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The top cause of mortality in patients with nonalcoholic fatty liver disease (NAFLD) is cardiovascular complications. However, mechanisms of NAFLD-associated vasculopathy remain understudied. Here, we show that blood outgrowth endothelial cells (BOECs) from NAFLD subjects exhibit global transcriptional upregulation of chemokines and human leukocyte antigens. In mouse models of diet-induced NAFLD, we confirm heightened endothelial expressions of CXCL12 in the aortas and the liver vasculatures, and increased retention of infiltrated leukocytes within the vessel walls. To elucidate endothelial-immune crosstalk, we performed immunoprofiling by single-cell analysis, uncovering T cell intensification in NAFLD patients. Functionally, treatment with a CXCL12-neutralizing antibody is effective at moderating the enhanced chemotactic effect of NAFLD BOECs in recruiting CD8+ T lymphocytes. Interference with the CXCL12-CXCR4 axis using a CXCR4 antagonist also averts the impact of immune cell transendothelial migration and restores endothelial barrier integrity. Clinically, we detect threefold more circulating damaged endothelial cells in NAFLD patients than in healthy controls. Our work provides insight into the modulation of interactions with effector immune cells to mitigate endothelial injury in NAFLD.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Movimiento Celular , Células Endoteliales/metabolismo , Humanos , Hígado/metabolismo , Linfocitos/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.
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Hialuronoglucosaminidasa , Degeneración Macular , Anciano , Coroides/irrigación sanguínea , Coroides/patología , Células Endoteliales , Angiografía con Fluoresceína , Glicocálix/patología , Humanos , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/uso terapéutico , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/patologíaRESUMEN
RATIONALE: Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association. OBJECTIVE: Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts. METHODS AND RESULTS: We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test1 to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in cis (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets. CONCLUSIONS: Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.
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Epigenoma , Variación Genética , Insuficiencia Cardíaca/genética , Histonas/genética , Acetilación , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Inmunoprecipitación de Cromatina , Bases de Datos Genéticas , Epigénesis Genética , Epigenómica , Femenino , Predisposición Genética a la Enfermedad , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/fisiopatología , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Sitios de Carácter CuantitativoRESUMEN
Age-related macular degeneration (AMD) is a global leading cause of visual impairment in older populations. 'Wet' AMD, the most common subtype of this disease, occurs when pathological angiogenesis infiltrates the subretinal space (choroidal neovascularization), causing hemorrhage and retinal damage. Gold standard anti-vascular endothelial growth factor (VEGF) treatment is an effective therapy, but the long-term prevention of visual decline has not been as successful. This warrants the need to elucidate potential VEGF-independent pathways. We generated blood out-growth endothelial cells (BOECs) from wet AMD and normal control subjects, then induced angiogenic sprouting of BOECs using a fibrin gel bead assay. To deconvolute endothelial heterogeneity, we performed single-cell transcriptomic analysis on the sprouting BOECs, revealing a spectrum of cell states. Our wet AMD BOECs share common pathways with choroidal neovascularization such as extracellular matrix remodeling that promoted proangiogenic phenotype, and our 'activated' BOEC subpopulation demonstrated proinflammatory hallmarks, resembling the tip-like cells in vivo. We uncovered new molecular insights that pathological angiogenesis in wet AMD BOECs could also be driven by interleukin signaling and amino acid metabolism. A web-based visualization of the sprouting BOEC single-cell transcriptome has been created to facilitate further discovery research.
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Neovascularización Coroidal , Degeneración Macular Húmeda , Humanos , Neovascularización Coroidal/tratamiento farmacológico , Transcriptoma , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales/metabolismo , Degeneración Macular Húmeda/tratamiento farmacológico , Factores de Crecimiento Endotelial Vascular , Interleucinas/uso terapéutico , Aminoácidos , Fibrina , Inhibidores de la Angiogénesis/uso terapéuticoRESUMEN
Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.
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Linfocitos T CD8-positivos , Herpesvirus Humano 4 , Humanos , Especificidad del Receptor de Antígeno de Linfocitos T , Antígenos Virales , FenotipoRESUMEN
Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available.
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BACKGROUND AND AIMS: Single nucleotide polymorphism rs6903956 has been identified as one of the genetic risk factors for coronary artery disease (CAD). However, rs6903956 lies in a non-coding locus on chromosome 6p24.1. We aim to interrogate the molecular basis of 6p24.1 containing rs6903956 risk alleles in endothelial disease biology. METHODS AND RESULTS: We generated induced pluripotent stem cells (iPSCs) from CAD patients (AA risk genotype at rs6903956) and non-CAD subjects (GG non-risk genotype at rs6903956). CRISPR-Cas9-based deletions (Δ63-89bp) on 6p24.1, including both rs6903956 and a short tandem repeat variant rs140361069 in linkage disequilibrium, were performed to generate isogenic iPSC-derived endothelial cells. Edited CAD endothelial cells, with removal of 'A' risk alleles, exhibited a global transcriptional downregulation of pathways relating to abnormal vascular physiology and activated endothelial processes. A CXC chemokine ligand on chromosome 10q11.21, CXCL12, was uncovered as a potential effector gene in CAD endothelial cells. Underlying this effect was the preferential inter-chromosomal interaction of 6p24.1 risk locus to a weak promoter of CXCL12, confirmed by chromatin conformation capture assays on our iPSC-derived endothelial cells. Functionally, risk genotypes AA/AG at rs6903956 were associated significantly with elevated levels of circulating damaged endothelial cells in CAD patients. Circulating endothelial cells isolated from patients with risk genotypes AA/AG were also found to have 10 folds higher CXCL12 transcript copies/cell than those with non-risk genotype GG. CONCLUSIONS: Our study reveals the trans-acting impact of 6p24.1 with another CAD locus on 10q11.21 and is associated with intensified endothelial injury.
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Enfermedad de la Arteria Coronaria , Células Endoteliales , Humanos , Enfermedad de la Arteria Coronaria/genética , Alelos , Genotipo , Polimorfismo de Nucleótido SimpleRESUMEN
Numerous reports of vascular events after an initial recovery from COVID-19 form our impetus to investigate the impact of COVID-19 on vascular health of recovered patients. We found elevated levels of circulating endothelial cells (CECs), a biomarker of vascular injury, in COVID-19 convalescents compared to healthy controls. In particular, those with pre-existing conditions (e.g., hypertension, diabetes) had more pronounced endothelial activation hallmarks than non-COVID-19 patients with matched cardiovascular risk. Several proinflammatory and activated T lymphocyte-associated cytokines sustained from acute infection to recovery phase, which correlated positively with CEC measures, implicating cytokine-driven endothelial dysfunction. Notably, we found higher frequency of effector T cells in our COVID-19 convalescents compared to healthy controls. The activation markers detected on CECs mapped to counter receptors found primarily on cytotoxic CD8+ T cells, raising the possibility of cytotoxic effector cells targeting activated endothelial cells. Clinical trials in preventive therapy for post-COVID-19 vascular complications may be needed.
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COVID-19/complicaciones , Enfermedades Cardiovasculares/etiología , Endotelio Vascular/patología , Activación de Linfocitos , Adulto , Anciano , COVID-19/inmunología , COVID-19/patología , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/patología , Citocinas/inmunología , Células Endoteliales/inmunología , Células Endoteliales/patología , Endotelio Vascular/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
COVID-19 management guidelines have largely attributed critically ill patients who develop acute respiratory distress syndrome, to a systemic overproduction of pro-inflammatory cytokines. Cardiovascular dysfunction may also represent a primary phenomenon, with increasing data suggesting that severe COVID-19 reflects a confluence of vascular dysfunction, thrombosis and dysregulated inflammation. Here, we first consolidate the information on localized microvascular inflammation and disordered cytokine release, triggering vessel permeability and prothrombotic conditions that play a central role in perpetuating the pathogenic COVID-19 cascade. Secondly, we seek to clarify the gateways which SARS-CoV-2, the causative COVID-19 virus, uses to enter host vascular cells. Post-mortem examinations of patients' tissues have confirmed direct viral endothelial infection within several organs. While there have been advances in single-cell RNA sequencing, endothelial cells across various vascular beds express low or undetectable levels of those touted SARS-CoV-2 entry factors. Emerging studies postulate alternative pathways and the apicobasal distribution of host cell surface factors could influence endothelial SARS-CoV-2 entry and replication. Finally, we provide experimental considerations such as endothelial polarity, cellular heterogeneity in organoids and shear stress dynamics in designing cellular models to facilitate research on viral-induced endothelial dysfunctions. Understanding the vascular underpinning of COVID-19 pathogenesis is crucial to managing outcomes and mortality.
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Betacoronavirus/metabolismo , Infecciones por Coronavirus/patología , Células Endoteliales/patología , Inflamación/patología , Neumonía Viral/patología , Trombosis/patología , Enzima Convertidora de Angiotensina 2 , COVID-19 , Permeabilidad Capilar/fisiología , Comorbilidad , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/mortalidad , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/patología , Citocinas/sangre , Células Endoteliales/virología , Humanos , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/inmunología , Neumonía Viral/mortalidad , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/patología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Internalización del VirusRESUMEN
We observed a unique bioelectric signal of human embryonic stem cells using direct current-voltage measurements facilitated by few-layered 2D-MoS2 sheets. A 1.828 mA cell signal was achieved (2 orders of magnitude higher than previous electrical-based detection methods) as well as multiple cell reading cycles demonstrating I â¼ 1.9 mA. Native stem cell proliferation, viability, and pluripotency were preserved. Molecular dynamics simulations elucidated the origin of the 2D-MoS2 sheet-assisted increase in current flow. This paves the way for the development of a broadly applicable, fast, and damage-free stem cell detection method capable of identifying pluripotency with virtually any complementary-metal-oxide-semiconductor circuits.
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Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease (HFMD) associated with severe neurological disease. EV71's pathogenesis remains poorly understood and the lack of approved antiviral has led to its emergence as a clinically important neurotropic virus. The goals of this study were to: (i) identify novel anti-EV71 compounds that may serve as lead molecules for therapeutics; and (ii) investigate their targets in downstream studies. We screened a 502-compound library of highly purified natural products for anti-EV71 activities in a cell-based immunofluorescence assay that were then confirmed in viral plaque reduction assays. Along with known antivirals, novel inhibitors of EV71 were also identified. We selected camptothecin for downstream studies and found that it is a limited spectrum enterovirus inhibitor that inhibits coxsackievirus A16 but not ECHOvirus 7. Camptothecin, a DNA topoisomerase 1 (TOP1) inhibitor, inhibits both viral RNA replication and translation based on luciferase replicon studies. Depletion of TOP1 using siRNA was then able to rescue EV71 infection from camptothecin inhibition. Interestingly, EV71 viral RNA replication and translation were also in TOP1 depleted cells. We found that nuclear TOP1 was relocalized to cytoplasmic replication vesicles during EV71 infection and localized with viral 3CD using confocal microscopy and proximity-ligation assays. Our findings reveal camptothecin to be a limited spectrum antiviral against enteroviruses that functions in a TOP1-dependent but cytotoxicity-independent manner. TOP1 is in turn needed for maximal EV71 viral RNA replication and viral protein synthesis.
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Antivirales/aislamiento & purificación , Antivirales/farmacología , Productos Biológicos/farmacología , Camptotecina/antagonistas & inhibidores , ADN-Topoisomerasas de Tipo I/efectos de los fármacos , Enterovirus Humano A/efectos de los fármacos , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Línea Celular , Supervivencia Celular , Evaluación Preclínica de Medicamentos , Enterovirus/efectos de los fármacos , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/tratamiento farmacológico , Técnicas de Silenciamiento del Gen , Cabras , Luciferasas , Ratones , Microscopía Confocal , ARN Interferente Pequeño/genética , Conejos , Replicón/efectos de los fármacos , Proteínas Virales/efectos de los fármacos , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.
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Enterovirus Humano A/crecimiento & desarrollo , Genoma Humano/genética , Interferencia de ARN , Replicación Viral , Línea Celular Tumoral , Resistencia a la Enfermedad/genética , Enterovirus Humano A/fisiología , Regulación Neoplásica de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Rabdomiosarcoma/virologíaRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogenic virus in the swine production. Current vaccines against PRRSV do not induce sterile immunity and the virus evolves at a rapid rate with frequent appearances of new strains. In this study, we screened a library of 502 highly purified natural product compounds to identify specific inhibitors of PRRSV replication cycle. Our observations showed that many of the inhibitory compounds identified have activity on the cellular ion transport mechanisms. We identified for the first time, four compounds which inhibit the PRRSV replication cycle at micro molar concentration or less, namely, 12-deoxyphorbol 13-phenylacetate 20-acetate, ouabain, bufalin and valinomycin. Further, we have identified 15 other compounds which can inhibit the PRRSV replication at the concentration of 8µM. This study provides a basis for further development of pharmacological agents to inhibit PRRSV replication.
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Antivirales/farmacología , Productos Biológicos/farmacología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Antivirales/aislamiento & purificación , Productos Biológicos/aislamiento & purificación , Línea Celular , Haplorrinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiologíaRESUMEN
BACKGROUND: Dengue virus (DENV) is a mosquito-borne virus that causes a spectrum of human diseases ranging from mild dengue fever to dengue haemorrhagic fever and dengue shock syndrome in severe cases. Currently, there is no effective antiviral therapy or vaccine against DENV infection. METHODS: In order to identify potential antiviral agents against DENV, we performed high-throughput cell-based screening on a highly purified natural products library. Among the screening hits, selected compounds which displayed 50-75% inhibition against DENV2 were validated using secondary assays. Time-of-addition studies, dose-dependent assays, real time quantitative reverse transcriptase (RT)-PCR, Western blot and ultrastructural imaging were conducted in an attempt to elucidate the potential antiviral mechanisms of narasin. RESULTS: In this study, an ionophore, narasin was selected for detailed analysis due to its strong inhibitory profile against DENV infection with minimal cytotoxicity (50% cytotoxic concentration >1,000 µM). A dose-dependent study revealed narasin to have an 50% inhibitory concentration of less than 1 µM against all four serotypes of DENV. Time-of-addition studies of narasin-treated, DENV2-infected Huh-7 cells suggested narasin to be involved in inhibiting the post-entry stages of viral replication during DENV infection. Proteomic and ultrastructural analyses revealed the antiviral mechanism of narasin as likely to be associated with the disruption of viral protein synthesis. In addition, quantitative RT-PCR studies showed no differences in viral RNA levels between narasin-treated and control DENV2-infected cells. CONCLUSIONS: Narasin was identified and characterized as a novel agent that inhibits DENV replication in vitro through non-cytotoxic mechanisms, thus indicating its potential to be further developed as a therapeutic anti-DENV agent.