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      International Journal of COPD (submit here)

      This international, peer-reviewed Open Access journal by Dove Medical Press focuses on pathophysiological processes underlying Chronic Obstructive Pulmonary Disease (COPD) interventions, patient focused education, and self-management protocols. Sign up for email alerts here.

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      Oxidative DNA damage in lung tissue from patients with COPD is clustered in functionally significant sequences

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          Abstract

          Lung tissue from COPD patients displays oxidative DNA damage. The present study determined whether oxidative DNA damage was randomly distributed or whether it was localized in specific sequences in either the nuclear or mitochondrial genomes. The DNA damage-specific histone, gamma-H2AX, was detected immunohistochemically in alveolar wall cells in lung tissue from COPD patients but not control subjects. A PCR-based method was used to search for oxidized purine base products in selected 200 bp sequences in promoters and coding regions of the VEGF, TGF-β1, HO-1, Egr1, and β-actin genes while quantitative Southern blot analysis was used to detect oxidative damage to the mitochondrial genome in lung tissue from control subjects and COPD patients. Among the nuclear genes examined, oxidative damage was detected in only 1 sequence in lung tissue from COPD patients: the hypoxic response element (HRE) of the VEGF promoter. The content of VEGF mRNA also was reduced in COPD lung tissue. Mitochondrial DNA content was unaltered in COPD lung tissue, but there was a substantial increase in mitochondrial DNA strand breaks and/or abasic sites. These findings show that oxidative DNA damage in COPD lungs is prominent in the HRE of the VEGF promoter and in the mitochondrial genome and raise the intriguing possibility that genome and sequence-specific oxidative DNA damage could contribute to transcriptional dysregulation and cell fate decisions in COPD.

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          Sequence-specific oxidative base modifications in hypoxia-inducible genes.

          Reactive oxygen species associated with hypoxic signaling in pulmonary arterial endothelial cells (PAECs) oxidatively modify specific nucleotides in the hypoxic response element (HRE) of the VEGF gene (FASEB J.19:387-394; 2005). In this study, we determined in PAECs if hypoxia caused genome-wide oxidative modifications or if they were restricted to the promoters of genes differentially regulated by hypoxia. Comet assays indicated that there were no differences between normoxic and hypoxic PAECs in terms of global DNA damage. However, a simple PCR-based method involving DNA amplification before and after treatment with formamidopyrimidine DNA glycosylase (Fpg), a bacterial DNA repair enzyme that cleaves at sites of purine base oxidation, revealed that hypoxia caused modifications in the HREs of the hypoxia-inducible VEGF, HO-1, and ET-1 genes which coincided with accumulation of their respective mRNA transcripts. Promoter sequences not involved with hypoxic induction and coding regions of these genes failed to display Fpg-sensitive sites. Oxidative modifications also were not detected in sequences of the hypoxia down-regulated ornithine decarboxylase and TFAM genes or the constitutively expressed beta-actin gene. These findings show that hypoxia-mediated oxidative DNA modifications cluster in functionally relevant promoter sequences in hypoxia-inducible genes and suggest that such oxidative modifications may be biologically significant.
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            Hypoxia-induced oxidative base modifications in the VEGF hypoxia-response element are associated with transcriptionally active nucleosomes.

            Reactive oxygen species (ROS) generated in hypoxic pulmonary artery endothelial cells cause transient oxidative base modifications in the hypoxia-response element (HRE) of the VEGF gene that bear a conspicuous relationship to induction of VEGF mRNA expression (K.A. Ziel et al., FASEB J. 19, 387-394, 2005). If such base modifications are indeed linked to transcriptional regulation, then they should be detected in HRE sequences associated with transcriptionally active nucleosomes. Southern blot analysis of the VEGF HRE associated with nucleosome fractions prepared by micrococcal nuclease digestion indicated that hypoxia redistributed some HRE sequences from multinucleosomes to transcriptionally active mono- and dinucleosome fractions. A simple PCR method revealed that VEGF HRE sequences harboring oxidative base modifications were found exclusively in mononucleosomes. Inhibition of hypoxia-induced ROS generation with myxathiozol prevented formation of oxidative base modifications but not the redistribution of HRE sequences into mono- and dinucleosome fractions. The histone deacetylase inhibitor trichostatin A caused retention of HRE sequences in compacted nucleosome fractions and prevented formation of oxidative base modifications. These findings suggest that the hypoxia-induced oxidant stress directed at the VEGF HRE requires the sequence to be repositioned into mononucleosomes and support the prospect that oxidative modifications in this sequence are an important step in transcriptional activation.
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              Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes.

              Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly repaired in MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA lesions at nucleotide resolution showed that damage patterns were similar between EC phenotypes, but there were disparities between XO and menadione in terms of the specific nucleotides damaged. These findings indicate that mtDNA in lung vascular ECs is damaged by XO- and menadione-derived free radicals and suggest that mtDNA damage and repair capacities differ between EC phenotypes.
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                Author and article information

                Journal
                Int J Chron Obstruct Pulmon Dis
                International Journal of COPD
                International Journal of Chronic Obstructive Pulmonary Disease
                Dove Medical Press
                1176-9106
                1178-2005
                2011
                2011
                13 March 2011
                : 6
                : 209-217
                Affiliations
                [1 ]Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL, USA;
                [2 ]Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado at Denver, Aurora, CO, USA
                Author notes
                Correspondence: Mark N Gillespie, Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA, Tel +1 (251) 460-6497, Fax +1 (251) 460-6798, Email mgillesp@ 123456jaguar1.usouthal.edu
                Article
                copd-6-209
                10.2147/COPD.S15922
                3107697
                21660298
                17fcdc9a-87f5-428d-9582-9b788cddeff4
                © 2011 Pastukh et al, publisher and licensee Dove Medical Press Ltd.

                This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

                History
                : 11 March 2011
                Categories
                Original Research

                Respiratory medicine
                vegf hypoxic response element,dna damage,copd,mtdna
                Respiratory medicine
                vegf hypoxic response element, dna damage, copd, mtdna

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