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1.
Liver Int ; 44(9): 2382-2395, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38847551

RESUMO

BACKGROUND & AIMS: Cystic fibrosis (CF) is considered a multisystemic disorder in which CF-associated liver disease (CFLD) is the third most common cause of mortality. Currently, no effective treatment is available for CFLD because its pathophysiology is still unclear. Interestingly, CFLD exhibits identical vascular characteristics as non-cirrhotic portal hypertension, recently classified as porto-sinusoidal vascular disorders (PSVD). METHODS: Since endothelial cells (ECs) are an important component in PSVD, we performed single-cell RNA sequencing (scRNA-seq) on four explant livers from CFLD patients to identify differential endothelial characteristics which could contribute to the disease. We comprehensively characterized the endothelial compartment and compared it with publicly available scRNA-seq datasets from cirrhotic and healthy livers. Key gene signatures were validated ex vivo on patient tissues. RESULTS: We found that ECs from CF liver explants are more closely related to healthy than cirrhotic patients. In CF patients we also discovered a distinct population of liver sinusoidal ECs-coined CF LSECs-upregulating genes involved in the complement cascade and coagulation. Finally, our immunostainings further validated the predominant periportal location of CF LSECs. CONCLUSIONS: Our work showed novel aspects of human liver ECs at the single-cell level thereby supporting endothelial involvement in CFLD, and reinforcing the hypothesis that ECs could be a driver of PSVD. Therefore, considering the vascular compartment in CF and CFLD may help developing new therapeutic approaches for these diseases.


Assuntos
Ativação do Complemento , Fibrose Cística , Células Endoteliais , Análise de Sequência de RNA , Análise de Célula Única , Humanos , Fibrose Cística/genética , Células Endoteliais/metabolismo , Fígado/patologia , Fígado/metabolismo , Masculino , Feminino , Adulto , Cirrose Hepática/genética , Cirrose Hepática/patologia , Hepatopatias/genética
2.
MethodsX ; 11: 102446, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37928105

RESUMO

Angiogenesis and lymphangiogenesis, the formation of new blood or lymphatic vessels, respectively, from preexisting vasculature is essential during embryonic development, but also occurs during tissue repair and in pathological conditions (cancer; ocular disease; ischemic, infectious and inflammatory disorders), which are all characterized to a certain extent by inflammatory conditions. Hence, a rapid, inexpensive, feasible / technically easy, reliable assay of inflammation-induced (lymph-)angiogenesis is highly valuable. In this context, the corneal thermal cauterization assay in mice is a simple, low-cost, reproducible, insightful and labor-saving assay to gauge the role of inflammation in angiogenesis and lymphangiogenesis. However, to the best of our knowledge, there is no standardized protocol to perform this assay. Here, we provide a step-by-step description of the model's procedures, which include:•The thermal cauterization of the corneas,•Enucleation and dissection of the corneas,•Subsequent immunofluorescence staining of the neovasculature, and morphometric analysis. We also discuss ethical considerations and aspects related to animal welfare guidelines. Altogether, this paper will help to increase the reproducibility of the corneal thermal cauterization model and facilitate its use for angiogenesis and lymphangiogenesis research.

3.
Cell Death Dis ; 14(9): 600, 2023 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-37684238

RESUMO

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we discovered that BAPTAi alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTAi provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTAi diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTAi. Notably, these effects were also induced by a BAPTAi analog with low affinity for Ca2+. Consequently, our findings uncover PFKFB3 inhibition as an Ca2+-independent mechanism through which BAPTAi impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.


Assuntos
Neoplasias , Monoéster Fosfórico Hidrolases , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Ácido Egtázico , Fosfofrutoquinase-2/genética
4.
Signal Transduct Target Ther ; 8(1): 305, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37591843

RESUMO

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.


Assuntos
Fator 2 de Crescimento de Fibroblastos , Fator B de Crescimento do Endotélio Vascular , Humanos , Fator 2 de Crescimento de Fibroblastos/genética , Imunoterapia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
5.
Acta Pharmacol Sin ; 44(3): 680-692, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36114272

RESUMO

The growth of solid tumors depends on tumor vascularization and the endothelial cells (ECs) that line the lumen of blood vessels. ECs generate a large fraction of ATP through glycolysis, and elevation of their glycolytic activity is associated with angiogenic behavior in solid tumors. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) positively regulates glycolysis via fructose-2/6-bisphosphate, the product of its kinase activity. Partial inhibition of glycolysis in tumor ECs by targeting PFKFB3 normalizes the otherwise abnormal tumor vessels, thereby reducing metastasis and improving the outcome of chemotherapy. Although a limited number of tool compounds exist, orally available PFKFB3 inhibitors are unavailable. In this study we conducted a high-throughput screening campaign against the kinase activity of PFKFB3, involving 250,240 chemical compounds. A total of 507 initial hits showing >50% inhibition at 20 µM were identified, 66 of them plus 1 analog from a similarity search consistently displayed low IC50 values (<10 µM). In vitro experiments yielded 22 nontoxic hits that suppressed the tube formation of primary human umbilical vein ECs at 10 µM. Of them, 15 exhibited binding affinity to PFKFB3 in surface plasmon resonance assays, including 3 (WNN0403-E003, WNN1352-H007 and WNN1542-F004) that passed the pan-assay interference compounds screening without warning flags. This study provides potential leads to the development of new PFKFB3 inhibitors.


Assuntos
Ensaios de Triagem em Larga Escala , Neoplasias , Fosfofrutoquinase-2 , Humanos , Glicólise , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo
6.
Cardiovasc Res ; 119(2): 520-535, 2023 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35998078

RESUMO

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.


Assuntos
COVID-19 , Fibrose Pulmonar Idiopática , Síndrome do Desconforto Respiratório , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Transcriptoma
7.
Nat Commun ; 13(1): 5511, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127427

RESUMO

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-γ and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-γ signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-γ link for BC treatment.


Assuntos
Neoplasias da Mama , Metformina , Neoplasias da Mama/patologia , Células Endoteliais/patologia , Feminino , Humanos , Imunidade , Ligantes , Lipídeos , Metformina/farmacologia , PPAR gama/genética , RNA , Estudos Retrospectivos
8.
STAR Protoc ; 3(4): 101691, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36173713

RESUMO

Tumor vessel co-option, a process in which cancer cells "hijack" pre-existing blood vessels to grow and invade healthy tissue, is poorly understood but is a proposed resistance mechanism against anti-angiogenic therapy (AAT). Here, we describe protocols for establishing murine renal (RENCA) and breast (4T1) cancer lung vessel co-option metastases models. Moreover, we outline a reproducible protocol for single-cell isolation from murine lung metastases using magnetic-activated cell sorting as well as immunohistochemical stainings to distinguish vessel co-option from angiogenesis. For complete details on the use and execution of this protocol, please refer to Teuwen et al. (2021).


Assuntos
Neoplasias Pulmonares , Neovascularização Patológica , Camundongos , Animais , Neovascularização Patológica/patologia , Células Endoteliais , Neoplasias Pulmonares/patologia , Modelos Animais de Doenças
9.
Autophagy ; 18(10): 2409-2426, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35258392

RESUMO

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.


Assuntos
Autofagia , Doenças Inflamatórias Intestinais , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , DNA Mitocondrial/metabolismo , Dextranos/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Formaldeído/metabolismo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Isotiocianatos , Lipopolissacarídeos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Fosfatidiletanolaminas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Respiração , Sirolimo
10.
Theranostics ; 11(19): 9243-9261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34646369

RESUMO

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown. Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF∆CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis. Results: Compared with wild-type mice, TF∆CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF∆CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF∆CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF∆CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF∆CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) - Rac1 axis. In particular, infarcted TF∆CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF∆CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF∆CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps. Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.


Assuntos
Infarto do Miocárdio/patologia , Tromboplastina/metabolismo , Remodelação Ventricular/fisiologia , Animais , Proliferação de Células/fisiologia , Inflamação/metabolismo , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miofibroblastos/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Domínios Proteicos/fisiologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transdução de Sinais/fisiologia , Tromboplastina/fisiologia , Função Ventricular Esquerda/fisiologia
11.
STAR Protoc ; 2(3): 100523, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34382011

RESUMO

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).


Assuntos
Células Endoteliais/citologia , Rim/citologia , Baço/citologia , Testículo/citologia , Animais , Citometria de Fluxo , Masculino , Camundongos
12.
Cell Metab ; 33(9): 1763-1776.e5, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34302744

RESUMO

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.


Assuntos
Hiperlactatemia , Sepse , Animais , Glucocorticoides , Ácido Láctico , Camundongos , Receptores de Glucocorticoides/metabolismo , Sepse/complicações , Sepse/metabolismo , Fator A de Crescimento do Endotélio Vascular
13.
Cell Rep ; 35(11): 109253, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34133923

RESUMO

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent.


Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/patologia , Análise de Célula Única , Animais , Linhagem Celular Tumoral , Células Endoteliais/patologia , Feminino , Neoplasias Renais/patologia , Neoplasias Pulmonares/secundário , Macrófagos/patologia , Camundongos Endogâmicos BALB C , Células Mieloides/patologia , Pericitos/patologia
14.
Eur Respir J ; 57(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33184117

RESUMO

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Endoteliais/metabolismo , Humanos , Fenótipo , Transcriptoma
15.
Nucleic Acids Res ; 48(W1): W385-W394, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32392297

RESUMO

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.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Software , Algoritmos , Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Gráficos por Computador , Células Endoteliais/metabolismo , Humanos , Metabolômica/métodos , Neoplasias/mortalidade , Proteômica/métodos , Análise de Sobrevida , Fluxo de Trabalho
16.
Cell Metab ; 31(4): 862-877.e14, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32268117

RESUMO

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets.


Assuntos
Células Endoteliais/metabolismo , Neoplasias Pulmonares/metabolismo , Degeneração Macular/metabolismo , Neovascularização Patológica/metabolismo , Transcriptoma , Animais , Células Endoteliais/citologia , Células Endoteliais/patologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Análise de Célula Única
19.
Cancer Cell ; 37(1): 21-36.e13, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31935371

RESUMO

Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.


Assuntos
Células Endoteliais/citologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Neovascularização Patológica , Inibidores da Angiogênese/farmacologia , Animais , Membrana Basal/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Análise por Conglomerados , Colágeno/química , Endotélio Vascular/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Fenótipo , Análise de Célula Única , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Cell Metab ; 28(6): 866-880.e15, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30146486

RESUMO

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASNKD) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASNKD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASNKD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade.


Assuntos
Ácido Graxo Sintase Tipo I/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Malonil Coenzima A/metabolismo , Neovascularização Retiniana/patologia , Serina-Treonina Quinases TOR/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Orlistate/uso terapêutico , Processamento de Proteína Pós-Traducional , Neovascularização Retiniana/tratamento farmacológico
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