Differential Responses of Targeted Lung Redox Enzymes to Rat Exposure to 60 or 85% Oxygen

Rat exposure to 60% O2 (hyper-60) or 85% O2 (hyper-85) for 7 days confers susceptibility or tolerance, respectively, of the otherwise lethal effects of exposure to 100% O2. The objective of this study was to determine whether activities of the antioxidant cytosolic enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III are differentially altered in hyper-60 and hyper-85 lungs. Duroquinone (DQ), an NQO1 substrate, or its hydroquinone (DQH2), a complex III substrate, was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of DQH2 and DQ were measured. Based on inhibitor effects and kinetic modeling, capacities of NQO1-mediated DQ reduction (Vmax1) and complex III-mediated DQH2 oxidation (Vmax2) increased by ∼140 and ∼180% in hyper-85 lungs, respectively, compared with rates in lungs of rats exposed to room air (normoxic). In hyper-60 lungs, Vmax1 increased by ∼80%, with no effect on Vmax2. Additional studies revealed that mitochondrial complex I activity in hyper-60 and hyper-85 lung tissue homogenates was ∼50% lower than in normoxic lung homogenates, whereas mitochondrial complex IV activity was ∼90% higher in only hyper-85 lung tissue homogenates. Thus NQO1 activity increased in both hyper-60 and hyper-85 lungs, whereas complex III activity increased in hyper-85 lungs only. This increase, along with the increase in complex IV activity, may counter the effects the depression in complex I activity might have on tissue mitochondrial function and/or reactive oxygen species production and may be important to the tolerance of 100% O2 observed in hyper-85 rats

Lung Circulation Modeling: Status and Prospect

Mathematical modeling has been used to interpret anatomical and physiological data obtained from metabolic and hemodynamic studies aimed at investigating structure-function relationships in the vasculature of the lung, and how these relationships are affected by lung injury and disease. The indicator dilution method was used to study the activity of redox processes within the lung. A steady-state model of the data was constructed and used to show that pulmonary endothelial cells may play an important role in reducing redox active compounds and that those reduction rates can be altered with oxidative stress induced by exposure to high oxygen environments. In addition, a morphometric model of the pulmonary vasculature was described and used to detect, describe,and predict changes in vascular morphology that occur in response to chronic exposure to low-oxygen environments, a common model of pulmonary hypertension. Finally, the model was used to construct simulated circulatory networks designed to aid in evaluation of competing hypotheses regarding the relative contribution of various morphological and biomechanical changes observed with hypoxia. These examples illustrate the role of mathematical modeling in the integration of the emerging metabolic, hemodynamic, and morphometric databases

Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs

We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time

Distribution of Capillary Transit Times in Isolated Lungs of Oxygen-Tolerant Rats

Rats pre-exposed to 85% O2 for 5–7 days tolerate the otherwise lethal effects of 100% O2. The objective was to evaluate the effect of rat exposure to 85% O2 for 7 days on lung capillary mean transit time (t¯c) and distribution of capillary transit times (h c(t)). This information is important for subsequent evaluation of the effect of this hyperoxia model on the redox metabolic functions of the pulmonary capillary endothelium. The venous concentration vs. time outflow curves of fluorescein isothiocyanate labeled dextran (FITC-dex), an intravascular indicator, and coenzyme Q1 hydroquinone (CoQ1H2), a compound which rapidly equilibrates between blood and tissue on passage through the pulmonary circulation, were measured following their bolus injection into the pulmonary artery of isolated perfused lungs from rats exposed to room air (normoxic) or 85% O2 for 7 days (hyperoxic). The moments (mean transit time and variance) of the measured FITC-dex and CoQ1H2 outflow curves were determined for each lung, and were then used in a mathematical model [Audi et al. J. Appl. Physiol. 77: 332–351, 1994] to estimate t¯c and the relative dispersion (RDc) of h c(t). Data analysis reveals that exposure to hyperoxia decreases lung t¯c by 42% and increases RDc, a measure h c(t) heterogeneity, by 40%

Understanding the in vivo Uptake Kinetics of a Phosphatidylethanolamine-binding Agent \u3csup\u3e99m\u3c/sup\u3eTc-Duramycin

Introduction 99mTc-Duramycin is a peptide-based molecular probe that binds specifically to phosphatidylethanolamine (PE). The goal was to characterize the kinetics of molecular interactions between 99mTc-Duramycin and the target tissue. Methods High level of accessible PE is induced in cardiac tissues by myocardial ischemia (30 min) and reperfusion (120 min) in Sprague–Dawley rats. Target binding and biodistribution of 99mTc-duramycin were captured using SPECT/CT. To quantify the binding kinetics, the presence of radioactivity in ischemic versus normal cardiac tissues was measured by gamma counting at 3, 10, 20, 60 and 180 min after injection. A partially inactivated form of 99mTc-Duramycin was analyzed in the same fashion. A compartment model was developed to quantify the uptake kinetics of 99mTc-Duramycin in normal and ischemic myocardial tissue. Results 99mTc-duramycin binds avidly to the damaged tissue with a high target-to-background radio. Compartment modeling shows that accessibility of binding sites in myocardial tissue to 99mTc-Duramycin is not a limiting factor and the rate constant of target binding in the target tissue is at 2.2 ml/nmol/min/g. The number of available binding sites for 99mTc-Duramycin in ischemic myocardium was estimated at 0.14 nmol/g. Covalent modification of D15 resulted in a 9-fold reduction in binding affinity. Conclusion 99mTc-Duramycin accumulates avidly in target tissues in a PE-dependent fashion. Model results reflect an efficient uptake mechanism, consistent with the low molecular weight of the radiopharmaceutical and the relatively high density of available binding sites. These data help better define the imaging utilities of 99mTc-Duramycin as a novel PE-binding agent

In vivo Detection of Hyperoxia-induced Pulmonary Endothelial Cell Death Using \u3csup\u3e99m\u3c/sup\u3eTc-Duramycin

Introduction 99mTc-duramycin, DU, is a SPECT biomarker of tissue injury identifying cell death. The objective of this study is to investigate the potential of DU imaging to quantify capillary endothelial cell death in rat lung injury resulting from hyperoxia exposure as a model of acute lung injury. Methods Rats were exposed to room air (normoxic) or \u3e 98% O2 for 48 or 60 hours. DU was injected i.v. in anesthetized rats, scintigraphy images were acquired at steady-state, and lung DU uptake was quantified from the images. Post-mortem, the lungs were removed for histological studies. Sequential lung sections were immunostained for caspase activation and endothelial and epithelial cells. Results Lung DU uptake increased significantly (p \u3c 0.001) by 39% and 146% in 48-hr and 60-hr exposed rats, respectively, compared to normoxic rats. There was strong correlation (r2 = 0.82, p = 0.005) between lung DU uptake and the number of cleaved caspase 3 (CC3) positive cells, and endothelial cells accounted for more than 50% of CC3 positive cells in the hyperoxic lungs. Histology revealed preserved lung morphology through 48 hours. By 60 hours there was evidence of edema, and modest neutrophilic infiltrate. Conclusions Rat lung DU uptake in vivo increased after just 48 hours of \u3e 98% O2 exposure, prior to the onset of any substantial evidence of lung injury. These results suggest that apoptotic endothelial cells are the primary contributors to the enhanced DU lung uptake, and support the utility of DU imaging for detecting early endothelial cell death in vivo

Optical Imaging of Lipopolysaccharide-induced Oxidative Stress in Acute Lung Injury from Hyperoxia and Sepsis

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many acute and chronic pulmonary disorders such as acute lung injury (ALI) in adults and bronchopulmonary dysplasia (BPD) in premature infants. Bacterial infection and oxygen toxicity, which result in pulmonary vascular endothelial injury, contribute to impaired vascular growth and alveolar simplification seen in the lungs of premature infants with BPD. Hyperoxia induces ALI, reduces cell proliferation, causes DNA damage and promotes cell death by causing mitochondrial dysfunction. The objective of this study was to use an optical imaging technique to evaluate the variations in fluorescence intensities of the auto-fluorescent mitochondrial metabolic coenzymes, NADH and FAD in four different groups of rats. The ratio of these fluorescence signals (NADH/FAD), referred to as NADH redox ratio (NADH RR) has been used as an indicator of tissue metabolism in injuries. Here, we investigated whether the changes in metabolic state can be used as a marker of oxidative stress caused by hyperoxia and bacterial lipopolysaccharide (LPS) exposure in neonatal rat lungs. We examined the tissue redox states of lungs from four groups of rat pups: normoxic (21% O2) pups, hyperoxic (90% O2) pups, pups treated with LPS (normoxic + LPS), and pups treated with LPS and hyperoxia (hyperoxic + LPS). Our results show that hyperoxia oxidized the respiratory chain as reflected by a ~ 31% decrease in lung tissue NADH RR as compared to that for normoxic lungs. LPS treatment alone or with hyperoxia had no significant effect on lung tissue NADH RR as compared to that for normoxic or hyperoxic lungs, respectively. Thus, NADH RR serves as a quantitative marker of oxidative stress level in lung injury caused by two clinically important conditions: hyperoxia and LPS exposure

Integrated Computational Model of Lung Tissue Bioenergetics

Altered lung tissue bioenergetics plays a key role in the pathogenesis of lung diseases. A wealth of information exists regarding the bioenergetic processes in mitochondria isolated from rat lungs, cultured pulmonary endothelial cells, and intact rat lungs under physiological and pathophysiological conditions. However, the interdependence of those processes makes it difficult to quantify the impact of a change in a single or multiple process(es) on overall lung tissue bioenergetics. Integrated computational modeling provides a mechanistic and quantitative framework for the bioenergetic data at different levels of biological organization. The objective of this study was to develop and validate an integrated computational model of lung bioenergetics using existing experimental data from isolated perfused rat lungs. The model expands our recently developed computational model of the bioenergetics of mitochondria isolated from rat lungs by accounting for glucose uptake and phosphorylation, glycolysis, and the pentose phosphate pathway. For the mitochondrial region of the model, values of kinetic parameters were fixed at those estimated in our recent model of the bioenergetics of mitochondria isolated from rat lungs. For the cytosolic region of the model, intrinsic parameters such as apparent Michaelis constants were determined based on previously published enzyme kinetics data, whereas extrinsic parameters such as maximal reaction and transport velocities were estimated by fitting the model solution to published data from isolated rat lungs. The model was then validated by assessing its ability to predict existing experimental data not used for parameter estimation, including relationships between lung nucleotides content, lung lactate production rate, and lung energy charge under different experimental conditions. In addition, the model was used to gain novel insights on how lung tissue glycolytic rate is regulated by exogenous substrates such as glucose and lactate, and assess differences in the bioenergetics of mitochondria isolated from lung tissue and those of mitochondria in intact lungs. To the best of our knowledge, this is the first model of lung tissue bioenergetics. The model provides a mechanistic and quantitative framework for integrating available lung tissue bioenergetics data, and for testing novel hypotheses regarding the role of different cytosolic and mitochondrial processes in lung tissue bioenergetics

Hypoxia Preconditioning Increases Survival and Decreases Expression of Toll-like Receptor 4 in Pulmonary Artery Endothelial Cells Exposed to Lipopolysaccharide

Pulmonary or systemic infections and hypoxemic respiratory failure are among the leading causes of admission to intensive care units, and these conditions frequently exist in sequence or in tandem. Inflammatory responses to infections are reproduced by lipopolysaccharide (LPS) engaging Toll-like receptor 4 (TLR4). Apoptosis is a hallmark of lung injury in sepsis. This study was conducted to determine whether preexposure to LPS or hypoxia modulated the survival of pulmonary artery endothelial cells (PAECs). We also investigated the role TLR4 receptor expression plays in apoptosis due to these conditions. Bovine PAECs were cultured in hypoxic or normoxic environments and treated with LPS. TLR4 antagonist TAK-242 was used to probe the role played by TLR4 receptors in cell survival. Cell apoptosis and survival were measured by caspase 3 activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) incorporation. TLR4 expression and tumor necrosis factor α (TNF-α) production were also determined. LPS increased caspase 3 activity in a TAK-242-sensitive manner and decreased MTT incorporation. Apoptosis was decreased in PAECs preconditioned with hypoxia prior to LPS exposure. LPS increased TNF-α production, and hypoxic preconditioning blunted it. Hypoxic preconditioning reduced LPS-induced TLR4 messenger RNA and TLR4 protein. TAK-242 decreased to baseline the LPS-stimulated expression of TLR4 messenger RNA regardless of environmental conditions. In contrast, LPS followed by hypoxia substantially increased apoptosis and cell death. In conclusion, protection from LPS-stimulated PAEC apoptosis by hypoxic preconditioning is attributable in part to reduction in TLR4 expression. If these signaling pathways apply to septic patients, they may account for differing sensitivities of individuals to acute lung injury depending on oxygen tensions in PAECs in vivo

Protection by Inhaled Hydrogen Therapy in a Rat Model of Acute Lung Injury can be Tracked \u3cem\u3ein vivo\u3c/em\u3e Using Molecular Imaging

Inhaled hydrogen gas (H2) provides protection in rat models of human acute lung injury (ALI). We previously reported that biomarker imaging can detect oxidative stress and endothelial cell death in vivo in a rat model of ALI. Our objective was to evaluate the ability of 99mTc-hexamethylpropyleneamineoxime (HMPAO) and 99mTc-duramycin to track the effectiveness of H2 therapy in vivo in the hyperoxia rat model of ALI. Rats were exposed to room air (normoxia), 98% O2 + 2% N2 (hyperoxia) or 98% O2 + 2% H2 (hyperoxia+H2) for up to 60 h. In vivo scintigraphy images were acquired following injection of 99mTc-HMPAO or 99mTc-duramycin. For hyperoxiarats, 99mTc-HMPAO and 99mTc-duramycin lung uptake increased in a time-dependent manner, reaching a maximum increase of 270% and 150% at 60 h, respectively. These increases were reduced to 120% and 70%, respectively, in hyperoxia+H2 rats. Hyperoxia exposure increased glutathione content in lung homogenate (36%) more than hyperoxia+H2 (21%), consistent with increases measured in 99mTc-HMPAO lung uptake. In 60-h hyperoxia rats, pleural effusion, which was undetectable in normoxia rats, averaged 9.3 gram/rat, and lung tissue 3-nitrotyrosine expression increased by 790%. Increases were reduced by 69% and 59%, respectively, in 60-h hyperoxia+H2 rats. This study detects and tracks the anti-oxidant and anti-apoptotic properties of H2 therapy in vivo after as early as 24 h of hyperoxia exposure. The results suggest the potential utility of these SPECT biomarkers for in vivo assessment of key cellular pathways in the pathogenesis of ALI and for monitoring responses to therapies