Utilization of 19F MRI for Identification of Iraq-Afghanistan War Lung Injury.

INTRODUCTION: There is mounting evidence of respiratory problems related to military service in the Middle East in the past two decades due to environmental exposures during deployment (eg, sand storms and burn pits). This pilot study tests the hypothesis that regional lung function in subjects with prior deployment in Iraq and/or Afghanistan with suspected War Lung Injury (WLI) would be worse than subjects with normal lung function. MATERIALS AND METHODS: Five subjects meeting the inclusion and exclusion criteria were recruited for this pilot study. All subjects underwent spirometry, high-resolution chest computed tomography imaging, and 19F MRI. RESULTS: While the WLI subjects had normal pulmonary function tests and normal high-resolution chest computed tomography evaluations, their regional lung function from 19F MRI was abnormal with compartments with poor function showing slower filling time constants for ventilation. The scans of suspected WLI subjects show higher fractional lung volume with slow filling compartments similar to patients with chronic obstructive pulmonary disease in contrast to normal subjects. CONCLUSIONS: This is consistent with our premise that WLI results in abnormal lung function and reflects small airways dysfunction and suggests that we may be able to provide a more sensitive tool for evaluation of WLI suspected cases.

RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling.

S-nitrosothiols derived from nitric oxide are known to regulate cell signaling through thiol modification. Since small G protein RhoA contains cysteine residues in the GTP-binding domain which is critical for its function, modification these thiols may alter RhoA activity and lead to changes in the downstream signaling such as myosin light chain phosphorylation. However, it is still unclear that if RhoA activity and its downstream signals might be modulated by S-nitrosothiols and if the two cysteine residues located in the GTP-binding domain are critical for the regulation. In this study we show that S-nitroso-L-cysteine (CSNO) blocked RhoA activation as determined by either GDP/GTP exchange, active RhoA binding to rhotekin or RhoA translocation. CSNO was shown to lead to RhoA nitrosylation and RhoA thiol oxidation status was found to be consistent with loss of its activity. Mutation of all 6 single cysteine residues to serine showed that purified recombinant C20S mutant and C26/20S mutant were resistant to CSNO, but interestingly, in the intact cells only the double C16/20S mutant was resistant to CSNO. Moreover, inhibition of RhoA activation led to Rho-kinase inhibition and inhibition of Rho pathway signaling by CSNO. In both smooth muscle cells and aortic tissue, the outcome was inhibition of agonist-stimulated MYPT1 phosphorylation and reduced levels of myosin light chain phosphorylation. These effects of CSNO on MYPT1 and myosin light chain phosphorylation appear to be cGMP-independent since they were unaffected by inhibition of guanylyl cyclase. In contrast to CSNO, spermine NONOate did not alter RhoA GDP/GTP exchange and the effects of this compound on myosin light chain phosphorylation were blocked by guanylyl cyclase inhibition. And importantly, in C16/20S overexpressed smooth muscle cells, MYPT1 phosphorylation was resistant to the inhibitory effect of CSNO. Together, these data suggest that S-nitrosothiols regulate myosin light chain phosphorylation by inhibiting RhoA/Rho-kinase signaling through modification of RhoA cysteine residues at 16 and 20 in its GTP-binding domain, which might be an important therapeutic target for diseases with imbalanced vascular resistance.

Erythropoietin activates mitochondrial biogenesis and couples red cell mass to mitochondrial mass in the heart.

RATIONALE: Erythropoietin (EPO) is often administered to cardiac patients with anemia, particularly from chronic kidney disease, and stimulation of erythropoiesis may stabilize left ventricular and renal function by recruiting protective effects beyond the correction of anemia. OBJECTIVE: We examined the hypothesis that EPO receptor (EpoR) ligand-binding, which activates endothelial NO synthase (eNOS), regulates the prosurvival program of mitochondrial biogenesis in the heart. METHODS AND RESULTS: We investigated the effects of EPO on mitochondrial biogenesis over 14 days in healthy mice. Mice expressing a mitochondrial green fluorescent protein reporter construct demonstrated sharp increases in myocardial mitochondrial density after 3 days of EPO administration that peaked at 7 days and surpassed hepatic or renal effects and anteceded significant increases in blood hemoglobin content. Quantitatively, in wild-type mice, complex II activity, state 3 respiration, and mtDNA copy number increased significantly; also, resting energy expenditure and natural running speed improved, with no evidence of an increase in left ventricular mass index. Mechanistically, EPO activated cardiac mitochondrial biogenesis by enhancement of nuclear respiratory factor-1, PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha), and mitochondrial transcription factor-A gene expression in wild-type but not in eNOS(-/-) or protein kinase B (Akt1)(-/-) mice. EpoR was required, because EpoR silencing in cardiomyocytes blocked EPO-mediated nuclear translocation of nuclear respiratory factor-1. CONCLUSIONS: These findings support a new physiological and protective role for EPO, acting through its cell surface receptor and eNOS-Akt1 signal transduction, in matching cardiac mitochondrial mass to the convective O(2) transport capacity as erythrocyte mass expands.

Nitric oxide synthase-2 regulates mitochondrial Hsp60 chaperone function during bacterial peritonitis in mice.

Nitric oxide synthase-2 (NOS2) plays a critical role in reactive nitrogen species generation and cysteine modifications that influence mitochondrial function and signaling during inflammation. Here, we investigated the role of NOS2 in hepatic mitochondrial biogenesis during Escherichia coli peritonitis in mice. NOS2(-/-) mice displayed smaller mitochondrial biogenesis responses than Wt mice during E. coli infection according to differences in mRNA levels for the PGC-1 alpha coactivator, nuclear respiratory factor-1, mitochondrial transcription factor-A (Tfam), and mtDNA polymerase (Pol gamma). NOS2(-/-) mice did not significantly increase mitochondrial Tfam and Pol gamma protein levels during infection in conjunction with impaired mitochondrial DNA (mtDNA) transcription, loss of mtDNA copy number, and lower State 3 respiration rates. NOS2 blockade in mitochondrial-GFP reporter mice disrupted Hsp60 localization to mitochondria after E. coli exposure. Mechanistically, biotin-switch and immunoprecipitation studies demonstrated NOS2 binding to and S-nitros(yl)ation of Hsp60 and Hsp70. Specifically, NOS2 promoted Tfam accumulation in mitochondria by regulation of Hsp60-Tfam binding via S-nitros(yl)ation. In hepatocytes, site-directed mutagenesis identified (237)Cys as a critical residue for Hsp60 S-nitros(yl)ation. Thus, the role of NOS2 in inflammation-induced mitochondrial biogenesis involves both optimal gene expression for nuclear-encoded mtDNA-binding proteins and functional regulation of the Hsp60 chaperone that enables their importation for mtDNA transcription and replication.

Mitochondrial biogenesis in the pulmonary vasculature during inhalational lung injury and fibrosis.

Cell survival and injury repair is facilitated by mitochondrial biogenesis; however, the role of this process in lung repair is unknown. We evaluated mitochondrial biogenesis in the mouse lung in two injuries that cause acute inflammation and in two that cause chronic inflammation and pulmonary fibrosis. By using reporter mice that express green fluorescent protein (GFP) exclusively in mitochondria, we tracked mitochondrial biogenesis and correlated it with histologic lung injury, proliferation, and fibrosis. At 72 hours after acute LPS or continuous exposure to hyperoxia (Fio2, 1.0), the lungs showed diffuse infiltration by inflammatory cells in the alveolar region. In reporter mice, patchy new mitochondrial fluorescence was found in the alveolar region but was most prominent and unexpected in perivascular regions. At 14 days after instillation of asbestos or bleomycin, diffuse chronic inflammation had developed, and green fluorescence appeared in inflammatory cells in the expanded interstitium and was most intense in smooth muscle cells of pulmonary vessels. In all four lung injuries, mitochondrial fluorescence colocalized with mitochondrial superoxide dismutase, but not with proliferating cell nuclear antigen. These data indicate that vascular mitochondrial biogenesis is activated in diverse inhalational lung injuries along with oxidative stress. This finding indicates a unique and unexpected mechanism of metabolic adaptation to pulmonary fibrotic injuries.

A new activating role for CO in cardiac mitochondrial biogenesis.

To investigate a possible new physiological role of carbon monoxide (CO), an endogenous gas involved in cell signaling and cytotoxicity, we tested the hypothesis that the mitochondrial generation of reactive oxygen species by CO activates mitochondrial biogenesis in the heart. In mice, transient elevations of cellular CO by five- to 20-fold increased the copy number of cardiac mitochondrial DNA, the content of respiratory complex I-V and interfibrillar mitochondrial density within 24 hours. Mitochondrial biogenesis is activated by gene and protein expression of the nuclear respiratory factor 1 (NRF1) and NRF2, of peroxisome proliferator-activated receptor gamma co-activator-1alpha, and of mitochondrial transcription factor A (TFAM), which augmented the copy number of mitochondrial DNA (mtDNA). This is independent of nitric oxide synthase (NOS), as demonstrated by the identical responses in wild-type and endothelial NOS (eNOS)-deficient mice, and by the inhibition of inducible NOS (iNOS). In the heart and in isolated cardiomyocytes, CO activation involved both guanylate cyclase and the pro-survival kinase Akt/PKB. Akt activation was facilitated by mitochondrial binding of CO and by production of hydrogen peroxide (H(2)O(2)). Interference with Akt activity by blocking PI 3-kinase and by mitochondrial targeting of catalase to scavenge H(2)O(2) prevented binding of NRF1 to the Tfam promoter, thereby connecting mitochondrial H(2)O(2) to the pathway leading to mtDNA replication. The findings disclose mitochondrial CO and H(2)O(2) as new activating factors in cardiac mitochondrial biogenesis.

Carbon monoxide, oxidative stress, and mitochondrial permeability pore transition.

The cellular effects of carbon monoxide (CO) are produced primarily by CO binding to iron or other transition metals, which may also promote prooxidant activities of the more reactive gases, oxygen and nitric oxide. We tested the hypothesis that prooxidant effects of CO deregulate the calcium-dependent mitochondrial pore transition (MPT), which disrupts membrane potential and releases apoptogenic proteins. Rats were exposed to either CO (50 ppm) or hypobaric hypoxia (HH) for 1, 3, or 7 days, and liver mitochondria harvested to study protein expression and sensitivity to MPT by calcium and oxidants. Both exposures induced hypoxia-sensitive protein expression: hypoxia-inducible factor 1alpha (HIF-1alpha), heme oxygenase-1 (HO-1), and manganese SOD (SOD2), but SOD2 induction was greater by CO than by HH, especially at 7 days. Relative to HH, CO also caused significant early mitochondrial oxidative and nitrosative stress shown by decreases in GSH/GSSG and increases in protein 3-nitrotyrosine (3-NT) and protein mixed disulfide formation. This altered MPT sensitivity to calcium through an effect on the "S-site," causing loss of pore protection by adenine nucleotides. By 7 days, despite continued CO, nitrosative stress decreased and adenine nucleotide protection was restored to preexposure levels. This is the first evidence of functional mitochondrial pore stress caused by CO independently of its hypoxic effect, as well as a compensatory response exemplifying a mitochondrial phenotype shift. The implications are that cellular CO can activate or deactivate mitochondria for initiation of apoptosis in vivo.

Blockade of tissue factor-factor X binding attenuates sepsis-induced respiratory and renal failure.

Tissue factor expression in sepsis activates coagulation in the lung, which potentiates inflammation and leads to fibrin deposition. We hypothesized that blockade of factor X binding to the tissue factor-factor VIIa complex would prevent sepsis-induced damage to the lungs and other organs. Acute lung injury was produced in 15 adult baboons primed with killed Escherichia coli [1 x 10(9) colony-forming units (CFU)/kg], and then 12 h later, they were given 1 x 10(10) CFU/kg live E. coli by infusion. Two hours after live E. coli, animals received antibiotics with or without monoclonal antibody to tissue factor intravenously to block tissue factor-factor X binding. The animals were monitored physiologically for 34 h before being killed and their tissue harvested. The antibody treatment attenuated abnormalities in gas exchange and lung compliance, preserved renal function, and prevented tissue neutrophil influx and bowel edema relative to antibiotics alone (all P < 0.05). It also attenuated fibrinogen depletion (P < 0.01) and decreased proinflammatory cytokines, e.g., IL-6 and -8 (P < 0.01), in systemic and alveolar compartments. Similar protective effects of the antibody on IL-6 and -8 expression and permeability were found in lipopolysaccharide-stimulated endothelial cells. Blockade of factor X binding to the tissue factor-factor VIIa complex attenuates lung and organ injuries in established E. coli sepsis by attenuating the neutrophilic response and inflammatory pathways.

Carbon monoxide actuates O(2)-limited heme degradation in the rat brain.

The biochemical paradigm for carbon monoxide (CO) is driven by the century-old Warburg hypothesis: CO alters O(2)-dependent functions by binding heme proteins in competitive relation to 1/oxygen partial pressure (PO(2)). High PO(2) thus hastens CO elimination and toxicity resolution, but with more O(2), CO-exposed tissues paradoxically experience less oxidative stress. To help resolve this paradox we tested the Warburg hypothesis using a highly sensitive gas-reduction method to track CO uptake and elimination in brain, heart, and skeletal muscle in situ during and after exogenous CO administration. We found that CO administration does increase tissue CO concentration, but not in strict relation to 1/PO(2). Tissue gas uptake and elimination lag behind blood CO as predicted, but 1/PO(2) vs. [CO] fails even at hyperbaric PO(2). Mechanistically, we established in the brain that cytosol heme concentration increases 10-fold after CO exposure, which sustains intracellular CO content by providing substrate for heme oxygenase (HO) activated after hypoxia when O(2) is resupplied to cells rich in reduced pyridine nucleotides. We further demonstrate by analysis of CO production rates that this heme stress is not due to HO inhibition and that heme accumulation is facilitated by low brain PO(2). The latter becomes rate limiting for HO activity even at physiological PO(2), and the heme stress leads to doubling of brain HO-1 protein. We thus reveal novel biochemical actions of both CO and O(2) that must be accounted for when evaluating oxidative stress and biological signaling by these gases.

Lipopolysaccharide stimulates mitochondrial biogenesis via activation of nuclear respiratory factor-1.

Exposure to bacterial lipopolysaccharide (LPS) in vivo damages mitochondrial DNA (mtDNA) and interferes with mitochondrial transcription and oxidative phosphorylation (OXPHOS). Because this damage accompanies oxidative stress and is reversible, we postulated that LPS stimulates mtDNA replication and mitochondrial biogenesis via expression of factors responsive to reactive oxygen species, i.e. nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor-A. In testing this hypothesis in rat liver, we found that LPS induces NRF-1 protein expression and activity accompanied by mRNA expression for mitochondrial transcription factor-A, mtDNA polymerase gamma, NRF-2, and single-stranded DNA-binding protein. These events restored the loss in mtDNA copy number and OXPHOS gene expression caused by LPS and increased hepatocyte mitotic index, nuclear cyclin D1 translocation, and phosphorylation of pro-survival kinase, Akt. Thus, NRF-1 was implicated in oxidant-mediated mitochondrial biogenesis to provide OXPHOS for proliferation. This implication was tested in novel mtDNA-deficient cells generated from rat hepatoma cells that overexpress NRF-1. Depletion of mtDNA (rhoo clones) diminished oxidant production and caused loss of NRF-1 expression and growth delay. NRF-1 expression and growth were restored by exogenous oxidant exposure indicating that oxidative stress stimulates biogenesis in part via NRF-1 activation and corresponding to recovery events after LPS-induced liver damage.

Postlipopolysaccharide oxidative damage of mitochondrial DNA.

Selected structural and functional alterations of mitochondria induced by bacterial lipopolysaccharide (LPS) were investigated on the basis of the hypothesis that LPS initiates hepatic mitochondrial DNA (mtDNA) damage by oxidative mechanisms. After a single intraperitoneal injection of Escherichia coli LPS, liver mtDNA copy number decreased, as determined by Southern analysis, within 24 hours relative to nuclear 18S rRNA (p < 0.05). LPS induced a novel oxidant-dependent 3.8-kb mtDNA deletion in the region encoding NADH dehydrogenase subunits 1 and 2 and cytochrome c oxidase subunit I, which correlated with mitochondrial glutathione depletion. Expression of mitochondrial mRNA and transcription of mitochondrial RNA were suppressed, whereas mRNA expression increased for selected nuclear-encoded mitochondrial proteins. Resolution of mtDNA damage was mediated by importation of mitochondrial transcription factor A protein, a central regulator of mtDNA copy number, accompanied by binding of mitochondrial protein extract to the mitochondrial transcription factor A DNA-binding site. Hence, mtDNA integrity and transcriptional capacity after LPS administration appeared to be reinstated by mitochondrial biogenesis. These data provide the first link between LPS-mediated hepatic injury and a specific oxidative mtDNA deletion, which inhibits mitochondrial transcription and is restored by activation of mechanisms that lead to biogenesis.

Coagulation and inflammation in acute lung injury.

The acute respiratory distress syndrome (ARDS) is a severe lung injury in patients with sepsis and other acute inflammatory insults, which is characterized by fibrin deposition in the pulmonary parenchyma, vasculature, and airspaces. Recent evidence suggests that progressive ARDS is closely linked to activation of inflammation and coagulation. Coagulation becomes activated by circulating endotoxin or bacteria, and a procoagulant state develops in the vascular and the alveolar compartments of the lung. This state is Tissue Factor (TF)-dependent and associated with increased elaboration of inflammatory cytokines. A similar procoagulant state is found in bronchoalveolar lavage of patients with ARDS, suggesting that extravascular coagulation contributes to lung inflammation. TF and other coagulation proteins, including Factor Xa, thrombin, and fibrin, also contribute to the pathogenesis of acute lung injury through multi-level interactions with inflammatory effectors, in which these proteins coordinately act as regulators of tissue injury responses. Each coagulation protein has direct and independent effects on inflammatory events that influences lung injury through changes in cytokine elaboration, inflammatory cell migration and activation, surfactant function, and repair mechanisms. New interventional strategies directed at procoagulant activity highlight the importance of the coagulation system to acute lung injury and suggest that blockade of initiation of coagulation may have therapeutic benefit in patients with ARDS.

Nitric oxide in the human respiratory cycle.

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O(2)) by subserving the classical physiological responses of hypoxic vasodilation and hyperoxic vasconstriction in the human respiratory cycle. Here we show that in in vitro and ex vivo systems as well as healthy adults alternately exposed to hypoxia or hyperoxia (to dilate or constrict pulmonary and systemic arteries in vivo), binding of NO to hemes (FeNO) and thiols (SNO) of Hb varies as a function of HbO(2) saturation (FeO(2)). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO(2) over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO(2). Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O(2) uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O(2) and carbon dioxide, may therefore prove useful in the diagnosis and treatment of many human conditions and in the development of new therapies. Our results also help elucidate the link between RBC dyscrasias and cardiovascular morbidity.