RESUMO
In the paper, the effect of spontaneous Brillouin scattering (SpBS) is analyzed as a noise source in distributed acoustic sensors (DAS). The intensity of the SpBS wave fluctuates over time, and these fluctuations increase the noise power in DAS. Based on experimental data, the probability density function (PDF) of the spectrally selected SpBS Stokes wave intensity is negative exponential, which corresponds to the known theoretical conception. Based on this statement, an estimation of the average noise power induced by the SpBS wave is given. This noise power equals the square of the average power of the SpBS Stokes wave, which in turn is approximately 18 dB lower than the Rayleigh backscattering power. The noise composition in DAS is determined for two configurations, the first for the initial backscattering spectrum and the second for the spectrum in which the SpBS Stokes and anti-Stokes waves are rejected. It is established that in the analyzed particular case, the SpBS noise power is dominant and exceeds the powers of the thermal, shot, and phase noises in DAS. Accordingly, by rejecting the SpBS waves at the photodetector input, it is possible to reduce the noise power in DAS. In our case, this rejection is carried out by an asymmetric Mach-Zehnder interferometer (MZI). The rejection of the SpBS wave is most relevant for broadband photodetectors, which are associated with the use of short probing pulses to achieve short gauge lengths in DAS.
Assuntos
Fertilização , Frequência Cardíaca , Funções VerossimilhançaRESUMO
A simple and cost-effective architecture of a distributed acoustic sensor (DAS) or a phase-OTDR for engineering geology is proposed. The architecture is based on the dual-pulse acquisition principle, where the dual probing pulse is formed via an unbalanced Michelson interferometer (MI). The necessary phase shifts between the sub-pulses of the dual-pulse are introduced using a 3 × 3 coupler built into the MI. Laser pulses are generated by direct modulation of the injection current, which obtains optical pulses with a duration of 7 ns. The use of an unbalanced MI for the formation of a dual-pulse reduces the requirements for the coherence of the laser source, as the introduced delay between sub-pulses is compensated in the fiber under test (FUT). Therefore, a laser with a relatively broad spectral linewidth of about 1 GHz can be used. To overcome the fading problem, as well as to ensure the linearity of the DAS response, the averaging of over 16 optical frequencies is used. The performance of the DAS was tested by recording a strong vibration impact on a horizontally buried cable and by the recording of seismic waves in a borehole in the seabed.
Assuntos
Engenharia , Geologia , Análise Custo-Benefício , Frequência Cardíaca , AcústicaRESUMO
This work presents a detailed review of the development of distributed acoustic sensors (DAS) and their newest scientific applications. It covers most areas of human activities, such as the engineering, material, and humanitarian sciences, geophysics, culture, biology, and applied mechanics. It also provides the theoretical basis for most well-known DAS techniques and unveils the features that characterize each particular group of applications. After providing a summary of research achievements, the paper develops an initial perspective of the future work and determines the most promising DAS technologies that should be improved.
Assuntos
Acústica , Tecnologia de Fibra Óptica , HumanosRESUMO
One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Lipopolissacarídeos , Pulmão/irrigação sanguínea , NF-kappa B/metabolismo , Edema Pulmonar/metabolismo , Fatores de Transcrição SOXF/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Sítios de Ligação , Células Cultivadas , Claudina-5/genética , Claudina-5/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Ácido Peroxinitroso/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/genética , Edema Pulmonar/patologia , Fatores de Transcrição SOXF/genética , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
A 3-channel biosensor based on spectral correlation interferometry (SCI) has been adapted for direct optical detection of antigens by measuring changes in thickness of a biolayer on functionalized glass slips employed as affordable single-use sensor chips. The instrument is insensitive to the bulk refractive index of a solution under test and provides signals in metrological units (pm or nm). Using real-time monitoring with the SCI, protocols for fabrication of sensor chips with different functional (epoxylated, carboxylated, and biotinylated) surfaces for antibody immobilization have been developed and optimized to minimize chip-to-chip variations and achieve better limit of detection (LOD), shorter assay time, and longer shelf life. The optimized coupling surfaces have been compared for detection of human serum albumin (HSA) used as a model agent of medical significance. The dynamic ranges for measuring the HSA concentration were 0.07-20, 0.12-30, and 0.25-10 µg/ml, and the assay durations were less than 20, 15, and 30 min for the epoxylated, carboxylated, and biotinylated chips, respectively. The advantages of each type of sensor chip have been shown, namely, the carboxylated chips feature the shortest assay time, the epoxylated ones demonstrate the best LOD, and the biotinylated chips exhibit the longest shelf life in an unprotected environment. The developed protocols of antibody immobilization can be used in different biosensors and assay techniques including those based on fluorescent, magnetic or plasmonic labels, etc. The SCI is well compatible with various partially transparent layers used in biosensing and with microarrays for multi-analyte detection.
Assuntos
Anticorpos Imobilizados , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Interferometria/métodos , Albumina Sérica/química , Humanos , Interferometria/instrumentação , Dispositivos Lab-On-A-Chip , Limite de DetecçãoRESUMO
RATIONALE: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.
Assuntos
Agonistas do Canal de Sódio Epitelial/metabolismo , Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/metabolismo , Alvéolos Pulmonares/metabolismo , Edema Pulmonar/metabolismo , Estreptolisinas , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteínas de Bactérias , Agonistas do Canal de Sódio Epitelial/química , Canais Epiteliais de Sódio/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Peptídeos Cíclicos/química , Alvéolos Pulmonares/microbiologia , Edema Pulmonar/microbiologia , Fator de Necrose Tumoral alfa/químicaRESUMO
Aggressive treatment with antibiotics in patients infected with Streptococcus pneumoniae induces release of the bacterial virulence factor pneumolysin (PLY). Days after lungs are sterile, this pore-forming toxin can still induce pulmonary permeability edema in patients, characterized by alveolar/capillary barrier dysfunction and impaired alveolar liquid clearance (ALC). ALC is mainly regulated through Na(+) transport by the apically expressed epithelial sodium channel (ENaC) and the basolaterally expressed Na(+)/K(+)-ATPase in type II alveolar epithelial cells. Because no standard treatment is currently available to treat permeability edema, the search for novel therapeutic candidates is of high priority. We detected mRNA expression for the active receptor splice variant SV1 of the hypothalamic polypeptide growth hormone-releasing hormone (GHRH), as well as for GHRH itself, in human lung microvascular endothelial cells (HL-MVEC). Therefore, we have evaluated the effect of the GHRH agonist JI-34 on PLY-induced barrier and ALC dysfunction. JI-34 blunts PLY-mediated endothelial hyperpermeability in monolayers of HL-MVEC, in a cAMP-dependent manner, by means of reducing the phosphorylation of myosin light chain and vascular endothelial (VE)-cadherin. In human airway epithelial H441 cells, PLY significantly impairs Na(+) uptake, but JI-34 restores it to basal levels by means of increasing cAMP levels. Intratracheal instillation of PLY into C57BL6 mice causes pulmonary alveolar epithelial and endothelial hyperpermeability as well as edema formation, all of which are blunted by JI-34. These findings point toward a protective role of the GHRH signaling pathway in PLY-induced permeability edema.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/agonistas , Edema Pulmonar/patologia , Estreptolisinas/toxicidade , Animais , Antígenos CD/metabolismo , Proteínas de Bactérias/toxicidade , Caderinas/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Ativação do Canal Iônico , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Cadeias Leves de Miosina/metabolismo , Permeabilidade , Fosforilação , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Edema Pulmonar/genética , Edema Pulmonar/fisiopatologia , Splicing de RNA/genética , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , Canais de Sódio/metabolismoRESUMO
Acute lung injury (ALI) is a severe hypoxemic respiratory insufficiency associated with lung leak, diffuse alveolar damage, inflammation, and loss of lung function. Decreased dimethylaminohydrolase (DDAH) activity and increases in asymmetric dimethylarginine (ADMA), together with exaggerated oxidative/nitrative stress, contributes to the development of ALI in mice exposed to LPS. Whether restoring DDAH function and suppressing ADMA levels can effectively ameliorate vascular hyperpermeability and lung injury in ALI is unknown, and was the focus of this study. In human lung microvascular endothelial cells, DDAH II overexpression prevented the LPS-dependent increase in ADMA, superoxide, peroxynitrite, and protein nitration. DDAH II also attenuated the endothelial barrier disruption associated with LPS exposure. Similarly, in vivo, we demonstrated that the targeted overexpression of DDAH II in the pulmonary vasculature significantly inhibited the accumulation of ADMA and the subsequent increase in oxidative/nitrative stress in the lungs of mice exposed to LPS. In addition, augmenting pulmonary DDAH II activity before LPS exposure reduced lung vascular leak and lung injury and restored lung function when DDAH activity was increased after injury. Together, these data suggest that enhancing DDAH II activity may prove a useful adjuvant therapy to treat patients with ALI.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Amidoidrolases/metabolismo , Células Endoteliais/enzimologia , Terapia Genética , Lipopolissacarídeos , Pulmão/irrigação sanguínea , Microvasos/enzimologia , Edema Pulmonar/prevenção & controle , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/genética , Amidoidrolases/genética , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Líquido da Lavagem Broncoalveolar/química , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Humanos , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Estresse Oxidativo , Ácido Peroxinitroso/metabolismo , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/enzimologia , Edema Pulmonar/genética , Superóxidos/metabolismo , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Permeability of the endothelial monolayer is increased when exposed to the bacterial endotoxin LPS. Our previous studies have shown that heat shock protein (Hsp) 90 inhibitors protect and restore LPS-mediated hyperpermeability in bovine pulmonary arterial endothelial cells. In this study, we assessed the effect of Hsp90 inhibition against LPS-mediated hyperpermeability in cultured human lung microvascular endothelial cells (HLMVECs) and delineated the underlying molecular mechanisms. We demonstrate that Hsp90 inhibition is critical in the early phase, to prevent LPS-mediated hyperpermeability, and also in the later phase, to restore LPS-mediated hyperpermeability in HLMVECs. Because RhoA is a well known mediator of endothelial hyperpermeability, we investigated the effect of Hsp90 inhibition on LPS-mediated RhoA signaling. RhoA nitration and activity were increased by LPS in HLMVECs and suppressed when pretreated with the Hsp90 inhibitor, 17-allylamino-17 demethoxy-geldanamycin (17-AAG). In addition, inhibition of Rho kinase, a downstream effector of RhoA, protected HLMVECs from LPS-mediated hyperpermeability and abolished LPS-induced myosin light chain (MLC) phosphorylation, a target of Rho kinase. In agreement with these findings, 17-AAG or dominant-negative RhoA attenuated LPS-induced MLC phosphorylation. MLC phosphorylation induced by constitutively active RhoA was also suppressed by 17-AAG, suggesting a role for Hsp90 downstream of RhoA. Inhibition of Src family kinases also suppressed RhoA activity and MLC phosphorylation. Together, these data indicate that Hsp90 inhibition prevents and repairs LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling.
Assuntos
Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Lipopolissacarídeos/efeitos adversos , Quinases Associadas a rho/metabolismo , Animais , Benzoquinonas/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) affect 200,000 people a year in the USA. Pulmonary vascular and specifically endothelial cell (EC) barrier compromise is a hallmark of these diseases. We have recently shown that extracellular adenosine enhances human pulmonary (EC) barrier via activation of adenosine receptors (ARs) in cell cultures. On the basis of these data, we hypothesized that activation of ARs might exert barrier-protective effects in a model of ALI/ARDS in mice. To test this hypothesis, we examined the effects of pre- and posttreatment of adenosine and 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective stable AR agonist, on LPS-induced lung injury. Mice were given vehicle or LPS intratracheally followed by adenosine, NECA, or vehicle instilled via the internal jugular vein. Postexperiment cell counts, Evans Blue Dye albumin (EBDA) extravasation, levels of proteins, and inflammatory cytokines were analyzed. Harvested lungs were used for histology and myeloperoxidase studies. Mice challenged with LPS alone demonstrated an inflammatory response typical of ALI. Cell counts, EBDA extravasation, as well as levels of proteins and inflammatory cytokines were decreased in adenosine-treated mice. Histology displayed reduced infiltration of neutrophils. NECA had a similar effect on LPS-induced vascular barrier compromise. Importantly, posttreatment with adenosine or NECA recovers lung vascular barrier and reduces inflammation induced by LPS challenge. Furthermore, adenosine significantly attenuated protein degradation of A2A and A3 receptors induced by LPS. Collectively, our results demonstrate that activation of ARs protects and restores vascular barrier functions and reduces inflammation in LPS-induced ALI.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Adenosina/metabolismo , Endotélio/metabolismo , Receptores Purinérgicos P1/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Adenosina-5'-(N-etilcarboxamida)/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Permeabilidade Capilar/efeitos dos fármacos , Contagem de Células , Citocinas/metabolismo , Células Endoteliais/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Agonistas do Receptor Purinérgico P1/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Vascular barrier dysfunction is characterized by increased permeability and inflammation of endothelial cells (ECs), which are prominent features of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and sepsis, and a major complication of the SARS-CoV-2 infection and COVID-19. Functional impairment of the EC barrier and accompanying inflammation arises due to microbial toxins and from white blood cells of the lung as part of a defensive action against pathogens, ischemia-reperfusion or blood product transfusions, and aspiration syndromes-based injury. A loss of barrier function results in the excessive movement of fluid and macromolecules from the vasculature into the interstitium and alveolae resulting in pulmonary edema and collapse of the architecture and function of the lungs, and eventually culminates in respiratory failure. Therefore, EC barrier integrity, which is heavily dependent on cytoskeletal elements (mainly actin filaments, microtubules (MTs), cell-matrix focal adhesions, and intercellular junctions) to maintain cellular contacts, is a critical requirement for the preservation of lung function. EC cytoskeletal remodeling is regulated, at least in part, by Ser/Thr phosphorylation/dephosphorylation of key cytoskeletal proteins. While a large body of literature describes the role of phosphorylation of cytoskeletal proteins on Ser/Thr residues in the context of EC barrier regulation, the role of Ser/Thr dephosphorylation catalyzed by Ser/Thr protein phosphatases (PPases) in EC barrier regulation is less documented. Ser/Thr PPases have been proposed to act as a counter-regulatory mechanism that preserves the EC barrier and opposes EC contraction. Despite the importance of PPases, our knowledge of the catalytic and regulatory subunits involved, as well as their cellular targets, is limited and under-appreciated. Therefore, the goal of this review is to discuss the role of Ser/Thr PPases in the regulation of lung EC cytoskeleton and permeability with special emphasis on the role of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) as major mammalian Ser/Thr PPases. Importantly, we integrate the role of PPases with the structural dynamics of the cytoskeleton and signaling cascades that regulate endothelial cell permeability and inflammation.
RESUMO
Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.
Assuntos
Arginase/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Pulmão/patologia , Proteína Quinase C-alfa/metabolismo , Estreptolisinas/farmacologia , Animais , Antígenos CD/metabolismo , Arginase/antagonistas & inibidores , Proteínas de Bactérias/farmacologia , Caderinas/metabolismo , Sinalização do Cálcio , Células Cultivadas , Células Endoteliais/enzimologia , Inibidores Enzimáticos/farmacologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Microvasos/patologia , Pneumonia/enzimologia , Pneumonia/imunologia , Pneumonia/patologia , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses that ERM proteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylation-deficient ERM mutants, exhibit less attenuation of 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubule-disrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Endotélio Vascular/metabolismo , Estradiol/análogos & derivados , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Moduladores de Tubulina/farmacologia , 2-Metoxiestradiol , Animais , Permeabilidade Capilar/genética , Células Cultivadas , Proteínas do Citoesqueleto/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Proteínas de Membrana/genética , Camundongos , Proteínas dos Microfilamentos/genética , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Pirróis/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Hepatitis B surface antigen (HBsAg) is the most clinically relevant serological marker of hepatitis B virus (HBV) infection. Its detection in blood is extremely important for identification of asymptomatic individuals or chronic HBV carriers, screening blood donors, and early seroconversion. Rapid point-of-care HBsAg tests are predominantly qualitative, and their analytical sensitivity does not meet the requirements of regulatory agencies. We present a highly sensitive lateral flow assay based on superparamagnetic nanoparticles for rapid quantification (within 30 min) of polyvalent HBsAg in serum. The demonstrated limit of detection (LOD) of 80 pg mL-1 in human serum is better than both the FDA recommendations for HBsAg assays (which is 0.5 ng mL-1) and the sensitivity of traditional laboratory-based methods such as enzyme linked immunosorbent assays. Along with the attractive LOD at lower concentrations and the wide linear dynamic range of more than 2.5 orders, the assay features rapidity, user-friendliness, on-site operation and effective performance in the complex biological medium. These are due to the combination of the immunochromatographic approach with a highly sensitive electronic registration of superparamagnetic nanolabels over the entire volume of a 3D test structure by their non-linear magnetization and selection of optimal antibodies by original optical label-free methods. The developed cost-efficient bioanalytical technology can be used in many socially important fields such as out-of-lab screening and diagnosis of HBV infection at a point-of-demand, especially in hard-to-reach or sparsely populated areas, as well as highly endemic regions.
Assuntos
Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Anticorpos Anti-Hepatite B , Vírus da Hepatite B/genética , Humanos , Nanopartículas Magnéticas de Óxido de Ferro , Sensibilidade e EspecificidadeRESUMO
Extracellular beta-NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5-trisphate and cAMP. Recently, beta-NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since beta-NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing beta-NAD-activated purinergic receptors upon beta-NAD stimulation. Our data demonstrate that extracellular beta-NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration-dependent manner indicating endothelial barrier enhancement. Importantly, beta-NAD significantly attenuated thrombin-induced EC permeability as well as the barrier-compromising effects of Gram-negative and Gram-positive bacterial toxins representing the barrier-protective function of beta-NAD. Immunofluorescence microscopy reveals more pronounced staining of cell-cell junctional protein VE-cadherin at the cellular periphery signifying increased tightness of the cell-cell contacts after beta-NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in beta-NAD-induced TER increase. beta-NAD-treatment attenuates the lipopolysaccharide (LPS)-induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP-dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in beta-NAD-induced EC barrier enhancement. With these results, we conclude that beta-NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1- and MLCP- dependent signaling pathways.
Assuntos
Actinas/metabolismo , Permeabilidade Capilar , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , NAD/metabolismo , Artéria Pulmonar/enzimologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Antígenos CD/metabolismo , Proteínas de Bactérias/farmacologia , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Junções Intercelulares/metabolismo , Lipopolissacarídeos/farmacologia , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y1 , Transdução de Sinais , Estreptolisinas/farmacologia , Trombina/metabolismo , Fatores de Tempo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with "normal" (similar to those in monolayer EC) and "fast" (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.
Assuntos
Células Endoteliais/fisiologia , Microtúbulos/fisiologia , Modelos Biológicos , Linhagem Celular , Centrossomo/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Microscopia de Vídeo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Bacterial toxins play a key role in the pathogenesis of lung disease. Based on their structural and functional properties, they employ various strategies to modulate lung barrier function and to impair host defense in order to promote infection. Although in general, these toxins target common cellular signaling pathways and host compartments, toxin- and cell-specific effects have also been reported. Toxins can affect resident pulmonary cells involved in alveolar fluid clearance (AFC) and barrier function through impairing vectorial Na+ transport and through cytoskeletal collapse, as such, destroying cell-cell adhesions. The resulting loss of alveolar-capillary barrier integrity and fluid clearance capacity will induce capillary leak and foster edema formation, which will in turn impair gas exchange and endanger the survival of the host. Toxins modulate or neutralize protective host cell mechanisms of both the innate and adaptive immunity response during chronic infection. In particular, toxins can either recruit or kill central players of the lung's innate immune responses to pathogenic attacks, i.e., alveolar macrophages (AMs) and neutrophils. Pulmonary disorders resulting from these toxin actions include, e.g., acute lung injury (ALI), the acute respiratory syndrome (ARDS), and severe pneumonia. When acute infection converts to persistence, i.e., colonization and chronic infection, lung diseases, such as bronchitis, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) can arise. The aim of this review is to discuss the impact of bacterial toxins in the lungs and the resulting outcomes for pathogenesis, their roles in promoting bacterial dissemination, and bacterial survival in disease progression.
Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Toxinas Bacterianas/metabolismo , Pulmão/microbiologia , Infecções Respiratórias/microbiologia , Imunidade Adaptativa , Animais , Bactérias/imunologia , Bactérias/metabolismo , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Progressão da Doença , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/patologia , Transdução de SinaisRESUMO
Heat shock protein 90 (hsp90) inhibitors inactivate and/or degrade various client proteins, including many involved in inflammation. Increased vascular permeability is a hallmark of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Thus, we tested the hypothesis that hsp90 inhibitors may prevent and/or restore endothelial cell (EC) permeability after injury. Exposure of confluent bovine pulmonary arterial endothelial cell (BPAEC) monolayer to TGF-beta1, thrombin, bacterial lipopolysaccharide (LPS), or vascular endothelial growth factor (VEGF) increased BPAEC permeability, as revealed by decreased transendothelial electrical resistance (TER). Treatment of injured endothelium with hsp90 inhibitors completely restored TER of BPAEC. Similarly, preincubation of BPAEC with hsp90 inhibitors prevented the decline in TER induced by the exposure to thrombin, LPS, VEGF, or TGF-beta1. In addition, hsp90 inhibitors restored the EC barrier function after PMA or nocodazole-induced hyperpermeability. These effects of the hsp90 inhibitors were associated with the restoration of TGF-beta1- or nocodazole-induced decrease in VE-cadherin and beta-catenin expression at EC junctions. The protective effect of hsp90 inhibitors on TGF-beta1-induced hyperpermeability was critically dependent upon preservation of F-actin cytoskeleton and was associated with the inhibition of agonist-induced myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, F-actin stress fibers formation, microtubule disassembly, increase in hsp27 phosphorylation, and association of hsp90 with hsp27, but independent of p38MAPK activity. We conclude that hsp90 inhibitors exert barrier protective effects on BPAEC, at least in part, via inhibition of hsp27-mediated, agonist-induced cytoskeletal rearrangement, and therefore may have useful therapeutic value in ALI, ARDS, and other pulmonary inflammatory disease.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Actinas/metabolismo , Animais , Bovinos , Permeabilidade da Membrana Celular , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , Macrolídeos/farmacologia , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pneumonia is a leading cause of death in children and the elderly worldwide, accounting for 15% of all deaths of children under 5 years old. Streptococcus pneumoniae is a common and aggressive cause of pneumonia and can also contribute to meningitis and sepsis. Despite the widespread use of antibiotics, mortality rates for pneumonia remain unacceptably high in part due to the release of bacterial toxins. Pneumolysin (PLY) is a cholesterol-dependent toxin that is produced by Streptococcus, and it is both necessary and sufficient for the development of the extensive pulmonary permeability edema that underlies acute lung injury. The mechanisms by which PLY disrupts the pulmonary endothelial barrier are not fully understood. Previously, we found that reactive oxygen species (ROS) contribute to the barrier destructive effects of PLY and identified an unexpected but potent role of Hsp70 in suppressing ROS production. The ability of Hsp70 to influence PLY-induced barrier dysfunction is not yet described, and the goal of the current study was to identify whether Hsp70 upregulation is an effective strategy to protect the lung microvascular endothelial barrier from G+ bacterial toxins. Overexpression of Hsp70 via adenovirus-mediated gene transfer attenuated PLY-induced increases in permeability in human lung microvascular endothelial cells (HLMVEC) with no evidence of cytotoxicity. To adopt a more translational approach, we employed a pharmacological approach using geranylgeranylacetone (GGA) to acutely upregulate endogenous Hsp70 expression. Following acute treatment (6 h) with GGA, HLMVECs exposed to PLY displayed improved cell viability and enhanced endothelial barrier function as measured by both Electric Cell-substrate Impedance Sensing (ECIS) and transwell permeability assays compared to control treated cells. PLY promoted increased mitochondrial ROS, decreased mitochondrial oxygen consumption, and increased caspase 3 cleavage and cell death, which were collectively improved in cells pretreated with GGA. In mice, IP pretreatment with GGA 24 h prior to IT administration of PLY resulted in significantly less Evans Blue Dye extravasation compared to vehicle, indicating preserved endothelial barrier integrity and suggesting that the acute upregulation of Hsp70 may be an effective therapeutic approach in the treatment of lung injury associated with pneumonia.
RESUMO
Pulmonary permeability edema is characterized by reduced alveolar Na⺠uptake capacity and capillary barrier dysfunction and is a potentially lethal complication of listeriosis. Apical Na⺠uptake is mainly mediated by the epithelial sodium channel (ENaC) and initiates alveolar liquid clearance. Here we examine how listeriolysin O (LLO), the pore-forming toxin of Listeria monocytogenes, impairs the expression and activity of ENaC. To that purpose, we studied how sub-lytic concentrations of LLO affect negative and positive regulators of ENaC expression in the H441 airway epithelial cell line. LLO reduced expression of the crucial ENaC-α subunit in H441 cells within 2 h and this was preceded by activation of PKC-α, a negative regulator of the channel's expression. At later time points, LLO caused a significant reduction in the phosphorylation of Sgk-1 at residue T256 and of Akt-1 at residue S473, both of which are required for full activation of ENaC. The TNF-derived TIP peptide prevented LLO-mediated PKC-α activation and restored phospho-Sgk-1-T256. The TIP peptide also counteracted the observed LLO-induced decrease in amiloride-sensitive Na⺠current and ENaC-α expression in H441 cells. Intratracheally instilled LLO caused profound pulmonary edema formation in mice, an effect that was prevented by the TIP peptide; thus indicating the therapeutic potential of the peptide for the treatment of pore-forming toxin-associated permeability edema.