RESUMO
In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GECs) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen. The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel(ENaC), which is expressed by GEC, upon binding to the channel's α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E2 and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Glomerulonefrite/tratamento farmacológico , Glomérulos Renais/efeitos dos fármacos , Peptídeos Cíclicos/administração & dosagem , Proteinúria/tratamento farmacológico , Animais , Nitrogênio da Ureia Sanguínea , Linhagem Celular , Dinoprostona/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Glomerulonefrite/sangue , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Humanos , Injeções Intraperitoneais , Glomérulos Renais/citologia , Glomérulos Renais/patologia , Camundongos , Óxido Nítrico/metabolismo , Técnicas de Patch-Clamp , Cultura Primária de Células , Proteinúria/sangue , Proteinúria/imunologia , Proteinúria/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dyslipidemia associated with triglyceride-rich lipoproteins (TRLs) represents an important residual risk factor for cardiovascular and chronic kidney disease in patients with type 1 diabetes (T1D). Levels of growth hormone (GH) are elevated in T1D, which aggravates both hyperglycemia and dyslipidemia. The hypothalamic growth hormone-releasing hormone (GHRH) regulates the release of GH by the pituitary but also exerts separate actions on peripheral GHRH receptors, the functional role of which remains elusive in T1D. In a rat model of streptozotocin (STZ)-induced T1D, GHRH receptor expression was found to be up-regulated in the distal small intestine, a tissue involved in chylomicron synthesis. Treatment of T1D rats with a GHRH antagonist, MIA-602, at a dose that did not affect plasma GH levels, significantly reduced TRL, as well as markers of renal injury, and improved endothelial-dependent vasorelaxation. Glucagon-like peptide 1 (GLP-1) reduces hyperglucagonemia and postprandial TRL, the latter in part through a decreased synthesis of apolipoprotein B-48 (ApoB-48) by intestinal cells. Although plasma GLP-1 levels were elevated in diabetic animals, this was accompanied by increased rather than reduced glucagon levels, suggesting impaired GLP-1 signaling. Treatment with MIA-602 normalized GLP-1 and glucagon to control levels in T1D rats. MIA-602 also decreased secretion of ApoB-48 from rat intestinal epithelial cells in response to oleic acid stimulation in vitro, in part through a GLP-1-dependent mechanism. Our findings support the hypothesis that antagonizing the signaling of GHRH in T1D may improve GLP-1 function in the small intestine, which, in turn, diminishes TRL and reduces renal and vascular complications.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Modelos Animais de Doenças , Dislipidemias/fisiopatologia , Hormônio Liberador de Hormônio do Crescimento/fisiologia , Animais , Dislipidemias/terapia , Hormônio Liberador de Hormônio do Crescimento/antagonistas & inibidores , Intestino Delgado/metabolismo , Masculino , Ratos , Ratos Wistar , Receptores de Neuropeptídeos/metabolismo , Receptores de Hormônios Reguladores de Hormônio Hipofisário/metabolismo , EstreptozocinaRESUMO
Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.
Assuntos
Agonistas do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/farmacologia , Linhagem Celular Tumoral , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Mutação Puntual , Domínios Proteicos/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
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
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
Approximately 5-10% of subjects with prediabetes become diabetic every year. Inflammation is involved in the development of obesity-related type 2 diabetes (T2D). However, to date, the relationship between inflammation and prediabetes, defined by hemoglobin A1c (HbA1c) ≥5.7 and <6.5%, remains largely unexplored, especially in African Americans. Therefore, in this study we examined a comprehensive panel of 13 cytokines involved in the inflammatory response in overweight/obese subjects with prediabetes. A total of 21 otherwise healthy, overweight/obese, young adult African American females with prediabetes, together with 20 matched overweight/obese controls, were selected for this study. Plasma cytokines were assessed by multiplex cytokine profiling. Plasma concentrations of interleukin (IL)-5, IL-6, IL-7, tumor necrosis factor-α (TNF-α), and granulocyte-monocyte colony-stimulating factor (GM-CSF) were significantly higher in the prediabetic group, as compared to the control group (all p<0.05). Plasma concentrations of all the other cytokines, interferon-γ (IFN-γ), IL-1ß, IL-2, IL-4, IL-8, IL-10, IL-12p70 and IL-13, seemed to be elevated in the prediabetic group, but failed to reach statistical significances. Upon merging both groups, HbA1c was found to be positively correlated with IFN-γ, IL-1ß, IL-2, IL-5, IL-7, IL-8, TNF-α and GM-CSF. This study demonstrates elevated levels of various pro-inflammatory cytokines in overweight/obese young subjects with prediabetes, which place them at higher risk of developing T2D and cardiovascular diseases. Our data also call for further investigations in animal models and population cohorts to establish the roles of a variety of pro-inflammatory cytokines in the early development of obesity-related T2D.
Assuntos
Citocinas/sangue , Diabetes Mellitus Tipo 2/metabolismo , Hemoglobinas Glicadas/metabolismo , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Adolescente , Adulto , Negro ou Afro-Americano , Tamanho Corporal , Citocinas/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Pessoa de Meia-Idade , Adulto JovemRESUMO
Introduction: Although both COVID-19 and non-COVID-19 ARDS can be accompanied by significantly increased levels of circulating cytokines, the former significantly differs from the latter by its higher vasculopathy, characterized by increased oxidative stress and coagulopathy in lung capillaries. This points towards the existence of SARS-CoV2-specific factors and mechanisms that can sensitize the endothelium towards becoming dysfunctional. Although the virus is rarely detected within endothelial cells or in the circulation, the S1 subunit of its spike protein, which contains the receptor binding domain (RBD) for human ACE2 (hACE2), can be detected in plasma from COVID-19 patients and its levels correlate with disease severity. It remains obscure how the SARS-CoV2 RBD exerts its deleterious actions in lung endothelium and whether there are mechanisms to mitigate this. Methods: In this study, we use a combination of in vitro studies in RBD-treated human lung microvascular endothelial cells (HL-MVEC), including electrophysiology, barrier function, oxidative stress and human ACE2 (hACE2) surface protein expression measurements with in vivo studies in transgenic mice globally expressing human ACE2 and injected with RBD. Results: We show that SARS-CoV2 RBD impairs endothelial ENaC activity, reduces surface hACE2 expression and increases reactive oxygen species (ROS) and tissue factor (TF) generation in monolayers of HL-MVEC, as such promoting barrier dysfunction and coagulopathy. The TNF-derived TIP peptide (a.k.a. solnatide, AP301) -which directly activates ENaC upon binding to its a subunit- can override RBD-induced impairment of ENaC function and hACE2 expression, mitigates ROS and TF generation and restores barrier function in HL-MVEC monolayers. In correlation with the increased mortality observed in COVID-19 patients co-infected with S. pneumoniae, compared to subjects solely infected with SARS-CoV2, we observe that prior intraperitoneal RBD treatment in transgenic mice globally expressing hACE2 significantly increases fibrin deposition and capillary leak upon intratracheal instillation of S. pneumoniae and that this is mitigated by TIP peptide treatment.
Assuntos
COVID-19 , Células Endoteliais , Animais , Camundongos , Humanos , Enzima de Conversão de Angiotensina 2/genética , RNA Viral , Espécies Reativas de Oxigênio , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2 , EndotélioRESUMO
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
The vascular endothelial injury occurs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, but the mechanisms are poorly understood. We sought to determine the frequency and type of cytokine elevations and their relationship to endothelial injury induced by plasma from patients with SARS-CoV-2 versus controls. Plasma from eight consecutively enrolled patients hospitalized with acute SARS-CoV-2 infection was compared to controls. Endothelial cell (EC) barrier integrity was evaluated using ECIS (electric cell-substrate impedance sensing) on human lung microvascular EC. Plasma from all SARS-CoV-2 but none from controls decreased transendothelial resistance to a greater degree than that produced by tumor necrosis factor-alpha (TNF-α), the positive control for the assay. Thrombin, angiopoietin 2 (Ang2), and vascular endothelial growth factor (VEGF), complement factor C3a and C5a, and spike protein increased endothelial permeability, but to a lesser extent and a shorter duration when compared to SARS-CoV-2 plasma. Analysis of Ang2, VEGF, and 15 cytokines measured in plasma revealed striking patient-to-patient variability within the SARS-CoV-2 patients. Pretreatment with thrombin inhibitors, single, or combinations of neutralizing antibodies against cytokines, Ca3 and C5a receptor antagonists, or with ACE2 antibody failed to lessen the SARS-CoV-2 plasma-induced EC permeability. The EC barrier destructive effects of plasma from patients with SARS-CoV-2 were susceptible to heat inactivation. Plasma from patients hospitalized with acute SARS-CoV-2 infection uniformly disrupts lung microvascular integrity. No predicted single, or set of, cytokine(s) accounted for the enhanced vascular permeability, although the factor(s) were heat-labile. A still unidentified but potent circulating factor(s) appears to cause the EC disruption in SARS-CoV-2 infected patients. IMPORTANCE Lung vascular endothelial injury in SARS-CoV-2 patients is one of the most important causes of morbidity and mortality and has been linked to more severe complications including acute respiratory distress syndrome (ARDS) and subsequent death due to multiorgan failure. We have demonstrated that in eight consecutive patients with SARS-CoV-2, who were not selected for evidence of endothelial injury, the diluted plasma-induced intense lung microvascular damage, in vitro. Known endothelial barrier-disruptive agents and proposed mediators of increased endothelial permeability in SARS-CoV-2, induced changes in permeability that were smaller in magnitude and shorter in duration than plasma from patients with SARS-CoV-2. The effect on endothelial cell permeability of plasma from patients with SARS-CoV-2 was heat-labile. The main plasma factor that causes the increased endothelial permeability remains to be identified. Our study provides a possible approach for future studies to understand the underlying mechanisms leading to vascular injury in SARS-CoV-2 infections.
Assuntos
COVID-19/sangue , Permeabilidade Capilar , Citocinas/sangue , Pulmão/irrigação sanguínea , SARS-CoV-2/fisiologia , Adulto , Idoso , COVID-19/fisiopatologia , COVID-19/virologia , Células Endoteliais/virologia , Feminino , Humanos , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/genética , Fator de Necrose Tumoral alfa/sangue , Fator A de Crescimento do Endotélio Vascular , Adulto JovemRESUMO
Solid-state nanopore sensors are highly versatile platforms for the rapid, label-free electrical detection and analysis of single molecules, applicable to next generation DNA sequencing. The versatility of this technology allows for both large scale device integration and interfacing with biological systems. Here we report on the development of a hybrid biological solid-state nanopore platform that incorporates a highly mobile lipid bilayer on a single solid-state Al(2)O(3) nanopore sensor, for the potential reconstitution of ion channels and biological nanopores. Such a system seeks to combine the superior electrical, thermal, and mechanical stability of Al(2)O(3) solid-state nanopores with the chemical specificity of biological nanopores. Bilayers on Al(2)O(3) exhibit higher diffusivity than those formed on TiO(2) and SiO(2) substrates, attributed to the presence of a thick hydration layer on Al(2)O(3), a key requirement to preserving the biological functionality of reconstituted membrane proteins. Molecular dynamics simulations demonstrate that the electrostatic repulsion between the dipole of the DOPC headgroup and the positively charged Al(2)O(3) surface may be responsible for the enhanced thickness of this hydration layer. Lipid bilayer coated Al(2)O(3) nanopore sensors exhibit excellent electrical properties and enhanced mechanical stability (GΩ seals for over 50 h), making this technology ideal for use in ion channel electrophysiology, the screening of ion channel active drugs and future integration with biological nanopores such as α-hemolysin and MspA for rapid single molecule DNA sequencing. This technology can find broad application in bio-nanotechnology.
Assuntos
Óxido de Alumínio/química , Técnicas Biossensoriais , Bicamadas Lipídicas/química , Nanoporos , Óxido de Alumínio/síntese química , DNA/química , Recuperação de Fluorescência Após Fotodegradação/métodos , Canais Iônicos/química , Bicamadas Lipídicas/síntese química , Modelos Biológicos , Simulação de Dinâmica Molecular , Nanotecnologia , Fosfatidilcolinas/química , Análise de Sequência de DNA , Dióxido de Silício/química , Coloração e Rotulagem , Titânio/químicaRESUMO
Alveolar-capillary leak is a hallmark of the acute respiratory distress syndrome (ARDS), a potentially lethal complication of severe sepsis, trauma and pneumonia, including COVID-19. Apart from barrier dysfunction, ARDS is characterized by hyper-inflammation and impaired alveolar fluid clearance (AFC), which foster the development of pulmonary permeability edema and hamper gas exchange. Tumor Necrosis Factor (TNF) is an evolutionarily conserved pleiotropic cytokine, involved in host immune defense against pathogens and cancer. TNF exists in both membrane-bound and soluble form and its mainly -but not exclusively- pro-inflammatory and cytolytic actions are mediated by partially overlapping TNFR1 and TNFR2 binding sites situated at the interface between neighboring subunits in the homo-trimer. Whereas TNFR1 signaling can mediate hyper-inflammation and impaired barrier function and AFC in the lungs, ligand stimulation of TNFR2 can protect from ventilation-induced lung injury. Spatially distinct from the TNFR binding sites, TNF harbors within its structure a lectin-like domain that rather protects lung function in ARDS. The lectin-like domain of TNF -mimicked by the 17 residue TIP peptide- represents a physiological mediator of alveolar-capillary barrier protection. and increases AFC in both hydrostatic and permeability pulmonary edema animal models. The TIP peptide directly activates the epithelial sodium channel (ENaC) -a key mediator of fluid and blood pressure control- upon binding to its α subunit, which is also a part of the non-selective cation channel (NSC). Activity of the lectin-like domain of TNF is preserved in complexes between TNF and its soluble TNFRs and can be physiologically relevant in pneumonia. Antibody- and soluble TNFR-based therapeutic strategies show considerable success in diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease, but their chronic use can increase susceptibility to infection. Since the lectin-like domain of TNF does not interfere with TNF's anti-bacterial actions, while exerting protective actions in the alveolar-capillary compartments, it is currently evaluated in clinical trials in ARDS and COVID-19. A more comprehensive knowledge of the precise role of the TNFR binding sites versus the lectin-like domain of TNF in lung injury, tissue hypoxia, repair and remodeling may foster the development of novel therapeutics for ARDS.
RESUMO
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
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.
Assuntos
Toxinas Bacterianas/toxicidade , Canais Epiteliais de Sódio/fisiologia , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Edema Pulmonar/tratamento farmacológico , Animais , Brônquios/citologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Humanos , Proteínas Imediatamente Precoces/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The α subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function. METHODS: The presence of α, ß, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α. RESULTS: HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance. CONCLUSION: Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.
RESUMO
Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that rises rapidly in response to nutrient ingestion. The GIP receptor is widely expressed in the brain including the brain stem, telencephalon, diencephalon, olfactory bulb, pituitary, and cerebellum. Until recently it was not clear what the endogenous ligand for this receptor was because no GIP expression had been demonstrated in the brain. GIP synthesis has now been documented in the dentate gyrus of the hippocampus. To define GIP effects on behavior we utilized a mouse model a GIP-overexpressing transgenic mouse (GIP Tg). Specifically, anxiety-related behavior, exploration, memory, and nociception were examined. Compared to age-matched adult male C57BI/6 controls GIP Tg mice displayed enhanced exploratory behavior in the open-field locomotor activity test. GIP Tg mice also demonstrated increased performance in some of the motor function tests. These data suggest that the GIP receptor plays a role in the regulation of locomotor activity and exploration. To our knowledge, this is the first report of effects of GIP on behavior.
Assuntos
Ansiedade , Polipeptídeo Inibidor Gástrico/metabolismo , Glucose/farmacologia , Aprendizagem em Labirinto , Memória , Animais , Ansiedade/genética , Polipeptídeo Inibidor Gástrico/sangue , Polipeptídeo Inibidor Gástrico/genética , Masculino , Camundongos , Camundongos Transgênicos , Nociceptores/efeitos dos fármacos , Regulação para CimaRESUMO
Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.
Assuntos
Osteoblastos/metabolismo , Receptores de Melanocortina/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , AMP Cíclico/metabolismo , Primers do DNA , Humanos , Imuno-Histoquímica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Pró-Opiomelanocortina/genética , Ligação Proteica , RNA Mensageiro/genética , Ratos , Receptores da Corticotropina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina/metabolismoRESUMO
Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Lipopolissacarídeos/efeitos adversos , Óxido Nítrico Sintase Tipo III/deficiência , Lesão Pulmonar Aguda/induzido quimicamente , Amidoidrolases/metabolismo , Animais , Citocinas/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Knockout , Testes de Função Respiratória , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
RATIONALE: Antibiotic treatment of patients infected with G(-) or G(+) bacteria promotes release of the toxins lipopolysaccharide (LPS) and pneumolysin (PLY) in their lungs. Growth Hormone-releasing Hormone (GHRH) agonist JI-34 protects human lung microvascular endothelial cells (HL-MVEC), expressing splice variant 1 (SV-1) of the receptor, from PLY-induced barrier dysfunction. We investigated whether JI-34 also blunts LPS-induced hyperpermeability. Since GHRH receptor (GHRH-R) signaling can potentially stimulate both cAMP-dependent barrier-protective pathways as well as barrier-disruptive protein kinase C pathways, we studied their interaction in GHRH agonist-treated HL-MVEC, in the presence of PLY, by means of siRNA-mediated protein kinase A (PKA) depletion. METHODS: Barrier function measurements were done in HL-MVEC monolayers using Electrical Cell substrate Impedance Sensing (ECIS) and VE-cadherin expression by Western blotting. Capillary leak was assessed by Evans Blue dye (EBD) incorporation. Cytokine generation in broncho-alveolar lavage fluid (BALF) was measured by multiplex analysis. PKA and PKC-α activity were assessed by Western blotting. RESULTS: GHRH agonist JI-34 significantly blunts LPS-induced barrier dysfunction, at least in part by preserving VE-cadherin expression, while not affecting inflammation. In addition to activating PKA, GHRH agonist also increases PKC-α activity in PLY-treated HL-MVEC. Treatment with PLY significantly decreases resistance in control siRNA-treated HL-MVEC, but does so even more in PKA-depleted monolayers. Pretreatment with GHRH agonist blunts PLY-induced permeability in control siRNA-treated HL-MVEC, but fails to improve barrier function in PKA-depleted PLY-treated monolayers. CONCLUSIONS: GHRH signaling in HL-MVEC protects from both LPS and PLY-mediated endothelial barrier dysfunction and concurrently induces a barrier-protective PKA-mediated and a barrier-disruptive PKC-α-induced pathway in the presence of PLY, the former of which dominates the latter.