Effect of Bariatric Surgery on Plasma Cell-Free Mitochondrial DNA, Insulin Sensitivity and Metabolic Changes in Obese Patients.

While improvement of mitochondrial function after bariatric surgery has been demonstrated, there is limited evidence about the effects of bariatric surgery on circulatory cell-free (cf) mitochondrial DNA (mtDNA) and intracellular mtDNA abundance. Plasma and peripheral blood mononuclear (PBM) cells were isolated from healthy controls (HC) and bariatric surgery patients before surgery and 2 weeks, 3 months, and 6 months after surgery. At baseline, the plasma level of short cf-mtDNA (ND6, ~100 bp) fragments was significantly higher in obese patients compared to HC. But there was no significant variation in mean ND6 values post-surgery. A significant positive correlation was observed between preop plasma ND6 levels and HgbA1c, ND6 and HOMA-IR 2 weeks post-surgery, and mtDNA content 6 months post-surgery. Interestingly, plasma from both HC and obese groups at all time points post-surgery contains long (~8 kb) cf-mtDNA fragments, suggesting the presence of near-intact and/or whole mitochondrial genomes. No significant variation was observed in mtDNA content post-surgery compared to baseline data in both PBM and skeletal muscle samples. Overall, bariatric surgery improved insulin sensitivity and other metabolic parameters without significant changes in plasma short cf-mtDNA levels or cellular mtDNA content. Our study provides novel insights about possible molecular mechanisms underlying the metabolic effects of bariatric surgery and suggests the development of new generalized approaches to characterize cf-mtDNA.

PFKFB3 Inhibits Fructose Metabolism in Pulmonary Microvascular Endothelial Cells.

Pulmonary microvascular endothelial cells contribute to the integrity of the lung gas exchange interface, and they are highly glycolytic. Although glucose and fructose represent discrete substrates available for glycolysis, pulmonary microvascular endothelial cells prefer glucose over fructose, and the mechanisms involved in this selection are unknown. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is an important glycolytic enzyme that drives glycolytic flux against negative feedback and links glycolytic and fructolytic pathways. We hypothesized that PFKFB3 inhibits fructose metabolism in pulmonary microvascular endothelial cells. We found that PFKFB3 knockout cells survive better than wild-type cells in fructose-rich medium under hypoxia. Seahorse assays, lactate and glucose measurements, and stable isotope tracing showed that PFKFB3 inhibits fructose-hexokinase-mediated glycolysis and oxidative phosphorylation. Microarray analysis revealed that fructose upregulates PFKFB3, and PFKFB3 knockout cells increase fructose-specific GLUT5 (glucose transporter 5) expression. Using conditional endothelial-specific PFKFB3 knockout mice, we demonstrated that endothelial PFKFB3 knockout increases lung tissue lactate production after fructose gavage. Last, we showed that pneumonia increases fructose in BAL fluid in mechanically ventilated ICU patients. Thus, PFKFB3 knockout increases GLUT5 expression and the hexokinase-mediated fructose use in pulmonary microvascular endothelial cells that promotes their survival. Our findings indicate that PFKFB3 is a molecular switch that controls glucose versus fructose use in glycolysis and help better understand lung endothelial cell metabolism during respiratory failure.

Enhanced Mitochondrial DNA Repair Resuscitates Transplantable Lungs Donated After Circulatory Death.

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.

Plasma mitochondrial DNA is elevated in obese type 2 diabetes mellitus patients and correlates positively with insulin resistance.

Cells damaged by mechanical or infectious injury release proinflammatory mitochondrial DNA (mtDNA) fragments into the circulation. We evaluated the relation between plasma levels of mtDNA fragments in obese type 2 diabetes mellitus (T2DM) patients and measures of chronic inflammation and insulin resistance. In 10 obese T2DM patients and 12 healthy control (HC) subjects, we measured levels of plasma cell-free mtDNA with quantitative real-time polymerase chain reaction, and mtDNA damage in skeletal muscle with quantitative alkaline Southern blot. Also, markers of systemic inflammation and oxidative stress in skeletal muscle were measured. Plasma levels of mtDNA fragments, mtDNA damage in skeletal muscle and plasma tumor necrosis factor α levels were greater in obese T2DM patients than HC subjects. Also, the abundance of plasma mtDNA fragments in obese T2DM patients levels positively correlated with insulin resistance. To the best of our knowledge, this is the first published evidence that elevated level of plasma mtDNA fragments is associated with mtDNA damage and oxidative stress in skeletal muscle and correlates with insulin resistance in obese T2DM patients. Plasma mtDNA may be a useful biomarker for predicting and monitoring insulin resistance in obese patients.

Plasma Transfusion Products and Contamination with Cellular and Associated Pro-Inflammatory Debris.

BACKGROUND: Stored plasma products are widely regarded as being functionally acellular, obviating the need for leukoreduction. We tested the hypothesis that donor plasma is contaminated by leukocytes and platelets, which, after frozen storage, would release cellular debris in quantities sufficient to elicit significant pro-inflammatory responses. STUDY DESIGN: Samples of never-frozen liquid plasma from 2 regional Level I trauma centers were analyzed for leukocyte and platelet contamination. To determine if the cellular contamination and associated debris found in liquid plasma were at levels sufficient to evoke an innate immune response, known quantities of leukocytes were subjected to a freeze-thaw cycle, added to whole blood, and the magnitude of the inflammatory response was determined by induction of interleukin-6. RESULTS: Units of never-frozen plasma from 2 regional Level I trauma centers located in Alabama and Louisiana contained significant amounts of leukocyte contamination (Louisiana, n = 22; 17.3 ± 4.5 million vs Alabama, n = 22; 11.3 ± 2.2 million) and platelet contamination (Louisiana, n = 21; 0.86 ± 0.20 billion vs Alabama, n = 22; 1.0 ± 0.3 billion). Cellular debris from as few as 1 million leukocytes induced significant increases in interleukin-6 levels (R2 = 0.74; p < 0.0001). CONCLUSIONS: Stored plasma units from trauma center blood banks were highly contaminated with leukocytes and platelets, at levels more than 15-fold higher than sufficient to elicit ex vivo inflammatory responses. In light of paradigm shifts toward the use of more empiric plasma for treatment of hypovolemia, this study suggests that new manufacturing and quality-control processes are needed to eliminate previously unrecognized cellular contamination present in stored plasma products.

Potential contribution of mitochondrial DNA damage associated molecular patterns in transfusion products to the development of acute respiratory distress syndrome after multiple transfusions.

BACKGROUND: Massive transfusions are accompanied by an increased incidence of a particularly aggressive and lethal form of acute lung injury (delayed transfusion-related acute lung injury) which occurs longer than 24 hours after transfusions. In light of recent reports showing that mitochondrial (mt)DNA damage-associated molecular patterns (DAMPs) are potent proinflammatory mediators, and that their abundance in the sera of severely injured or septic patients is predictive of clinical outcomes, we explored the idea that mtDNA DAMPs are present in transfusion products and are associated with the occurrence of delayed transfusion-related acute lung injury. METHODS: We prospectively enrolled fourteen consecutive severely injured patients that received greater than three units of blood transfusion products and determined if the total amount of mtDNA DAMPs delivered during transfusion correlated with serum mtDNA DAMPs measured after the last transfusion, and whether the quantity of mtDNA DAMPs in the serum-predicted development of acute respiratory distress syndrome (ARDS). RESULTS: We found detectable levels of mtDNA DAMPs in packed red blood cells (3 ± 0.4 ng/mL), fresh frozen plasma (213.7 ± 65 ng/mL), and platelets (94.8 ± 69.2), with the latter two transfusion products containing significant amounts of mtDNA fragments. There was a linear relationship between the mtDNA DAMPs given during transfusion and the serum concentration of mtDNA fragments (R = 0.0.74, p < 0.01). The quantity of mtDNA DAMPs in serum measured at 24 hours after transfusion predicted the occurrence of ARDS (9.9 ± 1.4 vs. 3.3 ± 0.9, p < 0.01). CONCLUSION: These data show that fresh frozen plasma and platelets contain large amounts of extracellular mtDNA, that the amount of mtDNA DAMPs administered during transfusion may be a determinant of serum mtDNA DAMP levels, and that serum levels of mtDNA DAMPs after multiple transfusions may predict the development of ARDS. Collectively, these findings support the idea that mtDNA DAMPs in transfusion products significantly contribute to the incidence of ARDS after massive transfusions. LEVEL OF EVIDENCE: Prognostic study, level II; therapeutic study, level II.

Mitochondrial DNA Damage Initiates Acute Lung Injury and Multi-Organ System Failure Evoked in Rats by Intra-Tracheal Pseudomonas Aeruginosa.

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 24 h 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 24 h mortality. Collectively, the present findings indicate that oxidative mtDNA damage to lung tissue initiates PA103-induced ALI and MOSF in rats.

Mitochondrial DNA damage associated molecular patterns in ventilator-associated pneumonia: Prevention and reversal by intratracheal DNase I.

BACKGROUND: Previous studies in isolated perfused rat lungs have revealed that endothelial barrier disruption after intratracheal administration of Pseudomonas aeruginosa (strain 103; PA103) only occurs after accumulation of extracellular mitochondrial DNA (mtDNA) damage-associated molecular patterns (DAMPs) in the perfusate and is suppressed by addition of DNase to the perfusion medium. Herein, we tested the hypothesis that intratracheal DNase-a route of administration readily translatable to patient with ventilator-associated pneumonia (VAP)-also enhances degradation of mtDNA and prevents bacteria-induced lung injury. METHODS: Intratracheal DNase was administered to isolated rat lungs either before or after intratracheal challenge with PA103 to determine if bacteria-induced mtDNA DAMP-dependent lung injury could be prevented or reversed by enhanced mtDNA degradation. To explore whether this concept is translatable to patients with VAP, consecutive patients suspected of VAP were prospectively enrolled. All patients suspected of VAP received a bronchoalveolar lavage (BAL) with quantitative culture for the diagnosis of VAP. Mitochondrial and nuclear DNAs were measured from the BAL. MtDNA DAMPs (i.e., ND6) were measured from serum at time of suspected diagnosis and at 24 to 48 hours afterward. RESULTS: Intratracheal PA103 caused significantly increased the vascular filtration coefficient (Kf) and perfusate mtDNA DAMPs. In contrast, lungs pretreated or posttreated with intratracheal DNase were protected from increases in Kf and mtDNA DAMPs. Patients with the diagnosis of VAP had significantly higher mtDNA DAMPs in the BAL (248.70 ± 109.7 vs. 43.91 ± 16.61, p < 0.05, respectively) and in the serum at 24 hours (159.60 ± 77.37 vs. 10.43 ± 4.36, p < 0.05; respectively) when compared with patients that did not have VAP. CONCLUSION: These findings in isolated perfused rat lungs and a cohort of severely injured patients reveal an association between bacterial pneumonia and accumulation of mtDNA DAMPs in the lung and serum. Furthermore, administration of intratracheal DNase I prevented and reversed pulmonary endothelial dysfunction evoked by PA103.

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells.

Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.

Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.

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.

An oxidative DNA "damage" and repair mechanism localized in the VEGF promoter is important for hypoxia-induced VEGF mRNA expression.

In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.

Mitochondrial DNA damage-associated molecular patterns mediate a feed-forward cycle of bacteria-induced vascular injury in perfused rat lungs.

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.

Role of protein phosphorylation in excitation-contraction coupling in taurine deficient hearts.

Taurine is a beta-amino acid found in very high concentration in the heart. Depletion of these intracellular stores results in the development of cardiomyopathy, thought to be mediated by abnormal sarcoplasmic reticular (SR) Ca(2+) transport. There is also evidence that taurine directly alters the Ca(2+) sensitivity of myofibrillar proteins. Major regulators of SR Ca(2+) ATPase (SERCA2a) are the phosphorylation status of a regulatory protein, phospholamban, and SERCA2a expression, which are diminished in the failing heart. The failing heart also exhibits reductions in myofibrillar Ca(2+) sensitivity, a property regulated by the phosphorylation of the muscle protein, troponin I. Therefore, we tested the hypothesis that taurine deficiency leads to alterations in SR Ca(2+) ATPase activity related to reduced phospholamban phosphorylation and expression of SERCA2a. We found that a sequence of events, which included elevated protein phosphatase 1 activity, reduced autophosphorylation of CaMKII, and reduced phospholamban phosphorylation, supports the reduction in SR Ca(2+) ATPase activity. However, the reduction in SR Ca(2+) ATPase activity was not caused by reduced SERCA2a expression. Taurine transporter knockout (TauTKO) hearts also exhibited a rightward shift in the Ca(2+) dependence of the myofibrillar Ca(2+) ATPase, a property that is associated with an elevation in phosphorylated troponin I. The findings support the observation that taurine deficient hearts develop systolic and diastolic defects related to reduced SR Ca(2+) ATPase activity, a change mediated in part by reduced phospholamban phosphorylation.

Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription.

Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA-mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1α (HIF-1α) or the binding of HIF-1α to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1α binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.

Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs.

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.

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

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.

The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells.

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.

Controlled DNA "damage" and repair in hypoxic signaling.

Hypoxia, a fundamental stimulus in biology and medicine, uses reactive oxygen species (ROS) as second messengers. A surprising target of hypoxia-generated ROS is specific bases within hypoxic response elements (HREs) of the VEGF and other hypoxia-inducible genes. Oxidative modifications coincide with the onset of mRNA accumulation and are localized to transcriptionally active mono-nucleosomes. The oxidative base modifications are removed by the base excision DNA repair pathway for which one of its components, the bifunctional transcriptional co-activator and DNA endonuclease Ref-1/Ape1, is critical for transcription complex assembly. Mimicking the effect of hypoxia by introducing an abasic site in an oligonucleotide model of the VEGF HRE, altered transcription factor binding, enhanced sequence flexibility, and engendered more robust reporter gene expression. These observations suggest that controlled DNA "damage" and repair, mediated by ROS used as second messengers and critically involving the base excision pathway of DNA repair, respectively, are important for hypoxia-induced transcriptional activation.

Effect of hypernatremia on injury caused by energy deficiency: role of T-type Ca2+ channel.

Hypernatremia exerts multiple cellular effects, many of which could influence the outcome of an ischemic event. To further evaluate these effects of hypernatremia, isolated neonatal cardiomyocytes were chronically incubated with medium containing either normal (142 mM) or elevated sodium (167 mM) and then transferred to medium containing deoxyglucose and the electron transport chain inhibitor amobarbital. Chronic hypernatremia diminished the degree of calcium accumulation and reactive oxygen species generation during the period of metabolic inhibition. The improvement in calcium homeostasis was traced in part to the downregulation of the Ca(V)3.1 T-type calcium channel, as deficiency in the Ca(V)3.1 subtype using short hairpin RNA or treatment with an inhibitor of the Ca(V)3.1 variant of the T-type calcium channel (i.e., diphenylhydantoin) attenuated energy deficiency-mediated calcium accumulation and cell death. Although hyperosmotically stressed cells (exposed to 50 mM mannitol) had no effect on T-type calcium channel activity, they were also resistant to death during metabolic inhibition. Both hyperosmotic stress and hypernatremia activated Akt, suggesting that they initiate the phosphatidylinositol 3-kinase/Akt cytoprotective pathway, which protects the cell against calcium overload and oxidative stress. Thus hypernatremia appears to protect the cell against metabolic inhibition by promoting the downregulation of the T-type calcium channel and stimulating cytoprotective protein kinase pathways.