Claude A. Piantadosi

Nitric oxide in the human respiratory cycle

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O2) 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 HbO2 saturation (FeO2). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO2 over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO2. Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O2 uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O2 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.

Mitochondrial Generation of Reactive Oxygen Species After Brain Ischemia in the Rat

BACKGROUND AND PURPOSE Brain mitochondria have a substantial capacity to generate reactive oxygen species after ischemia when the components of the respiratory chain are reduced and molecular oxygen is present. We tested the hypothesis that brain mitochondria in vivo produce reactive oxygen species after ischemia/reperfusion (IR) in rats at a rate sufficient to escape endogenous antioxidant defenses. METHODS Ischemia-dependent production of hydroxyl radical in the hippocampus of the anesthetized rat was monitored with the use of intracerebral microdialysis. Transient global ischemia was produced by bilateral carotid artery occlusion and hemorrhagic hypotension to a mean arterial pressure of 35 mm Hg for 15 minutes followed by reperfusion for 60 minutes. Salicylic acid was infused into the hippocampus during the experiments, and changes in the recovery of its hydroxylated product, 2,3-dihydroxybenzoic acid (2,3-DHBA), were used to assess the effects of inhibitors of mitochondrial complex I on formation of hydroxyl radical during IR. Hydroxylation data from control groups of animals were compared with data from animals undergoing IR during treatment with either a mitochondrial complex I inhibitor alone or the inhibitor plus succinic acid. RESULTS Transient ischemia led to a fivefold increase in the recovery of 2,3-DHBA by microdialysis after 1 hour relative to control animals (P < .05). Inhibition of mitochondrial complex I prevented 2,3-DHBA formation after IR; this effect could be reversed by infusion of succinic acid by microdialysis during IR. CONCLUSIONS The data indicate that reactive oxygen species generated by mitochondrial electron transport escape cellular antioxidant defenses and promote highly damaging hydroxyl radical activity after transient brain ischemia in the rat.

Brain Temperature Alters Hydroxyl Radical Production During Cerebral Ischemia/Reperfusion in Rats

Selective neuronal cell death in the CA, pyramidal cells of the hippocampus and neurons of the dorsolateral striatum as a consequence of brain ischemia/reperfusion (IR) can be ameliorated with brain hypothermia. Since postischemic injury is mediated partially by chemical production of reactive oxygen species (ROS), decreased ROS production may be one of the mechanisms responsible for cerebral protection by hypothermia. To determine if ischemic brain temperature alters ROS production, reversible IR was produced in rats by occlusion of both carotid arteries with hemorrhagic hypotension. After 15 min of ischemia, circulation was restored for 60 min. Brain temperature was maintained during ischemia at either 30, 36, or 39°C and kept at 36–37°C after reperfusion. Using cerebral microdialysis, we measured nonenzymatic hydroxylation of salicylate by HPLC with electrochemical detection in the hippocampus. CBF was also compared among the groups during IR. The results were that normothermic animals during reperfusion had persistently increased levels of the salicylate hydroxylation product, 2,3-dihydroxybenzoic acid (2,3-DHBA), reaching 251% of control at 60 min. This increase in 2,3-DHBA production was potentiated after 60 min of reperfusion (406% of control) with ischemic hyperthermia. In hypothermic ischemia, 2,3-DHBA production at 60 min was attenuated to 160% of control. CBF decreased to ∼5% of baseline value during ischemia, but increased three- to four-fold relative to control in all three groups. Therefore, the effects of ischemic brain temperature on 2,3-DHBA production did not correlate with changes in CBF during IR. We conclude that brain-temperature-related changes in OH · production are readily detected in the rat and decreased ROS generation may contribute to cerebral protection afforded by hypothermia during brain ischemia.

Mitochondrial oxidative stress after carbon monoxide hypoxia in the rat brain.

To better understand the mechanisms of tissue injury during and after carbon monoxide (CO) hypoxia, we studied the generation of partially reduced oxygen species (PROS) in the brains of rats subjected to 1% CO for 30 min, and then reoxygenated on air for 0-180 min. By determining H2O2-dependent inactivation of catalase in the presence of 3-amino-1,2,4-triazole (ATZ), we found increased H2O2 production in the forebrain after reoxygenation. The localization of catalase to brain microperoxisomes indicated an intracellular site of H2O2 production; subsequent studies of forebrain mitochondria isolated during and after CO hypoxia implicated nearby mitochondria as the source of H2O2. In the mitochondria, two periods of PROS production were indicated by decreases in the ratio of reduced to oxidized glutathione (GSH/GSSG). These periods of oxidative stress occurred immediately after CO exposure and 120 min after reoxygenation, as indicated by 50 and 43% decreases in GSH/GSSG, respectively. The glutathione depletion data were supported by studies of hydroxyl radical generation using a salicylate probe. The salicylate hydroxylation products, 2,3 and 2,5-dihydroxybenzoic acid (DHBA), were detected in mitochondria from CO exposed rats in significantly increased amounts during the same time intervals as decreases in GSH/GSSG. The DHBA products were increased 3.4-fold immediately after CO exposure, and threefold after 120 min reoxygenation. Because these indications of oxidative stress were not prominent in the postmitochondrial fraction, we propose that PROS generated in the brain after CO hypoxia originate primarily from mitochondria. These PROS may contribute to CO-mediated neuronal damage during reoxygenation after severe CO intoxication.

Heme Oxygenase-1 Regulates Cardiac Mitochondrial Biogenesis via Nrf2-Mediated Transcriptional Control of Nuclear Respiratory Factor-1

Heme oxygenase (HO)-1 is a protective antioxidant enzyme that prevents cardiomyocyte apoptosis, for instance, during progressive cardiomyopathy. Here we identify a fundamental aspect of the HO-1 protection mechanism by demonstrating that HO-1 activity in mouse heart stimulates the bigenomic mitochondrial biogenesis program via induction of NF-E2–related factor (Nrf)2 gene expression and nuclear translocation. Nrf2 upregulates the mRNA, protein, and activity for HO-1 as well as mRNA and protein for nuclear respiratory factor (NRF)-1. Mechanistically, in cardiomyocytes, endogenous carbon monoxide (CO) generated by HO-1 overexpression stimulates superoxide dismutase-2 upregulation and mitochondrial H2O2 production, which activates Akt/PKB. Akt deactivates glycogen synthase kinase-3&bgr;, which permits Nrf2 nuclear translocation and occupancy of 4 antioxidant response elements (AREs) in the NRF-1 promoter. The ensuing accumulation of nuclear NRF-1 protein leads to gene activation for mitochondrial biogenesis, which opposes apoptosis and necrosis caused by the cardio-toxic anthracycline chemotherapeutic agent, doxorubicin. In cardiac cells, Akt silencing exacerbates doxorubicin-induced apoptosis, and in vivo CO rescues wild-type but not Akt1−/− mice from doxorubicin cardiomyopathy. These findings consign HO-1/CO signaling through Nrf2 and Akt to the myocardial transcriptional program for mitochondrial biogenesis, provide a rationale for targeted mitochondrial CO therapy, and connect cardiac mitochondrial volume expansion with the inducible network of xenobiotic and antioxidant cellular defenses.

The Acute Respiratory Distress Syndrome

Clinical Principles Severe respiratory distress and 1 or more risk factors (including infection, aspiration, pancreatitis, and trauma) Impaired arterial oxygenation (hypoxemia) Bilateral pulmonary infiltrates on chest radiograph No clinical evidence of elevated left atrial pressure (or pulmonary artery occlusion pressure of 18 mm Hg if measurements are available) Physiologic Principles The cardinal feature of ARDS, refractory hypoxemia, is caused by formation of protein-rich alveolar edema after damage to the integrity of the lungs alveolar-capillary barrier. Alveolar-capillary damage in ARDS can be initiated by physical or chemical injury or by extensive activation of innate inflammatory responses. Such damage causes the lungs edema safety factor to decrease by about half, and edema develops at low capillary pressures. Widespread alveolar flooding in ARDS impairs alveolar ventilation, excludes oxygen, and inactivates surfactant; this, in turn, decreases lung compliance, increases dispersion of ventilation and perfusion, and produces intrapulmonary shunt. Intrapulmonary shunt is disclosed when hypoxemia does not improve despite oxygen administration; hypoxemia in ARDS does respond to positive end-expiratory pressure, which is applied carefully in accordance with strategies of lung-protective ventilation designed to avoid ventilator-associated lung injury and worsening ARDS. The acute respiratory distress syndrome (ARDS) is defined by noncardiogenic pulmonary edema and respiratory failure in the seriously ill patient. The diagnosis is clinical, established by the development of new bilateral pulmonary infiltrates and severe hypoxemia without congestive heart failure (1). The risk for ARDS also depends on both host and etiologic factors. The most common causes are sepsis, pneumonia, aspiration, trauma, pancreatitis, several blood transfusions, smoke or toxic gas inhalation, and certain types of drug toxicity (2, 3). Several etiologic factors often are present, and this increases the probability of developing the syndrome. The acute respiratory distress syndrome is a major cause of morbidity, death, and cost in intensive care units. Our review describes the clinical, etiologic, and physiologic basis of ARDS and summarizes our understanding of how its molecular pathogenesis leads to the physiologic alterations of respiratory failure, emphasizing factors known to be involved in the formation and resolution of permeability pulmonary edema. It also provides a physiologic basis for understanding and implementing modern strategies for the respiratory management of patients with ARDS. In 1967, Ashbaugh and colleagues (4) defined ARDS as an acute lung injury syndrome associated with trauma, sepsis, or aspiration. The syndromes similarities to shock lung and neonatal respiratory distress led to its original name, the adult respiratory distress syndrome, now the acute respiratory distress syndrome. Over the years, ARDS became associated with clinical risk factors that may cause lung injury either by direct involvement or by secondary processes that activate systemic inflammation and coincidentally damage the lung. The incidence of ARDS in at-risk populations is not certain, but prospective estimates range from 1.5 to 12.9 cases per 100 000 people per year depending on diagnostic criteria (5). The most common cause of ARDS, severe infection, accounts for approximately half of cases. These infections may involve localized disease (such as pneumonia) or systemic disease, including sepsis, sepsis syndrome, and septic shock. Sepsis-related conditions, particularly severe gram-negative infections, are also associated with multiple organ failure with or without progressive respiratory failure. The multiple organ failure syndrome is the major cause of death in ARDS, and the mortality rate of the syndrome with ARDS is about 40% (6-9). The AmericanEuropean Consensus Conference on ARDS formally defined ARDS (see Clinical Principles) to improve diagnostic consistency and interpretation of epidemiologic and therapeutic studies (1). The AmericanEuropean Consensus Conference also recommended a working definition for milder acute lung injury also based on the presence of hypoxemia and pulmonary infiltrates without elevated left atrial pressure. Usually, the compliance of the lungs also decreases. The acute respiratory distress syndrome is distinguished solely by pulmonary gas exchange defined by the ratio of Pao 2 to the inspired fraction of oxygen (Fio 2). A Pao 2Fio 2 ratio of 300 or less defines acute lung injury, and a ratio of 200 or less defines ARDS regardless of the amount of positive end-expiratory pressure (PEEP) needed to support oxygenation. Physiologic indexes of oxygenation are diagnostically useful, but the Pao 2Fio 2 ratio and physiologic scoring systems do not correlate with prognosis (9, 10). The consensus definitions recognize the clinical syndromes without attention to specific molecular, immune, or physical events that cause respiratory failure. The acute respiratory distress syndrome is thus the clinical expression of a group of diverse processes that produce widespread alveolar damage. Regardless of cause, lung damage causes fluid to leak across the alveolarcapillary barrier (despite relatively normal pulmonary circulatory pressures) and to produce enough alveolar edema to cause the cardinal physiologic manifestation of the syndrome, refractory hypoxemia (Figure 1). Figure 1. Relationships between extravascular lung water and development of hypoxemia in the acute respiratory distress syndrome. o PAP ClinicalPathologic Correlation in ARDS The lungs alveolarcapillary structure normally provides a large surface for gas exchange and a tight barrier between alveolar gas and pulmonary capillary blood. Diffuse damage to the alveolar region occurs in the acute or exudative phase of acute lung injury and ARDS (Figure 2). This damage involves both the endothelial and epithelial surfaces and disrupts the lungs barrier function, flooding alveolar spaces with fluid, inactivating surfactant, causing inflammation, and producing severe gas exchange abnormalities and loss of lung compliance. These events are reflected in the presence of bilateral infiltrates, which are indistinguishable by conventional radiology from cardiogenic pulmonary edema (11). Computed tomography of the chest often demonstrates heterogeneous areas of consolidation and atelectasis, predominantly in the dependent lung (12, 13), although areas of apparent sparing may still show inflammation. Pathologic findings consist of diffuse alveolar damage, including capillary injury, and areas of exposed alveolar epithelial basement membrane (14-16). The alveolar spaces are lined with hyaline membranes and are filled with protein-rich edema fluid and inflammatory cells. The interstitial spaces, alveolar ducts, small vessels, and capillaries also contain macrophages, neutrophils, and erythrocytes. Figure 2. Cellular and molecular events that interfere with gas exchange in the acute respiratory distress syndrome. The acute phase may resolve or progress to a fibrosis phase with persistent hypoxemia, increased dead space, pulmonary hypertension, and further loss of lung compliance. Chest radiographs may show new linear opacities consistent with evolving fibrosis. Computed tomography often shows diffuse interstitial thickening and blebs or honeycombing (13). Pathologic examination of the lung shows fibrosis with collagen deposition, acute and chronic inflammation, and incomplete resolution of edema (16). The recovery phase of ARDS is characterized by resolution of hypoxemia and improvement in dead space and lung compliance. Radiographic abnormalities usually resolve, but microscopic fibrosis remains. Clinically, failure to improve in the first week of treatment and the presence of extensive alveolar epithelial injury are poor prognostic signs (3, 10, 17). Survivors of ARDS tend to be young, and pulmonary function generally recovers gradually over a year, but residual abnormalities often remain (18-24), including mild restriction or obstruction, low diffusing capacity, and impaired gas exchange with exercise (18-21). Persistent abnormalities of pulmonary function occur more commonly after severe ARDS and a need for prolonged mechanical ventilation (20, 21). Survivors of ARDS also have diminished health-related and pulmonary diseasespecific quality of life, as well as systemic effects, such as muscle wasting, weakness, and fatigue (22-24). Causes of ARDS In North America and Europe, sepsis (including pneumonia) is the most common cause of ARDS, but multiple transfusions, severe trauma, and aspiration of gastric contents are also independent risk factors (1-3, 5). Patients with sepsis are at the highest risk, and many patients with severe sepsis develop respiratory failure (25). Many infectious organisms, as well as molecular components of gram-negative and gram-positive bacteria, can trigger intense pulmonary inflammation. The presence and duration of septic shock, particularly if circulating endotoxin (lipopolysaccharide) is present, are associated with a higher incidence of ARDS (1). However, many patients with sepsis never develop ARDS, and many patients with sepsis-induced ARDS survive. In all causes of ARDS, innate genetic differences regulate the lungs immune responses and are important in pathogenesis. Innate immunity provides the first-line host defense against pathogens and may play a key role in the development of ARDS in both infections and other conditions. The innate immune system identifies certain patterns of cell activation; therefore, a few pattern recognition receptors recognize a wide range of microbes and endogenous ligands (such as fibronectin and hyaluronic acid) (26, 27). For instance, pattern recognition receptors recognize the highly conserved lipid A portion of lipopolysaccharide, which produces a pathogen-associated molecular pattern. Some patte

Survival in critical illness is associated with early activation of mitochondrial biogenesis.

RATIONALE We previously reported outcome-associated decreases in muscle energetic status and mitochondrial dysfunction in septic patients with multiorgan failure. We postulate that survivors have a greater ability to maintain or recover normal mitochondrial functionality. OBJECTIVES To determine whether mitochondrial biogenesis, the process promoting mitochondrial capacity, is affected in critically ill patients. METHODS Muscle biopsies were taken from 16 critically ill patients recently admitted to intensive care (average 1-2 d) and from 10 healthy, age-matched patients undergoing elective hip surgery. MEASUREMENTS AND MAIN RESULTS Survival, mitochondrial morphology, mitochondrial protein content and enzyme activity, mitochondrial biogenesis factor mRNA, microarray analysis, and phosphorylated (energy) metabolites were determined. Ten of 16 critically ill patients survived intensive care. Mitochondrial size increased with worsening outcome, suggestive of swelling. Respiratory protein subunits and transcripts were depleted in critically ill patients and to a greater extent in nonsurvivors. The mRNA content of peroxisome proliferator-activated receptor γ coactivator 1-α (transcriptional coactivator of mitochondrial biogenesis) was only elevated in survivors, as was the mitochondrial oxidative stress protein manganese superoxide dismutase. Eventual survivors demonstrated elevated muscle ATP and a decreased phosphocreatine/ATP ratio. CONCLUSIONS Eventual survivors responded early to critical illness with mitochondrial biogenesis and antioxidant defense responses. These responses may partially counteract mitochondrial protein depletion, helping to maintain functionality and energetic status. Impaired responses, as suggested in nonsurvivors, could increase susceptibility to mitochondrial damage and cellular energetic failure or impede the ability to recover normal function. Clinical trial registered with clinical trials.gov (NCT00187824).

The CO/HO system reverses inhibition of mitochondrial biogenesis and prevents murine doxorubicin cardiomyopathy

The clinical utility of anthracycline anticancer agents, especially doxorubicin, is limited by a progressive toxic cardiomyopathy linked to mitochondrial damage and cardiomyocyte apoptosis. Here we demonstrate that the post-doxorubicin mouse heart fails to upregulate the nuclear program for mitochondrial biogenesis and its associated intrinsic antiapoptosis proteins, leading to severe mitochondrial DNA (mtDNA) depletion, sarcomere destruction, apoptosis, necrosis, and excessive wall stress and fibrosis. Furthermore, we exploited recent evidence that mitochondrial biogenesis is regulated by the CO/heme oxygenase (CO/HO) system to ameliorate doxorubicin cardiomyopathy in mice. We found that the myocardial pathology was averted by periodic CO inhalation, which restored mitochondrial biogenesis and circumvented intrinsic apoptosis through caspase-3 and apoptosis-inducing factor. Moreover, CO simultaneously reversed doxorubicin-induced loss of DNA binding by GATA-4 and restored critical sarcomeric proteins. In isolated rat cardiac cells, HO-1 enzyme overexpression prevented doxorubicin-induced mtDNA depletion and apoptosis via activation of Akt1/PKB and guanylate cyclase, while HO-1 gene silencing exacerbated doxorubicin-induced mtDNA depletion and apoptosis. Thus doxorubicin disrupts cardiac mitochondrial biogenesis, which promotes intrinsic apoptosis, while CO/HO promotes mitochondrial biogenesis and opposes apoptosis, forestalling fibrosis and cardiomyopathy. These findings imply that the therapeutic index of anthracycline cancer chemotherapeutics can be improved by the protection of cardiac mitochondrial biogenesis.

Apoptosis and Delayed Neuronal Damage after Carbon Monoxide Poisoning in the Rat

Delayed neurological damage after CO hypoxia was studied in rats to determine whether programmed cell death (PCD), in addition to necrosis, is involved in neuronal death. In rats exposed to either air or CO (2500 ppm), microdialysis in brain cortex and hippocampus was performed to determine the extent of glutamate release and hydroxyl radical generation during the exposures. Groups of control and CO-exposed rats also were tested in a radial maze to assess the effects of the CO exposures on learning and memory. At 3, 7, and 21 days after CO exposure brains were perfusion-fixed and hematoxylin-eosin (H&E) was used to assess injury and to select regions for further examination. DNA fragmentation was sought by examining cryosections with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) reaction. We found significant increases in glutamate release and .OH generation during and immediately after CO hypoxia. CO-exposed rats showed learning and memory deficits after exposure associated with heterogeneous cell loss in cortex, globus pallidus, and cerebellum. The frontal cortex was affected most seriously; the damage was slight at Day 3, increased at Day 7, and persistent at Day 21 after CO exposure. TUNEL staining was positive at all three time points, and TUNEL-labeled cells were distributed similarly to eosinophilic cells. The number of cells stained by TUNEL was less than by H&E and amounted to 2 to 5% of all cell nuclei in regions of injury. Ultrastructural features of both neuronal necrosis and apoptosis also were observed readily by electron microscopy. These findings indicate that both necrosis and apoptosis (PCD) contribute to CO poisoning-induced brain cell death.

Carbon monoxide, reactive oxygen signaling, and oxidative stress.

The ubiquitous gas, carbon monoxide (CO), is of substantial biological importance, but apart from its affinity for reduced transition metals, particularly heme-iron, it is surprisingly nonreactive-as is the ferrous-carbonyl-in living systems. CO does form strong complexes with heme proteins for which molecular O2 is the preferred ligand and to which are attributed diverse physiological, adaptive, and toxic effects. Lately, it has become apparent that both exogenous and endogenous CO produced by heme oxygenase engender a prooxidant milieu in aerobic mammalian cells which initiates signaling related to reactive oxygen species (ROS) generation. ROS signaling contingent on CO can be segregated by CO concentration-time effects on cellular function, by the location of heme proteins, e.g., mitochondrial or nonmitochondrial sites, or by specific oxidation-reduction (redox) reactions. The fundamental responses to CO involve overt physiological regulatory events, such as activation of redox-sensitive transcription factors or stress-activated kinases, which institute compensatory expression of antioxidant enzymes and other adaptations to oxidative stress. In contrast, responses originating from highly elevated or protracted CO exposures tend to be nonspecific, produce untoward biological oxidations, and interfere with homeostasis. This brief overview provides a conceptual framework for understanding CO biology in terms of this physiological-pathological hierarchy.