RESUMEN
Loss of endothelial integrity and vascular leakage are central features of sepsis pathogenesis; however, no effective therapeutic mechanisms for preserving endothelial integrity are available. Here we show that, compared to dermal microvessels, brain microvessels resist infection by Neisseria meningitidis, a bacterial pathogen that causes sepsis and meningitis. By comparing the transcriptional responses to infection in dermal and brain endothelial cells, we identified angiopoietin-like 4 as a key factor produced by the brain endothelium that preserves blood-brain barrier integrity during bacterial sepsis. Conversely, angiopoietin-like 4 is produced at lower levels in the peripheral endothelium. Treatment with recombinant angiopoietin-like 4 reduced vascular leakage, organ failure and death in mouse models of lethal sepsis and N. meningitidis infection. Protection was conferred by a previously uncharacterized domain of angiopoietin-like 4, through binding to the heparan proteoglycan, syndecan-4. These findings reveal a potential strategy to prevent endothelial dysfunction and improve outcomes in patients with sepsis.
Asunto(s)
Modelos Animales de Enfermedad , Células Endoteliales , Sepsis , Animales , Sepsis/microbiología , Ratones , Humanos , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Proteína 4 Similar a la Angiopoyetina/metabolismo , Proteína 4 Similar a la Angiopoyetina/genética , Barrera Hematoencefálica/metabolismo , Infecciones Meningocócicas/microbiología , Encéfalo/metabolismo , Encéfalo/microbiología , Encéfalo/patología , Ratones Endogámicos C57BL , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiologíaRESUMEN
Staphylococcus aureus α-hemolysin (Hla) is a pore-forming toxin critical for the pathogenesis of skin and soft tissue infections, which causes the pathognomonic lesion of cutaneous necrosis (dermonecrosis) in mouse models. To determine the mechanism by which dermonecrosis develops during S. aureus skin infection, mice were given control serum, Hla-neutralizing antiserum, or an inhibitor of Hla receptor [A-disintegrin and metalloprotease 10 (ADAM10) inhibitor] followed by subcutaneous infection by S. aureus, and the lesions were evaluated using immunohistochemistry and immunofluorescence. Hla induced apoptosis in the vascular endothelium at 6 hours post-infection (hpi), followed by apoptosis in keratinocytes at 24 hpi. The loss of vascular endothelial (VE)-cadherin expression preceded the loss of epithelial-cadherin expression. Hla also induced hypoxia in the keratinocytes at 24 hpi following vascular injury. Treatment with Hla-neutralizing antibody or ADAM10 inhibitor attenuated early cleavage of VE-cadherin, cutaneous hypoxia, and dermonecrosis. These findings suggest that Hla-mediated vascular injury with cutaneous hypoxia underlies the pathogenesis of S. aureus-induced dermonecrosis.
Asunto(s)
Proteína ADAM10 , Toxinas Bacterianas , Cadherinas , Proteínas Hemolisinas , Queratinocitos , Necrosis , Staphylococcus aureus , Animales , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidad , Ratones , Toxinas Bacterianas/toxicidad , Toxinas Bacterianas/metabolismo , Staphylococcus aureus/patogenicidad , Queratinocitos/microbiología , Queratinocitos/metabolismo , Proteína ADAM10/metabolismo , Cadherinas/metabolismo , Apoptosis , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antígenos CD/metabolismo , Proteínas de la Membrana/metabolismo , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/patología , Infecciones Cutáneas Estafilocócicas/inmunología , Piel/patología , Piel/microbiología , Femenino , Endotelio Vascular/patología , Endotelio Vascular/microbiología , Endotelio Vascular/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/patología , Modelos Animales de EnfermedadRESUMEN
The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection.
Asunto(s)
Arteriolas/microbiología , Dermis/irrigación sanguínea , Neisseria meningitidis/crecimiento & desarrollo , Neutrófilos/microbiología , Adulto , Animales , Arteriolas/patología , Adhesión Bacteriana , Capilares/microbiología , Capilares/patología , Moléculas de Adhesión Celular/metabolismo , Recuento de Colonia Microbiana , Selectina E/metabolismo , Endotelio Vascular/microbiología , Endotelio Vascular/patología , Femenino , Fimbrias Bacterianas/metabolismo , Xenoinjertos , Humanos , Inflamación/patología , Masculino , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/patología , Ratones SCID , Persona de Mediana Edad , Infiltración Neutrófila , Fagocitosis , Factores de Tiempo , Regulación hacia Arriba , Adulto JovenRESUMEN
METHODS: Sepsis was induced by cotton smoke inhalation followed by intranasal administration of Pseudomonas aeruginosa in female (> 6 months) Balb/c and syndecan-1 knockout mice. Survival of mice, lung capillary endothelial glycocalyx integrity, lung water content, and vascular hyper-permeability were determined with or without HMW-SH treatment in these mice. Effects of HMW-SH on endothelial permeability and neutrophil migration were tested in in vitro setting. RESULTS: In septic wildtype mice, we found a severely damaged pulmonary microvascular endothelial glycocalyx and elevated levels of shed syndecan-1 in the circulation. These changes were associated with significantly increased pulmonary vascular permeability. In septic syndecan-1 knockout mice, extravascular lung water content was higher, and early death was observed. The administration of HMW-SH significantly reduced mortality and lung water content in septic syndecan-1 knockout mice, but not in septic wildtype mice. In in vitro setting, HMW-SH inhibited neutrophil migration and reduced cultured endothelial cell permeability increases. However, these effects were reversed by the addition of recombinant syndecan-1 ectodomain. CONCLUSIONS: HMW-SH reduced lung tissue damage and mortality in the absence of syndecan-1 protein, possibly by reducing vascular hyper-permeability and neutrophil migration. Our results further suggest that increased shed syndecan-1 protein levels are linked with the inefficiency of HMW-SH in septic wildtype mice.
Asunto(s)
Antiinflamatorios/farmacología , Ácido Hialurónico/farmacología , Neutrófilos/efectos de los fármacos , Infecciones por Pseudomonas/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Lesión por Inhalación de Humo/tratamiento farmacológico , Sindecano-1/genética , Animales , Permeabilidad Capilar/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/microbiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Endotelio Vascular/microbiología , Femenino , Eliminación de Gen , Glicocálix/inmunología , Glicocálix/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/microbiología , Cultivo Primario de Células , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/mortalidad , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Sepsis/inmunología , Sepsis/microbiología , Sepsis/mortalidad , Lesión por Inhalación de Humo/inmunología , Lesión por Inhalación de Humo/microbiología , Lesión por Inhalación de Humo/mortalidad , Análisis de Supervivencia , Sindecano-1/deficiencia , Sindecano-1/inmunología , Agua/metabolismoRESUMEN
High blood pressure (BP) presents a significant public health challenge. Recent findings suggest that altered microbiota can exert a hypertensive effect on the host. One of the possible mechanisms involved is the chronic translocation of its components, mainly lipopolysaccharides (LPS) into systemic circulation leading to metabolic endotoxemia. In animal models, LPS has been commonly used to induce endothelial dysfunction and vascular inflammation. In human studies, plasma LPS concentration has been positively correlated with hypertension, however, the mechanistic link has not been fully elucidated. It is hypothesised here that the LPS-induced direct alterations to the vascular endothelium and resulting hypertension are possible targets for probiotic intervention. The methodology of this review involved a systematic search of the literature with critical appraisal of papers. Three tranches of search were performed: 1) existing review papers; 2) primary mechanistic animal, in vitro and human studies; and 3) primary intervention studies. A total of 70 peer-reviewed papers were included across the three tranches and critically appraised using SIGN50 for human studies and the ARRIVE guidelines for animal studies. The extracted information was coded into key themes and summarized in a narrative analysis. Results highlight the role of LPS in the activation of endothelial toll-like receptor 4 (TLR4) initiating a cascade of interrelated signalling pathways including: 1) Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/ Reactive oxygen species (ROS)/ Endothelial nitric oxide synthase (eNOS) pathway leading to endothelial dysfunction; and 2) Mitogen-Activated Protein Kinase (MAPK) and Nuclear factor kappa B (NF-κB) pathways leading to vascular inflammation. Findings from animal intervention studies suggest an improvement in vasorelaxation, vascular inflammation and hypertension following probiotic supplementation, which was mediated by downregulation of LPS-induced pathways. Randomised controlled trials (RCTs) and systematic reviews provided some evidence for the anti-inflammatory effect of probiotics with statistically significant antihypertensive effect in clinical samples and may offer a viable intervention for the management of hypertension.
Asunto(s)
Endotelio Vascular/metabolismo , Microbioma Gastrointestinal , Hipertensión/etiología , Hipertensión/terapia , Probióticos/uso terapéutico , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Animales , Endotelio Vascular/microbiología , Humanos , Hipertensión/microbiología , Inflamación/metabolismo , Inflamación/microbiología , Lipopolisacáridos/toxicidadRESUMEN
Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3-MT association. We discovered that-in addition to its known partners gp130 and JAK2-SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3-CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3-JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.
Asunto(s)
Inflamación/genética , Lesión Pulmonar/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Proteína 3 Supresora de la Señalización de Citocinas/genética , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/microbiología , Lesión Pulmonar Aguda/patología , Animales , Citoesqueleto/genética , Células Endoteliales , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Endotelio Vascular/patología , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Uniones Intercelulares/genética , Interleucina-6/genética , Lesión Pulmonar/metabolismo , Lesión Pulmonar/microbiología , Lesión Pulmonar/patología , Ratones , Ratones Noqueados , Permeabilidad , Staphylococcus aureus/patogenicidadRESUMEN
Aim: To investigate the mRNAs and noncoding RNAs (ncRNAs) expression in astrocytes upon meningitic-Escherichia coli infection. Materials & methods: The transcription of mRNAs and ncRNAs were fully investigated and profiled by whole transcriptome sequencing and bioinformatic approaches. Whole transcriptome differences between the infected astrocytes and brain microvascular endothelial cells were further compared and characterized. Results: A total of 2045 mRNAs, 74 long noncoding RNAs, 27 miRNAs and 418 circular RNAs were differentially transcribed in astrocytes upon infection. Competing endogenous RNAs regulatory networks were constructed and preliminary validated. Transcriptomic differences between astrocyte and brain microvascular endothelial cells revealed the cell-specific responses against the infection. Conclusion: Our study comprehensively characterized the ncRNAs and mRNAs profiles in astrocytes upon meningitic-E. coli infection, which will facilitate future functional studies.
Asunto(s)
Astrocitos/metabolismo , Astrocitos/microbiología , Escherichia coli , Transcriptoma , Línea Celular , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , MicroARNs/metabolismo , ARN Circular/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Glässer's disease, caused by Haemophilus parasuis (H. parasuis), is associated with vascular damage and vascular inflammation in pigs. Therefore, early assessment and treatment are essential to control the inflammatory disorder. MicroRNAs have been shown to be involved in the vascular pathology. Baicalin has important pharmacological functions, including anti-inflammatory, antimicrobial and antioxidant effects. In this study, we investigated the changes of microRNAs in porcine aortic vascular endothelial cells (PAVECs) induced by H. parasuis and the effect of baicalin in this model by utilizing high-throughput sequencing. The results showed that 155 novel microRNAs and 76 differentially expressed microRNAs were identified in all samples. Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the target genes of the differentially expressed microRNAs demonstrated that regulation of actin cytoskeleton, focal adhesion, ECM-receptor interaction, bacterial invasion of epithelial cells, and adherens junction were the most interesting pathways after PAVECs were infected with H. parasuis. In addition, when the PAVECs were pretreated with baicalin, mismatch repair, peroxisome, oxidative phosphorylation, DNA replication, and ABC transporters were the most predominant signaling pathways. STRING analysis showed that most of the target genes of the differentially expressed microRNAs were associated with each other. The expression levels of the differentially expressed microRNAs were negatively co-regulated with their target genes' mRNA following pretreatment with baicalin in the H. parasuis-induced PAVECs using co-expression networks analysis. This is the first report that microRNAs might have key roles in inflammatory damage of vascular tissue during H. parasuis infection. Baicalin regulated the microRNAs changes in the PAVECs following H. parasuis infection, which may represent useful novel targets to prevent or treat H. parasuis infection.
Asunto(s)
Aorta/metabolismo , Endotelio Vascular/metabolismo , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por Haemophilus/microbiología , MicroARNs/genética , Transcriptoma/efectos de los fármacos , Animales , Animales Recién Nacidos , Antiinflamatorios no Esteroideos/farmacología , Aorta/citología , Aorta/microbiología , Endotelio Vascular/citología , Endotelio Vascular/microbiología , Haemophilus parasuis/aislamiento & purificación , PorcinosRESUMEN
Bacterial neonatal meningitis results in high mortality and morbidity rates for those affected. Although improvements in diagnosis and treatment have led to a decline in mortality rates, morbidity rates have remained relatively unchanged. Bacterial resistance to antibiotics in this clinical setting further underlines the need for developing other technologies, such as phage therapy. We exploited an in vitro phage therapy model for studying bacterial neonatal meningitis based on Escherichia coli (E. coli) EV36, bacteriophage (phage) K1F and human cerebral microvascular endothelial cells (hCMECs). We show that phage K1F is phagocytosed and degraded by constitutive- and PAMP-dependent LC3-assisted phagocytosis and does not induce expression of inflammatory cytokines TNFα, IL-6, IL-8 or IFNß. Additionally, we observed that phage K1F temporarily decreases the barrier resistance of hCMEC cultures, a property that influences the barrier permeability, which could facilitate the transition of immune cells across the endothelial vessel in vivo. Collectively, we demonstrate that phage K1F can infect intracellular E. coli EV36 within hCMECs without themselves eliciting an inflammatory or defensive response. This study illustrates the potential of phage therapy targeting infections such as bacterial neonatal meningitis and is an important step for the continued development of phage therapy targeting antibiotic-resistant bacterial infections generally.
Asunto(s)
Bacteriófagos/fisiología , Encéfalo/citología , Endotelio Vascular/citología , Escherichia coli/virología , Encéfalo/metabolismo , Encéfalo/microbiología , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/microbiología , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/terapia , Adhesiones Focales/metabolismo , Humanos , Meningitis Bacterianas/metabolismo , Meningitis Bacterianas/terapia , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Terapia de Fagos , FagocitosisRESUMEN
Background Epidemiological studies have suggested an association between Helicobacter pylori (H pylori) infection and atherosclerosis through undefined mechanisms. Endothelial dysfunction is critical to the development of atherosclerosis and related cardiovascular diseases. The present study was designed to test the hypothesis that H pylori infection impaires endothelial function through exosome-mediated mechanisms. Methods and Results Young male and female patients (18-35 years old) with and without H pylori infection were recruited to minimize the chance of potential risk factors for endothelial dysfunction for the study. Endothelium-dependent flow-mediated vasodilatation of the brachial artery was evaluated in the patients and control subjects. Mouse infection models with CagA+H pylori from a gastric ulcer patient were created to determine if H pylori infection-induced endothelial dysfunction could be reproduced in animal models. H pylori infection significantly decreased endothelium-dependent flow-mediated vasodilatation in young patients and significantly attenuated acetylcholine-induced endothelium-dependent aortic relaxation without change in nitroglycerin-induced endothelium-independent vascular relaxation in mice. H pylori eradication significantly improved endothelium-dependent vasodilation in both patients and mice with H pylori infection. Exosomes from conditioned media of human gastric epithelial cells cultured with CagA+H pylori or serum exosomes from patients and mice with H pylori infection significantly decreased endothelial functions with decreased migration, tube formation, and proliferation in vitro. Inhibition of exosome secretion with GW4869 effectively preserved endothelial function in mice with H pylori infection. Conclusions H pylori infection impaired endothelial function in patients and mice through exosome-medicated mechanisms. The findings indicated that H pylori infection might be a novel risk factor for cardiovascular diseases.
Asunto(s)
Arteria Braquial/microbiología , Células Endoteliales/microbiología , Endotelio Vascular/microbiología , Exosomas/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Vasodilatación , Adolescente , Adulto , Compuestos de Anilina/farmacología , Animales , Antibacterianos/uso terapéutico , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Compuestos de Bencilideno/farmacología , Arteria Braquial/metabolismo , Arteria Braquial/fisiopatología , Estudios de Casos y Controles , Línea Celular , Movimiento Celular , Proliferación Celular , China , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Femenino , Fármacos Gastrointestinales/uso terapéutico , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/fisiopatología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/metabolismo , Interacciones Huésped-Patógeno , Humanos , Masculino , Ratones Endogámicos C57BL , Missouri , Neovascularización Fisiológica , Vasodilatación/efectos de los fármacos , Adulto JovenRESUMEN
Staphylococcus aureus is a leading cause of endovascular infections. This bacterial pathogen uses a diverse array of surface adhesins to clump in blood and adhere to vessel walls, leading to endothelial damage, development of intravascular vegetations and secondary infectious foci, and overall disease progression. In this work, we describe a novel strategy used by S. aureus to control adhesion and clumping through activity of the ArlRS two-component regulatory system, and its downstream effector MgrA. Utilizing a combination of in vitro cellular assays, and single-cell atomic force microscopy, we demonstrated that inactivation of this ArlRS-MgrA cascade inhibits S. aureus adhesion to a vast array of relevant host molecules (fibrinogen, fibronectin, von Willebrand factor, collagen), its clumping with fibrinogen, and its attachment to human endothelial cells and vascular structures. This impact on S. aureus adhesion was apparent in low shear environments, and in physiological levels of shear stress, as well as in vivo in mouse models. These effects were likely mediated by the de-repression of giant surface proteins Ebh, SraP, and SasG, caused by inactivation of the ArlRS-MgrA cascade. In our in vitro assays, these giant proteins collectively shielded the function of other surface adhesins and impaired their binding to cognate ligands. Finally, we demonstrated that the ArlRS-MgrA regulatory cascade is a druggable target through the identification of a small-molecule inhibitor of ArlRS signaling. Our findings suggest a novel approach for the pharmacological treatment and prevention of S. aureus endovascular infections through targeting the ArlRS-MgrA regulatory system.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Endotelio Vascular/microbiología , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/fisiología , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Fibrinógeno/genética , Fibrinógeno/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/patologíaRESUMEN
Mycobacterium tuberculosis (MTB) is a pathogen that infects and kills millions yearly. The mycobacterium's cell wall glycolipid trehalose 6,6'-dimycolate (TDM) has been used historically to model MTB induced inflammation and granuloma formation. Alterations to the model can significantly influence the induced pathology. One such method incorporates intraperitoneal pre-exposure, after which the intravenous injection of TDM generates pathological damage effectively mimicking the hypercoagulation, thrombus formation, and tissue remodeling apparent in lungs of infected individuals. The purpose of these experiments is to examine the histological inflammation involved in the TDM mouse model that induces development of the hemorrhagic response. TDM induced lungs of C57BL/6 mice to undergo granulomatous inflammation. Further histological examination of the peak response demonstrated tissue remodeling consistent with hypercoagulation. The observed vascular occlusion indicates that obstruction likely occurs due to subendothelial localized activity leading to restriction of blood vessel lumens. Trichrome staining revealed that associated damage in the hypercoagulation model is consistent with intra endothelial cell accumulation of innate cells, bordered by collagen deposition in the underlying parenchyma. Overall, the hypercoagulation model represents a comparative pathological instrument for understanding mechanisms underlying development of hemorrhage and vascular occlusion seen during MTB infection.
Asunto(s)
Factores Cordón/metabolismo , Endotelio Vascular/patología , Granuloma del Sistema Respiratorio/patología , Pulmón/irrigación sanguínea , Mycobacterium tuberculosis/metabolismo , Neumonía/patología , Tuberculosis Pulmonar/patología , Animales , Coagulación Sanguínea , Modelos Animales de Enfermedad , Endotelio Vascular/microbiología , Femenino , Granuloma del Sistema Respiratorio/sangre , Granuloma del Sistema Respiratorio/inducido químicamente , Granuloma del Sistema Respiratorio/microbiología , Pulmón/microbiología , Ratones Endogámicos C57BL , Neumonía/sangre , Neumonía/inducido químicamente , Neumonía/microbiología , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/inducido químicamente , Tuberculosis Pulmonar/microbiología , Remodelación VascularRESUMEN
Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA's Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ's capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB.
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Proteínas Bacterianas/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/patología , Endotelio Vascular/patología , Proteínas de Microfilamentos/metabolismo , Streptococcus/fisiología , Virulencia , Animales , Proteínas Bacterianas/genética , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/microbiología , Encéfalo/metabolismo , Encéfalo/microbiología , Permeabilidad de la Membrana Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
Bacterial meningitis is a serious, life-threatening infection of the central nervous system (CNS). To cause meningitis, bacteria must interact with and penetrate the meningeal blood-cerebrospinal fluid barrier (mB/CSFB), which comprises highly specialized brain endothelial cells. Neisseria meningitidis (meningococcus) is a leading cause of bacterial meningitis, and examination meningococcus' interaction with the BBB is critical for understanding disease progression. To examine specific interactions, in vitro mB/CSFB models have been developed and employed and are of great importance because in vivo models have been difficult to produce considering Neisseria meningitidis is exclusively a human pathogen. Most in vitro blood-brain barrier and mB/CSF models use primary and immortalized brain endothelial cells, and these models have been used to examine bacterial-mB/CSFB interactions by a variety of pathogens. This chapter describes the use of past and current in vitro brain endothelial cells to model Neisseria meningitidis interaction with the mB/CSFB, and inform on the standard operating procedure for their use.
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Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Endotelio Vascular/metabolismo , Interacciones Huésped-Patógeno , Infecciones Meningocócicas/metabolismo , Neisseria meningitidis/fisiología , Factores de Virulencia/metabolismo , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/microbiología , Encéfalo/citología , Encéfalo/microbiología , Endotelio Vascular/citología , Endotelio Vascular/microbiología , Humanos , Infecciones Meningocócicas/microbiología , Unión ProteicaRESUMEN
A wide variety of pathogens reach the circulatory system during viral, parasitic, fungal, and bacterial infections, causing clinically diverse pathologies. Such systemic infections are usually severe and frequently life-threatening despite intensive care, in particular during the age of antibiotic resistance. Because of its position at the interface between the blood and the rest of the organism, the endothelium plays a central role during these infections. Using several examples of systemic infections, we explore the diversity of interactions between pathogens and the endothelium. These examples reveal that bacterial pathogens target specific vascular beds and affect most aspects of endothelial cell biology, ranging from cellular junction stability to endothelial cell proliferation and inflammation.
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Bacterias/patogenicidad , Infecciones Bacterianas/microbiología , Endotelio/microbiología , Animales , Infecciones Bacterianas/sangre , Farmacorresistencia Bacteriana , Endotelio Vascular/microbiología , Interacciones Huésped-Patógeno , HumanosRESUMEN
Candidatus (Ca.) Neoehrlichia mikurensis is the cause of neoehrlichiosis, an emerging tick-borne infectious disease characterized by fever and vascular events. The bacterium belongs to the Anaplasmataceae, a family of obligate intracellular pathogens, but has not previously been cultivated, and it is uncertain which cell types it infects. The goals of this study were to cultivate Ca. N. mikurensis in cell lines and to identify possible target cells for human infection. Blood components derived from infected patients were inoculated into cell lines of both tick and human origin. Bacterial growth in the cell cultures was monitored by real-time PCR and imaging flow cytometry. Ca. N. mikurensis was successfully propagated from the blood of immunocompromised neoehrlichiosis patients in two Ixodes spp. tick cell lines following incubation periods of 7-20 weeks. Human primary endothelial cells derived from skin microvasculature as well as pulmonary artery were also susceptible to infection with tick cell-derived bacteria. Finally, Ca. N. mikurensis was visualized within circulating endothelial cells of two neoehrlichiosis patients. To conclude, we report the first successful isolation and propagation of Ca. N. mikurensis from clinical isolates and identify human vascular endothelial cells as a target of infection.
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Infecciones por Anaplasmataceae/microbiología , Anaplasmataceae/crecimiento & desarrollo , Anaplasmataceae/aislamiento & purificación , Células Endoteliales/microbiología , Endotelio Vascular/microbiología , Tropismo Viral , Animales , Técnicas de Cultivo de Célula , Citometría de Flujo , Humanos , Ixodes , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Interactions between the endothelium and infected erythrocytes play a major role in the pathogenesis of falciparum malaria, with microvascular dysfunction and parasite sequestration associated with worsening outcomes. The glycocalyx is a carbohydrate-rich layer that lines the endothelium, with multiple roles in vascular homeostasis. The role of the glycocalyx in falciparum malaria and the association with disease severity has not been investigated. METHODS: We prospectively enrolled Indonesian inpatients (aged ≥18 years) with severe (SM) or moderately severe (MSM) falciparum malaria, as defined by World Health Organization criteria, and healthy controls (HCs). On enrollment, blood and urine samples were collected concurrently with measurements of vascular nitric oxide (NO) bioavailability. Urine was assayed for glycocalyx breakdown products (glycosaminoglycans) using a dimethylmethylene blue (GAG-DMMB) and liquid chromatography-tandem mass spectrometry (GAG-MS) assay. RESULTS: A total of 129 patients (SM = 43, MSM = 57, HC=29) were recruited. GAG-DMMB and GAG-MS (g/mol creatinine) were increased in SM (mean, 95% confidence interval: 3.98, 2.44-5.53 and 6.82, 5.19-8.44) compared to MSM patients (1.78, 1.27-2.29 and 4.87, 4.27-5.46) and HCs (0.22, 0.06-0.37 and 1.24, 0.89-1.59; P < 0.001). In SM patients, GAG-DMMB and GAG-MS were increased in those with a fatal outcome (n = 3; median, interquartile range: 6.72, 3.80-27.87 and 12.15, 7.88-17.20) compared to survivors (n = 39; 3.10, 0.46-4.5 and 4.64, 2.02-15.20; P = 0.03). Glycocalyx degradation was significantly associated with parasite biomass in both MSM (r = 0.48, GAG-DMMB and r = 0.43, GAG-MS; P < 0.001) and SM patients (r = 0.47, P = 0.002 and r = 0.33, P = 0.04) and inversely associated with endothelial NO bioavailability. CONCLUSIONS: Increased endothelial glycocalyx breakdown is associated with severe disease and a fatal outcome in adults with falciparum malaria.
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Endotelio Vascular/metabolismo , Glicocálix/metabolismo , Interacciones Huésped-Parásitos , Malaria Falciparum/mortalidad , Malaria Falciparum/fisiopatología , Adolescente , Adulto , Endotelio Vascular/microbiología , Eritrocitos/metabolismo , Eritrocitos/parasitología , Femenino , Glicosaminoglicanos/orina , Humanos , Indonesia , Masculino , Persona de Mediana Edad , Óxido Nítrico/sangre , Plasmodium falciparum , Estudios Prospectivos , Adulto JovenRESUMEN
Sulforaphane (SFN) has been shown to protect the brain vascular system and effectively reduce ischemic injuries and cognitive deficits. Given the robust cerebrovascular protection afforded by SFN, the objective of this study was to profile these effects in vitro using primary mouse brain microvascular endothelial cells and focusing on cellular redox, metabolism and detoxification functions. We used a mouse MitoChip array developed and validated at the FDA National Center for Toxicological Research (NCTR) to profile a host of genes encoded by nuclear and mt-DNA following SFN treatment (0-5 µM). Corresponding protein expression levels were assessed (ad hoc) by qRT-PCR, immunoblots and immunocytochemistry (ICC). Gene ontology clustering revealed that SFN treatment (24 h) significantly up-regulated ~50 key genes (>1.5 fold, adjusted p < 0.0001) and repressed 20 genes (<0.7 fold, adjusted p < 0.0001) belonging to oxidative stress, phase 1 & 2 drug metabolism enzymes (glutathione system), iron transporters, glycolysis, oxidative phosphorylation (OXPHOS), amino acid metabolism, lipid metabolism and mitochondrial biogenesis. Our results show that SFN stimulated the production of ATP by promoting the expression and activity of glucose transporter-1, and glycolysis. In addition, SFN upregulated anti-oxidative stress responses, redox signaling and phase 2 drug metabolism/detoxification functions, thus elucidating further the previously observed neurovascular protective effects of this compound.
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Encéfalo/metabolismo , Endotelio Vascular/microbiología , Genómica/métodos , Isotiocianatos/farmacología , Proteómica/métodos , Adenosina Trifosfato/metabolismo , Animales , Antioxidantes/metabolismo , Western Blotting , Encéfalo/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Ratones , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , SulfóxidosRESUMEN
This review aims to elucidate the different mechanisms of blood brain barrier (BBB) disruption that may occur due to invasion by different types of bacteria, as well as to show the bacteria-host interactions that assist the bacterial pathogen in invading the brain. For example, platelet-activating factor receptor (PAFR) is responsible for brain invasion during the adhesion of pneumococci to brain endothelial cells, which might lead to brain invasion. Additionally, the major adhesin of the pneumococcal pilus-1, RrgA is able to bind the BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), thus leading to invasion of the brain. Moreover, Streptococcus pneumoniae choline binding protein A (CbpA) targets the common carboxy-terminal domain of the laminin receptor (LR) establishing initial contact with brain endothelium that might result in BBB invasion. Furthermore, BBB disruption may occur by S. pneumoniae penetration through increasing in pro-inflammatory markers and endothelial permeability. In contrast, adhesion, invasion, and translocation through or between endothelial cells can be done by S. pneumoniae without any disruption to the vascular endothelium, upon BBB penetration. Internalins (InlA and InlB) of Listeria monocytogenes interact with its cellular receptors E-cadherin and mesenchymal-epithelial transition (MET) to facilitate invading the brain. L. monocytogenes species activate NF-κB in endothelial cells, encouraging the expression of P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1), and Vascular cell adhesion protein 1 (VCAM-1), as well as IL-6 and IL-8 and monocyte chemoattractant protein-1 (MCP-1), all these markers assist in BBB disruption. Bacillus anthracis species interrupt both adherens junctions (AJs) and tight junctions (TJs), leading to BBB disruption. Brain microvascular endothelial cells (BMECs) permeability and BBB disruption are induced via interendothelial junction proteins reduction as well as up-regulation of IL-1α, IL-1ß, IL-6, TNF-α, MCP-1, macrophage inflammatory proteins-1 alpha (MIP1α) markers in Staphylococcus aureus species. Streptococcus agalactiae or Group B Streptococcus toxins (GBS) enhance IL-8 and ICAM-1 as well as nitric oxide (NO) production from endothelial cells via the expression of inducible nitric oxide synthase (iNOS) enhancement, resulting in BBB disruption. While Gram-negative bacteria, Haemophilus influenza OmpP2 is able to target the common carboxy-terminal domain of LR to start initial interaction with brain endothelium, then invade the brain. H. influenza type b (HiB), can induce BBB permeability through TJ disruption. LR and PAFR binding sites have been recognized as common routes of CNS entrance by Neisseria meningitidis. N. meningitidis species also initiate binding to BMECs and induces AJs deformation, as well as inducing specific cleavage of the TJ component occludin through the release of host MMP-8. Escherichia coli bind to BMECs through LR, resulting in IL-6 and IL-8 release and iNOS production, as well as resulting in disassembly of TJs between endothelial cells, facilitating BBB disruption. Therefore, obtaining knowledge of BBB disruption by different types of bacterial species will provide a picture of how the bacteria enter the central nervous system (CNS) which might support the discovery of therapeutic strategies for each bacteria to control and manage infection.
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Bacterias/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/microbiología , Encéfalo/metabolismo , Encéfalo/microbiología , Permeabilidad Capilar/fisiología , Animales , Bacterias/crecimiento & desarrollo , Bacterias/patogenicidad , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Interacciones Huésped-Parásitos/fisiología , HumanosRESUMEN
Mycobacterium tuberculosis (Mtb) has plagued humanity for tens of thousands of years, yet still remains a threat to human health. Its pathology is largely associated with pulmonary tuberculosis with symptoms including fever, hemoptysis, and chest pain. Mtb, however, also manifests in other extrapulmonary organs, such as the pleura, bones, gastrointestinal tract, central nervous system, and lymph nodes. Compared to the knowledge of pulmonary tuberculosis, extrapulmonary pathologies of Mtb are quite understudied. Lymph node tuberculosis is one of the most common extrapulmonary manifestations of tuberculosis, and presents significant challenges in its diagnosis, management, and treatment due to its elusive etiologies and pathologies. The objective of this review is to overview the current understanding of the tropism and pathogenesis of Mtb in endothelial cells of the extrapulmonary tissues, particularly, in lymph nodes. Lymphatic endothelial cells (LECs) are derived from blood vascular endothelial cells (BECs) during development, and these two types of endothelial cells demonstrate substantial molecular, cellular and genetic similarities. Therefore, systemic comparison of the differential and common responses of BECs vs. LECs to Mtb invasion could provide new insights into its pathogenesis, and may promote new investigations into this deadly disease.