RESUMEN
The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.
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Células Endoteliales/metabolismo , Análisis de la Célula Individual , Transcriptoma , Animales , Encéfalo/citología , Sistema Cardiovascular/citología , Células Endoteliales/clasificación , Células Endoteliales/citología , Tracto Gastrointestinal/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos/citología , Especificidad de Órganos , RNA-Seq , Testículo/citologíaRESUMEN
Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
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Células Endoteliales/enzimología , Células Endoteliales/patología , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/biosíntesis , Neovascularización Patológica , Actinas/metabolismo , Animales , Movimiento Celular , Células Endoteliales/metabolismo , Femenino , Glutamato-Amoníaco Ligasa/deficiencia , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Lipoilación , Ratones , Ácido Palmítico/metabolismo , Procesamiento Proteico-Postraduccional , Fibras de Estrés/metabolismo , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid ß-oxidation, impairs lymphatic development. LECs use fatty acid ß-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid ß-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.
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Ácidos Grasos/química , Ácidos Grasos/metabolismo , Linfangiogénesis , Vasos Linfáticos/citología , Vasos Linfáticos/metabolismo , Acetatos/farmacología , Acetilcoenzima A/metabolismo , Acetilación/efectos de los fármacos , Animales , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Epigénesis Genética , Femenino , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Linfangiogénesis/efectos de los fármacos , Linfangiogénesis/genética , Vasos Linfáticos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción/efectos de los fármacos , Biosíntesis de Proteínas , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Arterias Umbilicales/citología , Regulación hacia ArribaRESUMEN
BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.
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Próstata , Neoplasias de la Próstata , Animales , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Endoteliales/metabolismo , Humanos , Masculino , Ratones , Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Epoprostenol , Microambiente TumoralRESUMEN
Glioblastoma stem cells (GSCs) reside close to blood vessels (BVs) but vascular cues contributing to GSC stemness and the nature of GSC-BVs cross talk are not fully understood. Here, we dissected vascular cues influencing GSC gene expression and function to perfusion-based vascular cues, as well as to those requiring direct GSC-endothelial cell (EC) contacts. In light of our previous finding that perivascular tumor cells are metabolically different from tumor cells residing further downstream, cancer cells residing within a narrow, < 60 µm wide perivascular niche were isolated and confirmed to possess a superior tumor-initiation potential compared with those residing further downstream. To circumvent reliance on marker expression, perivascular GSCs were isolated from the respective locales based on their relative state of quiescence. Combined use of these procedures uncovered a large number of previously unrecognized differentially expressed GSC genes. We show that the unique metabolic milieu of the perivascular niche dominated by the highly restricted zone of mTOR activity is conducive for acquisition of GSC properties, primarily in the regulation of genes implicated in cell cycle control. A complementary role of vascular cues including those requiring direct glioma/EC contacts was revealed using glioma/EC co-cultures. Outstanding in the group of glioma cells impacted by nearby ECs were multiple genes responsible for maintaining GSCs in an undifferentiated state, a large fraction of which also relied on Notch-mediated signaling. Glioma-EC communication was found to be bidirectional, evidenced by extensive Notch-mediated EC reprogramming by contacting tumor cells, primarily metabolic EC reprogramming.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Señales (Psicología) , Glioblastoma/patología , Glioma/irrigación sanguínea , Glioma/genética , Humanos , Células Madre Neoplásicas/patologíaRESUMEN
The amount of biological data, generated with (single cell) omics technologies, is rapidly increasing, thereby exacerbating bottlenecks in the data analysis and interpretation of omics experiments. Data mining platforms that facilitate non-bioinformatician experimental scientists to analyze a wide range of experimental designs and data types can alleviate such bottlenecks, aiding in the exploration of (newly generated or publicly available) omics datasets. Here, we present BIOMEX, a browser-based software, designed to facilitate the Biological Interpretation Of Multi-omics EXperiments by bench scientists. BIOMEX integrates state-of-the-art statistical tools and field-tested algorithms into a flexible but well-defined workflow that accommodates metabolomics, transcriptomics, proteomics, mass cytometry and single cell data from different platforms and organisms. The BIOMEX workflow is accompanied by a manual and video tutorials that provide the necessary background to navigate the interface and get acquainted with the employed methods. BIOMEX guides the user through omics-tailored analyses, such as data pretreatment and normalization, dimensionality reduction, differential and enrichment analysis, pathway mapping, clustering, marker analysis, trajectory inference, meta-analysis and others. BIOMEX is fully interactive, allowing users to easily change parameters and generate customized plots exportable as high-quality publication-ready figures. BIOMEX is open source and freely available at https://www.vibcancer.be/software-tools/biomex.
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Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Programas Informáticos , Algoritmos , Neoplasias de los Conductos Biliares/genética , Colangiocarcinoma/genética , Gráficos por Computador , Células Endoteliales/metabolismo , Humanos , Metabolómica/métodos , Neoplasias/mortalidad , Proteómica/métodos , Análisis de Supervivencia , Flujo de TrabajoRESUMEN
Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine-derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine-deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.
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Asparagina/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Glutamina/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Medios de Cultivo/química , Células Endoteliales/metabolismo , Glutaminasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Redes y Vías Metabólicas , Neovascularización PatológicaRESUMEN
The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.
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Carbono/metabolismo , Células Endoteliales/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Nucleótidos/biosíntesis , Ácido Acético/farmacología , Adenosina Trifosfato/metabolismo , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclo del Ácido Cítrico , ADN/biosíntesis , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Silenciador del Gen , Glucosa/metabolismo , 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 , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Nucleótidos/química , Nucleótidos/farmacología , Oxidación-Reducción/efectos de los fármacos , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/patologíaRESUMEN
Endothelial cells (ECs) line blood vessels, regulate homeostatic processes (blood flow, immune cell trafficking), but are also involved in many prevalent diseases. The increasing use of high-throughput technologies such as gene expression microarrays and (single cell) RNA sequencing generated a wealth of data on the molecular basis of EC (dys-)function. Extracting biological insight from these datasets is challenging for scientists who are not proficient in bioinformatics. To facilitate the re-use of publicly available EC transcriptomics data, we developed the endothelial database EndoDB, a web-accessible collection of expert curated, quality assured and pre-analyzed data collected from 360 datasets comprising a total of 4741 bulk and 5847 single cell endothelial transcriptomes from six different organisms. Unlike other added-value databases, EndoDB allows to easily retrieve and explore data of specific studies, determine under which conditions genes and pathways of interest are deregulated and assess reprogramming of metabolism via principal component analysis, differential gene expression analysis, gene set enrichment analysis, heatmaps and metabolic and transcription factor analysis, while single cell data are visualized as gene expression color-coded t-SNE plots. Plots and tables in EndoDB are customizable, downloadable and interactive. EndoDB is freely available at https://vibcancer.be/software-tools/endodb, and will be updated to include new studies.
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Biología Computacional , Bases de Datos Genéticas , Transcriptoma/genética , Animales , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Análisis de Componente PrincipalRESUMEN
BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.
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Adaptación Fisiológica/genética , Células Endoteliales/metabolismo , Riñón/citología , Análisis de Secuencia de ARN , Privación de Agua/fisiología , Animales , Células Endoteliales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti-Ang-2-neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti-colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.
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Anticuerpos Antineoplásicos/farmacología , Glioblastoma , Macrófagos , Proteínas de Neoplasias , Neoplasias Experimentales , Neovascularización Patológica , Quinazolinas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular , Ribonucleasa Pancreática , Animales , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Ribonucleasa Pancreática/antagonistas & inhibidores , Ribonucleasa Pancreática/metabolismoRESUMEN
Clinically approved therapies that target angiogenesis in tumors and ocular diseases focus on controlling pro-angiogenic growth factors in order to reduce aberrant microvascular growth. Although research on angiogenesis has revealed key mechanisms that regulate tissue vascularization, therapeutic success has been limited owing to insufficient efficacy, refractoriness and tumor resistance. Emerging concepts suggest that, in addition to growth factors, vascular metabolism also regulates angiogenesis and is a viable target for manipulating the microvasculature. Recent studies show that endothelial cells rely on glycolysis for ATP production, and that the key glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) regulates angiogenesis by controlling the balance of tip versus stalk cells. As endothelial cells acquire a tip cell phenotype, they increase glycolytic production of ATP for sprouting. Furthermore, pharmacological blockade of PFKFB3 causes a transient, partial reduction in glycolysis, and reduces pathological angiogenesis with minimal systemic harm. Although further assessment of endothelial cell metabolism is necessary, these results represent a paradigm shift in anti-angiogenic therapy from targeting angiogenic factors to focusing on vascular metabolism, warranting research on the metabolic pathways that govern angiogenesis.
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Inhibidores de la Angiogénesis/uso terapéutico , Endotelio Vascular/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica , Adenosina Trifosfato/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Endotelio Vascular/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neovascularización Patológica/metabolismo , Fosfofructoquinasa-2/antagonistas & inhibidoresRESUMEN
Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/ß-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/ß-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/ß-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.
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Células Endoteliales , Factores de Transcripción Forkhead , Receptores Frizzled , Neoplasias Pulmonares , Animales , Humanos , Ratones , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/metabolismo , Neovascularización Patológica/genética , Vía de Señalización WntRESUMEN
Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y6. Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDAhigh cancer cells match with T cell cytotoxicity dysfunction and P2RY6high TAMs. Overall, we suggest CDA and P2Y6 as potential targets for cancer immunotherapy.
RESUMEN
Endothelial cells (ECs) are highly plastic, capable of differentiating into various cell types. Endothelial-to-mesenchymal transition (EndMT) is crucial during embryonic development and contributes substantially to vascular dysfunction in many cardiovascular diseases (CVDs). While targeting EndMT holds therapeutic promise, understanding its mechanisms and modulating its pathways remain challenging. Using single-cell RNA sequencing on three in vitro EndMT models, we identified conserved gene signatures. We validated original regulators in vitro and in vivo during embryonic heart development and peripheral artery disease. EndMT induction led to global expression changes in all EC subtypes rather than in mesenchymal clusters. We identified mitochondrial calcium uptake as a key driver of EndMT; inhibiting mitochondrial calcium uniporter (MCU) prevented EndMT in vitro, and conditional Mcu deletion in ECs blocked mesenchymal activation in a hind limb ischemia model. Tissues from patients with critical limb ischemia with EndMT features exhibited significantly elevated endothelial MCU. These findings highlight MCU as a regulator of EndMT and a potential therapeutic target.
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Señalización del Calcio , Células Endoteliales , Transición Epitelial-Mesenquimal , Mitocondrias , RNA-Seq , Análisis de la Célula Individual , Animales , Humanos , Mitocondrias/metabolismo , RNA-Seq/métodos , Ratones , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Canales de Calcio/metabolismo , Canales de Calcio/genética , Isquemia/metabolismo , Isquemia/patología , Calcio/metabolismo , Análisis de Expresión Génica de una Sola CélulaRESUMEN
BACKGROUND: Electronic health records (EHRs) are increasingly available and this was expected to reduce healthcare costs and medical errors. This promise has not been realized because healthcare professionals are unable to use EHRs in a manner that contributes to significant improvements in care, i.e. meaningful. Policymakers now acknowledge that training healthcare professionals in meaningful use is essential for successful EHR implementation. To help educators and policymakers design evidence based educational interventions (i.e. interventions that involve educational activities but no practical lessons) and training (i.e. interventions that involve practical components), we summarized all evidence regarding the efficacy of different educational interventions to improve meaningful use of EHRs. METHODS: We used a predefined search filter to search eight databases for studies that considered an educational intervention to promote meaningful use of EHRs by healthcare professionals. RESULTS: Seven of the 4507 reviewed articles met the in- and exclusion criteria. CONCLUSIONS: These studies suggest that a combination of classroom training, computer-based training and feedback is most effective to improve meaningful use. In addition, the training should be tailored to the needs of the trainees and they should be able to practice in their own time. However, the evidence is very limited and we recommend that governments, hospitals and other policymakers invest more in the development of evidence based educational interventions to improve meaningful use of EHRs.
Asunto(s)
Registros Electrónicos de Salud/organización & administración , Personal de Salud/educación , Uso Significativo/organización & administración , Análisis Costo-Beneficio , Consejo , Retroalimentación , Humanos , Políticas , Competencia Profesional , Mejoramiento de la Calidad/organización & administraciónRESUMEN
Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.
Asunto(s)
Autofagia , Enfermedades Inflamatorias del Intestino , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cisteína/metabolismo , ADN Mitocondrial/metabolismo , Dextranos/metabolismo , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Formaldehído/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Isotiocianatos , Lipopolisacáridos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Fosfatidiletanolaminas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Respiración , SirolimusRESUMEN
Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).