c-Kit-positive cardiac stem cells nested in hypoxic niches are activated by stem cell factor reversing the aging myopathy.

RATIONALE: Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. OBJECTIVE: A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. METHODS AND RESULTS: Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. CONCLUSIONS: Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.

Carbon monoxide negatively regulates NLRP3 inflammasome activation in macrophages.

Inflammasomes are cytosolic protein complexes that promote the cleavage of caspase-1, which leads to the maturation and secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß) and IL-18. Among the known inflammasomes, the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3)-dependent inflammasome is critically involved in the pathogenesis of various acute or chronic inflammatory diseases. Carbon monoxide (CO), a gaseous molecule physiologically produced in cells and tissues during heme catabolism, can act as an anti-inflammatory molecule and a potent negative regulator of Toll-like receptor signaling pathways. To date, the role of CO in inflammasome-mediated immune responses has not been fully investigated. Here, we demonstrated that CO inhibited caspase-1 activation and the secretion of IL-1ß and IL-18 in response to lipopolysaccharide (LPS) and ATP treatment in bone marrow-derived macrophages. CO also inhibited IL-18 secretion in response to LPS and nigericin treatment, another NLRP3 inflammasome activation model. In contrast, CO did not suppress IL-18 secretion in response to LPS and poly(dA:dT), an absent in melanoma 2 (AIM2)-mediated inflammasome model. LPS and ATP stimulation induced the formation of complexes between NLRP3 and apoptosis-associated speck-like protein, or NLRP3 and caspase-1. CO treatment inhibited these molecular interactions that were induced by LPS and ATP. Furthermore, CO inhibited mitochondrial ROS generation and the decrease of mitochondrial membrane potential induced by LPS and ATP in macrophages. We also observed that the inhibitory effect of CO on the translocation of mitochondrial DNA into the cytosol was associated with suppression of cytokine secretion. Our results suggest that CO negatively regulates NLRP3 inflammasome activation by preventing mitochondrial dysfunction.

Autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema.

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by chronic exposure to cigarette smoke (CS), which involves airway obstruction and alveolar loss (i.e., emphysema). The mechanisms of COPD pathogenesis remain unclear. Our previous studies demonstrated elevated autophagy in human COPD lung, and as a cellular and tissue response to CS exposure in an experimental model of emphysema in vivo. We identified the autophagic protein microtubule-associated protein 1 light chain-3B (LC3B) as a positive regulator of CS-induced lung epithelial cell death. We now extend these initial observations to explore the mechanism by which LC3B mediates CS-induced apoptosis and emphysema development in vivo. Here, we observed that LC3B(-/-) mice had significantly decreased levels of apoptosis in the lungs after CS exposure, and displayed resistance to CS-induced airspace enlargement, relative to WT littermate mice. We found that LC3B associated with the extrinsic apoptotic factor Fas in lipid rafts in an interaction mediated by caveolin-1 (Cav-1). The siRNA-dependent knockdown of Cav-1 sensitized epithelial cells to CS-induced apoptosis, as evidenced by enhanced death-inducing signaling complex formation and caspase activation. Furthermore, Cav-1(-/-) mice exhibited higher levels of autophagy and apoptosis in the lung in response to chronic CS exposure in vivo. In conclusion, we demonstrate a pivotal role for the autophagic protein LC3B in CS-induced apoptosis and emphysema, suggestive of novel therapeutic targets for COPD treatment. This study also introduces a mechanism by which LC3B, through interactions with Cav-1 and Fas, can regulate apoptosis.

TLR4 deficiency promotes autophagy during cigarette smoke-induced pulmonary emphysema.

Toll-like receptors (TLRs) exert important nonimmune functions in lung homeostasis. TLR4 deficiency promotes pulmonary emphysema. We examined the role of TLR4 in regulating cigarette smoke (CS)-induced autophagy, apoptosis, and emphysema. Lung tissue was obtained from chronic obstructive lung disease (COPD) patients. C3H/HeJ (Tlr4-mutated) mice and C57BL/10ScNJ (Tlr4-deficient) mice and their respective control strains were exposed to chronic CS or air. Human or mouse epithelial cells (wild-type, Tlr4-knockdown, and Tlr4-deficient) were exposed to CS-extract (CSE). Samples were analyzed for TLR4 expression, and for autophagic or apoptotic proteins by Western blot analysis or confocal imaging. Chronic obstructive lung disease lung tissues and human pulmonary epithelial cells exposed to CSE displayed increased TLR4 expression, and increased autophagic [microtubule-associated protein-1 light-chain-3B (LC3B)] and apoptotic (cleaved caspase-3) markers. Beas-2B cells transfected with TLR4 siRNA displayed increased expression of LC3B relative to control cells, basally and after exposure to CSE. The basal and CSE-inducible expression of LC3B and cleaved caspase-3 were elevated in pulmonary alveolar type II cells from Tlr4-deficient mice. Wild-type mice subjected to chronic CS-exposure displayed airspace enlargement;, however, the Tlr4-mutated or Tlr4-deficient mice exhibited a marked increase in airspace relative to wild-type mice after CS-exposure. The Tlr4-mutated or Tlr4-deficient mice showed higher levels of LC3B under basal conditions and after CS exposure. The expression of cleaved caspase-3 was markedly increased in Tlr4-deficient mice exposed to CS. We describe a protective regulatory function of TLR4 against emphysematous changes of the lung in response to CS.

Carbon monoxide reverses established pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by a progressive increase in pulmonary vascular resistance leading to right heart failure. Carbon monoxide (CO) has emerged as a potently protective, homeostatic molecule that prevents the development of vascular disorders when administered prophylactically. The data presented in this paper demonstrate that CO can also act as a therapeutic (i.e., where exposure to CO is initiated after pathology is established). In three rodent models of PAH, a 1 hour/day exposure to CO reverses established PAH and right ventricular hypertrophy, restoring right ventricular and pulmonary arterial pressures, as well as the pulmonary vascular architecture, to near normal. The ability of CO to reverse PAH requires functional endothelial nitric oxide synthase (eNOS/NOS3) and NO generation, as indicated by the inability of CO to reverse chronic hypoxia-induced PAH in eNOS-deficient (nos3-/-) mice versus wild-type mice. The restorative function of CO was associated with a simultaneous increase in apoptosis and decrease in cellular proliferation of vascular smooth muscle cells, which was regulated in part by the endothelial cells in the hypertrophied vessels. In conclusion, these data demonstrate that CO reverses established PAH dependent on NO generation supporting the use of CO clinically to treat pulmonary hypertension.

Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor beta1 (TGF-beta1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-beta1-induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad-cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.

Autophagic protein LC3B confers resistance against hypoxia-induced pulmonary hypertension.

RATIONALE: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown. OBJECTIVES: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH. METHODS: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B(-/-)), or early growth response-1 (Egr-1(-/-)), were evaluated for vascular morphology and hemodynamics. MEASUREMENTS AND MAIN RESULTS: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B(-/-) or Egr-1(-/-), but not Beclin 1(+/-), mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia. CONCLUSIONS: The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B(-/-) and Egr-1(-/-) mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.

Nitrite potently inhibits hypoxic and inflammatory pulmonary arterial hypertension and smooth muscle proliferation via xanthine oxidoreductase-dependent nitric oxide generation.

BACKGROUND: Pulmonary arterial hypertension is a progressive proliferative vasculopathy of the small pulmonary arteries that is characterized by a primary failure of the endothelial nitric oxide and prostacyclin vasodilator pathways, coupled with dysregulated cellular proliferation. We have recently discovered that the endogenous anion salt nitrite is converted to nitric oxide in the setting of physiological and pathological hypoxia. Considering the fact that nitric oxide exhibits vasoprotective properties, we examined the effects of nitrite on experimental pulmonary arterial hypertension. METHODS AND RESULTS: We exposed mice and rats with hypoxia or monocrotaline-induced pulmonary arterial hypertension to low doses of nebulized nitrite (1.5 mg/min) 1 or 3 times a week. This dose minimally increased plasma and lung nitrite levels yet completely prevented or reversed pulmonary arterial hypertension and pathological right ventricular hypertrophy and failure. In vitro and in vivo studies revealed that nitrite in the lung was metabolized directly to nitric oxide in a process significantly enhanced under hypoxia and found to be dependent on the enzymatic action of xanthine oxidoreductase. Additionally, physiological levels of nitrite inhibited hypoxia-induced proliferation of cultured pulmonary artery smooth muscle cells via the nitric oxide-dependent induction of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). The therapeutic effect of nitrite on hypoxia-induced pulmonary hypertension was significantly reduced in the p21-knockout mouse; however, nitrite still reduced pressures and right ventricular pathological remodeling, indicating the existence of p21-independent effects as well. CONCLUSIONS: These studies reveal a potent effect of inhaled nitrite that limits pathological pulmonary arterial hypertrophy and cellular proliferation in the setting of experimental pulmonary arterial hypertension.

Caveolin-1 regulates the secretion and cytoprotection of Cyr61 in hyperoxic cell death.

Cysteine-rich 61 (Cyr61) belongs to the CCN family and mediates cell proliferation, survival, and apoptosis. Our previous studies showed that Cyr61 protected against hyperoxia-induced lung cell death via Akt phosphorylation. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. Emerging data show that cav-1 regulates signal transduction-associated proteins that reside in the caveolae. Numerous integrin-related pathways, including PI3K/Akt-induced cell survival are controlled by cav-1-mediated signaling. Our data showed that recombinant Cyr61 promoted cell proliferation and resistance to hyperoxia-induced cell death in vitro. Neutralizing antibodies reversed the above effects, indicating functional role of secreted Cyr61 in response to hyperoxic stress. While deletion of cav-1 protected cells from hyperoxia-induced cell death, Cyr61-neutralizing antibodies abolished this protective effect. Furthermore, Cyr61 and cav-1 colocalized and physically interacted via integrins in bronchial epithelial cells. Deletion of cav-1 increased extracellular and decreased cytosolic Cyr61, both in vitro and in vivo. Pretreatment with Brefeldin A increased intracellular Cyr61 in cav-1(-/-) cells, while decreasing extracellular Cyr61. Taken together, Cav-1/Cyr61 interaction via integrins represents a novel pathway of Cyr61 signaling involving cav-1-dependent processes, which play a critical role in regulating hyperoxia-induced cell death.

Deletion of caveolin-1 protects hyperoxia-induced apoptosis via survivin-mediated pathways.

Hyperoxia-induced lung injury is an established model that mimics human acute respiratory distress syndrome. Cell death is a prominent feature in lungs following prolonged hyperoxia. Caveolae are omega-shaped invaginations of the plasma membrane. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. We have recently shown that deletion of cav-1 (cav-1-/-) protected against hyperoxia-induced cell death and lung injury both in vitro and in vivo; however, the mechanisms remain unclear. Survivin, a member of the inhibitor of apoptosis protein family, inhibits apoptosis in tumor cells. Although emerging evidence suggests that survivin is involved in wound healing, especially in vascular injuries, its role in hyperoxia-induced lung injury has not been investigated. Our current data demonstrated that hyperoxia induced apoptosis via suppressing survivin expression. Deletion of cav-1 abolished this suppression and subsequently protected against hyperoxia-induced apoptosis. Using "gain" and "loss" of function assays, we determined that survivin protected lung cells from hyperoxia-induced apoptosis via the inhibition of apoptosis executor caspase-3. Overexpression of survivin by deletion of cav-1 was regulated by Egr-1. Egr-1 functioned as a negative regulator of survivin expression. Deletion of cav-1 upregulated survivin via decreased Egr-1 binding of the survivin promoter region. Together, this study illustrates the effect of hyperoxia on survivin expression and the role of survivin in hyperoxia-induced apoptosis. We also demonstrate that deletion of cav-1 protects hyperoxia-induced apoptosis via modulation of survivin expression.

Carbon monoxide prevents ventilator-induced lung injury via caveolin-1.

OBJECTIVES: Carbon monoxide (CO) can confer anti-inflammatory protection in rodent models of ventilator-induced lung injury (VILI). Caveolin-1 exerts a critical role in cellular responses to mechanical stress and has been shown to mediate cytoprotective effects of CO in vitro. We sought to determine the role of caveolin-1 in lung susceptibility to VILI in mice. Furthermore, we assessed the role of caveolin-1 in the tissue-protective effects of CO in the VILI model. DESIGN: Prospective experimental study. SETTING: University laboratory. SUBJECTS: Wild type (wt) and caveolin-1 deficient (cav-1) mice. INTERVENTIONS: Mice were subjected to tracheostomy and arterial cannulation. Wt and cav-1 mice were ventilated with a tidal volume of 12 mL/kg body weight and a frequency of 80/minute for 5 minutes as control or for 8 hours with air in the absence or presence of CO (250 parts per million). Bronchoalveolar lavage and histology were used to determine lung injury. Lung sections or homogenates were analyzed for caveolin-1 expression by immunohistochemical staining or Western blotting, respectively. MEASUREMENTS AND MAIN RESULTS: Ventilation led to an increase in bronchoalveolar lavage protein concentration, cell count, neutrophil recruitment, and edema formation, which was prevented in the presence of CO. Although ventilation alone slightly induced caveolin-1 expression in epithelial cells, the application of CO during the ventilation significantly increased the expression of caveolin-1. In comparison with wt mice, mechanical ventilation of cav-1 mice led to a significantly higher degree of lung injury when compared with wt mice. In contrast to its effectiveness in wt mice, CO administration failed to reduce lung-injury markers in cav-1 mice. CONCLUSIONS: Caveolin-1 null mice are more susceptible to VILI. CO executes lung-protective effects during mechanical ventilation that are dependent, in part, on caveolin-1 expression.

Carbon monoxide protects against ventilator-induced lung injury via PPAR-gamma and inhibition of Egr-1.

RATIONALE: Ventilator-induced lung injury (VILI) leads to an unacceptably high mortality. In this regard, the antiinflammatory properties of inhaled carbon monoxide (CO) may provide a therapeutic option. OBJECTIVES: This study explores the mechanisms of CO-dependent protection in a mouse model of VILI. METHODS: Mice were ventilated (12 ml/kg, 1-8 h) with air in the absence or presence of CO (250 ppm). Airway pressures, blood pressure, and blood gases were monitored. Lung tissue was analyzed for inflammation, injury, and gene expression. Bronchoalveolar lavage fluid was analyzed for protein, cell and neutrophil counts, and cytokines. MEASUREMENTS AND MAIN RESULTS: Mechanical ventilation caused significant lung injury reflected by increases in protein concentration, total cell and neutrophil counts in the bronchoalveolar lavage fluid, as well as the induction of heme oxygenase-1 and heat shock protein-70 in lung tissue. In contrast, CO application prevented lung injury during ventilation, inhibited stress-gene up-regulation, and decreased lung neutrophil infiltration. These effects were preceded by the inhibition of ventilation-induced cytokine and chemokine production. Furthermore, CO prevented the early ventilation-dependent up-regulation of early growth response-1 (Egr-1). Egr-1-deficient mice did not sustain lung injury after ventilation, relative to wild-type mice, suggesting that Egr-1 acts as a key proinflammatory regulator in VILI. Moreover, inhibition of peroxysome proliferator-activated receptor (PPAR)-gamma, an antiinflammatory nuclear regulator, by GW9662 abolished the protective effects of CO. CONCLUSIONS: Mechanical ventilation causes profound lung injury and inflammatory responses. CO treatment conferred protection in this model dependent on PPAR-gamma and inhibition of Egr-1.

Deletion of caveolin-1 protects against oxidative lung injury via up-regulation of heme oxygenase-1.

Acute lung injury (ALI) is a major cause of morbidity and mortality in critically ill patients. Hyperoxia causes lung injury in animals and humans, and is an established model of ALI. Caveolin-1, a major constituent of caveolae, regulates numerous biological processes, including cell death and proliferation. Here we demonstrate that caveolin-1-null mice (cav-1(-/-)) were resistant to hyperoxia-induced death and lung injury. Cav-1(-/-) mice sustained reduced lung injury after hyperoxia as determined by protein levels in bronchoalveolar lavage fluid and histologic analysis. Furthermore, cav-1(-/-) fibroblasts and endothelial cells and cav-1 knockdown epithelial cells resisted hyperoxia-induced cell death in vitro. Basal and inducible expression of the stress protein heme oxygenase-1 (HO-1) were markedly elevated in lung tissue or fibroblasts from cav-1(-/-) mice. Hyperoxia induced the physical interaction between cav-1 and HO-1 in fibroblasts assessed by co-immunoprecipitation studies, which resulted in attenuation of HO activity. Inhibition of HO activity with tin protoporphyrin-IX abolished the survival benefits of cav-1(-/-) cells and cav-1(-/-) mice exposed to hyperoxia. The cav-1(-/-) mice displayed elevated phospho-p38 mitogen-activated protein kinase (MAPK) and p38beta expression in lung tissue/cells under basal conditions and during hyperoxia. Treatment with SB202190, an inhibitor of p38 MAPK, decreased hyperoxia-inducible HO-1 expression in wild-type and cav-1(-/-) fibroblasts. Taken together, our data demonstrated that cav-1 deletion protects against hyperoxia-induced lung injury, involving in part the modulation of the HO-1-cav-1 interaction, and the enhanced induction of HO-1 through a p38 MAPK-mediated pathway. These studies identify caveolin-1 as a novel component involved in hyperoxia-induced lung injury.

Carbon monoxide prevents multiple organ injury in a model of hemorrhagic shock and resuscitation.

The insult from severe hemorrhage is a multifactorial injury involving ischemia/reperfusion with inflammatory dysfunction. Our laboratories and others have demonstrated that the administration of exogenous carbon monoxide (CO) at low concentrations provides cytoprotection in vivo and in vitro. The purpose of these investigations was to test the hypothesis that CO protects against hemorrhagic shock- and resuscitation-induced systemic inflammation and end-organ damage. C57BL/6 mice underwent anesthesia and arterial cannulation. Mice were bled to reach a mean arterial pressure (MAP) of 25 mmHg and were maintained at this pressure for 2.5 h. Mice were then resuscitated with shed blood plus two times the volume of shed blood with Ringer's lactate. Sham animals were not bled. Additionally, mice were maintained in room air or in an environment of CO (250 parts per million). Primary mouse hepatocytes were harvested and used for in vitro cell viability and ATP measurement. These data demonstrate that delivery of a low concentration of inhaled CO protects against the development of end-organ injury decreases serum levels of inflammatory cytokines and increases serum levels of the anti-inflammatory cytokine IL-10. Additionally, CO paradoxically abrogates hemorrhage-induced hepatic cellular hypoxia. Furthermore, CO protected mouse hepatocytes from hypoxia-induced death while maintaining normal ATP levels. CO protects against systemic effects of hemorrhagic shock and resuscitation. The precise cellular mechanisms involved require further elucidation. CO may prove to be an adjunctive therapy that could be instituted rapidly and with ease as an out-of-hospital therapeutic modality for severe blood loss after trauma.

Carbon monoxide protection against endotoxic shock involves reciprocal effects on iNOS in the lung and liver.

Carbon monoxide (CO) has recently emerged as having potent cytoprotective properties; the mechanisms underlying these effects, however, are just beginning to be elucidated. In a rat model of lipopolysaccharide (LPS)-induced multiorgan failure, we demonstrate that exposure to a low concentration of CO for only 1 h imparts a potent defense against lethal endotoxemia and effectively abrogates the inflammatory response. Exposure to CO leads to long-term survival of >80% of animals vs. 20% in controls. In the lung, CO suppressed LPS-induced lung alveolitis and associated edema formation, while in the liver, it reduced expression of serum alanine aminotransferase, a marker of liver injury. This protection appears to be based in part on different mechanisms in the lung and liver in that CO had reciprocal effects on LPS-induced expression of iNOS and NO production, important mediators in the response to LPS. CO prevented the up-regulation of iNOS and NO in the lung while augmenting expression of iNOS and NO in the liver. Studies of primary lung macrophages and hepatocytes in vitro revealed a similar effect; CO inhibited LPS-induced cytokine production in lung macrophages while reducing LPS-induced iNOS expression and nitrite accumulation and protected hepatocytes from apoptosis while augmenting iNOS expression. Although it is unclear to which extent these changes in iNOS contribute to the cytoprotection conferred by CO, it is fascinating that in each organ CO influences iNOS in a manner known to be protective in that organ: NO is therapeutic in the liver while it is damaging in the lung.

Heme oxygenase-1-derived carbon monoxide protects hearts from transplant associated ischemia reperfusion injury.

Heme oxygenase-1 (HO-1) degrades heme into iron, biliverdin, and carbon monoxide (CO). HO-1 expression can be used therapeutically to ameliorate undesirable consequences of ischemia reperfusion injury (IRI), but the mechanism by which this occurs, remains to be established. Rat hearts, exposed to a prolonged period (24 h) of cold (4 degrees C) ischemia, failed to function upon transplantation into syngeneic recipients. Induction of HO-1 expression by administration of cobalt protoporphyrin IX (CoPPIX) to the graft donor restored graft function. Inhibition of HO-1 enzymatic activity, by administration of zinc protoporphyrin (ZnPPIX) at the time of transplantation, reversed the protective effect of HO-1. Exposure of the graft donor as well as the graft (during ischemia) to exogenous CO mimicked the protective effect of HO-1. This was associated with a significant reduction in the number of cells undergoing apoptosis in the graft with no apparent decrease of intravascular fibrin polymerization, platelet aggregation, or P-selectin expression. In conclusion, HO-1-derived CO prevents IRI associated with cardiac transplantation based on its antiapoptotic action. The observation that exposure of the donor and the graft to CO is sufficient to afford this protective effect should have important clinical implications in terms of preventing IRI associated with heart transplantation in humans.

Circadian rhythm reprogramming during lung inflammation.

Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian programme exhibits unique features, including a divergent group of rhythmic genes and metabolites compared with the basal state and a distinct periodicity and phase distribution. At the cellular level, endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex re-organization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.

Characterization of macroautophagic flux in vivo using a leupeptin-based assay.

Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.

Mitogen-activated protein kinases regulate susceptibility to ventilator-induced lung injury.

BACKGROUND: Mechanical ventilation causes ventilator-induced lung injury in animals and humans. Mitogen-activated protein kinases have been implicated in ventilator-induced lung injury though their functional significance remains incomplete. We characterize the role of p38 mitogen-activated protein kinase/mitogen activated protein kinase kinase-3 and c-Jun-NH(2)-terminal kinase-1 in ventilator-induced lung injury and investigate novel independent mechanisms contributing to lung injury during mechanical ventilation. METHODOLOGY AND PRINCIPLE FINDINGS: C57/BL6 wild-type mice and mice genetically deleted for mitogen-activated protein kinase kinase-3 (mkk-3(-/-)) or c-Jun-NH(2)-terminal kinase-1 (jnk1(-/-)) were ventilated, and lung injury parameters were assessed. We demonstrate that mkk3(-/-) or jnk1(-/-) mice displayed significantly reduced inflammatory lung injury and apoptosis relative to wild-type mice. Since jnk1(-/-) mice were highly resistant to ventilator-induced lung injury, we performed comprehensive gene expression profiling of ventilated wild-type or jnk1(-/-) mice to identify novel candidate genes which may play critical roles in the pathogenesis of ventilator-induced lung injury. Microarray analysis revealed many novel genes differentially expressed by ventilation including matrix metalloproteinase-8 (MMP8) and GADD45alpha. Functional characterization of MMP8 revealed that mmp8(-/-) mice were sensitized to ventilator-induced lung injury with increased lung vascular permeability. CONCLUSIONS: We demonstrate that mitogen-activated protein kinase pathways mediate inflammatory lung injury during ventilator-induced lung injury. C-Jun-NH(2)-terminal kinase was also involved in alveolo-capillary leakage and edema formation, whereas MMP8 inhibited alveolo-capillary protein leakage.

Protein kinase C alpha and zeta differentially regulate death-inducing signaling complex formation in cigarette smoke extract-induced apoptosis.

Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCalpha, zeta, epsilon, and eta, were activated by CSE exposure. Overexpression of wild-type PKCalpha inhibited, while PKCzeta promoted, CSE-induced cell death. Dominant-negative (dn)PKCzeta protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCalpha enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCalpha, but it was increased by dnPKCzeta. Expression of PKCalpha in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCzeta promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCalpha and PKCzeta via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.