Antony and Cleopatra

Analysis done 199

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles ameliorate airway inflammation in a rat model of Chronic Obstructive Pulmonary Disease (COPD)

Background Chronic obstructive pulmonary disease (COPD) is an incurable and debilitating chronic disease characterized by progressive airflow limitation associated with abnormal levels of tissue inflammation. Therefore, stem cell-based approaches to tackle the condition are currently a focus of regenerative therapies for COPD. Extracellular vesicles (EVs) released by all cell types are crucially involved in paracrine, extracellular communication. Recent advances in the field suggest that stem cell-derived EVs possess a therapeutic potential which is comparable to the cells of their origin. Methods In this study, we assessed the potential anti-inflammatory effects of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived EVs in a rat model of COPD. EVs were isolated from hUC-MSCs and characterized by the transmission electron microscope, western blotting, and nanoparticle tracking analysis. As a model of COPD, male Sprague-Dawley rats were exposed to cigarette smoke for up to 12 weeks, followed by transplantation of hUC-MSCs or application of hUC-MSC-derived EVs. Lung tissue was subjected to histological analysis using haematoxylin and eosin staining, Alcian blue-periodic acid-Schiff (AB-PAS) staining, and immunofluorescence staining. Gene expression in the lung tissue was assessed using microarray analysis. Statistical analyses were performed using GraphPad Prism 7 version 7.0 (GraphPad Software, USA). Student’s t test was used to compare between 2 groups. Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA). Data presented as median ± standard deviation (SD). Results Both transplantation of hUC-MSCs and application of EVs resulted in a reduction of peribronchial and perivascular inflammation, alveolar septal thickening associated with mononuclear inflammation, and a decreased number of goblet cells. Moreover, hUC-MSCs and EVs ameliorated the loss of alveolar septa in the emphysematous lung of COPD rats and reduced the levels of NF-κB subunit p65 in the tissue. Subsequent microarray analysis revealed that both hUC-MSCs and EVs significantly regulate multiple pathways known to be associated with COPD. Conclusions In conclusion, we show that hUC-MSC-derived EVs effectively ameliorate by COPD-induced inflammation. Thus, EVs could serve as a new cell-free-based therapy for the treatment of COPD

High glucose and oxidative/nitrosative stress conditions induce apoptosis in retinal endothelial cells by a caspase-independent pathway

http://www.sciencedirect.com/science/article/B6WFD-4V94X19-2/2/49680e499c2d31bd74eb7ad7fc99a8c

Heme oxygenase-1 protects retinal endothelial cells against high glucose- and oxidative/nitrosative stress-induced toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions

Alterations in phospholipidomic profile in the brain of mouse model of depression induced by chronic unpredictable stress

Depression is a worldwide disability disease associated with high morbidity and has increased dramatically in the last few years. The differential diagnosis and the definition of an individualized therapy for depression are hampered by the absence of specific biomarkers. The aim of this study was to evaluate the phospholipidomic profile of the brain and myocardium in a mouse model of depression induced by chronic unpredictable stress (CUS). The lipidomic profile was evaluated by thin layer and liquid chromatography and mass spectrometry and lipid oxidation was estimated by FOX II assay. Antioxidant enzyme activity and the oxidized/reduced glutathione (GSH/GSSG) ratio were also evaluated. Results showed that chronic stress affects primarily the lipid profile of the brain, inducing an increase in lipid hydroperoxides, which was not detected in the myocardium. A significant decrease in phosphatidylinositol (PI) and in cardiolipin (CL) relative contents and also oxidation of CL and a significant increase of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were observed in the brain of mice after unpredictable chronic stress conditions. In the myocardium only an increase in PC content was observed. Nevertheless, both organs present a decreased GSH/GSSG ratio when compared to control groups, corroborating the occurrence of oxidative stress. The enzyme activities of catalase (CAT) and superoxide dismutase (SOD) were found to be decreased in the myocardium and increased in the brain, while glutathione reductase (GR) was decreased in the brain. Our results indicate that in a mouse model for studying depression induced by CUS, the modification of the expression of oxidative stress-related enzymes did not prevent lipid oxidation in organs, particularly in the brain. These observations suggest that depression has an impact on the brain lipidome and that further studies are needed to better understand lipids role in depression and to evaluate their potential as future biomarkers

Exposure to high glucose, H₂O₂ or NOC-18 increases HO-1 protein levels in retinal endothelial cells.

<p>Cells were exposed to 30 mM glucose (A), mannitol (24.5 mM+5.5 mM glucose) (B), 100 µM H₂O₂ (C) or 250 µM NOC-18 (D) for 1, 3, 6, 12 or 24 h. HO-1 immunoreactivity was analysed by Western blotting (A-D) and by immunocytochemistry (E). Representative Western blots for HO-1 are presented above the graphs. The intensity of the bands was determined by quantitative densitometric analysis. The images in (E) were acquired in a confocal microscope (600x magnification). The results represent the mean ± SEM of at least three independent experiments, and are expressed as percentage of control. *p<0.05, **p<0.01; significantly different from control as determined by one-way ANOVA followed by <i>Dunnett’s post test</i>.</p