A hallmark of hyperoxic acute lung injury is the influx of inflammatory cells to lung tissue and the production of proinflammatory cytokines, such as IL-1β; however, the mechanisms connecting hyperoxia and the inflammatory response to lung damage is not clear. The inflammasome protein complex activates caspase-1 to promote the processing and secretion of proinflammatory cytokines. We hypothesized that hyperoxia-induced K+ efflux activates the inflammasome via the purinergic P2X7 receptor to cause inflammation and hyperoxic acute lung injury. To test this hypothesis, we characterized the expression and activation of inflammasome components in primary murine alveolar macrophages exposed to hyperoxia (95% oxygen and 5% CO2) in vitro, and in alveolar macrophages isolated from mice exposed to hyperoxia (100% oxygen). Our results showed that hyperoxia increased K+ efflux, inflammasome formation, release of proinflammatory cytokines, and induction of caspase-1 and IL-1β cleavage both in vitro and in vivo. The P2X7 agonist ATP enhanced hyperoxia-induced inflammasome activation, whereas the P2X7 antagonist, oxidized ATP, inhibited hyperoxia induced inflammasome activation. In addition, when ATP was scavenged with apyrase, hyperoxia-induced inflammasome activation was significantly decreased. Furthermore, short hairpin RNA silencing of inflammasome components abrogated hyperoxia-induced secretion of proinflammatory cytokines in vitro. These results suggest that hyperoxia induces K+ efflux through the P2X7 receptor, leading to inflammasome activation and secretion of proinflammatory cytokines. These events would affect the permeability of the alveolar epithelium and ultimately lead to epithelial barrier dysfunction and cell death.
Overexpression of IL-6 markedly diminishes hyperoxic lung injury, hyperoxia-induced cell death, and DNA fragmentation, and enhances Bcl-2 expression. We hypothesized that changes in the interactions between Bcl-2 family members play an important role in the IL-6–mediated protective response to oxidative stress. Consistent with this hypothesis, we found that IL-6 induced Bcl-2 expression, both in vivo and in vitro, disrupted interactions between proapoptotic and antiapoptotic factors, and suppressed H2O2-induced loss of mitochondrial membrane potential in vitro. In addition, IL-6 overexpression in mice protects against hyperoxia-induced lung mitochondrial damage. The overexpression of Bcl-2 in vivo prolonged the survival of mice exposed to hyperoxia and inhibited alveolar capillary protein leakage. In addition, apoptosis-associated DNA fragmentation was substantially reduced in these animals. This IL-6–mediated protection was lost when Bcl-2 was silenced, demonstrating that Bcl-2 is an essential mediator of IL-6 cytoprotection. Finally, Bcl-2 blocked the dissociation of Bak from mitofusion protein (Mfn) 2, and inhibited the interaction between Bak and Mfn1. Taken together, our results suggest that IL-6 induces Bcl-2 expression to perform cytoprotective functions in response to oxygen toxicity, and that this effect is mediated by alterations in the interactions between Bak and Mfns.
lung injury; mitochondria; apoptosis; cytochrome c; Bax
Hyperoxic acute lung injury (HALI) is characterized by a cell death response that is inhibited by IL-6. Suppressor of cytokine signaling-1 (SOCS-1) is an antiapoptotic negative regulator of the IL-6–mediated Janus kinase–signal transducer and activator of transcription signaling pathway. We hypothesized that SOCS-1 is a critical regulator and key mediator of IL-6–induced cytoprotection in HALI. To test this hypothesis, we characterized the expression of SOCS-1 and downstream apoptosis signal–regulating kinase (ASK)-1–Jun N-terminal kinase signaling molecules in small airway epithelial cells in the presence of H2O2, which induces oxidative stress. We also examined these molecules in wild-type and lung-specific IL-6 transgenic (Tg+) mice exposed to 100% oxygen for 72 hours. In control small airway epithelial cells exposed to H2O2 or in wild-type mice exposed to 100% oxygen, a marked induction of ASK-1 and pJun N-terminal kinase was observed. Both IL-6–stimulated endogenous SOCS-1 and SOCS-1 overexpression abolished H2O2-induced ASK-1 activation. In addition, IL-6 Tg+ mice exposed to 100% oxygen exhibited reduced ASK-1 levels and enhanced SOCS-1 expression compared with wild-type mice. Interestingly, no significant changes in activation of the key ASK-1 activator, tumor necrosis factor receptor-1/tumor necrosis factor receptor–associated factor-2 were observed between wild-type and IL-6 Tg+ mice. Furthermore, the interaction between SOCS-1 and ASK-1 promotes ubiquitin-mediated degradation both in vivo and in vitro. These studies demonstrate that SOCS-1 is an important regulator in IL-6–induced cytoprotection against HALI.
IL-6; apoptosis signal–regulating kinase-1; suppressor of cytokine signaling-1; lung injury; tumor necrosis factor receptor-1
Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA (miRNA) are potential candidates for coordinated regulation of these disease stimuli.
Methods and Results
Using a network biology approach, we identify miRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying miRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haplo insufficiency of the BMP Receptor Type 2 (BMPRII). To validate these predictions, we have found that hypoxia and BMPRII signaling independently up-regulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 down-regulates BMPRII expression. Furthermore, miR-21 directly represses RhoB expression and Rho kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is up-regulated in pulmonary tissue from several rodent models of PH and in humans with PH. Upon induction of disease in miR-21-null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH.
A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying miRNA in PH.
Pulmonary Heart Disease; microRNA; Network Biology; Molecular Biology; Vasculature
Pulmonary arterial hypertension (PAH) is a life threatening, progressive condition which eventually leads to fatal right heart failure. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, is increased in the pulmonary arteries of patients with pulmonary hypertension. Endothelin-1 acts through the stimulation of 2 subtypes of receptors (endothelin receptor subtypes A [ETA] and B [ETB]). In PAH patients, ETRAs block the deleterious vasoconstrictor effects of ET-1, and ETRA treatment in PAH patients has been shown to be safe and efficacious. Sitaxsentan is an orally active, highly ETA selective ETRA that, in clinical trials, has demonstrated improvements in exercise capacity, functional class and hemodynamics in PAH patients. Sitaxsentan has been shown to be safe, well tolerated, and associated with a lower incidence of liver toxicity than other approved ETRAs.
endothelin receptor antagonist; endothelin receptor inhibitor; endothelin A; sitaxsentan; pulmonary hypertension; endothelin
Pulmonary vasodilator testing is currently used to guide management of patients with pulmonary arterial hypertension (PAH). However, the utility of the pulmonary vascular response to inhaled nitric oxide (NO) and oxygen in predicting survival has not been established.
Eighty patients with WHO Group I PAH underwent vasodilator testing with inhaled NO (80 ppm with 90% O2 for 10 minutes) at the time of diagnosis. Changes in right atrial (RA) pressure, mean pulmonary artery pressure (mPAP), pulmonary capillary wedge pressure, Fick cardiac output, and pulmonary vascular resistance (PVR) were tested for associations to long-term survival (median follow-up 2.4 years).
Five-year survival was 56%. Baseline PVR (mean ± SD 850±580 dyne-sec/cm5) and mPAP (49±14 mmHg) did not predict survival, whereas the change in either PVR or mPAP while breathing NO & O2 was predictive. Patients with a ≥30% reduction in PVR with inhaled NO and O2 had a 53% relative reduction in mortality (Cox hazard ratio 0.47, 95% confidence interval (CI) 0.23-0.99, P=0.047), and those with a ≥12% reduction in mPAP with inhaled NO and O2 had a 55% relative reduction in mortality (hazard ratio 0.45, 95% CI 0.22-0.96, P=0.038). The same vasoreactive thresholds predicted survival in the subset of patients who never were treated with calcium channel antagonists (n=66). Multivariate analysis showed that decreases in PVR and mPAP with inhaled NO and O2 were independent predictors of survival.
Reduction in PVR or mPAP during short-term administration of inhaled NO and O2 predicts survival in PAH patients.
pulmonary arterial hypertension; vasodilator testing; nitric oxide; vasoreactivity
Pulmonary vasodilator testing is currently used to guide management of patients with pulmonary arterial hypertension (PAH). However, the utility of the pulmonary vascular response to inhaled nitric oxide (NO) and oxygen in predicting survival has not been established. Eighty patients with WHO Group I PAH underwent vasodilator testing with inhaled NO (80 ppm with 90% O2 for 10 minutes) at the time of diagnosis. Changes in right atrial (RA) pressure, mean pulmonary artery pressure (mPAP), pulmonary capillary wedge pressure, Fick cardiac output, and pulmonary vascular resistance (PVR) were tested for associations to long-term survival (median follow-up 2.4 years). Five-year survival was 56%. Baseline PVR (mean±SD 850±580 dyne-sec/cm5) and mPAP (49±14 mmHg) did not predict survival, whereas the change in either PVR or mPAP while breathing NO and O2 was predictive. Patients with a ≥30% reduction in PVR with inhaled NO and O2 had a 53% relative reduction in mortality (Cox hazard ratio 0.47, 95% confidence interval (CI) 0.23-0.99, P=0.047), and those with a ≥12% reduction in mPAP with inhaled NO and O2 had a 55% relative reduction in mortality (hazard ratio 0.45, 95% CI 0.22-0.96, P=0.038). The same vasoreactive thresholds predicted survival in the subset of patients who never were treated with calcium channel antagonists (n=66). Multivariate analysis showed that decreases in PVR and mPAP with inhaled NO and O2 were independent predictors of survival. Reduction in PVR or mPAP during short-term administration of inhaled NO and O2 predicts survival in PAH patients.
pulmonary arterial hypertension; vasodilator testing; nitric oxide; vasoreactivity
The clinical relevance of exercise-induced pulmonary arterial hypertension (EIPAH) is uncertain, and its existence has never been well-studied by direct measurements of central hemodynamics. Using invasive cardiopulmonary exercise testing, we hypothesized that EIPAH represents a symptomatic stage of PAH, physiologically intermediate between resting pulmonary arterial hypertension and normal.
Methods and Results
406 consecutive clinically indicated cardiopulmonary exercise tests with radial and pulmonary arterial catheters and radionuclide ventriculographic scanning were analyzed. The invasive hemodynamic phenotype of EIPAH (n=78) was compared to resting PAH (n=15), and normals (n=16). Log-log plots of mean pulmonary artery pressure vs. oxygen uptake (VO2) were obtained, and a “join-point” for a least residual sum-of-squares for two straight-line segments (slopes m1, m2) was determined; m2 < m1 = “plateau”, and m2 > m1 = “takeoff” pattern. At maximum exercise, VO2 (55.8±20.3 vs. 66.5±16.3 vs. 91.7±13.7 % predicted) was lowest in resting PAH, intermediate in EIPAH, and highest in normals, whereas mean pulmonary artery pressure (48.4±11.1 vs. 36.6±5.7 vs. 27.4+3.7 mmHg) and pulmonary vascular resistance (294±158 vs. 161±60 vs. 62±20 dynes-sec/cm5, respectively, p<0.05) followed an opposite pattern. An EIPAH plateau (n=32) was associated with lower VO2max (60.6±15.1 vs. 72.0±16.1 %predicted) and maximum cardiac output (78.2±17.1 vs. 87.8±18.3 %predicted), and a higher resting pulmonary vascular resistance (247±101 vs. 199±56 dynes-sec/cm5, p<0.05) than takeoff (n=40). The plateau pattern was most common in resting PAH, while the takeoff pattern was present in nearly all normals.
EIPAH is an early, mild, and clinically relevant phase of the PAH spectrum.
Circulation; Physiology; Hemodynamics; Pulmonary hypertension; Exercise
Portopulmonary hypertension is defined as the combination of pulmonary arterial
hypertension with portal hypertension and presents management complications in
patients awaiting liver transplantation. The combination of these vascular
disorders has a marked impact on mortality. At present the recommendations for
management are limited because of the paucity of definitive clinical trials. We
have reviewed the available data on prevalence, diagnosis and treatment. It is
clearly time to more formally approach the study of this patient population.
pulmonary hypertension; portal hypertension; liver disease; portopulmonary hypertension; vasodilators
Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in septic patients and animals. We hypothesized that the extent of pGSN reduction correlates with outcomes of septic patients and that circulating actin is a manifestation of sepsis.
We assayed pGSN in plasma samples from non-surgical septic patients identified from a pre-existing database which prospectively enrolled patients admitted to adult intensive care units at an academic hospital. We identified 21 non-surgical septic patients for the study. Actinemia was detected in 17 of the 21 patients, suggesting actin released into circulation from injured tissues is a manifestation of sepsis. Furthermore, we documented the depletion of pGSN in human clinical sepsis, and that the survivors had significantly higher pGSN levels than the non-survivors (163±47 mg/L vs. 89±48 mg/L, p = 0.01). pGSN levels were more strongly predictive of 28-day mortality than APACHE III scores. For every quartile reduction in pGSN, the odds of death increased 3.4-fold.
We conclude that circulating actin and pGSN deficiency are associated with early sepsis. The degree of pGSN deficiency correlates with sepsis mortality. Reversing pGSN deficiency may be an effective treatment for sepsis.
Inspired oxygen, an essential therapy for cardiorespiratory disorders, has the potential to generate reactive oxygen species that damage cellular DNA. Although DNA damage is implicated in diverse pulmonary disorders, including neoplasia and acute lung injury, the type and magnitude of DNA lesion caused by oxygen in vivo is unclear. We used single-cell gel electrophoresis (SCGE) to quantitate two distinct forms of DNA damage, base adduction and disruption of the phosphodiester backbone, in the lungs of mice. Both lesions were induced by oxygen, but a marked difference between the two was found. With 40 h of oxygen exposure, oxidized base adducts increased 3- to 4-fold in the entire population of lung cells. This lesion displayed temporal characteristics (a progressive increase over the first 24 h) consistent with a direct effect of reactive oxygen species attack upon DNA. DNA strand breaks, on the other hand, occurred in < 10% of pulmonary cells, which acquired severe levels of the lesion; dividing cells were preferentially affected. Characteristics of these cells suggested that DNA strand breakage was secondary to cell death, rather than a primary effect of reactive oxygen species attack on DNA. By analysis of IL-6– and IL-11–overexpressing transgenic animals, which are resistant to hyperoxia, we found that DNA strand breaks, but not base damage, correlated with acute lung injury. Analysis of purified alveolar type 2 preparations from hyperoxic mice indicated that strand breaks preferentially affected this cell type.
8-oxoguanine; acute lung injury; apoptosis; DNA damage; hyperoxia
Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11–induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11–induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11– and VEGF-induced cytoprotection.
Despite continuous advances in technologic and pharmacologic management, the mortality rate from septic shock remains high. Care of patients with sepsis includes measures to support the circulatory system and treat the underlying infection. There is a substantial body of knowledge indicating that fluid resuscitation, vasopressors, and antibiotics accomplish these goals. Recent clinical trials have provided new information on the addition of individual adjuvant therapies. Consensus on how current therapies should be prescribed is lacking. We present the reasoning and preferences of a group of intensivists who met to discuss the management of an actual case. The focus is on management, with emphasis on the criteria by which treatment decisions are made. It is clear from the discussion that there are areas where there is agreement and areas where opinions diverge. This presentation is intended to show how experienced intensivists apply clinical science to their practice of critical care medicine.
sepsis; septic shock; resuscitation; pneumonia
Hyperoxia is an important cause of acute lung injury. To determine whether IL-13 is protective in hyperoxia, we compared the survival in 100% O2 of transgenic mice that overexpress IL-13 in the lung and of nontransgenic littermate controls. IL-13 enhanced survival in 100% O2. One hundred percent of nontransgenic mice died in 4–5 days, whereas 100% of IL-13–overexpressing mice lived for more than 7 days, and many lived 10–14 days. IL-13 also stimulated VEGF accumulation in mice breathing room air, and it interacted with 100% 2 to increase VEGF accumulation further. The 164–amino acid isoform was the major VEGF moiety in bronchoalveolar lavage from transgenic mice in room air, whereas the 120– and 188–amino acid isoforms accumulated in these mice during hyperoxia. In addition, antibody neutralization of VEGF decreased the survival of IL-13–overexpressing mice in 100% 2. These studies demonstrate that IL-13 has protective effects in hyperoxic acute lung injury. They also demonstrate that IL-13, alone and in combination with 100% 2, stimulates pulmonary VEGF accumulation, that this stimulation is isoform-specific, and that the protective effects of IL-13 are mediated, in part, by VEGF.