Activation of AMP-Activated Protein Kinase by A769662 Ameliorates Sepsis-Induced Acute Lung Injury in Adult Mice.

A serious consequence of sepsis is acute lung injury, whose severity is particularly impacted by the age of the patient. AMP-activated protein kinase (AMPK) is a crucial regulator of cellular metabolism, which controls mitochondrial biogenesis and autophagy. Here, we investigated the effect of pharmacological activation of AMPK with A769662 on lung injury by using a model that would preferably mimic the clinical condition of adult patients. Male C57BL/6 retired breeder mice (7-9 months old) were subjected to sepsis by cecal ligation and puncture (CLP). Mice received vehicle or A769662 (10 mg/kg) intraperitoneally at 1 h after CLP. At 6 h after CLP, vehicle-treated mice exhibited severe lung injury and elevation of plasma pro-inflammatory cytokines when compared with control mice. At molecular analysis, lung injury was associated with downregulation of AMPKα1/α2 catalytic subunits and reduced phosphorylation of AMPKß1 regulatory subunit. Treatment with A769662 ameliorated lung architecture, reduced bacterial load in lung and blood, and attenuated plasma levels of interleukin-6. This protective effect was associated with nuclear phosphorylation of AMPKα1/α2 and AMPKß1, increased nuclear expression of peroxisome proliferator-activated receptor γ co-activator-α and increased autophagy, as evaluated by the light-chain (LC)3B-I and LC3B-II content, without changes in sirtuin-1 cellular dynamics. Treatment with A769662 alone or in combination with the antimicrobial agent imipenem (25 mg/kg) increased survival rate (29% and 51%, respectively) when compared with vehicle treatment (10%) at 7 days after CLP. These data suggest that pharmacological activation of AMPK might be a beneficial approach for the treatment of sepsis in adult population.

Age-dependent cardiac function during experimental sepsis: effect of pharmacological activation of AMP-activated protein kinase by AICAR.

Age represents a major risk factor for multiple organ failure, including cardiac dysfunction, in patients with sepsis. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis that controls mitochondrial biogenesis by activation of peroxisome proliferator-activated receptor-γ coactivator-1α and disposal of defective organelles by autophagy. We investigated whether AMPK dysregulation contributes to age-dependent cardiac injury in young (2-3 mo) and mature adult (11-13 mo) male mice subjected to sepsis by cecal ligation and puncture and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside affords cardioprotective effects. Plasma proinflammatory cytokines and myokine follistatin were similarly elevated in vehicle-treated young and mature adult mice at 18 h after sepsis. However, despite equivalent troponin I and T levels compared with similarly treated young mice, vehicle-treated mature adult mice exhibited more severe cardiac damage by light and electron microscopy analyses with more marked intercellular edema, inflammatory cell infiltration, and mitochondrial derangement. Echocardiography revealed that vehicle-treated young mice exhibited left ventricular dysfunction after sepsis, whereas mature adult mice exhibited a reduction in stroke volume without apparent changes in load-dependent indexes of cardiac function. At molecular analysis, phosphorylation of the catalytic subunits AMPK-α1/α2 was associated with nuclear translocation of peroxisome proliferator-activated receptor-γ coactivator-1α in vehicle-treated young but not mature adult mice. Treatment with 5-amino-4-imidazole carboxamide riboside ameliorated cardiac architecture derangement in mice of both ages. These cardioprotective effects were associated with attenuation of the systemic inflammatory response and amelioration of cardiac dysfunction in young mice only, not in mature adult animals. NEW & NOTEWORTHY Our data suggest that sepsis-induced cardiac dysfunction manifests with age-dependent characteristics, which are associated with a distinct regulation of AMP-activated protein kinase-dependent metabolic pathways. Consistent with this age-related deterioration, pharmacological activation of AMP-activated protein kinase may afford cardioprotective effects allowing a partial recovery of cardiac function in young but not mature age.

Metformin Exerts Beneficial Effects in Hemorrhagic Shock in An AMPKα1-Independent Manner.

Despite therapeutic advances in hemorrhagic shock, mortality from multiple organ failure remains high. AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Two catalytic subunits, α1 and α2, have been identified, with α1 subunit largely expressed in major organs. Here, we hypothesized that genetic deficiency of AMPKα1 worsens hemorrhage-induced multiple organ failure. We also investigated whether treatment with metformin, a clinically used drug for metabolic homeostasis, affords beneficial effects. AMPKα1 wild-type (WT) and knock-out mice (KO) were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with shed blood and Lactated Ringer's solution and treatment with vehicle or metformin. Mice were sacrificed at 3 h after resuscitation. Compared with vehicle-treated WT animals, KO animals exhibited a more severe hypotension, higher lung and liver injury and neutrophil infiltration, and higher levels of plasma inflammatory cytokines. Metformin treatment ameliorated organ injury and mean arterial blood pressure in both WT and KO mice, without affecting systemic cytokine levels. Furthermore, metformin treatment reduced liver lipid peroxidation and increased levels of complex II cosubstrate FAD and levels of ATP in WT and KO mice. Beneficial effects of metformin were associated with organ-specific nuclear-cytoplasmic shuttling and activation of liver kinase B1 and AMPKα2. Thus, our data suggest that AMPKα1 is an important regulator of hemodynamic stability and organ metabolic recovery during hemorrhagic shock. Our data also suggest that metformin affords beneficial effects, at least in part, independently of AMPKα1 and secondary to AMPKα2 activation, increase of Complex II function and reduction of oxidative stress.

Autophagy and mitochondrial biogenesis impairment contribute to age-dependent liver injury in experimental sepsis: dysregulation of AMP-activated protein kinase pathway.

Age is an independent risk factor of multiple organ failure in patients with sepsis. However, the age-related mechanisms of injury are not known. AMPK is a crucial regulator of energy homeostasis, which controls mitochondrial biogenesis by activation of peroxisome proliferator-activated receptor-γ coactivator-α (PGC-1α) and disposal of defective organelles by autophagy. We investigated whether AMPK dysregulation might contribute to age-dependent liver injury in young (2-3 mo) and mature male mice (11-13 mo) subjected to sepsis. Liver damage was higher in mature mice than in young mice and was associated with impairment of hepatocyte mitochondrial function, structure, and biogenesis and reduced autophagy. At molecular analysis, there was a time-dependent nuclear translocation of the active phosphorylated catalytic subunits AMPKα1/α2 and PGC-1α in young, but not in mature, mice after sepsis. Treatment with the AMPK activator 5-amino-4-imidazolecarboxamide riboside-1-ß-d-ribofuranoside (AICAR) improved liver mitochondrial structure in both age groups compared with vehicle. In loss-of-function studies, young knockout mice with systemic deficiency of AMPKα1 exhibited greater liver injury than did wild-type mice after sepsis. Our study suggests that AMPK is important for liver metabolic recovery during sepsis. Although its function may diminish with age, pharmacological activation of AMPK may be of therapeutic benefit.-Inata, Y., Kikuchi, S., Samraj, R. S., Hake, P. W., O'Connor, M., Ledford, J. R., O'Connor, J., Lahni, P., Wolfe, V., Piraino, G., Zingarelli, B. Autophagy and mitochondrial biogenesis impairment contribute to age-dependent liver injury in experimental sepsis: dysregulation of AMP-activated protein kinase pathway.

Metformin ameliorates gender-and age-dependent hemodynamic instability and myocardial injury in murine hemorrhagic shock.

Severity of multiple organ failure is significantly impacted by age and gender in patients with hemorrhagic shock. However, the molecular mechanisms underlying the enhanced organ injury are not fully understood. AMP-activated protein kinase (AMPK) is a pivotal orchestrator of metabolic responses during stress. We investigated whether hemorrhage-induced myocardial injury is age and gender dependent and whether treatment with metformin, an AMPK activator, affords cardioprotective effects. C57/BL6 young (3-5months) and mature (9-12months) male and female mice were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with blood and Lactated Ringer's solution. Vehicle-treated young and mature mice of both genders had a similar elevation of plasma inflammatory cytokines at 3h after resuscitation. However, vehicle-treated male mature mice experienced hemodynamic instability and higher myocardial damage than young male mice, as evaluated by echocardiography, histology and cardiovascular injury biomarkers. There was also a gender-dependent difference in cardiovascular injury in the mature group as vehicle-treated male mice exhibited more severe organ injury than female mice. At molecular analysis, vehicle-treated mature mice of both genders exhibited a marked downregulation of AMPKα activation and nuclear translocation of peroxisome proliferator-activated receptor γ co-activator α when compared with young mice. Treatment with metformin improved cardiovascular function and survival in mature animals of both genders. However, specific cardioprotective effects of metformin were gender-dependent. Metformin did not affect hemodynamic or inflammatory responses in young animals. Thus, our data suggest that targeting metabolic recovery with metformin may be a potential treatment approach in severe hemorrhage in adult population.

Age-Dependent Changes in AMPK Metabolic Pathways in the Lung in a Mouse Model of Hemorrhagic Shock.

The development of multiple organ failure in patients with hemorrhagic shock is significantly influenced by patient age. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which coordinates metabolic repair during cellular stress. We investigated whether AMPK-regulated signaling pathways are age-dependent in hemorrhage-induced lung injury and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside (AICAR) affords lung protective effects. Male C57/BL6 young mice (3-5 mo), mature adult mice (9-12 mo), and young AMPKα1 knockout mice (3-5 mo) were subjected to hemorrhagic shock by blood withdrawing, followed by resuscitation with shed blood and lactated Ringer's solution. Plasma proinflammatory cytokines were similarly elevated in C57/BL6 young and mature adult mice after hemorrhagic shock. However, mature adult mice exhibited more severe lung edema and neutrophil infiltration, and higher mitochondrial damage in alveolar epithelial type II cells, than did young mice. No change in autophagy was observed. At molecular analysis, the phosphorylation of the catalytic subunit AMPKα1 was associated with nuclear translocation of peroxisome proliferator-activated receptor γ co-activator-α in young, but not mature, adult mice. Treatment with AICAR ameliorated the disruption of lung architecture in mice of both ages; however, effects in mature adult mice were different than young mice and also involved inhibition of nuclear factor-κB. In young AMPKα1 knockout mice, AICAR failed to improve hypotension and lung neutrophil infiltration. Our data demonstrate that during hemorrhagic shock, AMPK-dependent metabolic repair mechanisms are important for mitigating lung injury. However, these mechanisms are less competent with age.

The AMPK Activator Aicar Ameliorates Age-Dependent Myocardial Injury in Murine Hemorrhagic Shock.

The development of myocardial dysfunction in patients with hemorrhagic shock is significantly impacted by the patient age. AMP-activated protein kinase (AMPK) is a pivotal orchestrator of energy homeostasis, which coordinates metabolic recovery after cellular stress. We investigated whether AMPK-regulated pathways are age-dependent in hemorrhage-induced myocardial injury and whether AMPK activation by 5-amino-4-imidazolecarboxamide riboside (AICAR) affords cardioprotective effects. Anesthetized C57/BL6 young (3-5 months old) and mature (9-12 months old) male mice were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with shed blood and Lactated Ringer's solution. Mice were sacrificed at 3 h after resuscitation, and plasma and hearts were harvested for biochemical assays. Vehicle-treated mature mice exhibited higher myocardial injury and higher levels of plasma biomarkers of cardiovascular injury (endocan and follistatin) when compared with young mice. Cardiac cell mitochondrial structure was also markedly impaired in vehicle-treated mature mice when compared with young mice. At molecular analysis, an increase of the phosphorylated catalytic subunit pAMPKα was associated with nuclear translocation of the peroxisome proliferator-activated receptor γ coactivator-α in young, but not mature mice. No changes in autophagy were observed as evaluated by the conversion of the light-chain (LC)3B-I protein to LC3B-II form. Treatment with AICAR ameliorated myocardial damage in both age groups. However, AICAR therapeutic effects were less effective in mature mice than young mice and involved distinct mechanisms of action. Thus, our data demonstrate that during hemorrhagic shock AMPK-dependent metabolic mechanisms are important for mitigating myocardial injury. However, these mechanisms are less competent with age.

Age-dependent therapeutic effects of liver X receptor-α activation in murine polymicrobial sepsis.

The severity of sepsis is significantly affected by advanced age; however, age-dependent molecular mechanisms of this susceptibility are unknown. Nuclear liver X receptor-α (LXRα) is a regulator of lipid metabolism with associated anti-inflammatory properties. Here, we investigated the role of LXRα in age-dependent lung injury and outcome of sepsis. Male C57BL/6, LXRα-deficient (LXRα(-/-)) and wild type (WT) (LXRα(+/+)) mice of different ages were subjected to sepsis by cecal ligation and puncture (CLP). In pharmacological studies, treatment with the LXRα ligand T0901317 reduced lung neutrophil infiltration in C57BL/6 mice aged from 1 to 8 mo when compared with vehicle-treated animals subjected to CLP. The LXRα ligand improved survival in young mice (2-3 mo old) but did not affect survival or neutrophil infiltration in mature adult mice (11-13 mo old). Immunoblotting revealed an age-dependent decrease of lung LXRα levels. Young LXRα(-/-) mice (2-3 mo old) exhibited earlier mortality than age-matched WT mice after CLP. Lung damage and neutrophil infiltration, lung activation of the pro-inflammatory NF-κB and plasma IL-6 levels were higher in LXRα(-/-) mice 18 h after CLP compared with LXRα(+/+) mice. This study suggests that the anti-inflammatory properties of LXRα in sepsis are age-dependent and severely compromised in mature adult animals.

Combined zinc supplementation with proinsulin C-peptide treatment decreases the inflammatory response and mortality in murine polymicrobial sepsis.

Zinc is a trace element vital for immune function during host response to infection. The proinsulin C-peptide has been shown to exert beneficial effects through activation of the anti-inflammatory peroxisome proliferator-activated receptor γ (PPARγ) in experimental endotoxemia. Some in vitro activities of C-peptide appear dependent on the presence of zinc. We investigated the effect of zinc supplementation before onset of sepsis on the anti-inflammatory properties of C-peptide. Male C57BL/6 mice were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Mice received zinc gluconate (1.3 mg/kg) intraperitoneally (i.p.) for 3 days before CLP. One hour after CLP, animals received C-peptide (280 nmol/kg i.p.) or the antimicrobial agent imipenem (25 mg/kg i.p.). Cecal ligation and puncture was associated with an 11% survival rate, pulmonary leukosequestration, and liver injury. Molecular analysis in lungs of septic mice showed increased nuclear activation of the proinflammatory extracellular signal-regulated kinases 1 and 2 and nuclear factor κB, but decreased PPARγ expression, when compared with sham animals. Combination of zinc supplementation with C-peptide posttreatment significantly improved survival rate (61%) similarly to antibiotic treatment (60%), ameliorated lung architecture and liver function, reduced tissue neutrophil infiltration, and increased bacterial clearance when compared with vehicle, C-peptide, or zinc treatment alone. These beneficial effects were associated with restored lung nuclear expression of PPARγ and reduction of phosphorylated extracellular signal-regulated kinases 1 and 2 and nuclear factor κB activities in comparison to vehicle or single treatment protocols. Our data demonstrate that short-term zinc prophylaxis before the infectious insult is a requisite for the anti-inflammatory properties of C-peptide by facilitating modulation of inflammatory pathways.

Conditional deletion of cardiomyocyte peroxisome proliferator-activated receptor γ enhances myocardial ischemia-reperfusion injury in mice.

The nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of the inflammatory response to an array of biologic insults. We have previously demonstrated that PPARγ ligands reduce myocardial ischemia-reperfusion injury in rodents. In the current study, we directly determined the role of cardiomyocyte PPARγ in ischemia-reperfusion injury, using a model of conditional cardiomyocyte-specific deletion of PPARγ in vivo. In mice, α-myosin heavy chain-restricted Cre-mediated PPARγ deficiency was induced by tamoxifen treatment (30 mg/kg intraperitoneally) for 4 days (PPARγ mice), whereas controls included mice treated with the oil diluent vehicle (PPARγ mice). Western blot and histochemical analyses confirmed that expression of PPARγ protein was abolished in cardiomyocytes of mice treated with tamoxifen, but not with vehicle. After tamoxifen or vehicle treatment, animals were subjected to 30-min ligation of the left anterior descending coronary artery followed by 2-h reperfusion. In PPARγ mice, myocardial ischemia and reperfusion induced extensive myocardial damage, which was associated with elevated tissue activity of myeloperoxidase, indicating infiltration of neutrophils, and elevated plasma levels of troponin I when compared with PPARγ mice. Upon echocardiographic analysis, PPARγ mice also demonstrated ventricular dilatation and systolic dysfunction. Plasma levels of the proinflammatory cytokines interleukin 1ß and interleukin 6 were higher in PPARγ mice when compared with PPARγ mice. These pathological events in PPARγ mice were associated with enhanced nuclear factor κB DNA binding in the infarcted hearts. Thus, our data suggest that cardiomyocyte PPARγ is a crucial protective receptor and may prevent reperfusion injury by modulating mechanisms of inflammation.

Short-term high fat feeding increases organ injury and mortality after polymicrobial sepsis.

The purpose of this study was to examine the effect of short-term high fat feeding on the inflammatory response in polymicrobial sepsis. Male C57BL/6 mice at 6 weeks of age were randomized to a high-fat diet (HFD) (60% kcal fat) or control diet (CD) (16% kcal fat) for 3 weeks. After 3 weeks of feeding, sepsis was induced by cecal ligation and puncture (CLP) and animals were monitored for survival. In a separate experiment, after 3 weeks of feeding mice underwent CLP and were sacrificed at various time points thereafter. Tissue was collected for biochemical studies. Mice fed a HFD gained more weight and had a greater fat mass compared to CD-fed mice. Mice on a HFD had a lower probability of survival and more severe lung injury compared with CD-fed mice following sepsis. Myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, was increased in the lung and liver after CLP in HFD-fed mice compared with CD (P < 0.05). The plasma cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were increased in both groups after CLP, however, TNF-α and IL-6 levels were lower in HFD mice at 3 h after CLP compared with CD and consistent with lung, but not liver, messenger RNA (mRNA) expression. Leptin levels were higher in HFD-fed mice at 18 h after sepsis compared to baseline levels (P < 0.05). Polymicrobial sepsis increased hepatic nuclear factor-κB (NF-κB) activation in HFD-fed mice after CLP vs. CD-fed mice. Short duration high fat feeding increases mortality and organ injury following polymicrobial sepsis. These effects correspond to changes in NF-κB.

C-peptide, a novel inhibitor of lung inflammation following hemorrhagic shock.

C-peptide is a 31-amino acid peptide cleaved from proinsulin during insulin synthesis. Initially thought to be inert, C-peptide may modulate the inflammatory response in the setting of endotoxemia and ischemia reperfusion. However, the spectrum of its biological effects is unclear. We hypothesized that exogenous administration of C-peptide would modulate pro- and anti-inflammatory signaling pathways and thereby attenuate lung inflammation in an in vivo model of hemorrhagic shock. Hemorrhagic shock was induced in male Wistar rats (aged 3-4 mo) by withdrawing blood to a mean arterial pressure of 50 mmHg. At 3 h after hemorrhage, rats were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, animals received C-peptide (280 nmol/kg) or vehicle parenterally. Animals were euthanized at 1 and 3 h after resuscitation. C-peptide administration at resuscitation following hemorrhagic shock ameliorated hypotension and blunted the systemic inflammatory response by reducing plasma levels of IL-1, IL-6, macrophage inflammatory protein-1α, and cytokine-induced neutrophil chemoattractant-1. This was associated with a reduction in lung neutrophil infiltration and plasma levels of receptor for advanced glycation end products. Mechanistically, C-peptide treatment was associated with reduced expression of proinflammatory transcription factors activator protein-1 and NF-κB and activation of the anti-inflammatory transcription factor peroxisome proliferator-activated receptor-γ. Our data suggest that C-peptide ameliorates the inflammatory response and lung inflammation following hemorrhagic shock. These effects may be modulated by altering the balance between pro- and anti-inflammatory signaling in the lung.

Liver X receptor α activation with the synthetic ligand T0901317 reduces lung injury and inflammation after hemorrhage and resuscitation via inhibition of the nuclear factor κB pathway.

Liver X receptor α (LXRα) is a nuclear transcription factor that regulates lipid metabolism. Recently, it has been shown that activation of LXRα with synthetic ligands has anti-inflammatory effects in atherosclerosis and chemical-induced dermatitis. We investigated the effect of the LXRα agonist, T0901317, on lung inflammation in a rodent model of hemorrhagic shock. Hemorrhagic shock was induced in male rats by withdrawing blood to a goal mean arterial blood pressure of 50 mmHg. Blood pressure was maintained at this level for 3 h, at which point rats were rapidly resuscitated with shed blood. Animals were then treated with T0901317 (50 mg · kg) or vehicle i.p. and sacrificed at 1, 2, and 3 h after resuscitation. Treatment with T0901317 significantly improved the cardiac and stroke volume indices as well as the heart rate of rats during the resuscitation period as compared with vehicle-treated rats. The T0901317-treated animals showed significant improvement in the plasma level of lactate, whereas base deficit and bicarbonate levels both trended toward improvement. The T0901317-treated animals also showed lower levels of plasma cytokines and chemokines monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, TNF-α, KC, and IL-6. Lung injury and neutrophil infiltration were reduced by treatment with T0901317, as evaluated by histology and myeloperoxidase assay. At molecular analysis, treatment with T0901317 increased nuclear LXRα expression and DNA binding while also inhibiting activation of nuclear factor κB, a proinflammatory transcription factor, in the lung. Thus, our data suggest that LXRα is an important modulator of the inflammatory response and lung injury after severe hemorrhagic shock, likely through the inhibition of the nuclear factor κB pathway.

Phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 Is associated with the downregulation of peroxisome proliferator-activated receptor (PPAR)-γ during polymicrobial sepsis.

Peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor and regulates inflammation. Posttranslational modifications regulate the function of PPARγ, potentially affecting inflammation. PPARγ contains a mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 leads to inhibition of PPARγ. This study investigated the kinetics of PPARγ expression and activation in parenchymal and immune cells in sepsis using the MAPK/ERK kinase (MEK)-1 inhibitor, an upstream kinase of ERK1/2. Adult male Sprague Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture. Rats received intraperitoneal injection of vehicle or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before cecal ligation and puncture. Rats were euthanized at 0, 1, 3, 6 and 18 h after cecal ligation and puncture. Control animals used were animals at time 0 h. Lung, plasma and peripheral blood mononuclear cells (PBMCs) were collected for biochemical assays. In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPARγ were decreased and associated with an increase in phosphorylated PPARγ and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPARγ expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPARγ expression and activation, in part, because of phosphorylation of PPARγ by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury.

Peroxisome proliferator-activated receptor {delta} regulates inflammation via NF-{kappa}B signaling in polymicrobial sepsis.

The nuclear peroxisome proliferator-activated receptor δ (PPARδ) is an important regulator of lipid metabolism. In contrast to its known effects on energy homeostasis, its biological role on inflammation is not well understood. We investigated the role of PPARδ in the modulation of the nuclear factor-κB (NF-κB)-driven inflammatory response to polymicrobial sepsis in vivo and in macrophages in vitro. We demonstrated that administration of GW0742, a specific PPARδ ligand, provided beneficial effects to rats subjected to cecal ligation and puncture, as shown by reduced systemic release of pro-inflammatory cytokines and neutrophil infiltration in lung, liver, and cecum, when compared with vehicle treatment. Molecular analysis revealed that treatment with GW0742 reduced NF-κB binding to DNA in lung and liver. In parallel experiments, heterozygous PPARδ-deficient mice suffered exaggerated lethality when subjected to cecal ligation and puncture and exhibited severe lung injury and higher levels of circulating tumor necrosis factor-α (TNFα) and keratinocyte-derived chemokine than wild-type mice. Furthermore, in lipopolysaccharide-stimulated J774.A1 macrophages, GW0742 reduced TNFα production by inhibiting NF-κB activation. RNA silencing of PPARδ abrogated the inhibitory effects of GW0742 on TNFα production. Chromatin immunoprecipitation assays revealed that PPARδ displaced the NF-κB p65 subunit from the κB elements of the TNFα promoter, while recruiting the co-repressor BCL6. These data suggest that PPARδ is a crucial anti-inflammatory regulator, providing a basis for novel sepsis therapies.

Ciglitazone, a novel inhibitor of lung apoptosis following hemorrhagic shock.

Apoptosis or programmed cell death has been demonstrated to play a role in the development of lung injury following hemorrhagic shock. A major pathway modulating the apoptotic response is the phosphatidylinositol 3-kinase/serine/threonine kinase (PI3K/Akt) pathway. Ciglitazone, a peroxisome proliferator-activated receptor-y (PPARy) ligand has previously been shown to attenuate lung inflammation following hemorrhagic shock. In vivo similar ligands have demonstrated anti-apoptotic effects with a reduction in organ injury in models of acute illness. In this study we examined the effect of ciglitazone on apoptosis and PI3K/Akt signaling in the lung following severe hemorrhage and resuscitation. Hemorrhagic shock was induced in male Wistar rats by withdrawing blood from the femoral artery to a mean arterial pressure of 50 mmHg. Animals were kept in shock for 3h at which time they were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, groups of animals received ciglitazone (10mg/kg) or DMSO intraperitoneally. Vehicle-treated rats had increased lung apoptosis following hemorrhage and resuscitation by Tunel staining. This was associated with increased activity of caspase-3. Ciglitazone treatment reduced lung apoptosis with a significant reduction in caspase-3 activity. This was associated with increased phosphorylation of the pro-survival kinase Akt. Thus, our data suggest that ciglitazone, a PPARy ligand, promotes cell survival in the lung following hemorrhagic shock.

Liver apoptosis is age dependent and is reduced by activation of peroxisome proliferator-activated receptor-gamma in hemorrhagic shock.

A clinical observation in pediatric and adult intensive care units is that the incidence of multiple organ failure in pediatric trauma victims is lower than in adult patients. However, the molecular mechanisms are not yet defined. Recent experimental studies have shown that the nuclear peroxisome proliferator-activated receptor-gamma (PPARgamma) modulates the inflammatory process. In this study, we hypothesized that severity of liver injury may be age dependent and PPARgamma activation may provide beneficial effects. Hemorrhagic shock was induced in anesthetized young (3-5 mo old) and mature male Wistar rats (11-13 mo old) by withdrawing blood to a mean arterial blood pressure of 50 mmHg. After 3 h, rats were rapidly resuscitated with shed blood. Animals were euthanized 3 h after resuscitation. In mature rats, liver injury appeared more pronounced compared with young rats and was characterized by marked hepatocyte apoptosis, extravasation of erythrocytes, and accumulation of neutrophils. The ratio between the antiapoptotic protein Bcl-2 and the proapoptotic protein BAX was lower, whereas activity of caspase-3, the executioner of apoptosis, was higher in liver of mature rats compared with young rats. Plasma alanine aminotransferase levels were not different between the two age groups. This heightened liver apoptosis was associated with a significant downregulation of PPARgamma DNA binding in mature rats compared with young rats. Treatment with the PPARgamma ligand ciglitazone significantly reduced liver apoptosis in mature rats. Our data suggest that liver injury after severe hemorrhage is age dependent and PPARgamma activation is a novel hepatoprotective mechanism.

Lung injury after hemorrhage is age dependent: role of peroxisome proliferator-activated receptor gamma.

OBJECTIVE: The incidence of multiple organ failure in pediatric trauma victims is lower than in the adult population. However, the molecular mechanisms are not yet defined. We investigated whether the pathophysiologic characteristics of hemorrhage-induced lung injury may be age dependent and may be regulated by the peroxisome proliferator-activated receptor gamma (PPARgamma). DESIGN: Prospective, laboratory investigation that used an established rodent model of hemorrhagic shock. SETTING: University hospital laboratory. SUBJECTS: Young (n = 67; 3-5 months old) and mature (n = 66; 11-13 months old) male rats. INTERVENTIONS: Hemorrhagic shock was induced in young and mature rats by withdrawing blood to a mean arterial blood pressure of 50 mm Hg. After 3 hours, rats were rapidly resuscitated by infusing the shed blood and killed 3 hours thereafter. MEASUREMENTS AND MAIN RESULTS: In young rats, lung injury was characterized by accumulation of red cells and neutrophils at the end of the resuscitation period; on Western blot analysis, lung expression of intercellular adhesion molecule-1 was increased. In contrast, the severity of lung injury was more pronounced in mature rats. Lung myeloperoxidase activity and expression of constitutive and inducible intercellular adhesion molecule-1 was significantly higher in mature rats compared with young rats. Mature rats also had higher plasma levels of cytokines and chemokines compared with young rats. This heightened inflammation was associated with higher degree of activation of nuclear factor-kappaB and down-regulation of PPARgamma and heat shock factor-1 in the lung of mature rats compared with young rats. Treatment with the PPARgamma ligand, the cyclopentenone prostaglandin 15-deoxy-Delta-prostaglandin J2, ameliorated lung injury in young, but not in mature animals. CONCLUSIONS: Lung injury after severe hemorrhage is age dependent and may be secondary to a diverse regulation of PPARgamma.

Ciglitazone ameliorates lung inflammation by modulating the inhibitor kappaB protein kinase/nuclear factor-kappaB pathway after hemorrhagic shock.

OBJECTIVE: Peroxisome proliferator-activated receptor-gamma is a ligand-activated transcription factor. Ciglitazone, a peroxisome proliferator-activated receptor-gamma ligand, has been shown to provide beneficial effects in experimental models of sepsis and ischemia/reperfusion injury. We investigated the effects of ciglitazone on lung inflammation after severe hemorrhage. DESIGN: Prospective, laboratory study, rodent model of hemorrhagic shock. SETTING: University hospital laboratory. SUBJECTS: Male rats. INTERVENTIONS: Hemorrhagic shock was induced by withdrawing blood to a mean arterial pressure of 50 mm Hg. At 3 hrs after hemorrhage, rats were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, animals received ciglitazone (10 mg/kg) or vehicle intraperitoneally. Heart rate and mean arterial pressure were measured throughout the experiment. Plasma and lung tissue were collected for analysis up to 3 hrs after resuscitation. MEASUREMENTS AND MAIN RESULTS: Ciglitazone treatment ameliorated mean arterial pressure, reduced lung injury, significantly blunted lung neutrophil infiltration, and lowered plasma interleukin-6, interleukin-10, and monocyte chemoattractant protein-1 levels. In a time course analysis, vehicle-treated rats had a significant increase in nuclear factor-kappaB DNA binding, which was preceded by increased inhibitor kappaB protein kinase activity and inhibitor kappaB alpha degradation in the lung. Treatment with ciglitazone significantly reduced inhibitor kappaB protein kinase activity and inhibitor kappaB alpha degradation and completely inhibited nuclear factor-kappaB DNA binding. This reduction of inhibitor kappaB protein kinase activity afforded by ciglitazone appeared to be a consequence of a physical interaction between peroxisome proliferator-activated receptor-gamma and increased inhibitor kappaB protein kinase. CONCLUSION: Ciglitazone ameliorates the inflammatory response and may reduce lung injury after hemorrhagic shock. These protective effects appear to be mediated through inhibition of the inhibitor kappaB protein kinase/nuclear factor-kappaB pathway.