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1.
Cell Stress Chaperones ; 25(5): 701-705, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32889638

RESUMO

Near the end of 2019, SARS-CoV-2, a novel highly contagious coronavirus phylogenetically related to the SARS virus, entered the human population with lethal consequences. This special issue devoted to the resulting disease COVID-19 was not planned but instead the articles accumulated organically as researchers in the cell stress response field noticed similarities among the pathophysiology of COVID-19 infections and the responses that they studied in contexts unrelated to viral infection. We preface the issue with an introductory article which begins with a brief review of the structure and biology of SARS-CoV-2. As we collected and compared the COVID-19 articles, several shared themes emerged. In the second part of the introduction, each article is summarized briefly and the common themes that link each into a spontaneously arising chain of ideas and hypotheses are emphasized. These themes include growing evidence of molecular mimicry among the viral proteins and the proteins of patients. The realization that much of the consequences of such immune mimicry may play out on the plasma membrane of vascular endothelial cells raised the specter of autoimmune-induced vascular endothelial damage in multiple organs. Proposals of new therapeutic approaches have coalesced around the theme of inducing protection of the vascular endothelium. New chemical treatments that are proposed include stannous chloride, inducers of the gasotransmitter hydrogen sulfide such as sodium thiosulfate and inducers of the cytoprotective stress protein heme oxygenase. Oxygen delivered by ventilators is already in extensive use to provide life support for patients with severe COVID-19. Two articles propose to advance the use of oxygen to the level of a therapeutic treatment early in the detection of the virus in infected patients by delivering oxygen under elevated pressure in hyperbaric chambers. At elevated blood plasma concentrations, hyperbaric oxygen is capable of achieving results far beyond the capability of ventilators as it promotes the activation of transcription factors that control the establishment of inducible cellular defense systems.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/fisiopatologia , Coronavirus , Células Endoteliais/imunologia , Oxigênio/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/fisiopatologia , Proteínas Virais/imunologia , Coronavirus/classificação , Coronavirus/imunologia , Células Endoteliais/citologia , Humanos , Pandemias
2.
PLoS One ; 15(9): e0238301, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881954

RESUMO

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) is a primary astrocytopathy driven by antibodies directed against the aquaporin-4 water channel located at the end-feet of the astrocyte. Although blood-brain barrier (BBB) breakdown is considered one of the key steps for the development and lesion formation, little is known about the molecular mechanisms involved. The aim of the study was to evaluate the effect of human immunoglobulins from NMOSD patients (NMO-IgG) on BBB properties. METHODS: Freshly isolated brain microvessels (IBMs) from rat brains were used as a study model. At first, analysis of the secretome profile from IBMs exposed to purified NMO-IgG, to healthy donor IgG (Control-IgG), or non-treated, was performed. Second, tight junction (TJ) proteins expression in fresh IBMs and primary cultures of brain microvascular endothelial cells (BMEC) was analysed by Western blotting (Wb) after exposition to NMO-IgG and Control-IgG. Finally, functional BBB properties were investigated evaluating the presence of rat-IgG in tissue lysate from brain using Wb in the rat-model, and the passage of NMO-IgG and sucrose in a bicameral model. RESULTS: We found that NMO-IgG induces functional and morphological BBB changes, including: 1) increase of pro-inflammatory cytokines production (CXCL-10 [IP-10], IL-6, IL-1RA, IL-1ß and CXCL-3) in IBMs when exposed to NMO-IgG; 2) decrease of Claudin-5 levels by 25.6% after treatment of fresh IBMs by NMO-IgG compared to Control-IgG (p = 0.002), and similarly, decrease of Claudin-5 by at least 20% when BMEC were cultured with NMO-IgG from five different patients; 3) a higher level of rat-IgG accumulated in periventricular regions of NMO-rats compared to Control-rats and an increase in the permeability of BBB after NMO-IgG treatment in the bicameral model. CONCLUSION: Human NMO-IgG induces both structural and functional alterations of BBB properties, suggesting a direct role of NMO-IgG on modulation of BBB permeability in NMOSD.


Assuntos
Aquaporina 4/imunologia , Barreira Hematoencefálica/metabolismo , Imunoglobulina G/farmacologia , Neuromielite Óptica/patologia , Permeabilidade/efeitos dos fármacos , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Claudina-5/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Imunoglobulina G/isolamento & purificação , Microvasos/citologia , Microvasos/metabolismo , Neuromielite Óptica/metabolismo , Ratos
3.
PLoS One ; 15(7): e0225351, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735563

RESUMO

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with Shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.


Assuntos
Barreira Hematorretiniana/metabolismo , Cílios/metabolismo , Células Endoteliais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/fisiologia , Células Endoteliais/citologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Larva/metabolismo , Mutagênese , Vasos Retinianos/citologia , Transdução de Sinais , Alcaloides de Veratrum/farmacologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Nat Commun ; 11(1): 3798, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732867

RESUMO

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis.


Assuntos
Células Endoteliais/citologia , Endotélio Vascular/citologia , Linfonodos/irrigação sanguínea , Linfócitos/imunologia , Células Mieloides/imunologia , Animais , Sequência de Bases , Movimento Celular/imunologia , Feminino , Perfilação da Expressão Gênica , Homeostase/imunologia , Inflamação/imunologia , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma/genética
5.
Arterioscler Thromb Vasc Biol ; 40(10): 2404-2407, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32762443

RESUMO

OBJECTIVE: Alveolar-capillary endothelial cells can be activated by severe acute respiratory syndrome coronavirus 2 infection leading to cytokine release. This could trigger endothelial dysfunction, pyroptosis, and thrombosis, which are the vascular changes, commonly referred to as coronavirus disease 2019 (COVID-19) endotheliopathy. Thus, this study aimed to identify tissue biomarkers associated with endothelial activation/dysfunction and the pyroptosis pathway in the lung samples of patients with COVID-19 and to compare them to pandemic influenza A virus H1N1 subtype 2009 and control cases. Approach and Results: Postmortem lung samples (COVID-19 group =6 cases; H1N1 group =10 cases, and control group =11 cases) were analyzed using immunohistochemistry and the following monoclonal primary antibodies: anti-IL (interleukin)-6, anti-TNF (tumor necrosis factor)-α, anti-ICAM-1 (intercellular adhesion molecule 1), and anticaspase-1. From the result, IL-6, TNF-α, ICAM-1, and caspase-1 showed higher tissue expression in the COVID-19 group than in the H1N1 and control groups. CONCLUSIONS: Our results demonstrated endothelial dysfunction and suggested the participation of the pyroptosis pathway in the pulmonary samples. These conditions might lead to systemic thrombotic events that could impair the clinical staff's efforts to avoid fatal outcomes. One of the health professionals' goals should be to identify the high risk of thrombosis patients early to block endotheliopathy and its consequences.


Assuntos
Infecções por Coronavirus/patologia , Células Endoteliais/citologia , Endotélio Vascular/patologia , Pneumonia Viral/patologia , Trombose/patologia , Doenças Vasculares/patologia , Autopsia , Biópsia por Agulha , Causas de Morte , Infecções por Coronavirus/mortalidade , Células Endoteliais/patologia , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pandemias , Pneumonia Viral/mortalidade , Medição de Risco , Trombose/etiologia , Trombose/mortalidade , Doenças Vasculares/mortalidade , Doenças Vasculares/fisiopatologia
6.
Circulation ; 142(12): 1190-1204, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32755395

RESUMO

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II, a potent vasoconstrictor, to angiotensin-(1-7) and is also a membrane protein that enables coronavirus disease 2019 (COVID-19) infectivity. AMP-activated protein kinase (AMPK) phosphorylation of ACE2 enhances ACE2 stability. This mode of posttranslational modification of ACE2 in vascular endothelial cells is causative of a pulmonary hypertension (PH)-protective phenotype. The oncoprotein MDM2 (murine double minute 2) is an E3 ligase that ubiquitinates its substrates to cause their degradation. In this study, we investigated whether MDM2 is involved in the posttranslational modification of ACE2 through its ubiquitination of ACE2, and whether an AMPK and MDM2 crosstalk regulates the pathogenesis of PH. METHODS: Bioinformatic analyses were used to explore E3 ligase that ubiquitinates ACE2. Cultured endothelial cells, mouse models, and specimens from patients with idiopathic pulmonary arterial hypertension were used to investigate the crosstalk between AMPK and MDM2 in regulating ACE2 phosphorylation and ubiquitination in the context of PH. RESULTS: Levels of MDM2 were increased and those of ACE2 decreased in lung tissues or pulmonary arterial endothelial cells from patients with idiopathic pulmonary arterial hypertension and rodent models of experimental PH. MDM2 inhibition by JNJ-165 reversed the SU5416/hypoxia-induced PH in C57BL/6 mice. ACE2-S680L mice (dephosphorylation at S680) showed PH susceptibility, and ectopic expression of ACE2-S680L/K788R (deubiquitination at K788) reduced experimental PH. Moreover, ACE2-K788R overexpression in mice with endothelial cell-specific AMPKα2 knockout mitigated PH. CONCLUSIONS: Maladapted posttranslational modification (phosphorylation and ubiquitination) of ACE2 at Ser-680 and Lys-788 is involved in the pathogenesis of pulmonary arterial hypertension and experimental PH. Thus, a combined intervention of AMPK and MDM2 in the pulmonary endothelium might be therapeutically effective in PH treatment.


Assuntos
Peptidil Dipeptidase A/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Hipertensão Arterial Pulmonar/patologia , Ubiquitinação , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Animais , Suscetibilidade a Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidil Dipeptidase A/genética , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos
7.
Proc Natl Acad Sci U S A ; 117(33): 19854-19865, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759214

RESUMO

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Claudina-5/genética , Claudina-5/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Edição de Genes , Genoma , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Gene ; 760: 144992, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32721474

RESUMO

BACKGROUND AND AIM: Diabetic retinopathy is a severe diabetic complication and a major cause of blindness. In this study, we explored the role of circ_0001879 in retinal vascular dysfunction under diabetic conditions. METHODS: Human retinal microvascular endothelial cells (HRMECs) were divided into normal glucose group (NG, 5.5 mmol/L d-glucose), high glucose group (HG, 25 mmol/L d-glucose), and osmotic control group (5.5 mmol/L d-glucose + 19.5 mmol/L mannitol). The expression of circ_0001879 and miR-30-3p was assessed via qRT-PCR. The circ_0001879/miR-30-3p roles in retinal vascular dysfunction were investigated through Cell Counting Kit-8 and Transwell assay. Bioinformatics analysis and luciferase reporter assays were applied to examine interactions between circ_0001879 and miR-30-3p in HRMECs. RESULTS: The relative circ_0001879 expression was remarkably increased in diabetic retinas group than that in the control group. Silencing circ_0001879 suppressed the proliferation and migration of HRMECs under high-glucose conditions. In addition, circ_0001879 acted as a binding platform and miRNA sponge for miR-30-3p. Circ_0001879 modulated the function of HRMECs via targeting miR-30-3p. CONCLUSION: Silencing circ_0001879 inhibited the proliferation and migration of HRMECs under high-glucose conditions via modulating miR-30-3p, which might shed new light on a novel potentially marker and molecular therapeutic target for diabetic retinopathy.


Assuntos
Retinopatia Diabética/patologia , Glucose/administração & dosagem , MicroRNAs/genética , Vasos Retinianos/patologia , Animais , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo
9.
Clin Med (Lond) ; 20(5): e146-e147, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601125

RESUMO

The novel coronavirus infectious disease (COVID-19) has rapidly spread and poses a great challenge to researchers, both in elucidating its pathogenic mechanism and developing effective treatments. It has been recently proposed that COVID-19 is an endothelial disease. Indeed, the COVID-19 virus binds to angiotensin-converting enzyme type 2 (ACE2), which is expressed in endothelial cells. ACE2 could be implicated in the production of reactive oxygen species (ROS) caused by endothelial dysfunction due to viral damage. Consequently, oxidative stress could prime these cells to acquire a pro-thrombotic and pro-inflammatory phenotype, predisposing patients to thromboembolic and vasculitic events and to disseminated intravascular coagulopathy (DIC). This implies a pivotal role played by oxygen in the pathogenetic mechanism of COVID-19 disease, in that its availability would tune the oxidant state and consequent damage.


Assuntos
Infecções por Coronavirus/fisiopatologia , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Pneumonia Viral/fisiopatologia , Síndrome Respiratória Aguda Grave/fisiopatologia , Infecções por Coronavirus/epidemiologia , Progressão da Doença , Humanos , Determinação de Necessidades de Cuidados de Saúde , Pandemias , Pneumonia Viral/epidemiologia , Medição de Risco , Síndrome Respiratória Aguda Grave/epidemiologia , Análise de Sobrevida
10.
PLoS One ; 15(6): e0233899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584883

RESUMO

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments.


Assuntos
Células Endoteliais/metabolismo , Xenoenxertos/citologia , Próstata/citologia , Transcriptoma , Animais , Células Endoteliais/citologia , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Animais , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo
11.
DNA Cell Biol ; 39(7): 1264-1273, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32584608

RESUMO

Transforming growth factor-beta 1 (TGF-ß1) plays important roles in the endothelial-to-mesenchymal transition (EndMT). Recently, long noncoding RNAs (lncRNAs) have been identified to be involved in the physiological and pathological processes of human diseases. However, the role of endothelial lncRNAs in the TGF-ß1-mediated control of angiogenesis and its underlying mechanism remains largely unclear. In this study, we first demonstrated that TGF-ß1 induced EndMT; promoted cell viability, proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Second, our study displayed that TGF-ß1 upregulated the lncRNA UCA1 expression in HUVECs, knocked down UCA1 with small interfering RNAs, and inhibited the function of TGF-ß1 in HUVECs. Third, our study showed that UCA1 was located in the cytoplasm and absorbed miR-455 in TGF-ß1-treated HUVECs. Further, the miR-455 inhibitor restored the role of the inhibited UCA1 in HUVECs treated with TGF-ß1. Fourth, our study revealed that miR-455 inhibited ZEB1 expression, and overexpression of ZEB1 restored the role of miR-455 in HUVECs treated with TGF-ß1. Finally, our study revealed that UCA1 exerted its role via regulating the UCA1/miR-455/ZEB1 regulatory axis in HUVECs treated with TGF-ß1. Collectively, our study identified the role of the UCA1/miR-455/ZEB1 pathway in HUVECs treated with TGF-ß1 and indicated the potential therapeutic role of this regulatory axis in angiogenesis.


Assuntos
Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Mesoderma/citologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Fator de Crescimento Transformador beta1/farmacologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Sequência de Bases , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Regulação para Cima/efeitos dos fármacos
12.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510519

RESUMO

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network. By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules. The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.


Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Células Endoteliais/metabolismo , Microfluídica/métodos , Adesividade Plaquetária , Agregação Plaquetária , Trombose/fisiopatologia , Células Endoteliais/citologia , Fibrinólise , Hemostasia , Humanos , Microfluídica/instrumentação
13.
Parasitol Res ; 119(8): 2563-2577, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32548739

RESUMO

Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24-30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that-in terms of host cell cycle modulation-infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti-infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii-related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti-infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.


Assuntos
Doenças dos Bovinos/fisiopatologia , Coccidiose/veterinária , Pontos de Checagem da Fase G2 do Ciclo Celular , Pontos de Checagem da Fase M do Ciclo Celular , Sarcocystidae/fisiologia , Animais , Apoptose , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/parasitologia , Coccidiose/fisiopatologia , Células Endoteliais/citologia , Células Endoteliais/parasitologia
14.
J Vis Exp ; (160)2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32597877

RESUMO

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.


Assuntos
Procedimentos Cirúrgicos Cardíacos/métodos , Células Endoteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miocárdio/metabolismo , Engenharia Tecidual/métodos , Células Endoteliais/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
15.
Mol Pharmacol ; 98(1): 61-71, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358165

RESUMO

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.


Assuntos
Anoctamina-1/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Animais , Anoctamina-1/antagonistas & inibidores , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Ácido Niflúmico/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia
16.
Zhonghua Gan Zang Bing Za Zhi ; 28(4): 357-360, 2020 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-32403890

RESUMO

Liver sinusoidal endothelial cells (LSECs) are not only important intermediary cells for substance exchange between blood and hepatocytes, but also important hepatic non-parenchymal cells to cause liver fibrosis and cirrhosis because of chronic liver injury factors. It mainly regulates the liver microcirculation and participates in the development of hepatic fibrosis by interacting with hepatic stellate cells (HSCs), hepatocytes, Kupffer cells and mediating hepatic stiffness and hepatic angiogenesis. Hence, clarifying these mechanisms will help to explore new targets and strategies for the treatment of liver fibrosis.


Assuntos
Células Endoteliais/citologia , Cirrose Hepática/patologia , Fígado/citologia , Células Estreladas do Fígado , Hepatócitos , Humanos , Macrófagos do Fígado , Fígado/patologia
17.
Am J Physiol Renal Physiol ; 319(1): F41-F51, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390509

RESUMO

Endothelial cells (ECs) from different human organs possess organ-specific characteristics that support specific tissue regeneration and organ development. EC specificity is identified by both intrinsic and extrinsic cues, among which the parenchyma and organ-specific microenvironment are critical contributors. These extrinsic cues are, however, largely lost during ex vivo cultures. Outstanding challenges remain to understand and reestablish EC organ specificity for in vitro studies to recapitulate human organ-specific physiology. Here, we designed an open microfluidic platform to study the role of human kidney tubular epithelial cells in supporting EC specificity. The platform consists of two independent cell culture regions segregated with a half wall; culture media are added to connect the two culture regions at a desired time point, and signaling molecules can travel across the half wall (paracrine signaling). Specifically, we report that in the microscale coculture device, primary human kidney proximal tubule epithelial cells (HPTECs) rescued primary human kidney peritubular microvascular EC (HKMEC) monolayer integrity and fenestra formation and that HPTECs upregulated key HKMEC kidney-specific genes (hepatocyte nuclear factor 1 homeobox B, adherens junctions-associated protein 1, and potassium voltage-gated channel subfamily J member 16) and endothelial activation genes (vascular cell adhesion molecule-1, matrix metalloproteinase-7, and matrix metalloproteinase-10) in coculture. Coculturing with HPTECs also promoted kidney-specific genotype expression in human umbilical vein ECs and human pluripotent stem cell-derived ECs. Compared with culture in HPTEC conditioned media, coculture of ECs with HPTECs showed increased upregulation of kidney-specific genes, suggesting potential bidirectional paracrine signaling. Importantly, our device is compatible with standard pipettes, incubators, and imaging readouts and could also be easily adapted to study cell signaling between other rare or sensitive cells.


Assuntos
Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Comunicação Parácrina/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Epiteliais/citologia , Humanos , Rim/citologia , Microfluídica
18.
Adv Exp Med Biol ; 1233: 279-302, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274762

RESUMO

Given the low mitotic activity of cardiomyocytes, the contractile unit of the heart, these cells strongly rely on efficient and highly regulated mechanisms of protein degradation to eliminate unwanted potentially toxic proteins. This is particularly important in the context of disease, where an impairment of protein quality control mechanisms underlies the onset and development of diverse cardiovascular maladies. One of the biological processes which is tightly regulated by proteolysis mechanisms is intercellular communication. The different types of cells that form the heart, including cardiomyocytes, endothelial cells, fibroblasts, and macrophages, can communicate directly, through gap junctions (GJ) or tunneling nanotubes (TNT), or at long distances, via extracellular vesicles (EV) or soluble factors.The direct communication between cardiomyocytes is vital to ensure the anisotropic propagation of the electrical impulse, which allows the heart to beat in a coordinated and synchronized manner, as a functional syncytium. The rapid and efficient propagation of the depolarization wave is mainly conducted by low resistance channels called GJ, formed by six subunits of a family of proteins named Cxs. Dysfunctional GJ intercellular communication, due to increased degradation and/or redistribution of connexin43 (Cx43), the main Cx present in the heart, has been associated with several cardiac disorders, such as myocardial ischemia, hypertrophy, arrhythmia, and heart failure. Besides electrical coupling, a fine-tuned exchange of information, namely proteins and microRNAs, conveyed by EV is important to ensure organ function and homeostasis. Disease-induced deregulation of EV-mediated communication between cardiac cells has been implicated in diverse processes such as inflammation, angiogenesis, and fibrosis. Therefore, a better understanding of the mechanisms whereby proteolysis modulates the cross talk between cardiac cells is of utmost importance to develop new strategies to tackle diseases caused by defects in intercellular communication.


Assuntos
Comunicação Celular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteostase , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Junções Comunicantes/metabolismo , Humanos
19.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(1): 20-25, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32314720

RESUMO

Objective To investigate the role of CXCR2 in the cerebral endothelial activation and migration of neutrophils into the brain in septic encephalopathy (SE) induced by lipopolysaccharide (LPS). Methods C57BL/6J mice and CXCR2-knockout mice were randomly divided into a normal control group, a wildtype mice group with LPS treatment and CXCR2-knockout mice group with LPS treatment. Mouse SE models were induced by intraperitoneal LPS injection. Naphthol AS-D chloroacetate histochemical staining of the brain section was performed to quantitate the neutrophils infiltrating into the cerebral cortex. TNF-α and CXCL1 concentrations in the brain and plasma were determined by ELISA. After the stimulation of LPS (1 µg/mL) and TNF-α (200 ng/mL), the levels of CXCR2 protein in the primary mouse brain microvascular endothelial cells isolated from the cerebral cortex were detected by Western blotting. The levels of F-actin and vascular cell adhesion molecule 1 (VCAM-1) protein in the primary mouse brain microvascular endothelial cells stimulated by CXCL1 (100 ng/mL) were detected by Western blotting. Results After intraperitoneal LPS injection, there was a significant increase in the level of TNF-α in the brain and plasma and there was also an evident increase in the level of CXCL1 in the brain of wild type mice (C57BL/6J mice). And intraperitoneal LPS injection caused increased neutrophil infiltration into the cerebral cortex in the wild type mice (C57BL/6J mice). But CXCR2-knockout mice displayed evidently reduced neutrophil infiltration into the cerebral cortex compared with the wildtype mice. In vitro LPS and TNF-α upregulated the expression of CXCR2 in the primary brain microvascular endothelial cells. CXCL1 increased remarkably the expression of endothelial F-actin and VCAM-1. Conclusion In the SE model, CXCR2 participates in the cerebral endothelial activation and neutrophil migration into the brain.


Assuntos
Encefalopatias/metabolismo , Movimento Celular , Células Endoteliais/citologia , Neutrófilos/citologia , Receptores de Interleucina-8B/metabolismo , Sepse/metabolismo , Actinas/metabolismo , Animais , Encefalopatias/patologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Sepse/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
20.
PLoS One ; 15(4): e0231634, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298350

RESUMO

The WST-1 assay is the most common test to assess the in vitro cytotoxicity of chemicals. Tetrazolium-based assays can, however, be affected by the interference of tested chemicals, including carbon nanotubes or Mg particles. Here, we report a new interference of Mn materials with the WST-1 assay. Endothelial cells exposed to Mn particles (Mn alone or Fe-Mn alloy from 50 to 1600 µg/ml) were severely damaged according to the WST-1 assay, but not the ATP content assay. Subsequent experiments revealed that Mn particles interfere with the reduction of the tetrazolium salt to formazan. Therefore, the WST-1 assay is not suitable to evaluate the in vitro cytotoxicity of Mn-containing materials, and luminescence-based assays such as CellTiter-Glo® appear more appropriate.


Assuntos
Citotoxinas/toxicidade , Células Endoteliais/efeitos dos fármacos , Manganês/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Medições Luminescentes/métodos , Oxirredução , Sais de Tetrazólio/química , Testes de Toxicidade/métodos
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