Michael Zieger

Endothelial cell preservation at 10 degrees C minimizes catalytic iron, oxidative stress, and cold-induced injury.

There is growing evidence that oxidative stress plays an important role in mediating the injury induced by hypothermia during the preservation of cells and tissues for clinical or research use. In cardiovascular allografts, endothelial cell loss or injury may lead to impaired control of vascular permeability and tone, thrombosis, and inflammation. We hypothesized that hypothermia-induced damage to the endothelium is linked to increases in intracellular catalytic iron pools and oxidative stress. In this study, bovine aortic endothelial cells and cell culture methods were used to model the response of the endothelium of cardiovascular tissues to hypothermia. Confluent cells were stored at 0°C to 25°C and cell damage was measured by lipid peroxidation (LPO) and lactate dehydrogenase release. Varying the bleomycin-detectible iron (BDI) in cells modulated cold-induced LPO and cell injury. In untreated cells, injury was highest at 0°C and a minimum at 10°C. A similar temperature-dependent trend was found in BDI levels and cell plating efficiencies. Arrhenius plots of cell killing and iron accumulation rates showed biphasic temperature dependence, with minima at 10°C and matching activation energies above and below 10°C. These findings imply that the mechanisms underlying the hypothermic increase in catalytic iron, oxidative stress, and cell killing are the same and that preservation of the endothelium may be optimized at temperatures above those routinely used.

Proteomic analysis of endothelial cold-adaptation

BackgroundUnderstanding 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.ResultsCells 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.ConclusionsEndothelial 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.

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

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.

Endothelial cell fatty acid unsaturation mediates cold-induced oxidative stress.

Ultraprofound hypothermia (< 5°C) induces changes to cell membranes such as liquid‐to‐gel lipid transitions and oxidative stress that have a negative effect on membrane function and cell survival. We hypothesized that fatty acid substitution of endothelial cell lipids and alterations in their unsaturation would modify cell survival at 0°C, a temperature commonly used during storage and transportation of isolated cells or tissues and organs used in transplantation. Confluent bovine aortic endothelial cells were treated with 18‐carbon fatty acids (C18:0, C18:1n‐9, C18:2n‐6, or C18:3n‐3), C20:5n‐3 or C22:6n‐3 (DHA), and then stored at 0°C without fatty acid supplements. Storage of control cells caused the release of lactate dehydrogenase (LDH) and a threefold increase in lipid peroxidation (LPO) when compared to control cells not exposed to cold. Pre‐treating cells with C18:0 decreased the unsaturation of cell lipids and reduced LDH release at 0°C by 50%, but all mono‐ or poly‐unsaturated fatty acids increased injury in a concentration‐dependent manner and as the extent of fatty acid unsaturation increased. DHA‐treatment increased cell fatty acid unsaturation and caused maximal injury at 0°C, which was prevented by lipophilic antioxidants BHT or vitamin E, the iron chelator deferoxamine, and to a lesser extent by vitamin C. Furthermore, the cold‐induced increase in LPO was reduced by C18:0, vitamin E, or DFO but enhanced by DHA. In conclusion, the findings implicate iron catalyzed free radicals and LPO as a predominant mechanism of endothelial cell injury at 0°C, which may be reduced by increasing lipid saturation or treating cells with antioxidants. J. Cell. Biochem. 99: 784–796, 2006.

A paper-based in vitro model for on-chip investigation of the human respiratory system

Culturing cells at the air-liquid interface (ALI) is essential for creating functional in vitro models of lung tissues. We present the use of direct-patterned laser-treated hydrophobic paper as an effective semi-permeable membrane, ideal for ALI cell culture. The surface properties of the paper are modified through a selective CO2 laser-assisted treatment to create a unique porous substrate with hydrophilic regions that regulate fluid diffusion and cell attachment. To select the appropriate model, four promising hydrophobic films were compared with each other in terms of gas permeability and long-term strength in an aqueous environment (wet-strength). Among the investigated substrates, parchment paper showed the fastest rate of oxygen permeability (3 times more than conventional transwell cell culture membranes), with the least variation in its dry and wet tensile strengths (124 MPa and 58 MPa, remaining unchanged after 7 days of submersion in PBS).The final paper-based platform provides an ideal, robust, and inexpensive device for generating monolayers of lung epithelial cells on-chip in a high-throughput fashion for disease modelling and in vitro drug testing.

Distinct Effects of Adipose-derived Stem Cells and Adipocytes on Normal and Cancer Cell Hierarchy

Adipose-derived stem cells (ASC) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix composition and stiffness, migration, and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A, ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225, and MCF10A-overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly, ASCs promoted the self-renewal of all cell types except SUM225. ASC coculture or treatment with ASC conditioned media altered the number of CD49fhigh/EpCAMlow basal/stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells. Among multiple factors secreted by ASCs, IFNγ and hepatocyte growth factor (HGF) displayed unique actions on epithelial cell hierarchy. IFNγ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres, whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF, whereas adipocytes expressed higher levels of IFNγ. As luminal progenitor cells are believed to be prone for transformation, IFNγ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells. Implications: This study suggests that the ratio of ASCs to adipocytes influences cancer cell hierarchy, which may impact incidence and progression. Mol Cancer Res; 14(7); 660–71. ©2016 AACR.

A wireless strain sensor for wound monitoring with direct laser-defined patterning on a commercial dressing

Controlled mechanical strain or stress on a wound site can promote accelerated neovascularization and cellular proliferation for improved wound healing; however, these mechanical forces have not been properly quantified due to a lack of standardized technique. As a solution, we developed a wireless strain sensor on a commercial wound dressing. The sensor consists of a flexible antenna coil whose resonant frequency changes in response to applied strain. The frequency change of the sensor is observed to be a linear function of applied strain in the range of 0-35%, with an average sensitivity of 150 kHz/%strain and negligible hysteresis. The sensor is fabricated through a simple process that consist of defining a screen-printing mask directly over the wound dressing using laser machining. The fabrication technique can be scaled up for mass production using roll-to-roll methods.