Gary R. Smerdon

Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions

Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2 kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90 min (97.5% O(2) at 2.4 ATA; PO(2) ~237 kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.

Production of human gastric lipase in the fission yeast Schizosaccharomyces pombe.

A cDNA encoding human gastric lipase (hGL) has been expressed on multicopy plasmids in the fission yeast Schizosaccharomyces pombe (Sp). Active lipase is secreted from transformants containing the hGL cDNA under the control of either the Sp adh1 promoter (Padh1) or the plant cauliflower mosaic virus (CaMV) 35S promoter. Cell-wall-associated lipase activities are greatest in the early logarithmic growth phase and with Padh1. Western blot analysis indicates that a protein of identical molecular mass to natural hGL is secreted by Sp, although the major secreted product is of a higher molecular mass than either native hGL or recombinant hGL produced in the budding yeast Saccharomyces cerevisiae (Sc). Several distinct hGL are present within cells at all growth phases. Treatment of these proteins with endoglycosidase H gives rise to a single species equivalent in size to deglycosylated natural hGL, indicating that most of these are glycosylation intermediates. An hGL of similar molecular mass accumulates intracellularly in Sp when a modified version of cDNA is used which lacks the sequence encoding the natural secretory signal peptide. Production of hGL markedly slows the growth rate of Sp. The average copy number per cell of the plasmid expressing the hGL cDNA from the recombinant Padh1 is 2-3, as compared with 11-12 for the control plasmid.

Effects of hyperbaric oxygen treatment on antimicrobial function and apoptosis of differentiated HL-60 (neutrophil-like) cells.

AIMSnNeutrophil apoptosis is important in the resolution of inflammation in chronic wounds. Hyperbaric oxygen (HBO) therapy, an intermittent inhalation of 100% oxygen at greater than atmospheric pressure, appears to be an effective treatment for chronic wounds. The aim was to use HL-60 cells differentiated using all-trans retinoic acid (ATRA) (neutrophil-like cells) to test the hypothesis that an HBO-induced increase in antimicrobial activity might lead to an increase in apoptosis, thereby contributing to neutrophil clearance from chronic wounds.nnnMAIN METHODSnATRA differentiated HL-60 cells, an in vitro neutrophil model, were used to test the effects of normoxia, hypoxia (5% O2), hyperoxia (95% O2), hyperbaric normoxia (pressure) (8.8% O2 at 2.4 ATA) and HBO (97.9% O2 at 2.4 ATA) on antimicrobial function [NBT staining, superoxide and H2O2 production, and phagocytosis activity] and apoptosis (caspase 3/7 activity and morphological changes observed using SYBR Safe staining).nnnKEY FINDINGSnA single 90min HBO exposure caused an increase in the respiratory burst activity of neutrophil-like cells post exposure. Phagocytosis of Staphylococcus aureus was also increased. HBO pre-treatment had a pro-apoptotic effect, increasing caspase 3/7 activity and causing morphological changes associated with apoptosis.nnnSIGNIFICANCEnThe potential detrimental effect of enhanced antimicrobial activity induced by HBO may be offset by enhanced apoptosis. Both hyperoxia and pressure alone seemed to contribute to the HBO-induced increases in antimicrobial activity and apoptosis, although there was no consistent pattern. These data contribute to explaining the effectiveness of HBO in the treatment of chronic wounds.

Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor‐alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule‐1 and vascular cell adhesion molecule‐1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide–related protein modifications. The transnitrosating agent S‐nitroso‐L‐cysteine ethyl ester (600u2009μM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400W (10u2009μM) and HgCl2, which promotes the decomposition of S‐nitrosothiols (1u2009mM), reversed the effect of HBO, suggesting that S‐nitrosation may inhibit neutrophil‐endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S‐nitrosation.

Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe

Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid.

Safety and efficacy of hyperbaric oxygen therapy in chronic wound management: current evidence

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Chronic Wound Care Management and Research 2015:2 81–93 Chronic Wound Care Management and Research Dovepress

Hyperbaric oxygen treatment induces platelet aggregation and protein release, without altering expression of activation molecules

OBJECTIVESnTo investigate the effect of hyperbaric oxygen (HBO) on platelet physiology.nnnDESIGN AND METHODSnHuman platelets were exposed to HBO (97.7% O(2), balance CO(2) at 2.2 ata) or control (CON; 5% CO(2), balance air at 1 ata) for 90 min, and analyzed for aggregation, protein release, ()NO production, and activation.nnnRESULTSnHBO induced 29.8+/-3.0% of platelets to aggregate compared with CON (5.5+/-0.9%). Proteins observed to be released in greater abundance from HBO- compared with CON-treated platelets included 14-3-3 zeta and alpha-2-macroglobulin. Release of ()NO by platelets was unaffected following exposure to HBO, as was platelet activation as measured by surface expression of PECAM-1, CD62P and the activated form of alpha(IIB)beta(IIIa).nnnCONCLUSIONSnExposure to HBO induces both platelet aggregation and protein release. Further study will better define the precise mechanisms and effects of HBO on platelet activation.

Hyperbaric oxygen enhances neutrophil apoptosis and their clearance by monocyte-derived macrophages

Neutrophil apoptosis and clearance by macrophages are essential for wound healing. Evidence suggests that hyperbaric oxygen (HBO) exposure may enhance neutrophil apoptosis, but HBO effects leading to neutrophil clearance by macrophages are still unclear. In the current study, bovine neutrophils and monocyte-derived macrophages (MDMΦ) were co-cultured under HBO (97.9% O2, 2.1% CO2 at 2.4 atm absolute (ATA)) (1 atm = 101.325 kPa), hyperbaric normoxia (8.8% O2 at 2.4 ATA), normobaric hyperoxia (95% O2, 5% CO2), normoxia (air), and normobaric hypoxia (5% O2, 5% CO2). Phagocytosis of fresh and 22 h aged neutrophils by MDMΦ was increased after HBO pre-treatment, assessed using flow cytometry and light microscopy. Enhanced clearance of neutrophils was accompanied by an increase in H2O2 levels following HBO pre-treatment with upregulation of IL-10 (anti-inflammatory cytokine) mRNA expression in LPS-stimulated MDMΦ that had ingested aged neutrophils. TNF-α (pro-inflammatory cytokine) gene expression did not change in LPS-stimulated MDMΦ that had ingested fresh or aged neutrophils after HBO, pressure, and hyperoxia. These findings suggest that HBO-activated MDMΦ participate in the clearance of apoptotic cells. Uptake of neutrophils by MDMΦ exposed to HBO may contribute to resolution of inflammation, because HBO induced up-regulation of IL-10 mRNA expression.

The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species.

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

Abstract In vivo, mammalian cells reside in an environment of 0.5–10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment‐induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long‐term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long‐term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long‐term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress‐induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.