Hypoxia relieves X-ray-induced delayed effects in normal human embryo cells

We studied the effect of hypoxia on X-ray-induced delayed effects in normal human embryo cells to elucidate the role of oxidative stress in the susceptibility of cells to induction of genetic instability by radiation. We examined X-ray-induced delayed cell death, giant cell formation, and chromosome aberrations under normally oxygenated (20%) and hypoxic (2%) conditions at 28-38 population doublings postirradiation. The results revealed that hypoxia reduced the X-ray-induced delayed effects, suggesting that radiation enhances cellular oxidative stress, which plays a significant role in determining the susceptibility of irradiated cells to genetic instability. The present study emphasizes the biological significance of epigenetic effects, such as oxygen tension, as well as direct DNA damage in the induction of genetic instability by radiation.     

Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals?

The reactive oxygen species that are generated by mitochondrial respiration, including hydrogen peroxide (H2O2), are potent inducers of oxidative damage and mediators of ageing. It is not clear, however, whether oxidative stress is the result of a genetic programme or the by-product of physiological processes. Recent findings demonstrate that a fraction of mitochondrial H2O2, produced by a specialized enzyme as a signalling molecule in the pathway of apoptosis, induces intracellular oxidative stress and accelerates ageing. We propose that genes that control H2O2 production are selected determinants of lifespan.     

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

Vascular superoxide and hydrogen peroxide production and oxidative stress resistance in two closely related rodent species with disparate longevity

Vascular aging is characterized by increased oxidative stress, impaired nitric oxide (NO) bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower production of reactive oxygen species (ROS) and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), that show a more than twofold difference in maximum lifespan potential (MLSP = 8 and 3.5 years, respectively). We compared interspecies differences in endothelial superoxide (O2-) and hydrogen peroxide (H2O2) production, NAD(P)H oxidase activity, mitochondrial ROS generation, expression of pro- and antioxidant enzymes, NO production, and resistance to oxidative stress-induced apoptosis. In aortas of P. leucopus, NAD(P)H oxidase expression and activity, endothelial and H2O2 production, and ROS generation by mitochondria were less than in mouse vessels. In P. leucopus, there was a more abundant expression of catalase, glutathione peroxidase 1 and hemeoxygenase-1, whereas expression of Cu/Zn-SOD and Mn-SOD was similar in both species. NO production and endothelial nitric oxide synthase expression was greater in P. leucopus. In mouse aortas, treatment with oxidized low-density lipoprotein (oxLDL) elicited substantial oxidative stress, endothelial dysfunction and endothelial apoptosis (assessed by TUNEL assay, DNA fragmentation and caspase 3 activity assays). According to our prediction, vessels of P. leucopus were more resistant to the proapoptotic effects of oxidative stressors (oxLDL and H2O2). Primary fibroblasts from P. leucopus also exhibited less H2O2-induced DNA damage (comet assay) than mouse cells. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accords with the prediction of the oxidative stress hypothesis of aging.     

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.     

Low oxygen tension alleviates oxidative damage and delays cellular senescence in G6PD-deficient cells

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are under increased oxidative stress and undergo premature cellular senescence. The present study demonstrates that G6PD-deficient cells cultured under 3% oxygen concentration had an extended replicative lifespan, as compared with those cultured under atmospheric oxygen level. This was accompanied by a reduction in the number of senescence-associated β-galactosidase (SA-β-Gal) positive and morphologically senile cells at comparable population doubling levels (PDL). Concomitant with the extension of lifespan was decreased production of reactive oxygen species. Additionally, lifespan extension was paralleled by the greatly abated formation of such oxidative damage markers as 8-hydroxy-deoxyguanosine (8-OHdG) as well as the oxidized and cross-linked proteins. Moreover, the mitochondrial mass increased, but the mitochondrial membrane potential ΔΨm decreased in cells upon serial propagation. These changes were inhibited by lowering the oxygen tension. Our findings provide additional support to the notion that oxidative damage contributes to replicative senescence of G6PD-deficient cells and reduction of oxidative damage by lowering oxygen tension can delay the onset of cellular senescence.     

Alpha-tocopherol ameliorates cypermethrin-induced toxicity and oxidative stress in the nematode Caenorhabdtis elegans

Oxidative stress and other effects induced by cypermethrin (CYP, 15 mM) and their amelioration by alpha-tocopherol (400 microM) was studied in the nematode Caenorhabditis elegans. The worms exposed for 4 h to CYP showed increased levels of reactive oxygen species (46%), H2O2 (37%) and protein carbonyls (29%), accompanied by decreased lifespan and brood size. However, exposure to both CYP and alpha-tocopherol resulted in diminution of above alterations with the worms exhibiting relatively lower levels of ROS (30%), H2O2 (15%), protein carbonyls (14%), altered antioxidant enzyme activities and normal lifespan and brood size. The results suggest that CYP induces oxidative stress in C. elegans and the strategy of intervention with alpha-tocopherol could be exploited to offset this induced oxidative stress.     

Trichostatin a modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow‐mesenchymal stem cells

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow‐mesenchymal stem cells (hBM‐MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H₂O₂), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1–5 μM TSA. Cell protective effects against oxidative stress were significantly increased in TSA‐MSCs after treatment with low doses of TSA (50–500 nM) and decreased with high doses of TSA (5–10 μM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress‐induced MSCs demonstrated that expression of phosphorylated‐FOXO1 and phosphorylated‐SOD2 decreased in H₂O₂‐treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM‐MSCs against oxidative stress. Copyright © 2014 John Wiley & Sons, Ltd.     

Low oxygen tension alleviates oxidative damage and delays cellular senescence in G6PD-deficient cells

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are under increased oxidative stress and undergo premature cellular senescence. The present study demonstrates that G6PD-deficient cells cultured under 3% oxygen concentration had an extended replicative lifespan, as compared with those cultured under atmospheric oxygen level. This was accompanied by a reduction in the number of senescence-associated β-galactosidase (SA-β-Gal) positive and morphologically senile cells at comparable population doubling levels (PDL). Concomitant with the extension of lifespan was decreased production of reactive oxygen species. Additionally, lifespan extension was paralleled by the greatly abated formation of such oxidative damage markers as 8-hydroxy-deoxyguanosine (8-OHdG) as well as the oxidized and cross-linked proteins. Moreover, the mitochondrial mass increased, but the mitochondrial membrane potential ΔΨm decreased in cells upon serial propagation. These changes were inhibited by lowering the oxygen tension. Our findings provide additional support to the notion that oxidative damage contributes to replicative senescence of G6PD-deficient cells and reduction of oxidative damage by lowering oxygen tension can delay the onset of cellular senescence.     

Melanin as a target for melanoma chemotherapy: pro-oxidant effect of oxygen and metals on melanoma viability

Melanoma cells have a poor ability to mediate oxidative stress, which may be attributed to constitutive abnormalities in their melanosomes. We hypothesize that disorganization of the melanosomes will allow chemical targeting of the melanin within. Chemical studies show that under oxidative conditions, synthetic melanins demonstrate increased metal affinity and a susceptibility to redox cycling with oxygen to form reactive oxygen species. The electron paramagnetic resonance (EPR)-active 5,5'-dimethyl-pyrollidine N-oxide spin adduct was used to show that binding of divalent Zn or Cu to melanin induces a pro-oxidant response under oxygen, generating superoxide and hydroxyl radicals. A similar pro-oxidant behaviour is seen in melanoma cell lines under external peroxide stress. Melanoma cultures grown under 95% O2/5% CO2 atmospheres show markedly reduced viability as compared with normal melanocytes. Cu- and Zn-dithiocarbamate complexes, which induce passive uptake of the metal ions into cells, show significant antimelanoma activity. The antimelanoma effect of metal- and oxygen-induced stress appears additive rather than synergistic; both treatments are shown to be significantly less toxic to melanocytes.     

Regulation of chronological aging in Schizosaccharomyces pombe by the protein kinases Pka1 and Sck2

Budding yeast shows a progressive decline in viability after entering stationary phase, a phenomenon known as chronological aging. We show here that the fission yeast Schizosaccharomyces pombe also undergoes chronological aging and that the process is regulated by genes controlling two related nutrient signalling pathways. The first pathway includes the serine/threonine cAMP-activated protein kinase Pka1 and the second pathway comprises the serine/threonine kinase Sck2, a homologue of Saccharomyces cerevisiae SCH9. A double mutant for pka1 and sck2 displayed an additive effect on prolonging the fission yeast lifespan, suggesting that these genes regulate related but independent pathways. These long-lived mutants also accumulated less reactive oxygen species and had a delayed initiation of apoptosis compared with wild-type cells. We also found that strains carrying pka1 deletion but not those with sck2 deletion gained resistance to oxidative stress due to exposure to H(2)O(2) or menadione. On the other hand, the additional increase in lifespan shown by the Deltapka1Deltasck2 double-mutant strain correlated with an increased resistance to both oxidative stress and heat shock. These results underscore the importance of nutrient signalling pathways and reactive oxygen species on organismal lifespan and establish S. pombe as a new model organism to study the molecular mechanisms underlying aging.     

Inhibition of activated/memory (CD45RO(+)) T cells by oxidative stress associated with block of NF-kappaB activation

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-gamma release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO(+)) T cells and correlated with a block in NF-kappaB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-gamma, TNF-alpha, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO(+), T cells found in the tumor milieu are functionally suppressed.     

Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions

Here, we enriched a human cell population from adipose tissue that exhibited both mesenchymal plasticity, self-renewal capacity, and a cell-surface marker profile indistinguishable from that of bone marrow-derived mesenchymal stem cells. In addition to adipogenic and osteogenic differentiation, these adipose-derived stem cells displayed skeletal myogenic potential when co-cultured with mouse skeletal myocytes in reduced serum conditions. Physical incorporation of stem cells into multinucleated skeletal myotubes was determined by genetic lineage tracing, whereas human-specific antibody staining was employed to demonstrate functional contribution of the stem cells to a myogenic lineage. To investigate the effects of hypoxia, cells were maintained and differentiated at 2% O(2). In contrast with reports on bone marrow-derived stem cells, both osteogenic and adipogenic differentiation were significantly attenuated. In summary, the relative accessibility of adipose-derived mesenchymal stem cells from human donors provides opportunity for molecular investigation of mechanistic dysfunction in disease settings and may introduce new prospects for cell-based therapy.     

Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts

Chewing betel quid (BQ) is a popular habit worldwide. A causal association between BQ chewing and oral cancer has been well documented. Emerging evidence indicates that sustained exposure to stress induces epigenetic reprogramming of some mammalian cells and increases the mutation rate to accelerate adaptation to stressful environments. In this study, we first confirmed that 24-h treatment with areca nut extracts (ANE; a major component of BQ) at doses over 40 microg/ml induced mutations at the hypoxanthine phosphoribisyltransferase (HPRT) locus in human keratinocytes (HaCaT cells). We then investigated whether the stress of long-term exposure to sublethal doses of ANE (0, 5 and 20 microg/ml for 35 passages) could enhance genetic damage to HaCaT cells. Compared to cells exposed to 0 or 5 microg/ml ANE, cells exposed to 20 microg/ml ANE were slightly but significantly more resistant to a 72-h treatment with ANE and its major ingredients, arecoline and arecaidine, but did not develop cross-resistance to other BQ ingredients or alcohol. The cells that received 20 microg/ml ANE for 35 passages also had a significantly increased mutation frequency at the HPRT locus and an increased frequency in the appearance of micronuclei compared to lower doses. Moreover, increased intracellular levels of reactive oxygen species and 8-hydroxyguanosine in cells exposed to 20 microg/ml ANE suggested that long-term ANE exposure results in the accumulation of oxidative damage. However, cells subjected to long-term treatment of 20 microg/ml ANE contained higher levels of glutathione than unexposed cells. Therefore, after long-term exposure to sublethal doses of ANE, intracellular antioxidative activity may also be enhanced in response to increased oxidative stress. These results suggest that stress caused by long-term ANE exposure enhances oxidative stress and genetic damage in human keratinocytes.     

Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation

Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose-deprivation-induced cytotoxicity. To determine whether oxidative stress mediated by O(2)(*-) and hydroperoxides contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, the oxidation of DHE (dihydroethidine; for O(2)(*-)) and CDCFH(2) [5- (and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate; for hydroperoxides] was measured in human colon and breast cancer cells (HT29, HCT116, SW480 and MB231) and compared with that in normal human cells [FHC cells, 33Co cells and HMECs (human mammary epithelial cells)]. Cancer cells showed significant increases in DHE (2-20-fold) and CDCFH(2) (1.8-10-fold) oxidation, relative to normal cells, that were more pronounced in the presence of the mitochondrial electron-transport-chain blocker, antimycin A. Furthermore, HCT116 and MB231 cells were more susceptible to glucose-deprivation-induced cytotoxicity and oxidative stress, relative to 33Co cells and HMECs. HT29 cells were also more susceptible to 2DG (2-deoxyglucose)-induced cytotoxicity, relative to FHC cells. Overexpression of manganese SOD (superoxide dismutase) and mitochondrially targeted catalase significantly protected HCT116 and MB231 cells from glucose-deprivation-induced cytotoxicity and oxidative stress and also protected HT29 cells from 2DG-induced cytotoxicity. These results show that cancer cells (relative to normal cells) demonstrate increased steady-state levels of ROS (reactive oxygen species; i.e. O(2)(*-) and H(2)O(2)) that contribute to differential susceptibility to glucose-deprivation-induced cytotoxicity and oxidative stress. These studies support the hypotheses that cancer cells increase glucose metabolism to compensate for excess metabolic production of ROS and that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.     

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.     

Increased spontaneous DNA damage in Cu/Zn superoxide dismutase (SOD1) deficient Drosophila.

The superoxide dismutases (SODs) protect oxygen-using cells against reactive oxygen species, the potentially toxic by-products of respiration, oxidative metabolism, and radiation. We have previously shown that genetic disruption of CuZn SOD (SOD1) in Drosophila imparts a recessive phenotype of reduced lifespan, infertility, and hypersensitivity to oxidative stress. We now show that the absence of SOD1 increases spontaneous genomic damage. The increase in spontaneous mutation rate occurs in SOD1-null mutants in somatic cells as well as in the germ line. Further, we show that specific DNA repair-defective mutations, which are easily tolerated in SOD1(+) flies, lead to high mortality when introduced into the SOD1-null homozygous mutant background.     

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.