RESUMO
BACKGROUND: Transplantation of lungs procured after donation after circulatory death (DCD) is challenging because postmortem metabolic degradation may engender susceptibility to ischemia-reperfusion (IR) injury. Because oxidative mitochondrial DNA (mtDNA) damage has been linked to endothelial barrier disruption in other models of IR injury, here we used a fusion protein construct targeting the DNA repair 8-oxoguanine DNA glycosylase-1 (OGG1) to mitochondria (mtOGG1) to determine if enhanced repair of mtDNA damage attenuates endothelial barrier dysfunction after IR injury in a rat model of lung procurement after DCD. MATERIALS AND METHODS: Lungs excised from donor rats 1 h after cardiac death were cold stored for 2 h after which they were perfused ex vivo in the absence and presence of mt-OGG1 or an inactive mt-OGG1 mutant. Lung endothelial barrier function and mtDNA integrity were determined during and at the end of perfusion, respectively. RESULTS AND CONCLUSIONS: Mitochondria-targeted OGG1 attenuated indices of lung endothelial dysfunction incurred after a 1h post-mortem period. Oxidative lung tissue mtDNA damage as well as accumulation of proinflammatory mtDNA fragments in lung perfusate, but not nuclear DNA fragments, also were reduced by mitochondria-targeted OGG1. A repair-deficient mt-OGG1 mutant failed to protect lungs from the adverse effects of DCD procurement. CONCLUSIONS: These findings suggest that endothelial barrier dysfunction in lungs procured after DCD is driven by mtDNA damage and point to strategies to enhance mtDNA repair in concert with EVLP as a means of alleviating DCD-related lung IR injury.
Assuntos
DNA Glicosilases/administração & dosagem , Endotélio Vascular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Recombinantes de Fusão/administração & dosagem , Traumatismo por Reperfusão/prevenção & controle , Aloenxertos/irrigação sanguínea , Aloenxertos/citologia , Aloenxertos/efeitos dos fármacos , Animais , DNA Glicosilases/genética , Reparo do DNA/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/citologia , Pulmão/efeitos dos fármacos , Transplante de Pulmão , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Perfusão/métodos , Ratos , Proteínas Recombinantes de Fusão/genética , Traumatismo por Reperfusão/patologia , Coleta de Tecidos e Órgãos/métodosRESUMO
Oxidative stress results in mtDNA damage and contributes to myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We investigated a novel, mitochondria-targeted fusion protein (Exscien1-III) containing endonuclease III in myocardial ischemia-reperfusion injury and transverse aortic constriction (TAC)-induced heart failure. Male C57/BL6J mice (10-12 wk) were subjected to 45 min of myocardial ischemia and either 24 h or 4 wk of reperfusion. Exscien1-III (4 mg/kg ip) or vehicle was administered at the time of reperfusion. Male C57/BL6J mice were subjected to TAC, and Exscien1-III (4 mg/kg i.p) or vehicle was administered daily starting at 3 wk post-TAC and continued for 12 wk. Echocardiography was performed to assess left ventricular (LV) structure and function. Exscien1-III reduced myocardial infarct size ( P < 0.01) at 24 h of reperfusion and preserved LV ejection fraction at 4 wk postmyocardial ischemia. Exscien1-III attenuated TAC-induced LV dilation and dysfunction at 6-12 wk post-TAC ( P < 0.05). Exscien1-III reduced ( P < 0.05) cardiac hypertrophy and maladaptive remodeling after TAC. Assessment of cardiac mitochondria showed that Exscien1-III localized to mitochondria and increased mitochondrial antioxidant and reduced apoptotic markers. In conclusion, our results indicate that administration of Exscien1-III provides significant protection against myocardial ischemia and preserves myocardial structure and LV performance in the setting of heart failure. NEW & NOTEWORTHY Oxidative stress-induced mitochondrial DNA damage is a prominent feature in the pathogenesis of cardiovascular diseases. In the present study, we demonstrate the efficacy of a novel, mitochondria-targeted fusion protein that traffics endonuclease III specifically for mitochondrial DNA repair in two well-characterized murine models of cardiac injury and failure.
Assuntos
Fármacos Cardiovasculares/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Fibrose , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Volume Sistólico/efeitos dos fármacos , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Fragments of the mitochondrial genome released into the systemic circulation after mechanical trauma, termed mitochondrial DNA damage-associated molecular patterns (mtDNA DAMPs), are thought to mediate the systemic inflammatory response syndrome. The close association between circulating mtDNA DAMP levels and outcome in sepsis suggests that bacteria also might be a stimulus for mtDNA DAMP release. To test this hypothesis, we measured mtDNA DAMP abundance in medium perfusing isolated rat lungs challenged with an intratracheal instillation of 5 × 10(7) colony-forming units of Pseudomonas aeruginosa (strain 103; PA103). Intratracheal PA103 caused rapid accumulation of selected 200-bp sequences of the mitochondrial genome in rat lung perfusate accompanied by marked increases in both lung tissue oxidative mtDNA damage and in the vascular filtration coefficient (Kf). Increases in lung tissue mtDNA damage, perfusate mtDNA DAMP abundance, and Kf were blocked by addition to the perfusion medium of a fusion protein targeting the DNA repair enzyme Ogg1 to mitochondria. Intra-arterial injection of mtDNA DAMPs prepared from rat liver mimicked the effect of PA103 on both Kf and lung mtDNA integrity. Effects of mtDNA and PA103 on Kf were also attenuated by an oligodeoxynucleotide inhibitor of Toll-like receptor 9 (TLR-9) by mitochondria-targeted Ogg1 and by addition of DNase1 to the perfusion medium. Collectively, these findings are consistent with a model wherein PA103 causes oxidative mtDNA damage leading to a feed-forward cycle of mtDNA DAMP formation and TLR-9-dependent mtDNA damage that culminates in acute lung injury.
Assuntos
Lesão Pulmonar Aguda , Dano ao DNA , DNA Mitocondrial/metabolismo , Pulmão , Infecções por Pseudomonas , Pseudomonas aeruginosa , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , DNA Glicosilases/metabolismo , Desoxirribonuclease I/farmacologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Masculino , Oligodesoxirribonucleotídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Perfusão , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia , Ratos , Ratos Sprague-Dawley , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismoRESUMO
This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.
Assuntos
DNA Glicosilases/uso terapêutico , Reparo do DNA/fisiologia , DNA Mitocondrial/efeitos dos fármacos , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Animais , Quimiocina CXCL2/metabolismo , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/fisiologia , Glutationa/metabolismo , Interleucina-6/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Mitocôndrias/enzimologia , Edema Pulmonar/tratamento farmacológico , Edema Pulmonar/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/mortalidadeRESUMO
In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.
Assuntos
DNA Mitocondrial/genética , Células Endoteliais/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Animais , Fracionamento Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Dano ao DNA , DNA Glicosilases/farmacologia , DNA Glicosilases/fisiologia , Endodesoxirribonucleases/farmacologia , Endodesoxirribonucleases/fisiologia , Células Endoteliais/metabolismo , Endotélio/metabolismo , Glucose Oxidase/química , Glucose Oxidase/farmacologia , Glucose Oxidase/fisiologia , Técnicas In Vitro , Pulmão/citologia , Pulmão/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Permeabilidade , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
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.
Assuntos
Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Animais , Sequência de Bases , Células Cultivadas , DNA/química , DNA/genética , Dano ao DNA , Primers do DNA/genética , Células Endoteliais/metabolismo , Endotelina-1/genética , Regulação da Expressão Gênica , Heme Oxigenase (Desciclizante)/genética , Oxirredução , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Although studies in rat cultured pulmonary artery endothelial cells, perfused lungs, and intact mice support the concept that oxidative mitochondrial (mt) DNA damage triggers acute lung injury (ALI), it has not yet been determined whether enhanced mtDNA repair forestalls development of ALI and its progression to multiple organ system failure (MOSF). Accordingly, here we examined the effect of a fusion protein construct targeting the DNA glycosylase, Ogg1, to mitochondria in a rat model intra-tracheal Pseudomonas aeruginosa (strain 103; PA103)-induced ALI and MOSF. Relative to controls, animals given PA103 displayed increases in lung vascular filtration coefficient accompanied by transient lung tissue oxidative mtDNA damage and variable changes in mtDNA copy number without evidence of nuclear DNA damage. The approximate 40% of animals surviving 24âh after bacterial administration exhibited multiple organ dysfunction, manifest as increased serum and tissue-specific indices of kidney and liver failure, along with depressed heart rate and blood pressure. While administration of mt-targeted Ogg1 to control animals was innocuous, the active fusion protein, but not a DNA repair-deficient mutant, prevented bacteria-induced increases in lung tissue oxidative mtDNA damage, failed to alter mtDNA copy number, and attenuated lung endothelial barrier degradation. These changes were associated with suppression of liver, kidney, and cardiovascular dysfunction and with decreased 24âh mortality. Collectively, the present findings indicate that oxidative mtDNA damage to lung tissue initiates PA103-induced ALI and MOSF in rats.
Assuntos
Lesão Pulmonar Aguda/genética , Dano ao DNA/genética , DNA Mitocondrial/genética , Insuficiência de Múltiplos Órgãos/genética , Lesão Pulmonar Aguda/microbiologia , Animais , DNA Glicosilases/genética , Masculino , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Pseudomonas aeruginosa/patogenicidade , Ratos , Ratos Sprague-Dawley , Traqueia/microbiologiaRESUMO
Lung ischemia-reperfusion (IR) injury contributes to post-transplant complications, including primary graft dysfunction. Decades of reports show that reactive oxygen species generated during lung IR contribute to pulmonary vascular endothelial barrier disruption and edema formation, but the specific target molecule(s) that "sense" injury-inducing oxidant stress to activate signaling pathways culminating in pathophysiologic changes have not been established. This review discusses evidence that mitochondrial DNA (mtDNA) may serve as a molecular sentinel wherein oxidative mtDNA damage functions as an upstream trigger for lung IR injury. First, the mitochondrial genome is considerably more sensitive than nuclear DNA to oxidant stress. Multiple studies suggest that oxidative mtDNA damage could be transduced to physiologic dysfunction by pathways that are either a direct consequence of mtDNA damage per se or involve formation of proinflammatory mtDNA damage-associated molecular patterns. Second, transgenic animals or cells overexpressing components of the base excision DNA repair pathway in mitochondria are resistant to oxidant stress-mediated pathophysiologic effects. Finally, published and preliminary studies show that pharmacologic enhancement of mtDNA repair or mtDNA damage-associated molecular pattern degradation suppresses reactive oxygen species-induced or IR injury in multiple organs, including preclinical models of lung procurement for transplant. Collectively, these findings point to the interesting prospect that pharmacologic enhancement of DNA repair during procurement or ex vivo lung perfusion may increase the availability of lungs for transplant and reduce the IR injury contributing to primary graft dysfunction.
Assuntos
DNA Mitocondrial/efeitos dos fármacos , Pulmão/irrigação sanguínea , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/prevenção & controle , Animais , Dano ao DNA , Reparo do DNA , Humanos , Transplante de Pulmão , Disfunção Primária do Enxerto , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Despite improvements in the management of severely injured patients, development of multiple organ dysfunction syndrome (MODS) remains a morbid complication of traumatic shock. One of the key attributes of MODS is a profound bioenergetics crisis, for which the mediators and mechanisms are poorly understood. We hypothesized that metabolic uncoupling using an experimental phosphoinositol-3 kinase (PI3-K) inhibitor, LY294002 (LY), may prevent mitochondrial abnormalities that lead to the generation of mitochondrial DNA (mtDNA) damage and the release of mtDNA damage-associated molecular patterns (DAMPs). METHODS: Sixteen swine were studied using LY, a nonselective PI3-K inhibitor. Animals were assigned to trauma only (TO, n = 3), LY drug only (LYO, n = 3), and experimental (n = 10), trauma + drug (LY + T) groups. Both trauma groups underwent laparotomy, 35% hemorrhage, severe ischemia-reperfusion injury, and protocolized resuscitation. A battery of hemodynamic, laboratory, histological, and bioenergetics parameters were monitored. Mitochondrial DNA damage was determined in lung, liver, and kidney using Southern blot analyses, whereas plasma mtDNA DAMP analysis used polymerase chain reaction amplification of a 200-bp sequence of the mtDNA D-loop region. RESULTS: Relative to control animals, H + I/R (hemorrhage and ischemia/reperfusion) produced severe, time-dependent decrements in hepatic, renal, cardiovascular, and pulmonary function accompanied by severe acidosis and lactate accumulation indicative of bioenergetics insufficiency. The H-I/R animals displayed prominent oxidative mtDNA damage in all organs studied, with the most prominent damage in the liver. Mitochondrial DNA damage was accompanied by accumulation of mtDNA DAMPs in plasma. Pretreatment of H + I/R animals with LY resulted in profound metabolic suppression, with approximately 50% decreases in O2 consumption and CO2 production. In addition, it prevented organ and bioenergetics dysfunction and was associated with a significant decrease in plasma mtDNA DAMPs to the levels of control animals. CONCLUSIONS: These findings show that H + I/R injury in anesthetized swine is accompanied by MODS and by significant mitochondrial bioenergetics dysfunction, including oxidative mtDNA damage and accumulation in plasma of mtDNA DAMPs. Suppression of these changes with the PI3-K inhibitor LY indicates that pharmacologically induced metabolic uncoupling may comprise a new pharmacologic strategy to prevent mtDNA damage and DAMP release and prevent or treat trauma-related MODS. LEVEL OF EVIDENCE: Therapeutic study, level III.
Assuntos
Cromonas/uso terapêutico , Dano ao DNA , DNA Mitocondrial , Inibidores Enzimáticos/uso terapêutico , Morfolinas/uso terapêutico , Insuficiência de Múltiplos Órgãos/prevenção & controle , Choque Traumático/terapia , Animais , Modelos Animais de Doenças , Metabolismo Energético , Insuficiência de Múltiplos Órgãos/etiologia , Choque Traumático/complicações , SuínosRESUMO
Mitochondria of mammalian cells contain multiple copies of mitochondrial (mt) DNA. Although mtDNA copy number can fluctuate dramatically depending on physiological and pathophysiologic conditions, the mechanisms regulating mitochondrial genome replication remain obscure. Hypoxia, like many other physiologic stimuli that promote growth, cell proliferation and mitochondrial biogenesis, uses reactive oxygen species as signaling molecules. Emerging evidence suggests that hypoxia-induced transcription of nuclear genes requires controlled DNA damage and repair in specific sequences in the promoter regions. Whether similar mechanisms are operative in mitochondria is unknown. Here we test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region of the mitochondrial genome are required for mitochondrial DNA replication and transcription in hypoxia. We found that hypoxia had little impact on expression of mitochondrial proteins in pulmonary artery endothelial cells, but elevated mtDNA content. The increase in mtDNA copy number was accompanied by oxidative modifications in the D-loop region of the mitochondrial genome. To investigate the role of this sequence-specific oxidation of mitochondrial genome in mtDNA replication, we overexpressed mitochondria-targeted 8-oxoguanine glycosylase Ogg1 in rat pulmonary artery endothelial cells, enhancing the mtDNA repair capacity of transfected cells. Overexpression of Ogg1 resulted in suppression of hypoxia-induced mtDNA oxidation in the D-loop region and attenuation of hypoxia-induced mtDNA replication. Ogg1 overexpression also reduced binding of mitochondrial transcription factor A (TFAM) to both regulatory and coding regions of the mitochondrial genome without altering total abundance of TFAM in either control or hypoxic cells. These observations suggest that oxidative DNA modifications in the D-loop region during hypoxia are important for increased TFAM binding and ensuing replication of the mitochondrial genome.
Assuntos
Hipóxia Celular/genética , DNA Glicosilases/genética , Mitocôndrias/genética , Estresse Oxidativo/genética , Fatores de Transcrição/genética , Animais , Proliferação de Células/genética , Dano ao DNA/genética , Replicação do DNA/genética , DNA Mitocondrial/genética , Proteínas de Ligação a DNA , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Genoma Mitocondrial/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Biogênese de Organelas , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA) damage and ventilator induced lung injury (VILI). In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII) which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP) was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH) and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group.
RESUMO
Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.
Assuntos
Citoproteção , DNA Glicosilases/metabolismo , Células Endoteliais/enzimologia , Oxidantes/efeitos adversos , Animais , Apoptose , Técnicas de Cultura de Células , Citoproteção/genética , Dano ao DNA , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/genética , Reparo do DNA , DNA Mitocondrial/metabolismo , Células Endoteliais/citologia , Expressão Gênica , Inativação Gênica , Guanina/análogos & derivados , Guanina/metabolismo , Hipoxantina/efeitos adversos , Hipoxantina/metabolismo , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologia , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Xantina Oxidase/efeitos adversos , Xantina Oxidase/metabolismoRESUMO
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-ß1, HO-1, Egr1, and ß-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.
Assuntos
Dano ao DNA , Pulmão/química , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/genética , Análise de Variância , Baltimore , Southern Blotting , Estudos de Casos e Controles , Colorado , DNA Mitocondrial/análise , Histonas/análise , Humanos , Imuno-Histoquímica , Pulmão/patologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , RNA Mensageiro/análise , Índice de Gravidade de Doença , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Hipóxia Celular , Células Endoteliais/metabolismo , Nucleossomos/metabolismo , Elementos de Resposta/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Southern Blotting , Fracionamento Celular , Células Cultivadas , Células Endoteliais/patologia , Epigênese Genética/genética , Nucleossomos/genética , Estresse Oxidativo/genética , Reação em Cadeia da Polimerase , Artéria Pulmonar/patologia , Ratos , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Oxidant-induced death and dysfunction of pulmonary vascular cells play important roles in the evolution of acute lung injury. In pulmonary artery endothelial cells (PAECs), oxidant-mediated damage to mitochondrial DNA (mtDNA) seems to be critical in initiating cytotoxicity inasmuch as overexpression of the mitochondrially targeted human DNA repair enzyme, human Ogg1 (hOgg1), prevents both mtDNA damage and cell death (Dobson AW, Grishko V, LeDoux SP, Kelley MR, Wilson GL, and Gillespie MN. Am J Physiol Lung Cell Mol Physiol 283: L205-L210, 2002). The mechanism by which mtDNA damage leads to PAEC death is unknown, and the present study tested the specific hypothesis that enhanced mtDNA repair suppresses PAEC mitochondrial dysfunction and apoptosis evoked by xanthine oxidase (XO). PAECs transfected either with an adenoviral vector encoding hOgg1 linked to a mitochondrial targeting sequence or with empty vector were challenged with ascending doses of XO plus hypoxanthine. Quantitative Southern blot analyses revealed that, as expected, hOgg1 overexpression suppressed XO-induced mtDNA damage. Mitochondrial overexpression of hOgg1 also suppressed the XO-mediated loss of mitochondrial membrane potential. Importantly, hOgg1 overexpression attenuated XO-induced apoptosis as detected by suppression of caspase-3 activation, by reduced DNA fragmentation, and by a blunted appearance of condensed, fragmented nuclei. These observations suggest that mtDNA damage serves as a trigger for mitochondrial dysfunction and apoptosis in XO-treated PAECs.