Hypothermic preconditioning of endothelial cells attenuates cold-induced injury by a ferritin-dependent process

Methodist Research Institute, Indianapolis, IN 46202, USA.
Free Radical Biology and Medicine (Impact Factor: 5.74). 03/2009; 46(5):680-91. DOI: 10.1016/j.freeradbiomed.2008.12.004
Source: PubMed


Hypothermia for myocardial protection or storage of vascular grafts may damage the endothelium and impair vascular function upon reperfusion/rewarming. Catalytic iron pools and oxidative stress are important mediators of cold-induced endothelial injury. Because endothelial cells are highly adaptive, we hypothesized that hypothermic preconditioning (HPC) protects cells at 0 degrees C by a heme oxygenase-1 (HO-1) and ferritin-dependent mechanism. Storage of human coronary artery endothelial cells at 0 degrees C caused the release of lactate dehydrogenase, increases in bleomycin-detectible iron (BDI), and increases in the ratio of oxidized/reduced glutathione, signifying oxidative stress. Hypoxia increased injury at 0 degrees C but did not increase BDI or oxidative stress further. HPC at 25 degrees C for 15-72 h attenuated these changes by an amount achievable by pretreating cells with 10-20 microM deferoxamine, an iron chelator, and protected cell viability. Treating cells with hemin chloride at 37 degrees C transiently increased intracellular heme, HO-1, BDI, and ferritin. Elevated heme/iron sensitized cells to 0 degrees C but ferritin was protective. HPC increased iron maximally after 2 h at 25 degrees C and ferritin levels peaked after 15 h. HO-1 was not induced. When HPC-mediated increases in ferritin were blocked by deferoxamine, protection at 0 degrees C was diminished. We conclude that HPC-mediated endothelial protection from hypothermic injury is an iron- and ferritin-dependent process.

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    ABSTRACT: It is now well established that reactive oxygen species (ROS), reactive nitrogen species (RNS), and a basal level of oxidative stress are essential for cell survival. It is also well known that while severe oxidative stress often leads to widespread oxidative damage and cell death, a moderate level of oxidative stress, induced by a variety of stressors, can yield great beneficial effects on adaptive cellular responses to pathological challenges in aging and aging-associated disease tolerance such as ischemia tolerance. Here in this review, I term this moderate level of oxidative stress as positive oxidative stress, which usually involves imprinting molecular signatures on lipids and proteins via formation of lipid peroxidation by-products and protein oxidation adducts. As ROS/RNS are short-lived molecules, these molecular signatures can thus execute the ultimate function of ROS/RNS. Representative examples of lipid peroxidation products and protein oxidation adducts are presented to illustrate the role of positive oxidative stress in a variety of pathological settings, demonstrating that positive oxidative stress could be a valuable prophylactic and/or therapeutic approach targeting aging and aging-associated diseases.
    01/2014; 2(1). DOI:10.1016/j.redox.2014.01.002
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    • "In our studies of cold-adaptation, human coronary artery endothelial cells (HCAECs) cultured at 25°C become progressively more resistant over time to 0°C-injury and in particular to the oxidative stress induced by exposure to 0°C and rewarming [16]. The molecular basis of the adaptation remains largely unknown but the resulting protection at 0°C is due, in part, to the sequestration of catalytically active iron [16]. The protection may also be associated with an increase in intracellular glutathione, an important antioxidant and signaling molecule of the cell, at 25°C. "
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    ABSTRACT: Understanding how human cells in tissue culture adapt to hypothermia may aid in developing new clinical procedures for improved ischemic and hypothermic protection. Human coronary artery endothelial cells grown to confluence at 37°C and then transferred to 25°C become resistant over time to oxidative stress and injury induced by 0°C storage and rewarming. This protection correlates with an increase in intracellular glutathione at 25°C. To help understand the molecular basis of endothelial cold-adaptation, isolated proteins from cold-adapted (25°C/72 h) and pre-adapted cells were analyzed by quantitative proteomic methods and differentially expressed proteins were categorized using the DAVID Bioinformatics Resource. Cells adapted to 25°C expressed changes in the abundance of 219 unique proteins representing a broad range of categories such as translation, glycolysis, biosynthetic (anabolic) processes, NAD, cytoskeletal organization, RNA processing, oxidoreductase activity, response-to-stress and cell redox homeostasis. The number of proteins that decreased significantly with cold-adaptation exceeded the number that increased by 2:1. Almost half of the decreases were associated with protein metabolic processes and a third were related to anabolic processes including protein, DNA and fatty acid synthesis. Changes consistent with the suppression of cytoskeletal dynamics provided further evidence that cold-adapted cells are in an energy conserving state. Among the specific changes were increases in the abundance and activity of redox proteins glutathione S-transferase, thioredoxin and thioredoxin reductase, which correlated with a decrease in oxidative stress, an increase in protein glutathionylation, and a recovery of reduced protein thiols during rewarming from 0°C. Increases in S-adenosylhomocysteine hydrolase and nicotinamide phosphoribosyltransferase implicate a central role for the methionine-cysteine transulfuration pathway in increasing glutathione levels and the NAD salvage pathway in increasing the reducing capacity of cold-adapted cells. Endothelial adaptation to mild-moderate hypothermia down-regulates anabolic processes and increases the reducing capacity of cells to enhance their resistance to oxidation and injury associated with 0°C storage and rewarming. Inducing these characteristics in a clinical setting could potentially limit the damaging effects of energy insufficiency due to ischemia and prevent the disruption of integrated metabolism at low temperatures.
    BMC Genomics 12/2011; 12(1):630. DOI:10.1186/1471-2164-12-630 · 3.99 Impact Factor
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    ABSTRACT: Objectives: The effect of hypothermia on cardiomyocyte injury induced by oxidative stress remains unclear. The authors investigated the effects of hypothermia on apoptosis and mitochondrial dysfunction in cardiomyocytes exposed to oxidative stress. Methods: Cardiomyocytes (H9c2) derived from embryonic rat heart cell culture were exposed to either normothermic (37°C) or hypothermic (31°C) environments before undergoing oxidative stress via treatment with hydrogen peroxide (H2O2). The degree of apoptosis was determined by annexin V and terminal deoxynucleotidyl transferase (TUNEL) staining. The amount of reactive oxygen species (ROS) was compared after H 2O2 exposure between normo- and hypothermic-pretreated groups. Mitochondrial dysfunction in both groups was measured by differential reductase activity and transmembrane potential (ΔΨm). Results: Hydrogen peroxide induced significant apoptosis in both normothermic and hypothermic cardiomyocytes. Hypothermia ameliorated apoptosis as demonstrated by decreased annexin V staining (33 ± 1% vs. 49 ± 4%; p < 0.05) and TUNEL staining (27 ± 17% vs. 80 ±25%; p < 0.01). The amount of intracellular ROS increased after H2O2 treatment and was higher in the hypothermic group than that in the normothermic group (237.9 ± 31.0% vs. 146.6 ± 20.6%; p < 0.05). In the hypothermic group, compared with the normothermic group, after H2O2 treatment mitochondrial reductase activity was greater (72.0 ± 17.9% vs. 27.0 ± 13.3%; p < 0.01) and the mitochondria ΔΨm was higher (101.0 ± 22.6% vs. 69.7 ± 12.9%; p < 0.05). Pretreatment of cardiomyocytes with the antioxidant ascorbic acid diminished the hypothermia-induced increase in intracellular ROS and prevented the beneficial effects of hypothermia on apoptosis and mitochondrial function. Conclusions: Hypothermia at 31°C can protect cardiomyocytes against oxidative stress-induced injury by decreasing apoptosis and mitochondrial dysfunction through intracellular ROS-dependent pathways.
    Academic Emergency Medicine 09/2009; 16(9):872-80. DOI:10.1111/j.1553-2712.2009.00495.x · 2.01 Impact Factor
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