scholarly journals Enhanced mitophagy in bronchial fibroblasts from severe asthmatic patients

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242695
Author(s):  
Rakhee K. Ramakrishnan ◽  
Khuloud Bajbouj ◽  
Mahmood Y. Hachim ◽  
Andrea K. Mogas ◽  
Bassam Mahboub ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Severe Asthma ◽  
Metabolic Activity ◽  
Mitochondrial Membrane ◽  
Bioinformatics Analysis ◽  
Lysosomal Protease ◽  
Endogenous Expression ◽  
Severe Asthmatic ◽  
Pepstatin A

Background Sub-epithelial fibrosis is a characteristic feature of airway remodeling in asthma which correlates with disease severity. Current asthma medications are ineffective in treating fibrosis. In this study, we aimed to investigate the mitochondrial phenotype in fibroblasts isolated from airway biopsies of non-asthmatic and severe asthmatic subjects by examining mitophagy as a mechanism contributing to fibroblast persistence and thereby, fibrosis in severe asthma. Methods Bioinformatics analysis of publicly available transcriptomic data was performed to identify the top enriched pathways in asthmatic fibroblasts. Endogenous expression of mitophagy markers in severe asthmatic and non-asthmatic fibroblasts was determined using qRT-PCR, western blot and immunofluorescence. Mitophagy flux was examined by using lysosomal protease inhibitors, E64d and pepstatin A. Mitochondrial membrane potential and metabolic activity were also evaluated using JC-1 assay and MTT assay, respectively. Results Bioinformatics analysis revealed the enrichment of Pink/Parkin-mediated mitophagy in asthmatic fibroblasts compared to healthy controls. In severe asthmatic fibroblasts, the differential expression of mitophagy genes, PINK1 and PRKN, was accompanied by the accumulation of PINK1, Parkin and other mitophagy proteins at baseline. The further accumulation of endogenous LC3BII, p62 and PINK1 in the presence of E64d and pepstatin A in severe asthmatic fibroblasts reinforced their enhanced mitophagy flux. Significantly reduced mitochondrial membrane potential and metabolic activity were also demonstrated at baseline confirming the impairment in mitochondrial function in severe asthmatic fibroblasts. Interestingly, these fibroblasts displayed neither an apoptotic nor senescent phenotype but a pro-fibrotic phenotype with an adaptive survival mechanism triggered by increased AMPKα phosphorylation and mitochondrial biogenesis. Conclusions Our results demonstrated a role for mitophagy in the pathogenesis of severe asthma where the enhanced turnover of damaged mitochondria may contribute to fibrosis in severe asthma by promoting the persistence and pro-fibrotic phenotype of fibroblasts.

scholarly journals Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease

2020 ◽  
Vol 117 (37) ◽  
pp. 23113-23124
Author(s):  
Helena M. Viola ◽  
Ashay A. Shah ◽  
Victoria P. A. Johnstone ◽  
Henrietta Cserne Szappanos ◽  
Mark P. Hodson ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Hypertrophic Cardiomyopathy ◽  
Mitochondrial Membrane Potential ◽  
Metabolic Activity ◽  
Murine Model ◽  
Mitochondrial Membrane ◽  
Cardiac Myocyte ◽  
Human Condition ◽  
Preventative Therapy

Currently there is an unmet need for treatments that can prevent hypertrophic cardiomyopathy (HCM). Using a murine model we previously identified that HCM causing cardiac troponin I mutation Gly203Ser (cTnI-G203S) is associated with increased mitochondrial metabolic activity, consistent with the human condition. These alterations precede development of the cardiomyopathy. Here we examine the efficacy of in vivo treatment of cTnI-G203S mice with a peptide derived against the α-interaction domain of the cardiac L-type calcium channel (AID-TAT) on restoring mitochondrial metabolic activity, and preventing HCM. cTnI-G203S or age-matched wt mice were treated with active or inactive AID-TAT. Following treatment, targeted metabolomics was utilized to evaluate myocardial substrate metabolism. Cardiac myocyte mitochondrial metabolic activity was assessed as alterations in mitochondrial membrane potential and flavoprotein oxidation. Cardiac morphology and function were examined using echocardiography. Cardiac uptake was assessed using an in vivo multispectral imaging system. We identified alterations in six biochemical intermediates in cTnI-G203S hearts consistent with increased anaplerosis. We also reveal that AID-TAT treatment of precardiomyopathic cTnI-G203S mice, but not mice with established cardiomyopathy, restored cardiac myocyte mitochondrial membrane potential and flavoprotein oxidation, and prevented myocardial hypertrophy. Importantly, AID-TAT was rapidly targeted to the heart, and not retained by the liver or kidneys. Overall, we identify biomarkers of HCM resulting from the cTnI mutation Gly203Ser, and present a safe, preventative therapy for associated cardiomyopathy. Utilizing AID-TAT to modulate cardiac metabolic activity may be beneficial in preventing HCM in “at risk” patients with identified Gly203Ser gene mutations.

scholarly journals Egr2 upregulation induced mitochondrial iron overload implicating in sevoflurane-induced cognitive deficits in developing mice

2020 ◽  
Author(s):  
Piao Zhang ◽  
Yeru Chen ◽  
Shuxia Zhang ◽  
Man Fang ◽  
Xianyi Lin ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cognitive Function ◽  
Mitochondrial Dysfunction ◽  
Prussian Blue ◽  
Mitochondrial Membrane Potential ◽  
Cognitive Deficits ◽  
Mitochondrial Membrane ◽  
Bioinformatics Analysis ◽  
Blue Staining ◽  
Behavioral Tests

Abstract Background Sevoflurane inhalation initiated cognitive deficits implicated in mitochondrial dysfunction and synaptogenesis impairment. Bioinformatics analysis indicated that Egr2 may play a crucial role in maintaining cognitive function. Therefore, we attempted to clarify the potential mechanism regarding Egr2 expression and cognitive deficits induced by sevoflurane administration.Methods Animals received sevoflurane anesthesia, and the behavioral tests including Morris water maze, novel object recognition test and trace fear conditioning were performed. Then, the immunofluorescent staining was employed to detect the effect of sevoflurane inhalation in hippocampal neurons. Meanwhile, bioinformatics analysis was implemented, and the level of lipid peroxidation, mitochondrial membrane potential, morphology and membrane permeability, and cytoplasm calcium levels were investigated after Egr2 interference by using JC-1 probe, MitoTracker staining, Mitochondrial permeability transition pore (mPTP) assay, and Fluo calcium indicators, respectively. Additionally, Prussian blue staining was used to evaluate the iron content.Results The behavioral tests indicated that the cognitive function was significantly attenuated after sevoflurane administration. The Golgi-Cox staining displayed that the dendritic length, density and nodes were significantly reduced following sevoflurane inhalation. The bioinformatics analysis showed that sevoflurane administration results in the Egr2 expression upregulation. Additionally, the results suggested that sevoflurane administration elevated the cytoplasm calcium levels, reduced the mitochondrial membrane potential and triggered the opening of mPTP. Prussian blue staining showed that the iron deposition was apparently increased. However, Egr2 level downregulation partly reversed these above changes. Moreover, the behavioral performance was effectively improved after deferiprone (DFP) administration.Conclusion These findings demonstrated that sevoflurane administration elicited mitochondrial dysfunction and iron dyshomeostasis, and eventually resulted in cognitive impairments, whereas suppressing Egr2 expression partly improved this pathological process.

scholarly journals MiR-4673 Modulates Paclitaxel-Induced Oxidative Stress and Loss of Mitochondrial Membrane Potential by Targeting 8-Oxoguanine-DNA Glycosylase-1

2017 ◽  
Vol 42 (3) ◽  
pp. 889-900 ◽  
Author(s):  
Hai-Li Huang ◽  
Ya-Peng Shi ◽  
Hui-Juan He ◽  
Ya-Hong Wang ◽  
Ting Chen ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Bioinformatics Analysis ◽  
Dna Glycosylase ◽  
Ros Generation ◽  
Oxygen Species ◽  
Induced Apoptosis

Background: Our previous study identified a novel microRNA, miR-4673, which is upregulated in A549 cells exposed to paclitaxel (PTX). In this study, we investigated the role of miR-4673 in PTX-induced cytotoxicity. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, apoptosis assay, 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide (JC-1) staining and 2’,7’-Dichlorofluorescein (DCFH) staining were used to evaluate cell viability, apoptosis, mitochondrial membrane potential (MMP) loss and reactive oxygen species (ROS) generation in A549 and H1299 cells. Bioinformatics analysis and Luciferase reporter assay were used to explore whether 8-oxoguanine-DNA glycosylase-1 (OGG1) is a target gene of miR-4673. Results: Enforced expression of miR-4673 decreased cell viability and increased PTX-induced apoptosis, MMP loss and reactive oxygen species (ROS) generation in A549 and H1299 cells. Bioinformatics analysis, which was used to identify potential target of miR-4673, revealed a binding site of miR-4673 in 3’UTR of OGG1. Luciferase reporters assays showed that miR-4673 specifically binds to ‘CUGUUGA’ in 3’UTR of OGG1. Enforced expression of miR-4673 decreased accumulation of OGG1. In addition, silencing OGG1 enhanced inhibitory effects of PTX on apoptosis, MMP loss and ROS generation, which is similar to effects of miR-4673. Moreover, enforced expression of OGG1 compromised promoting effects of miR-4673 on PTX-induced apoptosis, MMP loss and ROS generation. Conclusion: miR-4673 modulates PTX-induced apoptosis, MMP loss and ROS generation by targeting OGG1.

scholarly journals Differential effects of cisplatin on cybrid cells with varying mitochondrial DNA haplogroups

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9908
Author(s):  
Sina Abedi ◽  
Gregory Yung ◽  
Shari R. Atilano ◽  
Kunal Thaker ◽  
Steven Chang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Side Effects ◽  
Mitochondrial Membrane Potential ◽  
Metabolic Activity ◽  
Mitochondrial Membrane ◽  
Nucleotide Polymorphisms ◽  
Mt Dna ◽  
Expression Levels ◽  
Ethnic Populations ◽  
Cytoplasmic Hybrids

Background Drug therapy yields different results depending on its recipient population. Cisplatin, a commonly used chemotherapeutic agent, causes different levels of resistance and side effects for different patients, but the mechanism(s) are presently unknown. It has been assumed that this variation is a consequence of differences in nuclear (n) DNA, epigenetics, or some external factor(s). There is accumulating evidence that an individual’s mitochondrial (mt) DNA may play a role in their response to medications. Variations within mtDNA can be observed, and an individual’s mtDNA can be categorized into haplogroups that are defined by accumulations of single nucleotide polymorphisms (SNPs) representing different ethnic populations. Methods The present study was conducted on transmitochondrial cytoplasmic hybrids (cybrids) that possess different maternal-origin haplogroup mtDNA from African (L), Hispanic [A+B], or Asian (D) backgrounds. Cybrids were created by fusing Rho0 ARPE-19 cells (lacking mtDNA) with platelets, which contain numerous mitochondria but no nuclei. These cybrid cells were cultured to passage five, treated with cisplatin, incubated for 48 h, then analyzed for cell metabolic activity (tetrazolium dye (MTT) assay), mitochondrial membrane potential (JC-1 assay), cytotoxicity (lactate dehydrogenase (LDH) assay), and gene expression levels for ALK, BRCA1, EGFR, and ERBB2/HER2. Results Results indicated that untreated cybrids with varying mtDNA haplogroups had similar relative metabolic activity before cisplatin treatment. When treated with cisplatin, (1) the decline in metabolic activity was greatest in L (27.4%, p < 0.012) < D (24.86%, p = 0.0001) and [A+B] cybrids (24.67%, p = 0.0285) compared to untreated cybrids; (2) mitochondrial membrane potential remained unchanged in all cybrids (3) LDH production varied between cybrids (L >[A+B], p = 0.0270). (4) The expression levels decreased for ALK in L (p < 0.0001) and [A+B] (p = 0.0001) cybrids but not in D cybrids (p = 0.285); and decreased for EGFR in [A+B] cybrids (p = 0.0246) compared to untreated cybrids. Conclusion Our findings suggest that an individual’s mtDNA background may be associated with variations in their response to cisplatin treatment, thereby affecting the efficiency and the severity of side effects from the treatment.

scholarly journals Effects of Reduced Graphene Oxides on Apoptosis and Cell Cycle of Glioblastoma Multiforme

2018 ◽  
Vol 19 (12) ◽  
pp. 3939 ◽  
Author(s):  
Jaroslaw Szczepaniak ◽  
Barbara Strojny ◽  
Ewa Sawosz Chwalibog ◽  
Slawomir Jaworski ◽  
Joanna Jagiello ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
Glioblastoma Multiforme ◽  
Mitochondrial Membrane Potential ◽  
Metabolic Activity ◽  
Mitochondrial Membrane ◽  
Graphene Oxides ◽  
Graphene Derivatives ◽  
Electron Clouds

Graphene (GN) and its derivatives (rGOs) show anticancer properties in glioblastoma multiforme (GBM) cells in vitro and in tumors in vivo. We compared the anti-tumor effects of rGOs with different oxygen contents with those of GN, and determined the characteristics of rGOs useful in anti-glioblastoma therapy using the U87 glioblastoma line. GN/ExF, rGO/Term, rGO/ATS, and rGO/TUD were structurally analysed via transmission electron microscopy, Raman spectroscopy, FTIR, and AFM. Zeta potential, oxygen content, and electrical resistance were determined. We analyzed the viability, metabolic activity, apoptosis, mitochondrial membrane potential, and cell cycle. Caspase- and mitochondrial-dependent apoptotic pathways were investigated by analyzing gene expression. rGO/TUD induced the greatest decrease in the metabolic activity of U87 cells. rGO/Term induced the highest level of apoptosis compared with that induced by GN/ExF. rGO/ATS induced a greater decrease in mitochondrial membrane potential than GN/ExF. No significant changes were observed in the cytometric study of the cell cycle. The effectiveness of these graphene derivatives was related to the presence of oxygen-containing functional groups and electron clouds. Their cytotoxicity mechanism may involve electron clouds, which are smaller in rGOs, decreasing their cytotoxic effect. Overall, cytotoxic activity involved depolarization of the mitochondrial membrane potential and the induction of apoptosis in U87 glioblastoma cells.

Sphingosine-induced apoptosis is dependent on lysosomal proteases

2001 ◽  
Vol 359 (2) ◽  
pp. 335-343 ◽  
Author(s):  
Katarina KÅGEDAL ◽  
Ming ZHAO ◽  
Iréne SVENSSON ◽  
Ulf T. BRUNK
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Cysteine Proteases ◽  
Caspase Activation ◽  
Lysosomal Hydrolases ◽  
Lysosomal Protease ◽  
General Caspase Inhibitor ◽  
Pre Treatment ◽  
Induced Apoptosis

We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester (‘E-64d’), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (‘Z-VAD-FMK’), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

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