysis of the melanoma cell secretome indicated that CHI3L1 increases the abundance of various cytokines, such as CC-chemokine ligand 2 (CCL2), and growth factors, such as vascular endothelial growth factor A (VEGF-A). Using a solid-phase binding assay, we found that heparan sulfate-bound VEGF-A and CCL2 were displaced by recombinant CHI3L1 in a dose-dependent manner. Microfluidic experiments indicated that the CHI3L1 altered melanoma cell secretome promoted immune cell recruitment to the vascular endothelium. In line with the elevated VEGF-A levels, CHI3L1 was also able to promote angiogenesis through the release of extracellular matrix-bound pro-angiogenic factors. In conclusion, we showed that CHI3L1 is able to affect the tumor cell secretome, which in turn can regulate immune cell recruitment and blood vessel formation. Accordingly, our data suggest that the molecular targeting of CHI3L1 in the course of cancer immunotherapies can tune patients' response and antitumoral inflammation.
Asunto(s)
Quimiocina CCL2/genética , Proteína 1 Similar a Quitinasa-3/genética , Melanoma/genética , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/inmunología , Vasos Sanguíneos/patología , Línea Celular Tumoral , Células Endoteliales/inmunología , Células Endoteliales/patología , Endotelio Vascular/crecimiento & desarrollo , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Matriz Extracelular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/farmacología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Melanoma/inmunología , Melanoma/patología , Técnicas Analíticas Microfluídicas , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Unión Proteica/genética , Unión Proteica/inmunologíaRESUMEN
BACKGROUND: The interleukin-1-receptor antagonist IL1RA (encoded by the IL1RN gene) is a potent competitive antagonist to interleukin-1 (IL1) and thereby is mainly involved in the regulation of inflammation. Previous data indicated a role of IL1RA in muscle-invasive urothelial carcinoma of the bladder (UCB) as well as an IL1-dependent decrease in tissue barrier function, potentially contributing to cancer cell invasion. OBJECTIVE: Based on these observations, here we investigated the potential roles of IL1RA, IL1A, and IL1B in bladder cancer cell invasion in vitro. METHODS: Cell culture, real-time impedance sensing, invasion assays (Boyden chamber, pig bladder model), qPCR, Western blot, ELISA, gene overexpression. RESULTS: We observed a loss of IL1RA expression in invasive, high-grade bladder cancer cell lines T24, UMUC-3, and HT1197 while IL1RA expression was readily detectable in the immortalized UROtsa cells, the non-invasive bladder cancer cell line RT4, and in benign patient urothelium. Thus, we modified the invasive human bladder cancer cell line T24 to ectopically express IL1RA, and measured changes in cell migration/invasion using the xCELLigence Real-Time-Cell-Analysis (RTCA) system and the Boyden chamber assay. The real-time observation data showed a significant decrease of cell migration and invasion in T24 cells overexpressing IL1RA (T24-IL1RA), compared to cells harboring an empty vector (T24-EV). Concurrently, tumor cytokines, e.g., IL1B, attenuated the vascular endothelial barrier, which resulted in a reduction of the Cell Index (CI), an impedance-based dimensionless unit. This reduction could be reverted by the simultaneous incubation with IL1RA. Moreover, we used an ex vivo porcine organ culture system to evaluate cell invasion capacity and showed that T24-IL1RA cells showed significantly less invasive capacity compared to parental T24 cells or T24-EV. CONCLUSIONS: Taken together, our results indicate an inverse correlation between IL1RA expression and tumor cell invasive capacity and migration, suggesting that IL1RA plays a role in bladder carcinogenesis, while the exact mechanisms by which IL1RA influences tumor cells migration/invasion remain to be clarified in future studies. Furthermore, we confirmed that real-time impedance sensing and the porcine ex vivo organ culture methods are powerful tools to discover differences in cancer cell migration and invasion.
Asunto(s)
Movimiento Celular/genética , Células Epiteliales/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/genética , Interleucina-1alfa/genética , Interleucina-1beta/genética , Neoplasias de la Vejiga Urinaria/genética , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Invasividad Neoplásica , Transducción de Señal , Porcinos , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Chitosan-caseinate nanoparticles were synthesized by polyelectrolyte complex (PEC) formation. Caseinate is an anionic micellar nanocolloid in aqueous solutions, which association with the polycationic chitosan yielded polyelectrolyte complexes with caseinate cores surrounded by a chitosan corona. The pre-structuration of caseinate micelles facilitates the formation of natural polyelectrolyte nanoparticles with good stability and sizes around 200 nm. Such natural nanoparticles can be loaded with molecules for applications in drug-controlled release. In the nanoparticles processing, parameters such as the chitosan degree of acetylation (DA) and molecular weight, order of addition of the polyelectrolytes chitosan (polycation) and caseinate (polyanion), and added weight ratio of polycation:polyanion were varied, which were shown to influence the structure of the polyelectrolyte association, the nanoparticle size and zeta potential. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) analyses revealed the chemical structure of hydrogel colloidal systems consisting of nanoparticles that contain chitosan and caseinate. Transmission electron microscopy (TEM) allowed further characterization of the spherical morphology of the nanoparticles. Furtherly, insulin was chosen as a model drug to study the application of the nanoparticles as a safe biodegradable nanocarrier system for drug-controlled release. An insulin entrapment efficiency of 75% was achieved in the chitosan-caseinate nanoparticles.
Asunto(s)
Quitosano/química , Liberación de Fármacos , Hidrogeles/farmacología , Nanopartículas/química , Caseínas/química , Quitosano/farmacología , Coloides/química , Coloides/farmacología , Humanos , Hidrogeles/química , Concentración de Iones de Hidrógeno , Tamaño de la Partícula , Polielectrolitos/químicaRESUMEN
The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE-/-), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Reparación del ADN , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Animales , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Senescencia Celular , ADN/metabolismo , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Homeostasis , Pulmón/fisiopatología , Proteína Homóloga de MRE11 , Ratones Endogámicos C57BL , Ratones Noqueados , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/fisiopatología , Receptor para Productos Finales de Glicación Avanzada/genética , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Transducción de SeñalRESUMEN
Microvascular endothelial cells are an essential part of many biological barriers, such as the bloodâ»brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB could lead to improved therapies due to enhanced drug bioavailability. Vanilloids, such as capsaicin, are known to reversibly open tight junctions of epithelial and endothelial cells. In this study, we used several in vitro assays with the murine endothelial capillary brain cells (line cEND) as a BBB model to characterize the interaction between capsaicin and endothelial tight junctions.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Capsaicina/farmacología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Animales , Barrera Hematoencefálica/citología , Capilares/citología , Capilares/efectos de los fármacos , Células Cultivadas , Células Endoteliales/citología , Endotelio Vascular/citología , RatonesRESUMEN
Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation.
Asunto(s)
Melanoma/metabolismo , Agregación Plaquetaria , Factor de von Willebrand/metabolismo , Proteínas ADAM/sangre , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Animales , Coagulación Sanguínea , Plaquetas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Activación Enzimática , Fibrinolíticos/farmacología , Heparina de Bajo-Peso-Molecular/farmacología , Humanos , Melanoma/sangre , Melanoma/patología , Ratones , Ratones Transgénicos , Microvasos/metabolismo , Neovascularización Patológica/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-ret/metabolismo , Tinzaparina , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Leda-1/Pianp is a type-I transmembrane protein which is sorted to the basolateral membrane domain of polarized epithelial cells. Here, we investigated trafficking mechanisms and functions of Leda-1/Pianp in MDCK and MCF-7 cells. Basolateral sorting and posttranslational modifications depended on the intracellular juxtamembrane region. Functionally, Leda-1/Pianp increased the transepithelial electrical resistance generated by a polarized cell sheet. Furthermore, resistance to junctional destabilization by tumor cells was enhanced by Leda-1/Pianp indicating increased stability and tightness of intercellular junctions. While Claudin 1 and 4 expression and activities of small GTPases were not affected, γ-Secretase-mediated cleavage of E-Cadherin was attenuated by Leda-1/Pianp. Regulation of proteolytic processing is thus a molecular mechanism by which Leda-1/Pianp can affect junctional integrity and function.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cadherinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Antígenos CD , Perros , Impedancia Eléctrica , Células Epiteliales/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Células de Riñón Canino Madin DarbyRESUMEN
OBJECTIVE: Inflammatory conditions provoke essential processes in the human vascular system. It leads to the formation of ultralarge von Willebrand factor (VWF) fibers, which are immobilized on the endothelial cell surface and transform to highly adhesive strings under shear conditions. Furthermore, leukocytes release a meshwork of DNA (neutrophil extracellular traps) during the process of the recently discovered cell death program NETosis. In the present study, we characterized the interaction between VWF and DNA and possible binding sites to underline the role of VWF in thrombosis and inflammation besides its function in platelet adhesion. APPROACH AND RESULTS: Both functionalized surfaces and intact cell layers of human umbilical vein endothelial cells were perfused with isolated, protein-free DNA or leukocytes from whole blood at distinct shear rates. DNA-VWF interaction was monitored using fluorescence microscopy, ELISA-based assays, molecular dynamics simulations, and electrostatic potential calculations. Isolated DNA, as well as DNA released by stimulated leukocytes, was able to bind to shear-activated, but not inactivated, VWF. However, DNA-VWF binding does not alter VWF degradation by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. Moreover, DNA-VWF interaction can be blocked using unfractionated and low-molecular-weight heparin, and DNA-VWF complexes attenuate platelet binding to VWF. These findings were supported using molecular dynamics simulations and electrostatic calculations of the A1- and A2-domains. CONCLUSIONS: Our findings suggest that VWF directly binds and immobilizes extracellular DNA released from leukocytes. Therefore, we hypothesize that VWF might act as a linker for leukocyte adhesion to endothelial cells, supporting leukocyte extravasation and inflammation.
Asunto(s)
Adhesión Celular , ADN/metabolismo , Neutrófilos/metabolismo , Factor de von Willebrand/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Sitios de Unión , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Heparina de Bajo-Peso-Molecular/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Modelos Moleculares , Neutrófilos/efectos de los fármacos , Adhesividad Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteolisis , Flujo Sanguíneo Regional , Estrés Mecánico , Factores de Tiempo , Factor de von Willebrand/químicaRESUMEN
BACKGROUND: During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. METHODS AND RESULTS: Perfusion of activated human endothelial cells with fluorescent bacteria under high-shear-rate conditions revealed 95% attachment of the S aureus by ultralarge von Willebrand factor (ULVWF). Flow experiments with VWF deletion mutants and heparin indicate a contribution of the A-type domains of VWF to bacterial binding. In this context, analyses of different bacterial deletion mutants suggest the involvement of wall teichoic acid but not of staphylococcal protein A. The presence of inactivated platelets and serum increased significantly ULVWF-mediated bacterial adherence. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) caused a dose-dependent reduction of bacterial binding and a reduced length of ULVWF, but single cocci were still tethered by ULVWF at physiological levels of ADAMTS13. To further prove the role of VWF in vivo, we compared wild-type mice with VWF knockout mice. Binding of fluorescent bacteria was followed in tumor necrosis factor-α-stimulated tissue by intravital microscopy applying the dorsal skinfold chamber model. Compared with wild-type mice (n=6), we found less bacteria in postcapillary (60±6 versus 32±5 bacteria) and collecting venules (48±5 versus 18±4 bacteria; P<0.05) of VWF knockout mice (n=5). CONCLUSIONS: Our data provide the first evidence that ULVWF contributes to the initial pathogenic step of S aureus-induced endocarditis in patients with an apparently intact endothelium. An intervention reducing the ULVWF formation with heparin or ADAMTS13 suggests novel therapeutic options to prevent infective endocarditis.
Asunto(s)
Endocarditis Bacteriana/metabolismo , Células Endoteliales/microbiología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Factor de von Willebrand/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Animales , Anticoagulantes/metabolismo , Anticoagulantes/farmacología , Adhesión Bacteriana/fisiología , Plaquetas/metabolismo , Plaquetas/microbiología , Endocarditis Bacteriana/microbiología , Endocarditis Bacteriana/prevención & control , Células Endoteliales/citología , Células Endoteliales/fisiología , Fibrinógeno/metabolismo , Fibrinógeno/farmacología , Heparina/metabolismo , Heparina/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Metaloendopeptidasas/metabolismo , Ratones , Ratones Noqueados , Tamaño de la Partícula , Piel/citología , Piel/microbiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/patogenicidad , Estrés Mecánico , Factores de Virulencia/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/genéticaRESUMEN
In this study, it is shown that the cytotoxic response of cells as well as the uptake kinetics of nanoparticles (NPs) is cell type dependent. We use silica NPs with a diameter of 310 nm labeled with perylene dye and 304 nm unlabeled particles to evaluate cell type-dependent uptake and cytotoxicity on human vascular endothelial cells (HUVEC) and cancer cells derived from the cervix carcinoma (HeLa). Besides their size, the particles are characterized concerning homogeneity of the labeling and their zeta potential. The cellular uptake of the labeled NPs is quantified by imaging the cells via confocal microscopy in a time-dependent manner, with subsequent image analysis via a custom-made and freely available digital method, Particle_in_Cell-3D. We find that within the first 4 h of interaction, the uptake of silica NPs into the cytoplasm is up to 10 times more efficient in HUVEC than in HeLa cells. Interestingly, after 10 or 24 h of interaction, the number of intracellular particles for HeLa cells by far surpasses the one for HUVEC. Inhibitor studies show that these endothelial cells internalize 310 nm SiO2 NPs via the clathrin-dependent pathway. Remarkably, the differences in the amount of taken up NPs are not directly reflected by the metabolic activity and membrane integrity of the individual cell types. Interaction with NPs leads to a concentration-dependent decrease in mitochondrial activity and an increase in membrane leakage for HUVEC, whereas HeLa cells show only a reduced mitochondrial activity and no membrane leakage. In addition, silica NPs lead to HUVEC cell death while HeLa cells survive. These findings indicate that HUVEC are more sensitive than HeLa cells upon silica NP exposure.
Asunto(s)
Nanopartículas/metabolismo , Nanopartículas/toxicidad , Dióxido de Silicio/química , Muerte Celular/efectos de los fármacos , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Cinética , Nanopartículas/químicaRESUMEN
PURPOSE: We developed and validated an electrophysiological method for standardized preclinical assessment of the invasive potential of urothelial carcinoma of the bladder. MATERIALS AND METHODS: Human UMUC-3, RT-112, HT-1197 and T24/83 bladder urothelial carcinoma cells, and UROtsa benign urothelial cells were co-cultivated with high resistance MDCK-C7 cells seeded below a 0.4 µm pore membrane of an insert to avoid physical contact and cellular migration. Transepithelial electrical resistance in Ω cm(2) across the MDCK-C7 monolayer was measured longitudinally. Invasive potential coefficients were calculated based on the secretion of proteolytic factors by invading cells. RESULTS: Consistent transepithelial electrical resistance breakdown patterns were reproduced in 14 or more independent samples of each cell line. Coefficients of invasive potential were significantly higher in bladder urothelial carcinoma than UROtsa cells, including a mean ± SD of 1.5 ± 0.32 vs 9.9 ± 4.97 in UMUC-3, 12.5 ± 6.61 in T24/83, 20.5 ± 4.24 in RT-112 and 21.0 ± 5.15 in HT-1197 cells (p <0.001). No correlation was found between the secretion patterns of matrix metalloproteinase-1, 2 and 9, and invasive potential. Stimulation of UROtsa cells with recombinant human epidermal growth factor up-regulated matrix metalloproteinase-9 secretion and significantly increased invasive potential a mean of 1.3 ± 0.22 vs 14.6 ± 3.28 after stimulation with 10 ng/ml epidermal growth factor (p <0.001). CONCLUSIONS: We developed a highly sensitive translational tool to study the initial process of metastatic spread of urothelial carcinoma of the bladder. The presented electrophysiological invasion assay enables reliable quantification of the invasive potential of bladder urothelial carcinoma cells before physical transmigration. It can be used to identify key molecules for bladder urothelial carcinoma invasion and develop new therapeutic strategies.
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Carcinoma de Células Transicionales/patología , Neoplasias de la Vejiga Urinaria/patología , Bioensayo , Impedancia Eléctrica , Humanos , Invasividad Neoplásica , Células Tumorales CultivadasRESUMEN
Cancer metastasis is a complex process. After their intravasation into the circulation, the cancer cells are exposed to a harsh environment of physical and biochemical hazards. Whether circulating tumor cells (CTCs) survive and escape from blood flow defines their ability to metastasize. CTCs sense their environment with surface-exposed receptors. The recognition of corresponding ligands, e.g., fibrinogen, by integrins can induce intracellular signaling processes driving CTCs' survival. Other receptors, such as tissue factor (TF), enable CTCs to induce coagulation. Cancer-associated thrombosis (CAT) is adversely connected to patients' outcome. However, cancer cells have also the ability to inhibit coagulation, e.g., through expressing thrombomodulin (TM) or heparan sulfate (HS), an activator of antithrombin (AT). To that extent, individual CTCs can interact with plasma proteins, and whether these interactions are connected to metastasis or clinical symptoms such as CAT is largely unknown. In the present review, we discuss the biological and clinical relevance of cancer-cell-expressed surface molecules and their interaction with plasma proteins. We aim to encourage future research to expand our knowledge of the CTC interactome, as this may not only yield new molecular markers improving liquid-biopsy-based diagnostics but also additional targets for better cancer therapies.
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Advances in understanding the molecular mechanisms of tumor progression have achieved impressive progress in the treatment of cancer and so-called immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. Indeed, antibody-based drugs blocking immune escape of tumor cells by modulation of T cell responses are increasingly utilized for a wide range of tumor entities. Nonetheless, response rates remain limited, and the development of secondary resistance is a common problem. In addition, by increasing the immune response a variety of severe side effects are provoked. Next to autoimmune responses, activation of the complement system and skin toxicity, an increased incidence for thrombotic complications has been observed associated with an increased mortality rate. Based on this, it can be postulated that the interplay of coagulation with inflammation in the tumor microenvironment is relevant for each step in the tumor life cycle. This review focuses on the coagulation as central player fostering mechanisms associated with tumor progression. Thus, a better understanding of the molecular pathways involved in the complex interaction of circulating tumor cells, the plasmatic coagulation and immune cells may help to improve therapeutic concepts reducing mortality and morbidity associated with cancer.
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Coagulación Sanguínea/inmunología , Heparina de Bajo-Peso-Molecular/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inflamación/sangre , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Anticoagulantes/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Humanos , Inflamación/inmunología , Inflamación/patología , Neoplasias/inmunología , Trombosis/sangre , Trombosis/tratamiento farmacológico , Trombosis/patología , Escape del Tumor/efectos de los fármacosRESUMEN
Introduction: The intravascular formation of neutrophil extracellular traps (NETs) is a trigger for coagulation and blood vessel occlusion. NETs are released from neutrophils as a response to strong inflammatory signals in the course of different diseases such as COVID-19, cancer or antiphospholipid syndrome. NETs are composed of large, chromosomal DNA fibers decorated with a variety of proteins such as histones. Previous research suggested a close mechanistic crosstalk between NETs and the coagulation system involving the coagulation factor XII (FXII), von Willebrand factor (VWF) and tissue factor. However, the direct impact of NET-related DNA fibers on blood flow and blood aggregation independent of the coagulation cascade has remained elusive. Methods: In the present study, we used different microfluidic setups in combination with fluorescence microscopy to investigate the influence of neutrophil-derived extracellular DNA fibers on blood rheology, intravascular occlusion and activation of the complement system. Results: We found that extended DNA fiber networks decelerate blood flow and promote intravascular occlusion of blood vessels independent of the plasmatic coagulation. Associated with the DNA dependent occlusion of the flow channel was the strong activation of the complement system characterized by the production of complement component 5a (C5a). Vice versa, we detected that the local activation of the complement system at the vascular wall was a trigger for NET release. Discussion: In conclusion, we found that DNA fibers as the principal component of NETs are sufficient to induce blood aggregation even in the absence of the coagulation system. Moreover, we discovered that complement activation at the endothelial surface promoted NET formation. Our data envisions DNA degradation and complement inhibition as potential therapeutic strategies in NET-induced coagulopathies.
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COVID-19 , Trampas Extracelulares , Humanos , Trampas Extracelulares/metabolismo , COVID-19/metabolismo , Neutrófilos/metabolismo , ADN/metabolismo , Activación de ComplementoRESUMEN
Breast cancer (BC) is the second-most common cause of brain metastases (BM) and BCBM patients have a reduced quality of life and a poor prognosis. Hyaluronan (HA), and in particular the hyaluronidase Hyal-1, has been already linked to the development of BCBM, and therefore presents an interesting opportunity to develop new effective therapeutic options. HA metabolism was further discovered by the CRISPR/Cas9-mediated knockout of HYAL1 and the shRNA-mediated down-regulation of HA-receptor CD44 in the brain-seeking triple-negative breast cancer (TNBC) cell line MDA-MB-231-BR. Therefore, the impact of Hyal-1 on adhesion, disruption, and invasion through the brain endothelium, both in vitro and in vivo, was studied. Our analysis points out a key role of Hyal-1 and low-molecular-weight HA (LMW-HA) in the formation of a pericellular HA-coat in BC cells, which in turn promotes tumor cell adhesion, disruption, and migration through the brain endothelium in vitro as well as the extent of BM in vivo. CD44 knockdown in MDA-MB-231-BR significantly reduced the pericellular HA-coat on these cells, and, consequently, tumor cell adhesion and invasion through the brain endothelium. Thus, the interaction between Hyal-1-generated LMW-HA fragments and the HA-receptor CD44 might represent a potential target for future therapeutic options in BC patients with a high risk of cerebral metastases formation.