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1.
Am J Respir Crit Care Med ; 207(11): 1464-1474, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-36480958

RESUMEN

Rationale: Mechanical ventilation (MV) is life-saving but may evoke ventilator-induced lung injury (VILI). Objectives: To explore how the circadian clock modulates severity of murine VILI via the core clock component BMAL1 (basic helix-loop-helix ARNT like 1) in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM (lysozyme 2 promoter/enhancer driving cre recombinase expression)Bmal1-/-) or wild-type control (LysMBmal1+/+) mice were subjected to 4 hours MV (34 ml/kg body weight) to induce lung injury. Ventilation was initiated at dawn or dusk or in complete darkness (circadian time [CT] 0 or CT12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers, and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared with dusk. Experiments at CT showed deterioration of pulmonary function, worsening of oxygenation, and increased mortality at CT0 compared with CT12. Wild-type neutrophils isolated at dawn showed higher activation and reactive oxygen species production compared with dusk, whereas these day-night differences were dampened in LysMBmal1-/- neutrophils. In LysMBmal1-/- mice, circadian variations in VILI severity were dampened and VILI-induced mortality at CT0 was reduced compared with LysMBmal1+/+ mice. Conclusions: Inflammatory response and lung barrier dysfunction upon MV exhibit diurnal variations, regulated by the circadian clock. LysMBmal1-/- mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysMBmal1+/+ mice. Our data suggest that the internal clock in myeloid cells is an important modulator of VILI.


Asunto(s)
Relojes Circadianos , Lesión Pulmonar Inducida por Ventilación Mecánica , Ratones , Animales , Relojes Circadianos/genética , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Pulmón , Lesión Pulmonar Inducida por Ventilación Mecánica/genética , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Ritmo Circadiano/genética , Ratones Endogámicos C57BL
2.
Kidney Int ; 100(5): 1071-1080, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34332958

RESUMEN

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.


Asunto(s)
Relojes Circadianos , Eritropoyetina , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relojes Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Criptocromos/metabolismo , Regulación de la Expresión Génica , Homocigoto , Riñón/metabolismo , Ratones , Ratones Noqueados , Eliminación de Secuencia
3.
Am J Respir Crit Care Med ; 207(12): 1643, 2023 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-36731053
4.
Crit Care ; 19: 23, 2015 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-25888164

RESUMEN

INTRODUCTION: Lung-protective ventilation reduced acute respiratory distress syndrome (ARDS) mortality. To minimize ventilator-induced lung injury (VILI), tidal volume is limited, high plateau pressures are avoided, and positive end-expiratory pressure (PEEP) is applied. However, the impact of specific ventilatory patterns on VILI is not well defined. Increasing inspiratory time and thereby the inspiratory/expiratory ratio (I:E ratio) may improve oxygenation, but may also be harmful as the absolute stress and strain over time increase. We thus hypothesized that increasing inspiratory time and I:E ratio aggravates VILI. METHODS: VILI was induced in mice by high tidal-volume ventilation (HVT 34 ml/kg). Low tidal-volume ventilation (LVT 9 ml/kg) was used in control groups. PEEP was set to 2 cm H2O, FiO2 was 0.5 in all groups. HVT and LVT mice were ventilated with either I:E of 1:2 (LVT 1:2, HVT 1:2) or 1:1 (LVT 1:1, HVT 1:1) for 4 hours or until an alternative end point, defined as mean arterial blood pressure below 40 mm Hg. Dynamic hyperinflation due to the increased I:E ratio was excluded in a separate group of animals. Survival, lung compliance, oxygenation, pulmonary permeability, markers of pulmonary and systemic inflammation (leukocyte differentiation in lung and blood, analyses of pulmonary interleukin-6, interleukin-1ß, keratinocyte-derived chemokine, monocyte chemoattractant protein-1), and histopathologic pulmonary changes were analyzed. RESULTS: LVT 1:2 or LVT 1:1 did not result in VILI, and all individuals survived the ventilation period. HVT 1:2 decreased lung compliance, increased pulmonary neutrophils and cytokine expression, and evoked marked histologic signs of lung injury. All animals survived. HVT 1:1 caused further significant worsening of oxygenation, compliance and increased pulmonary proinflammatory cytokine expression, and pulmonary and blood neutrophils. In the HVT 1:1 group, significant mortality during mechanical ventilation was observed. CONCLUSION: According to the "baby lung" concept, mechanical ventilation-associated stress and strain in overinflated regions of ARDS lungs was simulated by using high tidal-volume ventilation. Increase of inspiratory time and I:E ratio significantly aggravated VILI in mice, suggesting an impact of a "stress/strain × time product" for the pathogenesis of VILI. Thus increasing the inspiratory time and I:E ratio should be critically considered.


Asunto(s)
Espiración , Inhalación , Pulmón/patología , Respiración Artificial/efectos adversos , Volumen de Ventilación Pulmonar , Lesión Pulmonar Inducida por Ventilación Mecánica/fisiopatología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Respiración Artificial/métodos , Síndrome de Dificultad Respiratoria/complicaciones , Síndrome de Dificultad Respiratoria/fisiopatología , Lesión Pulmonar Inducida por Ventilación Mecánica/complicaciones , Lesión Pulmonar Inducida por Ventilación Mecánica/patología
5.
FEBS J ; 291(1): 114-131, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-37690456

RESUMEN

The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2 (ZP2), a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding, and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing ovastacin-substrate interactions as well as the individual characteristics that distinguish ovastacin from similar proteases, such as meprins and tolloid. Eventually, we identified several substrates whose cleavage could affect mammalian fertilization. Consequently, these substrates indicate newly identified functions of ovastacin in mammalian fertilization beyond zona pellucida hardening.


Asunto(s)
Fibroblastos , Semen , Masculino , Animales , Ratones , Glicoproteínas de la Zona Pelúcida/metabolismo , Fibroblastos/metabolismo , Semen/metabolismo , Metaloproteasas/metabolismo , Mamíferos/metabolismo , Endopeptidasas , Fertilización/fisiología
6.
Acta Physiol (Oxf) ; 238(1): e13962, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36916859

RESUMEN

Patients admitted to the intensive care unit (ICU) are in need of continuous organ replacement strategies and specialized care, for example because of neurological dysfunction, cardio-pulmonary instability, liver or kidney failure, trauma, hemorrhagic or septic shock or even preterm birth. The 24-h nursing and care interventions provided to critically ill patients significantly limit resting and/or recovery phases. Consecutively, the patient's endogenous circadian rhythms are misaligned and disrupted, which in turn may interfere with their critical condition. A more thorough understanding of the complex interactions of circadian effectors and tissue-specific molecular clocks could therefore serve as potential means for enhancing personalized treatment in critically ill patients, conceivably restoring their circadian network and thus accelerating their physical and neurocognitive recovery. This review addresses the overarching issue of how circadian rhythms are affected and disturbed in critically ill newborns and adults in the ICU, and whether the conflicting external or environmental cues in the ICU environment further promote disruption and thus severity of illness. We direct special attention to the influence of cell-type specific molecular clocks on with severity of organ dysfunctions such as severity of brain dysfunction, pneumonia- or ventilator-associated lung inflammation, cardiovascular instability, liver and kidney failure, trauma, and septic shock. Finally, we address the potential of circadian rhythm stabilization to enhance and accelerate clinical recovery.


Asunto(s)
Nacimiento Prematuro , Insuficiencia Renal , Choque Séptico , Recién Nacido , Adulto , Femenino , Humanos , Enfermedad Crítica , Ritmo Circadiano
7.
NAR Genom Bioinform ; 4(4): lqac097, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36601580

RESUMEN

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms.

8.
Cells ; 10(12)2021 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-34944089

RESUMEN

Pneumolysin (PLY) is a pore-forming toxin of Streptococcus pneumoniae that contributes substantially to the inflammatory processes underlying pneumococcal pneumonia and lung injury. Host responses against S. pneumoniae are regulated in part by neutrophils and platelets, both individually and in cooperative interaction. Previous studies have shown that PLY can target both neutrophils and platelets, however, the mechanisms by which PLY directly affects these cells and alters their interactions are not completely understood. In this study, we characterize the effects of PLY on neutrophils and platelets and explore the mechanisms by which PLY may induce neutrophil-platelet interactions. In vitro studies demonstrated that PLY causes the formation of neutrophil extracellular traps (NETs) and the release of extracellular vesicles (EVs) from both human and murine neutrophils. In vivo, neutrophil EV (nEV) levels were increased in mice infected with S. pneumoniae. In platelets, treatment with PLY induced the cell surface expression of P-selectin (CD62P) and binding to annexin V and caused a significant release of platelet EVs (pl-EVs). Moreover, PLY-induced nEVs but not NETs promoted platelet activation. The pretreatment of nEVs with proteinase K inhibited platelet activation, indicating that the surface proteins of nEVs play a role in this process. Our findings demonstrate that PLY activates neutrophils and platelets to release EVs and support an important role for neutrophil EVs in modulating platelet functions in pneumococcal infections.


Asunto(s)
Vesículas Extracelulares/metabolismo , Neutrófilos/metabolismo , Activación Plaquetaria/efectos de los fármacos , Estreptolisinas/farmacología , Animales , Proteínas Bacterianas/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Trampas Extracelulares/efectos de los fármacos , Trampas Extracelulares/metabolismo , Humanos , Ratones , Activación Neutrófila/efectos de los fármacos
9.
Sci Rep ; 9(1): 546, 2019 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-30679641

RESUMEN

Vertebrate fetuins are multi-domain plasma-proteins of the cystatin-superfamily. Human fetuin-A is also known as AHSG, α2-Heremans-Schmid-glycoprotein. Gene-knockout in mice identified fetuin-A as essential for calcified-matrix-metabolism and bone-mineralization. Fetuin-B deficient mice, on the other hand, are female infertile due to zona pellucida 'hardening' caused by the metalloproteinase ovastacin in unfertilized oocytes. In wildtype mice fetuin-B inhibits the activity of ovastacin thus maintaining oocytes fertilizable. Here we asked, if fetuins affect further proteases as might be expected from their evolutionary relation to single-domain-cystatins, known as proteinase-inhibitors. We show that fetuin-A is not an inhibitor of any tested protease. In stark contrast, the closely related fetuin-B selectively inhibits astacin-metalloproteinases such as meprins and ovastacin, but not astacins of the tolloid-subfamily, nor any other proteinase. The analysis of fetuin-B expressed in various mammalian cell types, insect cells, and truncated fish-fetuin expressed in bacteria, showed that the cystatin-like domains alone are necessary and sufficient for inhibition. This report highlights fetuin-B as a specific antagonist of ovastacin and meprin-metalloproteinases. Control of ovastacin was shown to be indispensable for female fertility. Meprin inhibition, on the other hand, renders fetuin-B a potential key-player in proteolytic networks controlling angiogenesis, immune-defense, extracellular-matrix-assembly and general cell-signaling, with implications for inflammation, fibrosis, neurodegenerative disorders and cancer.


Asunto(s)
Fetuína-B/metabolismo , Mamíferos/sangre , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Plasma/metabolismo , Animales , Astacoidea , Bovinos , Fertilización/fisiología , Fibrinolisina/metabolismo , Glicosilación , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteasas/antagonistas & inhibidores , Ratones , Proteolisis , Proteínas Recombinantes/metabolismo , alfa-2-Glicoproteína-HS/metabolismo
10.
PLoS One ; 11(4): e0153290, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27054568

RESUMEN

GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFß (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFß and GARP and connection of this propeptide to αvß6 or αvß8 integrins of target cells during mechanical TGFß release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFß and a soluble variant of GARP. Surprisingly, soluble GARP and TGFß formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFß activity. TGFß activation is enhanced by the non-covalent GARP-TGFß complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo.


Asunto(s)
Proliferación Celular , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Dicroismo Circular , Clonación Molecular , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genética
11.
Case Rep Med ; 2014: 651683, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25580130

RESUMEN

Mediastinal cysts are extremely rare clinical disorders. They usually have a pericardial origin. In this report, we present a 27-year-old male patient with a mediastinal bronchogenic cyst together with clinical presentation and management of the pathology.

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