Extracellular histones trigger oxidative stress-dependent induction of the NF-kB/CAM pathway via TLR4 in endothelial cells.

Extracellular histones have been reported to aggravate different pathophysiological processes by increasing vascular permeability, coagulopathy, and inflammation. In the present study, we elucidate how extracellular histones (10-100 µg/mL) concentration dependently increase cytosolic reactive oxygen species (ROS) production using human umbilical vein endothelial cells (HUVECs). Furthermore, we identify cyclooxygenase (COX) and NADPH oxidase (NOX) activity as sources of ROS production in extracellular histone-treated HUVEC. This COX/NOX-mediated ROS production is also involved in enhanced NF-kB activity and cell adhesion molecules (VCAM1 and ICAM1) expression in histone-treated HUVEC. Finally, by using different toll-like receptor (TLR) antagonists, we demonstrate the role of TLR4 in CAMs overexpression triggered by extracellular histones in endothelial cells. In conclusion, our data suggest that through TLR4 signaling, extracellular histones increase endothelial cell activation, a mechanism involving increased COX- and NOX-mediated ROS. These findings increase our understanding on how extracellular histones enhance systemic inflammatory responses in diseases in which histone release occurs as part of the pathological processes

The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

BACKGROUND:Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate. PRINCIPAL FINDINGS:We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation. CONCLUSIONS:Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle

Extracellular Histones Activate Endothelial NLRP3 Inflammasome and are Associated with a Severe Sepsis Phenotype

Jesús Beltrán-García,1– 3 Rebeca Osca-Verdegal,1,3 Daniel Pérez-Cremades,2,3 Susana Novella,2,3 Carlos Hermenegildo,2,3 Federico V Pallardó,1– 3 José Luis García-Giménez1– 3 1Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain; 2Instituto de Investigación Sanitaria INCLIVA, Valencia, Spain; 3Departamento de Fisiología, Facultad de Medicina y Odontología, Universitat de València, València, SpainCorrespondence: José Luis García-Giménez, Departamento de Fisiología, Facultad de Medicina y Odontología, Universitat de València, València, 46010, Spain, Tel +34 963 864 646, Email [email protected]: Circulating extracellular histones acquire relevance as cytotoxic mediators in sepsis. Extracellular histones act as damage-associated molecular patterns (DAMPs), which induce oxidative stress and NLRP3 inflammasome activation. Inflammasome mediates pyroptosis, a programmed cell death mechanism that produces inflammation. Despite evidence for inflammasome activation in immune cells during sepsis, it was unknown whether extracellular histones can produce endothelial inflammasomes activation.Methods: We used human umbilical vein endothelial cells (HUVEC) to explore the activation of pyroptosis, endothelial function and inflammation by extracellular histones. We evaluated pyroptosis by flow cytometry, caspase-1 activity assay, and gene and protein expression analysis by RT-qPCR and Western blot, respectively. The upstream molecular responses involved in pyroptosis activation by extracellular histones were validated by means of using antioxidant glutathione ethyl ester and NLRP3 inflammasome inhibitors. Finally, using mass spectrometry, we measured circulating histones in blood from critically-ill patients and demonstrated that circulating histone levels correlated with the expression of pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity.Results: We found that extracellular histones mediate the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how the hyperacetylation of extracellular histones or the use of antioxidants decreased pyroptosis. In addition, we showed that pyroptosis is a feasible process occurring in septic shock patients.Discussion: Circulating histone levels correlated with the expression of pro-inflammatory and pyroptosis-related cytokines, the release of endothelial adhesion factors and septic shock severity. We propose to block histone-mediated pyroptosis as a feasible therapeutic strategy in sepsis.Graphical Abstract: Keywords: sepsis, extracellular histones, histone acetylation, endothelium, inflammasome, NLRP

From clinical description, to in vitro and animal studies, and backward to patients: Oxidative stress and mitochondrial dysfunction in Fanconi anemia.

Fanconi anemia (FA) is a rare genetic disease associated with deficiencies in DNA repair pathways. A body of literature points to a pro-oxidant state in FA patients, along with evidence for oxidative stress (OS) in the FA phenotype reported by in vitro, molecular, and animal studies. A highlight arises from the detection of mitochondrial dysfunction (MDF) in FA cell lines of complementation groups A, C, D2, and G. As yet lacking, in vivo studies should focus on FA-associated MDF, which may help in the understanding of the mitochondrial basis of OS detected in cells and body fluids from FA patients. Beyond the in vitro and animal databases, the available analytical devices may prompt the direct observation of metabolic and mitochondrial alterations in FA patients. These studies should evaluate a set of MDF-related endpoints, to be related to OS endpoints. The working hypothesis is raised that, parallel to OS, nitrosative stress might be another, so far unexplored, hallmark of the FA phenotype. The expected results may shed light on the FA pathogenesis and might provide grounds for pilot chemoprevention trials using mitochondrial nutrients

Lymphocyte respiration in children with Trisomy 21

<p>Abstract</p> <p>Background</p> <p>This study measured lymphocyte mitochondrial O₂ consumption (cellular respiration) in children with trisomy 21.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells were isolated from whole blood of trisomy 21 and control children and these cells were immediately used to measure cellular respiration rate. [O₂] was determined as a function of time from the phosphorescence decay rates (1/τ) of Pd (II)-<it>meso</it>-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials containing lymphocytes and glucose as a respiratory substrate, [O₂] declined linearly with time, confirming the zero-order kinetics of O₂ conversion to H₂O by cytochrome oxidase. The rate of respiration (<it>k</it>, in μM O₂ min^-1), thus, was the negative of the slope of [O₂] <it>vs.</it> time. Cyanide inhibited O₂ consumption, confirming that oxidation occurred in the mitochondrial respiratory chain.</p> <p>Results</p> <p>For control children (age = 8.8 ± 5.6 years, n = 26), the mean (± SD) value of <it>k</it>_<it>c</it> (in μM O₂ per min per 10⁷ cells) was 1.36 ± 0.79 (coefficient of variation, Cv = 58%; median = 1.17; range = 0.60 to 3.12; -2SD = 0.61). For children with trisomy 21 (age = 7.2 ± 4.6 years, n = 26), the values of <it>k</it>_<it>c</it> were 0.82 ± 0.62 (Cv = 76%; median = 0.60; range = 0.20 to 2.80), <it>p</it><0.001. Similar results (<it>p</it><0.000) were obtained after excluding the five trisomy 21 children with elevated serum TSH (values >6.1 mU/L). Fourteen of 26 (54%) children with trisomy 21 had <it>k</it>_<it>c</it> values of 0.20 to 0.60 (i.e., <−2SD). The values of <it>k</it>_<it>c</it> positively correlated with body-mass index (BMI, <it>R</it> >0.302), serum creatinine (<it>R</it> >0.507), blood urea nitrogen (BUN, <it>R</it> >0.535) and albumin (<it>R</it> >0.446).</p> <p>Conclusions</p> <p>Children with trisomy 21 in this study have reduced lymphocyte bioenergetics. The clinical importance of this finding requires further studies.</p