RESUMEN
T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).
Asunto(s)
Vacuna BNT162 , COVID-19 , Animales , Cricetinae , Humanos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/prevención & control , Epítopos , SARS-CoV-2/genéticaRESUMEN
Neoantigens arising from mutations in tumor DNA provide targets for immune-based therapy. Here, we report the clinical and immune data from a Phase Ib clinical trial of a personalized neoantigen-vaccine NEO-PV-01 in combination with pemetrexed, carboplatin, and pembrolizumab as first-line therapy for advanced non-squamous non-small cell lung cancer (NSCLC). This analysis of 38 patients treated with the regimen demonstrated no treatment-related serious adverse events. Multiple parameters including baseline tumor immune infiltration and on-treatment circulating tumor DNA levels were highly correlated with clinical response. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination. Epitope spread to non-vaccinating neoantigens, including responses to KRAS G12C and G12V mutations, were detected post-vaccination. Neoantigen-specific CD4+ T cells generated post-vaccination revealed effector and cytotoxic phenotypes with increased CD4+ T cell infiltration in the post-vaccine tumor biopsy. Collectively, these data support the safety and immunogenicity of this regimen in advanced non-squamous NSCLC.
Asunto(s)
Vacunas contra el Cáncer , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antígenos de Neoplasias , Linfocitos T CD8-positivos , Vacunas contra el Cáncer/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Inmunoterapia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genéticaRESUMEN
Neoantigens arise from mutations in cancer cells and are important targets of T cell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).
Asunto(s)
Vacunas contra el Cáncer/inmunología , Inmunoterapia/métodos , Medicina de Precisión/métodos , Anciano , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Persona de Mediana Edad , Mutación , Nivolumab/uso terapéutico , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/inmunologíaRESUMEN
PURPOSE: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. EXPERIMENTAL DESIGN: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. RESULTS: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFß1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFß1 expression evoke gene-regulatory networks and pathways (TGFß signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand-VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFß1 axis reduces tumor cell growth and overcomes drug resistance. CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFß1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Resistencia a Antineoplásicos , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Neoplasias de la Tiroides/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Vemurafenib/farmacología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Transducción de Señal/efectos de los fármacos , Sorafenib/farmacología , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Factor de Crecimiento Transformador beta1/genética , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hemostasis in vertebrates involves both a cellular and a protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa We found a single vwf transcript that encodes a simpler protein compared with higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high-flow conditions. Immunohistochemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies in the endothelium. Hagfish Vwf formed high-molecular-weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.
Asunto(s)
Anguila Babosa , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismo , Proteína ADAMTS13/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Cricetulus , ADN Complementario , Endotelio Vascular/metabolismo , Evolución Molecular , Expresión Génica , Homeostasis , Humanos , Modelos Moleculares , Agregación Plaquetaria , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteolisis , Relación Estructura-Actividad , Vertebrados , Cuerpos de Weibel-Palade/metabolismo , Cuerpos de Weibel-Palade/ultraestructura , Factor de von Willebrand/químicaRESUMEN
Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Vasos Sanguíneos/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Adenoviridae/metabolismo , Animales , Bibencilos/farmacología , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Hipertensión/patología , Ratones Endogámicos C57BL , Ratones Desnudos , Microvasos/efectos de los fármacos , Microvasos/patología , Modelos Biológicos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.
Asunto(s)
Aorta/metabolismo , Capilares/metabolismo , Metilación de ADN , Células Endoteliales/metabolismo , Mosaicismo , ARN Mensajero/metabolismo , Factor de von Willebrand/genética , Animales , Inmunoprecipitación de Cromatina , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Células 3T3 NIH , Fenotipo , Regiones Promotoras Genéticas , Arteria Pulmonar/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de von Willebrand/metabolismoRESUMEN
Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Clatrina/inmunología , Dinaminas/inmunología , Células Endoteliales/inmunología , Proteínas de Neoplasias/inmunología , Fracciones Subcelulares/inmunología , Células Cultivadas , Células Endoteliales/ultraestructura , HumanosRESUMEN
Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1's larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (1) preculturing human ECFC on laminin, fibronectin, and collagen-coated plates, and (2) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together, these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.
Asunto(s)
Antígenos de Superficie/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Línea Celular Tumoral , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Mesenquimatosas , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Neovascularización Patológica , Ingeniería de Tejidos/métodos , Transcripción GenéticaRESUMEN
Immune cell trafficking requires the frequent breaching of the endothelial barrier either directly through individual cells ('transcellular' route) or through the inter-endothelial junctions ('paracellular' route). What determines the loci or route of breaching events is an open question with important implications for overall barrier regulation. We hypothesized that basic biomechanical properties of the endothelium might serve as crucial determinants of this process. By altering junctional integrity, cytoskeletal morphology and, consequently, local endothelial cell stiffness of different vascular beds, we could modify the preferred route of diapedesis. In particular, high barrier function was associated with predominantly transcellular migration, whereas negative modulation of junctional integrity resulted in a switch to paracellular diapedesis. Furthermore, we showed that lymphocytes dynamically probe the underlying endothelium by extending invadosome-like protrusions (ILPs) into its surface that deform the nuclear lamina, distort actin filaments and ultimately breach the barrier. Fluorescence imaging and pharmacologic depletion of F-actin demonstrated that lymphocyte barrier breaching efficiency was inversely correlated with local endothelial F-actin density and stiffness. Taken together, these data support the hypothesis that lymphocytes are guided by the mechanical 'path of least resistance' as they transverse the endothelium, a process we term 'tenertaxis'.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Linfocitos/metabolismo , Animales , Fenómenos Biomecánicos , Células Endoteliales/citología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Leucocitos/metabolismo , Linfocitos/citología , RatasRESUMEN
RATIONALE: Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FoxO1 in the endothelium remains enigmatic. OBJECTIVE: To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis. METHODS AND RESULTS: We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FoxO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor-induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling. CONCLUSIONS: Our findings suggest that in mice, endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.
Asunto(s)
Células Endoteliales/metabolismo , Factores de Transcripción Forkhead/fisiología , Insuficiencia Cardíaca/genética , Complejos Multiproteicos/fisiología , Neovascularización Fisiológica/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Inducción Enzimática , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Insuficiencia Cardíaca/fisiopatología , Homeostasis , Células Endoteliales de la Vena Umbilical Humana , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Ratones Transgénicos , Neovascularización Fisiológica/genética , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo III/genética , Especificidad de Órganos , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión , Transducción de Señal/fisiología , Saco Vitelino/irrigación sanguíneaRESUMEN
Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/fisiología , Seudópodos/fisiología , Migración Transendotelial y Transepitelial , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Fenómenos Biomecánicos , Adhesión Celular , Células Cultivadas , Técnicas de Cocultivo , Cortactina/metabolismo , Humanos , Linfocitos/fisiología , Microscopía Fluorescente , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Imagen de Lapso de Tiempo , Cicatrización de Heridas , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Systemically administered adult mesenchymal stem cells (MSCs), which are being explored in clinical trials to treat inflammatory disease, exhibit the critical ability to extravasate at sites of inflammation. We aimed to characterize the basic cellular processes mediating this extravasation and compare them to those involved in leukocyte transmigration. Using high-resolution confocal and dynamic microscopy, we show that, like leukocytes, human bone marrow-derived MSC preferentially adhere to and migrate across tumor necrosis factor-α-activated endothelium in a vascular cell adhesion molecule-1 (VCAM-1) and G-protein-coupled receptor signaling-dependent manner. As several studies have suggested, we observed that a fraction of MSC was integrated into endothelium. In addition, we observed two modes of transmigration not previously observed for MSC: Paracellular (between endothelial cells) and transcellular (directly through individual endothelial cells) diapedesis through discrete gaps and pores in the endothelial monolayer, in association with VCAM-1-enriched "transmigratory cups". Contrasting leukocytes, MSC transmigration was not preceded by significant lateral migration and occurred on the time scale of hours rather than minutes. Interestingly, rather than lamellipodia and invadosomes, MSC exhibited nonapoptotic membrane blebbing activity that was similar to activities previously described for metastatic tumor and embryonic germ cells. Our studies suggest that low avidity binding between endothelium and MSC may grant a permissive environment for MSC blebbing. MSC blebbing was associated with early stages of transmigration, in which blebs could exert forces on underlying endothelial cells indicating potential functioning in breaching the endothelium. Collectively, our data suggest that MSC transmigrate actively into inflamed tissues via both leukocyte-like and novel mechanisms.
Asunto(s)
Células Endoteliales/fisiología , Células Madre Mesenquimatosas/fisiología , Migración Transendotelial y Transepitelial , Factor de Necrosis Tumoral alfa/fisiología , Animales , Adhesión Celular , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/inmunología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Leucocitos/fisiología , Células Madre Mesenquimatosas/ultraestructura , Microvasos/citología , Ratas , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
VEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cß3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCß3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.
Asunto(s)
Permeabilidad Capilar , Endotelio Vascular/metabolismo , Fosfolipasa C beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Modificados Genéticamente , Señalización del Calcio/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Embrión no Mamífero , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Ensayos Analíticos de Alto Rendimiento , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Ratones Noqueados , Morfolinos/farmacología , Fosfolipasa C beta/antagonistas & inhibidores , Fosfolipasa C beta/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Adaptive immunity requires that T cells efficiently scan diverse cell surfaces to identify cognate Ag. However, the basic cellular mechanisms remain unclear. In this study, we investigated this process using vascular endothelial cells, APCs that possess a unique and extremely advantageous, planar morphology. High-resolution imaging revealed that CD4 memory/effector T cells dynamically probe the endothelium by extending submicron-scale, actin-rich "invadosome/podosome-like protrusions" (ILPs). The intimate intercellular contacts enforced by ILPs consistently preceded and supported T cell activation in response to endothelial MHC class II/Ag. The resulting calcium flux stabilized dense arrays of ILPs (each enriched in TCR, protein kinase C-θ, ZAP70, phosphotyrosine, and HS1), forming what we term a podo-synapse. Similar findings were made using CD8 CTLs on endothelium. Furthermore, careful re-examination of both traditional APC models and professional APCs suggests broad relevance for ILPs in facilitating Ag recognition. Together, our results indicate that ILPs function as sensory organelles that serve as actuators of immune surveillance.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Extensiones de la Superficie Celular/inmunología , Células Endoteliales/inmunología , Inmunidad Adaptativa , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/citología , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Células CHO , Calcio/inmunología , Calcio/metabolismo , Señalización del Calcio , Comunicación Celular/inmunología , Extensiones de la Superficie Celular/ultraestructura , Cricetinae , Células Endoteliales/citología , Genes MHC Clase II , Humanos , Memoria Inmunológica , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/inmunología , Migración Transendotelial y Transepitelial , TransfecciónRESUMEN
Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 µm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and ß-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.
Asunto(s)
Antígenos de Superficie/metabolismo , Estructuras de la Membrana Celular/metabolismo , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Proteínas de Neoplasias/metabolismo , Seudópodos/metabolismo , Antígenos de Superficie/genética , Estructuras de la Membrana Celular/genética , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/metabolismo , Células Endoteliales/citología , Fibroblastos/citología , Humanos , Proteínas de Neoplasias/genética , Seudópodos/genéticaRESUMEN
Vascular endothelium provides a selective barrier between the blood and tissues, participates in wound healing and angiogenesis, and regulates tissue recruitment of inflammatory cells. Nuclear factor (NF)-κB transcription factors are pivotal regulators of survival and inflammation, and have been suggested as potential therapeutic targets in cancer and inflammatory diseases. Here we show that mice lacking IKKß, the primary kinase mediating NF-κB activation, are smaller than littermates and born at less than the expected Mendelian frequency in association with hypotrophic and hypovascular placentae. IKKß-deleted endothelium manifests increased vascular permeability and reduced migration. Surprisingly, we find that these defects result from loss of kinase-independent effects of IKKß on activation of the serine-threonine kinase, Akt. Together, these data demonstrate essential roles for IKKß in regulating endothelial permeability and migration, as well as an unanticipated connection between IKKß and Akt signalling.
Asunto(s)
Endotelio Vascular/enzimología , Quinasa I-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Movimiento Celular , Endotelio Vascular/citología , Femenino , Quinasa I-kappa B/genética , Masculino , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Caveolin-1, the signature protein of endothelial cell caveolae, has many important functions in vascular cells. Caveolae are thought to be the transcellular pathway by which plasma proteins cross normal capillary endothelium, but, unexpectedly, cav-1(-/-) mice, which lack caveolae, have increased permeability to plasma albumin. The acute increase in vascular permeability induced by agents such as vascular endothelial growth factor (VEGF)-A occurs through venules, not capillaries, and particularly through the vesiculo-vacuolar organelle (VVO), a unique structure composed of numerous interconnecting vesicles and vacuoles that together span the venular endothelium from lumen to ablumen. Furthermore, the hyperpermeable blood vessels found in pathological angiogenesis, mother vessels, are derived from venules. The present experiments made use of cav-1(-/-) mice to investigate the relationship between caveolae and VVOs and the roles of caveolin-1 in VVO structure in the acute vascular hyperpermeability induced by VEGF-A and in pathological angiogenesis and associated chronic vascular hyperpermeability. We found that VVOs expressed caveolin-1 variably but, in contrast to caveolae, were present in normal numbers and with apparently unaltered structure in cav-1(-/-) mice. Nonetheless, VEGF-A-induced hyperpermeability was strikingly reduced in cav-1(-/-) mice, as was pathological angiogenesis and associated chronic vascular hyperpermeability, whether induced by VEGF-A(164) or by a tumor. Thus, caveolin-1 is not necessary for VVO structure but may have important roles in regulating VVO function in acute vascular hyperpermeability and angiogenesis.
Asunto(s)
Permeabilidad Capilar/fisiología , Caveolina 1/deficiencia , Neovascularización Patológica/fisiopatología , Adenoviridae , Animales , Caveolina 1/metabolismo , Línea Celular Tumoral , Proliferación Celular , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/metabolismo , Piel/irrigación sanguínea , Piel/patología , Piel/ultraestructura , Vacuolas/metabolismo , Vacuolas/ultraestructura , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Diapedesis is critical for immune system function and inflammatory responses. This occurs by migration of blood leukocytes either directly through individual microvascular endothelial cells (the "transcellular" route) or between them (the "paracellular" route). Mechanisms for transcellular pore formation in endothelium remain unknown. Here we demonstrate that lymphocytes used podosomes and extended "invasive podosomes" to palpate the surface of, and ultimately form transcellular pores through, the endothelium. In lymphocytes, these structures were dependent on Src kinase and the actin regulatory protein WASP; inhibition of podosome formation selectively blocked the transcellular route of diapedesis. In endothelium, membrane fusion events dependent on the SNARE-containing membrane fusion complex and intracellular calcium were required for efficient transcellular pore formation in response to podosomes. These findings provide insights into basic mechanisms for leukocyte trafficking and the functions of podosomes.
Asunto(s)
Movimiento Celular , Endotelio Vascular/inmunología , Linfocitos/inmunología , Seudópodos/fisiología , Capilares/citología , Capilares/inmunología , Membrana Celular/química , Endotelio Vascular/citología , Humanos , Leucocitos/inmunología , Linfocitos/ultraestructura , Fusión de Membrana , Porosidad , Seudópodos/ultraestructura , Proteínas SNARE/análisis , Proteínas SNARE/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Mast cells are important effector cells in IgE-associated immune responses, but also can contribute to host defense in certain examples of bacterial infection. We found that genetically mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice exhibited more bacterial CFUs per spleen by 6 days after intraperitoneal injection of bioluminescent Salmonella typhimurium, and died more rapidly after infection, than did the congenic WBB6F1-Kit(+/+) wild type mice. Adoptive transfer of bone marrow-derived cultured mast cells of Kit(+/+) origin to the peritoneal cavity of Kit(W)/Kit(W-v) mice resulted in engraftment of mast cells in the peritoneal cavity and mesentery of the recipient mice, and the development of large numbers of mast cells in the spleen. However, such mast cell-engrafted Kit(W)/Kit(W-v) mice appeared sicker after intraperitoneal injection with S. typhimurium than did mast cell-deficient Kit(W)/Kit(W-v) mice, and exhibited numbers of CFUs of bacteria per spleen, and a survival curve, that were not significantly different than those of Kit(W)/Kit(W-v) mice. These results, when taken together with prior studies investigating the roles of mast cells in innate immunity, strongly suggest that whether mast cells can be shown to have a significant role in enhancing survival during bacterial infections may depend critically on the details of the particular experimental systems examined.