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1.
Cells ; 10(7)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34359824

RESUMO

Activation of Transient Receptor Potential (TRP) channels can disrupt endothelial barrier function, as their mediated Ca2+ influx activates the CaM (calmodulin)/MLCK (myosin light chain kinase)-signaling pathway, and thereby rearranges the cytoskeleton, increases endothelial permeability and thus can facilitate activation of inflammatory cells and formation of pulmonary edema. Interestingly, TRP channel subunits can build heterotetramers, whereas heteromeric TRPC1/4, TRPC3/6 and TRPV1/4 are expressed in the lung endothelium and could be targeted as a protective strategy to reduce endothelial permeability in pulmonary inflammation. An update on TRP heteromers and their role in lung inflammation will be provided with this review.


Assuntos
Pneumonia/metabolismo , Multimerização Proteica , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Ativação do Canal Iônico , Modelos Biológicos , Pneumonia/patologia , Pneumonia/fisiopatologia
2.
Kidney Int ; 100(5): 1071-1080, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34332958

RESUMO

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.


Assuntos
Relógios Circadianos , Eritropoetina , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Criptocromos/metabolismo , Regulação da Expressão Gênica , Homozigoto , Rim/metabolismo , Camundongos , Camundongos Knockout , Deleção de Sequência
3.
ESC Heart Fail ; 8(4): 3130-3144, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34002482

RESUMO

AIMS: Heart failure with preserved ejection fraction (HFpEF) is frequently (30%) associated with right ventricular (RV) dysfunction, which increases morbidity and mortality in these patients. Yet cellular mechanisms of RV remodelling and RV dysfunction in HFpEF are not well understood. Here, we evaluated RV cardiomyocyte function in a rat model of metabolically induced HFpEF. METHODS AND RESULTS: Heart failure with preserved ejection fraction-prone animals (ZSF-1 obese) and control rats (Wistar Kyoto) were fed a high-caloric diet for 13 weeks. Haemodynamic characterization by echocardiography and invasive catheterization was performed at 22 and 23 weeks of age, respectively. After sacrifice, organ morphometry, RV histology, isolated RV cardiomyocyte function, and calcium (Ca2+ ) transients were assessed. ZSF-1 obese rats showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV diastolic dysfunction (including increased LV end-diastolic pressures and E/e' ratio), and preserved LV ejection fraction. ZSF-1 obese animals developed RV dilatation (50% increased end-diastolic area) and mildly impaired RV ejection fraction (42%) with evidence of RV hypertrophy. In isolated RV cardiomyocytes from ZSF-1 obese rats, cell shortening amplitude was preserved, but cytosolic Ca2+ transient amplitude was reduced. In addition, augmentation of cytosolic Ca2+ release with increased stimulation frequency was lost in ZSF-1 obese rats. Myofilament sensitivity was increased, while contractile kinetics were largely unaffected in intact isolated RV cardiomyocytes from ZSF-1 obese rats. Western blot analysis revealed significantly increased phosphorylation of cardiac myosin-binding protein C (Ser282 cMyBP-C) but no change in phosphorylation of troponin I (Ser23, 24 TnI) in RV myocardium from ZSF-1 obese rats. CONCLUSIONS: Right ventricular dysfunction in obese ZSF-1 rats with HFpEF is associated with intrinsic RV cardiomyocyte remodelling including reduced cytosolic Ca2+ amplitudes, loss of frequency-dependent augmentation of Ca2+ release, and increased myofilament Ca2+ sensitivity.


Assuntos
Insuficiência Cardíaca , Disfunção Ventricular Direita , Animais , Insuficiência Cardíaca/etiologia , Homeostase , Humanos , Miócitos Cardíacos , Miofibrilas , Ratos , Volume Sistólico , Disfunção Ventricular Direita/etiologia
5.
Cell Syst ; 11(1): 11-24.e4, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32619549

RESUMO

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.


Assuntos
Proteínas Sanguíneas/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Proteômica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Biomarcadores/sangue , Proteínas Sanguíneas/análise , COVID-19 , Infecções por Coronavirus/classificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/classificação , Pneumonia Viral/classificação , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Adulto Jovem
6.
Front Immunol ; 11: 413, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210976

RESUMO

Transient receptor potential vanilloid-type 4 (TRPV4) cation channel is widely expressed in all tissues as well as in immune cells and its function as mechanosensitive Ca2+ channel seems to be conserved throughout all mammalian species. Of late, emerging evidence has implicated TRPV4 in the activation and differentiation of innate immune cells, especially in neutrophils, monocytes, and macrophages. As such, TRPV4 has been shown to mediate neutrophil adhesion and chemotaxis, as well as production of reactive oxygen species in response to pro-inflammatory stimuli. In macrophages, TRPV4 mediates formation of both reactive oxygen and nitrogen species, and regulates phagocytosis, thus facilitating bacterial clearance and resolution of infection. Importantly, TRPV4 may present a missing link between mechanical forces and immune responses. This connection has been exemplary highlighted by the demonstrated role of TRPV4 in macrophage activation and subsequent induction of lung injury following mechanical overventilation. Mechanosensation via TRPV4 is also expected to activate innate immune cells and establish a pro-inflammatory loop in fibrotic diseases with increased deposition of extracellular matrix (ECM) and substrate stiffness. Likewise, TRPV4 may be activated by cell migration through the endothelium or the extracellular matrix, or even by circulating immune cells squeezing through the narrow passages of the pulmonary or systemic capillary bed, a process that has recently been linked to neutrophil priming and depriming. Here, we provide an overview over the emerging role of TRPV4 in innate immune responses and highlight two distinct modes for the activation of TRPV4 by either mechanical forces ("mechanoTRPV4") or by pathogens ("immunoTRPV4").


Assuntos
Lesão Pulmonar/imunologia , Macrófagos/imunologia , Mecanotransdução Celular/imunologia , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Fibrose , Humanos , Imunidade Inata , Fagocitose
7.
Sci Rep ; 8(1): 4878, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29559678

RESUMO

Transient receptor potential vanilloid 4 (TRPV4) cation channels are functional in all renal vascular segments and mediate endothelium-dependent vasorelaxation. Moreover, they are expressed in distinct parts of the tubular system and activated by cell swelling. Ischaemia/reperfusion injury (IRI) is characterized by tubular injury and endothelial dysfunction. Therefore, we hypothesised a putative organ protective role of TRPV4 in acute renal IRI. IRI was induced in TRPV4 deficient (Trpv4 KO) and wild-type (WT) control mice by clipping the left renal pedicle after right-sided nephrectomy. Serum creatinine level was higher in Trpv4 KO mice 6 and 24 hours after ischaemia compared to WT mice. Detailed histological analysis revealed that IRI caused aggravated renal tubular damage in Trpv4 KO mice, especially in the renal cortex. Immunohistological and functional assessment confirmed TRPV4 expression in proximal tubular cells. Furthermore, the tubular damage could be attributed to enhanced necrosis rather than apoptosis. Surprisingly, the percentage of infiltrating granulocytes and macrophages were comparable in IRI-damaged kidneys of Trpv4 KO and WT mice. The present results suggest a renoprotective role of TRPV4 during acute renal IRI. Further studies using cell-specific TRPV4 deficient mice are needed to clarify cellular mechanisms of TRPV4 in IRI.


Assuntos
Túbulos Renais/metabolismo , Traumatismo por Reperfusão/metabolismo , Canais de Cátion TRPV/deficiência , Injúria Renal Aguda/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Isquemia/patologia , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Reperfusão/métodos , Traumatismo por Reperfusão/genética , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
8.
Crit Care Med ; 46(3): e258-e267, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29298188

RESUMO

OBJECTIVES: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. INTERVENTIONS: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. MEASUREMENTS AND MAIN RESULTS: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. CONCLUSIONS: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Inflamação/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pneumonia Pneumocócica/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/etiologia , Animais , Feminino , Humanos , Inflamação/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/complicações , Pneumonia Pneumocócica/enzimologia , Receptores de Esfingosina-1-Fosfato , Streptococcus pneumoniae
9.
Anesthesiology ; 126(2): 300-311, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27861175

RESUMO

BACKGROUND: Mechanical ventilation can cause lung endothelial barrier failure and inflammation cumulating in ventilator-induced lung injury. Yet, underlying mechanotransduction mechanisms remain unclear. Here, the authors tested the hypothesis that activation of the mechanosensitive Ca channel transient receptor potential vanilloid (TRPV4) by serum glucocorticoid-regulated kinase (SGK) 1 may drive the development of ventilator-induced lung injury. METHODS: Mice (total n = 54) were ventilated for 2 h with low (7 ml/kg) or high (20 ml/kg) tidal volumes and assessed for signs of ventilator-induced lung injury. Isolated-perfused lungs were inflated with continuous positive airway pressures of 5 or 15 cm H2O (n = 7 each), and endothelial calcium concentration was quantified by real-time imaging. RESULTS: Genetic deficiency or pharmacologic inhibition of TRPV4 or SGK1 protected mice from overventilation-induced vascular leakage (reduction in alveolar protein concentration from 0.84 ± 0.18 [mean ± SD] to 0.46 ± 0.16 mg/ml by TRPV4 antagonization), reduced lung inflammation (macrophage inflammatory protein 2 levels of 193 ± 163 in Trpv4 vs. 544 ± 358 pmol/ml in wild-type mice), and attenuated endothelial calcium responses to lung overdistension. Functional coupling of TRPV4 and SGK1 in lung endothelial mechanotransduction was confirmed by proximity ligation assay demonstrating enhanced TRPV4 phosphorylation at serine 824 at 18% as compared to 5% cyclic stretch, which was prevented by SGK1 inhibition. CONCLUSIONS: Lung overventilation promotes endothelial calcium influx and barrier failure through a mechanism that involves activation of TRPV4, presumably due to phosphorylation at its serine 824 residue by SGK1. TRPV4 and SGK1 may present promising new targets for prevention or treatment of ventilator-induced lung injury.


Assuntos
Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Respiração Artificial/efeitos adversos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Animais , Western Blotting , Modelos Animais de Doenças , Pulmão/metabolismo , Masculino , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Canais de Cátion TRPV/economia , Lesão Pulmonar Induzida por Ventilação Mecânica/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo
10.
Am J Respir Cell Mol Biol ; 54(3): 370-83, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26222277

RESUMO

The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Pulmão/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Canais de Cátion TRPV/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/prevenção & controle , Animais , Transplante de Medula Óssea , Sinalização do Cálcio , Permeabilidade Capilar , Modelos Animais de Doenças , Humanos , Ácido Clorídrico , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Masculino , Camundongos Knockout , Morfolinas/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Pneumonia/metabolismo , Edema Pulmonar/metabolismo , Pirróis/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética
11.
J Cell Sci ; 127(Pt 17): 3794-804, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25002400

RESUMO

Skeletal and heart muscle-specific variant of the α subunit of nascent polypeptide associated complex (skNAC; encoded by NACA) is exclusively found in striated muscle cells. Its function, however, is largely unknown. Previous reports have demonstrated that skNAC binds to m-Bop/Smyd1, a multi-functional protein that regulates myogenesis both through the control of transcription and the modulation of sarcomerogenesis, and that both proteins undergo nuclear-to-cytoplasmic translocation at the later stages of myogenic differentiation. Here, we show that skNAC binds to the E3 SUMO ligase mammalian Mms21/Nse2 and that knockdown of Nse2 expression inhibits specific aspects of myogenic differentiation, accompanied by a partial blockade of the nuclear-to-cytoplasmic translocation of the skNAC-Smyd1 complex, retention of the complex in promyelocytic leukemia (PML)-like nuclear bodies and disturbed sarcomerogenesis. In addition, we show that the skNAC interaction partner Smyd1 contains a putative sumoylation motif and is sumoylated in muscle cells, with depletion of Mms21/Nse2 leading to reduced concentrations of sumoylated Smyd1. Taken together, our data suggest that the function, specifically the balance between the nuclear and cytosolic roles, of the skNAC-Smyd1 complex might be regulated by sumoylation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Morfogênese/genética , Desenvolvimento Muscular/genética , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Camundongos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Sumoilação/genética
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