Assessing Mitochondrial Redox Status by Flow Cytometric Methods: Vascular Response to Fluid Shear Stress

Li, Rongsong; Jen, Nelson; Yu, Fei; Hsiai, Tzung K.

doi:10.1002/0471142956.cy0937s58

Cited by 18 publications

(18 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mitochondrial superoxide formation was determined by the uptake of MitoSoxÔ Red reagent (Invitrogen) in live cells, as previously described [36]. Briefly, cells were plated at a density of 10,000 cells/cm 2 in six-well plates, with or without EET-A, 10 mM, overnight and then incubated with Hoechst nuclear stain (5 mg/mL) for 30 min in complete medium.…”

Section: Flow Cytometer Measurement Of Mitosoxmentioning

confidence: 99%

Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function

Waldman

Bellner

Vanella

et al. 2016

Stem Cells and Development

View full text Add to dashboard Cite

Epoxyeicosatrienoic acid (EET) contributes to browning of white adipose stem cells to ameliorate obesity/ diabetes and insulin resistance. In the current study, we show that EET altered preadipocyte function, enhanced peroxisome proliferation-activated receptor g coactivator a (PGC-1a) expression, and increased mitochondrial function in the 3T3-L1 preadipocyte subjected to adipogenesis. Cells treated with EET resulted in an increase, P < 0.05, in PGC-1a and a decrease in mitochondria-derived ROS (MitoSox), P < 0.05. The EET increase in heme oxygenase-1 (HO-1) levels is dependent on activation of PGC-1a as cells deficient in PGC-1a (PGC-1a knockout adipocyte cell) have an impaired ability to express HO-1, P < 0.02. Additionally, adipocytes treated with EET exhibited an increase in mitochondrial superoxide dismutase (SOD) in a PGC-1a-dependent manner, P < 0.05. The increase in PGC-1a was associated with an increase in b-catenin, P < 0.05, adiponectin expression, P < 0.05, and lipid accumulation, P < 0.02. EET decreased heme levels and mitochondria-derived ROS (MitoSox), P < 0.05, compared to adipocytes that were untreated. EET also decreased mesoderm-specific transcript (MEST) mRNA and protein levels (P < 0.05). Adipocyte secretion of EET act in an autocrine/ paracrine manner to increase PGC-1a is required for activation of HO-1 expression. This is the first study to dissect the mechanism by which the antiadipogenic and anti-inflammatory lipid, EET, induces the PGC-1a signaling cascade and reprograms the adipocyte phenotype by regulating mitochondrial function and HO-1 expression, leading to an increase in healthy, that is, small, adipocytes and a decrease in adipocyte enlargement and terminal differentiation. This is manifested by an increase in mitochondrial function and an increase in the canonical Wnt signaling cascade during adipocyte proliferation and terminal differentiation.

show abstract

Section: Flow Cytometer Measurement Of Mitosoxmentioning

confidence: 99%

Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function

Waldman

Bellner

Vanella

et al. 2016

Stem Cells and Development

View full text Add to dashboard Cite

show abstract

“…Mitochondrial superoxide (mtO 2 -) production was assessed by staining with MitoSOX Red, as previously described (73,74), and was quantified using flow cytometry as previously described (52).…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis

Jen

et al. 2015

Antioxidants & Redox Signaling

Self Cite

View full text Add to dashboard Cite

Aim: Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Autophagy is tightly regulated in intracellular bulk degradation/recycling system for maintaining cellular homeostasis. We postulated that disturbed flow modulates autophagy with an implication in mitochondrial superoxide (mtO 2 -) production. Results: In the disturbed flow or oscillatory shear stress (OSS)-exposed aortic arch, we observed prominent staining of p62, a reverse marker of autophagic flux, whereas in the pulsatile shear stress (PSS)-exposed descending aorta, p62 was attenuated. OSS significantly increased (i) microtubuleassociated protein light chain 3 (LC3) II to I ratios in human aortic endothelial cells, (ii) autophagosome formation as quantified by green fluorescent protein (GFP)-LC3 dots per cell, and (iii) p62 protein levels, whereas manganese superoxide dismutase (MnSOD) overexpression by recombinant adenovirus, N-acetyl cysteine treatment, or c-Jun N-terminal kinase ( JNK) inhibition reduced OSS-mediated LC3-II/LC3-I ratios and mitochondrial DNA damage. Introducing bafilomycin to Earle's balanced salt solution or to OSS condition incrementally increased both LC3-II/LC3-I ratios and p62 levels, implicating impaired autophagic flux. In the OSS-exposed aortic arch, both anti-phospho-JNK and anti-8-hydroxy-2¢-deoxyguanosine (8-OHdG) staining for DNA damage were prominent, whereas in the PSS-exposed descending aorta, the staining was nearly absent. Knockdown of ATG5 with siRNA increased OSS-mediated mtO 2 -, whereas starvation or rapamycin-induced autophagy reduced OSS-mediated mtO 2 -, mitochondrial respiration, and complex II activity. Innovation: Disturbed flow-mediated oxidative stress and JNK activation induce autophagy. Conclusion: OSS impairs autophagic flux to interfere with mitochondrial homeostasis.

show abstract

“…The fold change in mean florescence intensity both of SLE and control samples were measured. 19 ÁÉm Mitochondrial oxidant generation is regulated by Ácm, which is a marker for ROS-mediated apoptosis. The JC-1 MitoScreen assay (BD Biosciences) was used to assess Ácm.…”

Section: Mitochondrial Oxidative Stressmentioning

confidence: 99%

Role of altered mitochondria functions in the pathogenesis of systemic lupus erythematosus

Leishangthem

Sharma

Bhatnagar

2015

Lupus

View full text Add to dashboard Cite

Mitochondria, main producers of reactive-oxygen species (ROS), were studied to examine their role in the pathogenesis of systemic lupus erythematosus (SLE). PBMCs and mitochondria were isolated from SLE patients and healthy volunteers for various parameters. Mitochondrial ROS, swelling, hyperpolarization and levels of cytochrome c, caspase3 in the cells were assessed by flow cytometry. ROS was significantly increased in SLE patients (SLE vs controls: 1.83 ± 1.03 vs 1.10 ± 0.35; p < 0.0001). Depolarized state of mitochondria was greater in patients (SLE vs controls: 7.10 ± 5.50% vs 2.5 ± 1.8%; p < 0.05). Mitochondria swelling was found to be significantly altered in patients (SLE vs controls: 112.65 ± 36.56 vs 60.49 ± 20.69; p < 0.001). Expression of cytochrome c and caspase 3 (SLE vs controls: 1.37 ± 0.37% vs 1.01 ± 0.03%; 1.57 ± 0.46% vs 1.06 ± 0.07%; p < 0.05) respectively was found to be significantly increased in SLE. Further, the enzymatic activity of mitochondrial complex was assessed in isolated mitochondria. A significant decrease in activity of Complex I (SLE vs controls: 11.79 ± 3.18 vs 15.10 ± 6.38 nmol NADH oxidized/min/mg protein, p < 0.05); Complex IV (SLE vs control: 9.41 ± 5.16 vs 13.56 ± 5.92 nmol cytochrome c oxidized/min/mg protein, p < 0.05) and Complex V (SLE vs controls: 4.85 ± 1.39 vs 6.17 ± 2.02 nmol ATP hydrolyzed/min/mg protein, p < 0.05) was found in SLE patients in comparison to healthy controls. However, Complex II did not show significant variation in either group (SLE vs controls: 42.2 ± 28.6 vs 61.71 ± 42.3 nmol succinate oxidized/min/mg protein; ns). The decrease in enzyme activities of mitochondrial Complexes I, IV and V on one hand and ROS, hyperpolarization and apoptosis on the other points toward a possible role of mitochondria in the pathogenesis of lupus.

show abstract

Assessing Mitochondrial Redox Status by Flow Cytometric Methods: Vascular Response to Fluid Shear Stress

Cited by 18 publications

References 51 publications

Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function

Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function

Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis

Role of altered mitochondria functions in the pathogenesis of systemic lupus erythematosus

Contact Info

Product

Resources

About