The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients

Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.

ITRAQ-Based Proteomics Analysis of Acute Lung Injury Induced by Oleic Acid in Mice.

BACKGROUND/AIMS: This study was conducted to investigate the relationship between differentially expressed proteins (DEPs) and the pathogenesis of oleic acid (OA)-induced acute lung injury (ALI) in mice. METHODS: Eight-week-old male C57BL/6 mice were injected with OA through the tail vein and sacrificed 6 hours after OA administration to identify protein expression levels in lung tissue using isobaric tags for relative and absolute quantification (iTRAQ) technology. Then, DEPs such as antithrombin III (AT III), 12-lipoxygenase (12-LO), dedicator of cytokinesis 2 (DOCK2), polycystin-2 and plasminogen were identified by western blotting. Subsequently, we focused on investigating the effect of AT III on endothelial integrity using siRNA interference technology. The levels of IL-6, IL-1ß, TNF-α and TGF-ß expression were detected using an enzyme-linked immunosorbent assay (ELISA). Alterations in the tight junction component ZO-1 and the phosphorylation of myosin light chain (pMLC) were determined by western blotting. The stress fiber F-actin were also detected by immunofluorescence staining. In addition, endothelial permeability was determined via a transwell permeability assay. RESULTS: A total of 5152 proteins were found to be expressed in lung tissues from the OA-treated and saline-treated mice. Among these proteins, 849 were differentially expressed between the two groups, including 545 upregulated and 304 downregulated proteins. After AT III knockdown, the levels of inflammatory factors and endothelial permeability were elevated, the expression of ZO-1 was decreased, and the expression of F-actin and pMLC was increased. All these results illustrated that AT III knockdown exaggerated the disruption of endothelial integrity mediated by OA. CONCLUSION: These findings using iTRAQ technology demonstrate, for the first time, differences in the lung tissue expression levels of proteins between OA-treated mice and saline-treated mice. This study reveals that 12-LO, DOCK2 and especially AT III may be candidate biomarkers for OA-induced acute lung injury.

12/15-lipoxygenase expression is increased in oligodendrocytes and microglia of periventricular leukomalacia.

Oxidative stress involving premyelinating oligodendrocytes (OLs) is a major factor in the pathogenesis of preterm white matter injury. In animal and cell culture studies, activation of the lipid-oxidizing enzyme 12/15-lipoxygenase (12/15-LOX) plays a central role as an inflammatory mediator in the pathology of oxidative stress and OL cell death, as well as ischemia and neuronal death. The role of 12/15-LOX, however, is unclear in the developing human brain. The mechanism of 12/15-LOX involves the production of reactive oxygen species through the metabolism of arachidonic acid, as well as direct detrimental effects on organelle membranes. Here we tested the hypothesis that the density of 12/15-LOX-expressing cells is increased in periventricular leukomalacia (PVL). Using immunocytochemistry (ICC) in human paraffin-embedded tissue, 12/15-LOX expression was seen in macrophages of the focally necrotic lesions in the periventricular white matter, as well as in glial cells throughout the surrounding white matter with reactive gliosis. Interestingly, no significant 12/15-LOX expression was detected in neurons in the cerebral cortex overlying the damaged white matter. Using a scoring system from 0 to 3, we assessed the density of 12/15-LOX-expressing cells in diffusely gliotic white matter from 20 to 43 postconceptional (PC) weeks in 19 PVL cases (median = 36 PC weeks) and 10 control (non-PVL) cases (median = 34 PC weeks). The density of 12/15-LOX-positive cells was significantly increased in the diffuse component of PVL (score = 1.17 ± 0.15) compared to controls (score = 0.48 ± 0.21; p = 0.014). Using double-label ICC, 12/15-LOX was observed in PVL in OLs of the O4 and O1 premyelinating stages, as well as in mature OLs as determined with the mature OL marker adenomatous polyposis coli (APC). In addition, 12/15-LOX expression was present in a population of CD68-positive activated microglia. There was no 12/15-LOX expression in reactive astrocytes. Finally we observed terminal deoxynucleotide transferase dUTP nick end-labeling-positive cells within the white matter of PVL that expressed 12/15-LOX and/or within close proximity of 12/15-LOX-positive cells. Our data support a role for 12/15-LOX activation as an inflammatory mediator of injury in PVL, with a contribution of 12/15-LOX to PVL-induced damage to or cell death of OLs, including those at the O1 and O4 stages.

Expression of cyclooxygenase and lipoxygenase enzymes in sinonasal mucosa of patients with cystic fibrosis.

OBJECTIVE: To evaluate the expression of cyclooxygenase (COX) and lipoxygenase (LO) enzymes in the sinonasal mucosa of patients with cystic fibrosis (CF). DESIGN: Immunohistochemical staining of archived tissue. PARTICIPANTS: Specimens from 9 patients with CF were analyzed; control specimens were obtained from 4 patients without a history of CF or rhinosinusitis. INTERVENTIONS: Expression of the enzymes COX-1, COX-2, 5-LO, 12-LO, and 15-LO was evaluated with the use of immunohistochemical techniques in archived sinonasal mucosal tissue from patients with CF. These results were compared with those of the control group. RESULTS: We noted the characteristic staining patterns of epithelium and submucosal glands for each enzyme. Statistically significant (P < .05) differences between control and CF specimens were noted in the staining intensity of columnar epithelium for COX-2 (cytoplasm) and 12-LO (cytoplasm and nucleus) and of submucosal glands for COX-2 (cytoplasm) and 12-LO (cytoplasm). No significant differences were noted for the staining intensity of COX-1, 5-LO, or 15-LO between the groups. CONCLUSIONS: Significant differences in sinonasal mucosal expression of COX-2 and 12-LO enzymes exist between patients with CF and controls. This suggests a difference in arachidonic acid metabolism between these 2 groups.

Detection and cellular localization of 12R-lipoxygenase in human tonsils.

Formation of the 12R-lipoxygenase product, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), has been detected previously only in human skin (Boeglin et al. (1998) Proc. Natl. Acad. Sci. USA 95, 6744). The unexpected appearance of an EST sequence (AA649213) for human 12R-lipoxygenase from germinal center B lymphocytes purified from human tonsils prompted our search for the existence of the enzyme in this novel source. Incubation of [1-14C]arachidonic acid with homogenates of human tonsillar tissue yielded mixtures of radiolabeled 12-HETE and 15-HETE. Stereochemical analysis showed varying ratios of 12S- and 12R-HETE, while 15-HETE was exclusively of the S-configuration. Using stereospecifically labeled [10S-3H]- and [10R-3H]arachidonic acid substrates we detected pro-R hydrogen abstraction at carbon 10 associated with formation of 12R-HETE. This mechanistic evidence implicates a 12R-lipoxygenase in the biosynthesis of 12R-HETE. The mRNA for the enzyme was identified in tonsils by RT-PCR and Northern analysis. The cellular distribution was established by in situ hybridization. Unexpectedly, hybridization was not observed in the lymphocytes of the germinal centers. Specific reaction was restricted to squamous epithelial cells, including the epithelium lining the tonsillar crypts. In this location the 12R-lipoxygenase might help regulate differentiation of the epithelium or participate in lymphocyte- epithelial cell interactions.

12-Lipoxygenase in A431 cells: genetic identity, modulation of expression, and intracellular localization.

Human A431 epidermoid carcinoma cells express 12-lipoxygenase enzymatic activity. However, the isoform identity based on cDNA sequence data is not known. Further, the simultaneous characterization of the intracellular distribution of 12-lipoxygenase protein and activity is lacking. Here we report that the cDNA sequence from RT-PCR-amplified 12-lipoxygenase mRNA is identical with the platelet-type 12-lipoxygenase isoform, and the leukocyte-type isoform of 12-lipoxygenase is not expressed in A431 cells. The predominant amount (78%) of 12-lipoxygenase protein resides in the cytosol. In contrast, the predominant (98%) 12-lipoxygenase activity is localized in the membrane fraction. Western blot and immunofluorescence data demonstrate that epidermal growth factor increases total cellular 12-lipoxygenase protein and enhances the association of 12-lipoxygenase protein with perinuclear or nuclear membrane sites. In addition, epidermal growth factor stimulates 12-lipoxygenase activity resulting in generation of 12(S)-hydroxyeicosatetraenoic acid from cellular arachidonate. In contrast, both 12-lipoxygenase protein and activity decrease approximately 80% within 24 h during serum starvation. The recovery of 12-lipoxygenase expression in serum-deprived cells can be induced by readdition of epidermal growth factor or serum. Further, the basal expression of 12-lipoxygenase depends on signal pathways requiring protein tyrosine kinase activity, since genistein, herbimycin A, and tyrphostin 25 reduce the expression of 12-lipoxygenase protein in A431 cells.

Arachidonate 12-lipoxygenase in porcine anterior pituitary cells: its localization and possible function in gonadotrophs.

Arachidonate 12-lipoxygenase, which oxygenates positions 12 and 13 of arachidonic and linoleic acids, is present in porcine anterior pituitary cells. Colocalization of the 12-lipoxygenase with various pituitary hormones was examined by immunohistochemical double-staining using antibodies against 12-lipoxygenase and various anterior pituitary hormones. Under light microscopy, approximately 7% of the cells producing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were positive for 12-lipoxygenase, whereas the enzyme was detected in less than 2% of the cells producing thyrotrophin, prolactin, growth hormone (GH), and adrenocorticotrophin. In an attempt to examine the participation of 12-lipoxygenase metabolites in pituitary hormone release, we incubated the primary culture of porcine anterior pituitary cells with 12-hydroperoxy-arachidonic acid or 13-hydroperoxy-linoleic acid. Significant stimulation of LH and FSH release by these hydroperoxides was observed at 10 microM in a time-dependent manner. At doses around 10 microM these compounds produced responses of similar magnitude to 1 nM gonadotrophin-releasing hormone (GnRH), but higher concentrations (30 microM) of the compounds were required for GH release. In contrast, 12-hydroxy-arachidonic and 13-hydroxy-linoleic acids were almost ineffective. Furthermore, the gonadotrophin release by 1 nM GnRH was inhibited by nordihydroguaiaretic acid (a lipoxygenase inhibitor) with an IC50 of about 5 microM. Thus, the hydroperoxy (but not hydroxy) products of 12-lipoxygenase may be involved in the release of pituitary hormones especially LH and FSH.

Immunocytochemical localization of platelet-type arachidonate 12-lipoxygenase in mouse blood cells.

Arachidonate 12-lipoxygenase is an enzyme that oxygenates the 12 position of arachidonic acid to produce its 12-hydroperoxy derivative. We were interested in the tissue distribution and subcellular localization of the platelet-type 12-lipoxygenase, which is distinguished from the leukocyte type by several criteria. Antiserum was raised in rabbits against purified recombinant arachidonate 12-lipoxygenase of human platelets. When mouse bone marrow cells and lung were immunostained and observed by light and electron microscopy, the positively stained cells were platelets, megakaryocytes, and eosinophils. 12-Lipoxygenase was localized in the cytoplasm of platelets but was hardly detectable in the plasma membrane and intracellular organelles. The enzyme was found in the cytoplasm of immature megakaryocytes with kidney-shaped nuclei and a few profiles of demarcation membranes, as well as in the mature form with well-developed demarcation membranes. These results indicated the expression of 12-lipoxygenase at an early stage in the course of megakaryocytopoiesis.

12-Lipoxygenase in Lewis lung carcinoma cells: molecular identity, intracellular distribution of activity and protein, and Ca(2+)-dependent translocation from cytosol to membranes.

Recently we demonstrated that Lewis lung (3LL) tumor cells express 12-lipoxygenase (12-LOX) mRNA and protein, respectively. In this study we partially sequenced the 12-LOX cDNA after reverse-transcription polymerase chain reaction amplification of 12-LOX mRNA from cultured 3LL cells. Comparison with platelet and leukocyte 12-LOX indicates that 3LL 12-LOX is identical with the platelet-type enzyme at least within the sequenced region. Further, we investigated the intracellular distribution of both 12-LOX enzyme protein and its activity which are prerequisites for understanding 12-LOX regulation. 12-LOX activity was monitored via the production of 12-hyroxyeicosatetraenoic acid from 3LL cells and their subcellular fractions using reverse-phase high performance liquid chromatography. 12-LOX protein was measured by direct slot blot and by Western Blotting. In 3LL cells, both 12-LOX activity and 12-LOX protein were predominantly localized in the cytosol. This 12-LOX activity was optimal at 37 degrees C. However at 24 degrees C and 10 degrees C, it showed 87% and 61% of this activity, respectively, thus differing distinctly from 12-LOX in platelets or rat basophilic leukemia cells. Incubation of 3LL cell homogenates with 0-100 microM free Ca2+ and subsequent separate analyses of cytosol and membrane fractions indicated that, as in platelets, an increase in intracellular free Ca2+ caused a loss of cytosolic 12-LOX activity. However, no significant Ca(2+)-induced increase in membrane-associated 12-LOX activity was observed under these conditions in 3LL cells. In contrast, at the 12-LOX protein level we observed a Ca(2+)-dependent loss in the cytosol and a concomitant increase in the membrane fraction. Thus, we suggest that 12-LOX in 3LL cells undergoes rapid translocation from cytosol to membrane in a Ca(2+)-dependent manner, but is no longer active or becomes inactivated at the membrane site.

Endogenous 12(S)-HETE production by tumor cells and its role in metastasis.

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

Porcine leukocyte 5- and 12-lipoxygenases are iron enzymes.

5- and 12-lipoxygenases isolated from porcine leukocytes were investigated by electron paramagnetic resonance at X-band and atomic absorption spectroscopy. For comparison potato 5-lipoxygenase was studied under identical experimental conditions. All three lipoxygenases contained between 0.7 and 0.9 Fe atoms/enzyme molecule. As isolated, both mammalian enzymes exhibited a characteristic EPR signal at low magnetic field with a maximum at g = 5.20 indicative of a high-spin ferric iron center. The signal was not affected by the oxidants 12-hydroperoxyeicosatetraenoic acid or arachidonic acid, nor was it affected by the reductant nordihydroguaiaretic acid. In the case of the potato enzyme an intense EPR signal with resonances at g = 7.50, 6.39 and 5.84 was only observed after addition of an oxidant, such as 9-hydroperoxyoctadecadienoic acid.

Eicosanoid forming enzyme mRNA in human tissues. Analysis by quantitative polymerase chain reaction.

The key enzymes in the formation of eicosanoids, including leukocyte 5-lipoxygenase (5LX), platelet 12-lipoxygenase (12LX), reticulocyte 15-lipoxygenase (15LX), prostaglandin G/H synthase cyclooxygenase, and leukotriene A4 (LTA) hydrolase have been studied extensively in recent years. Little is known, however, about the regulation of these enzymes at the gene level. We have developed a quantitative polymerase chain reaction (PCR) assay to quantify the mRNAs for these five enzymes, as well as for cytoplasmic beta-actin (bACT) mRNA. Human erythroleukemia (HEL) cells, which display megakaryocytic/erythroid characteristics, were selected as a source of RNA to characterize the assay. These cells expressed mRNA for bACT, LTA, cyclooxygenase, and 12LX (in decreasing order). mRNA for 5LX and 15LX was undetectable. Bronchoalveolar lavage fluid cells obtained from asthmatic patients, primarily alveolar macrophages, contained mRNA for bACT, LTA, 5LX, cyclooxygenase, and 15LX (in decreasing order). Treatment of HEL cells with phorbol 12-myristate 13-acetate or steroid administration to asthmatic patients apparently selectively regulated certain of these target genes. The utility of this assay in quantifying mRNA for the various target genes in blood cells, including platelets from patients with chronic myelogenous leukemia, has also been demonstrated. Studies on the regulation of genes for enzymes involved in the leukotriene and prostaglandin biosynthetic pathways, especially when only small tissue samples are available, will be facilitated with this approach.