SERUM HUMANIN IN PEDIATRIC SEPTIC SHOCK-ASSOCIATED MULTIPLE-ORGAN DYSFUNCTION SYNDROME.

ABSTRACT: Background: Multiple-organ dysfunction syndrome disproportionately contributes to pediatric sepsis morbidity. Humanin (HN) is a small peptide encoded by mitochondrial DNA and thought to exert cytoprotective effects in endothelial cells and platelets. We sought to test the association between serum HN (sHN) concentrations and multiple-organ dysfunction syndrome in a prospectively enrolled cohort of pediatric septic shock. Methods: Human MT-RNR2 ELISA was used to determine sHN concentrations on days 1 and 3. The primary outcome was thrombocytopenia-associated multiorgan failure (TAMOF). Secondary outcomes included individual organ dysfunctions on day 7. Associations across pediatric sepsis biomarker (PERSEVERE)-based mortality risk strata and correlation with platelet and markers of endothelial activation were tested. Results: One hundred forty subjects were included in this cohort, of whom 39 had TAMOF. The concentration of sHN was higher on day 1 relative to day 3 and among those with TAMOF phenotype in comparison to those without. However, the association between sHN and TAMOF phenotype was not significant after adjusting for age and illness severity in multivariate models. In secondary analyses, sHN was associated with presence of day 7 sepsis-associated acute kidney injury ( P = 0.049). Furthermore, sHN was higher among those with high PERSEVERE-mortality risk strata and correlated with platelet counts and several markers of endothelial activation. Conclusion: Future investigation is necessary to validate the association between sHN and sepsis-associated acute kidney injury among children with septic shock. Furthermore, mechanistic studies that elucidate the role of HN may lead to therapies that promote organ recovery through restoration of mitochondrial homeostasis among those critically ill.

PROTECTIVE EFFECTS OF HUMANIN-G IN HEMORRHAGIC SHOCK IN FEMALE MICE VIA AMPKα1-INDEPENDENT MECHANISMS.

ABSTRACT: Introduction: Despite therapeutic advances in hemorrhagic shock, mortality from multiple organ failure remains high. We previously showed that the α1 subunit of AMP-activated protein kinase (AMPK), a crucial regulator of mitochondrial function, exerts a protective role in hemorrhagic shock. Humanin is a mitochondrial peptide with cytoprotective properties against cellular stress. Here, we investigated whether AMPKα1 influences systemic levels of endogenous humanin in hemorrhagic shock and whether treatment with the synthetic analog humanin-G affords beneficial effects. Methods: AMPKα1 wild-type (WT) and knockout (KO) female mice were subjected to hemorrhagic shock followed by resuscitation with blood and lactated Ringer's solution. In short-term studies, mice were treated with humanin-G or vehicle and sacrificed at 3 h after resuscitation; in survival studies, mice were treated with PEGylated humanin-G and monitored for 7 days. Results: Compared with the vehicle WT group, KO mice exhibited severe hypotension, cardiac mitochondrial damage, and higher plasma levels of Th17 cytokines but had similar lung injury and similar plasma elevation of endogenous humanin. Treatment with humanin-G improved lung injury, mean arterial blood pressure, and survival in both WT and KO mice, without affecting systemic cytokine or humanin levels. Humanin-G also ameliorated cardiac mitochondrial damage and increased adenosine triphosphate levels in KO mice. Beneficial effects of humanin-G were associated with lung cytoplasmic and nuclear activation of the signal transducer and activator of transcription-3 (STAT3) in AMPKα1-independent manner with marginal or no effects on mitochondrial STAT3 and complex I subunit GRIM-19. Conclusions: Our data indicate that circulating levels of humanin increase during hemorrhagic shock in AMPKα1-independent fashion as a defense mechanism to counteract metabolic derangement and that administration of humanin-G affords beneficial effects through STAT3 activation even in the absence of a functional AMPKα1.

SEX-DEPENDENT EFFECTS OF ADIPOCYTE STAT3 INHIBITION ON THE INFLAMMATORY RESPONSE DURING SEVERE SEPSIS.

ABSTRACT: Introduction: Sepsis is a dysregulated host response to infection that can lead to life-threatening organ dysfunction. Clinical and animal studies consistently demonstrate that female subjects are less susceptible to the adverse effects of sepsis, demonstrating the importance of understanding how sex influences sepsis outcomes. The signal transducer and activator of transcription 3 (STAT3) pathway are a major signaling pathway that facilitates inflammation during sepsis. STAT3 is abundantly expressed in white adipose tissue; however, little is known about the contribution of white adipose tissue STAT3 activation during sepsis. We hypothesize that adipocyte STAT3 inhibition during severe sepsis will exaggerate the inflammatory response and impact organ injury, in a sex-dependent manner. Methods: We generated STAT3 flox/flox (wild-type [WT]) and adipocyte STAT3 knock out (A-STAT3 KO) mice using Cre-lox technology. Studies were done in 12- to 16-week-old male and female mice. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Control nonseptic mice did not undergo CLP (0 h CLP). Tissues were harvested 18 h after CLP. Body composition was determined by echo magnetic resonance imaging. Energy metabolism was determined by indirect calorimetry. White adipose tissue morphology was determined by hematoxylin and eosin staining, while STAT3 activation in the white adipose tissue was determined by western blot analysis and immunohistochemistry staining of STAT3 activation/phosphorylation at tyrosine 705. Plasma cytokines (TNF-α, IL-6, and leptin) were determined by luminex assay. Neutrophil infiltration of the lung and liver was assessed by myeloperoxidase activity assay. Histological signs of organ injury on lung and liver tissue were assessed by hematoxylin and eosin staining. Liver injury was further assessed by measuring plasma alanine and aspartate aminotransferase. In a separate cohort of mice, sepsis was induced by CLP and mice were monitored every 6-12 h over a 7-day period to assess survival rate. Results: We demonstrate that neither body composition nor energy metabolism is altered with adipocyte STAT3 inhibition in male or female mice, under nonseptic conditions. Sepsis was associated with reduced adipocyte size in female WT and A-STAT3 KO mice, suggesting that this event is STAT3 independent. Sepsis did not alter adipocyte size in male WT and A-STAT3 KO mice, suggesting that this event is also sex dependent. Although STAT3 phosphorylation at tyrosine 705 expression is negligible in male and female A-STAT3 KO mice, septic female WT and A-STAT3 KO mice have higher white adipose tissue STAT3 activation than male WT and A-STAT3 KO mice. Adipocyte STAT3 inhibition did not alter the proinflammatory cytokine response during sepsis in male or female mice, as measured by plasma TNF-α, IL-6, and leptin levels. Adipocyte STAT3 inhibition reduced lung neutrophil infiltration and histological signs of lung injury during sepsis in male mice. On the contrary, adipocyte STAT3 inhibition had no effect on lung neutrophil infiltration or lung injury in female mice. We further demonstrate that neither liver neutrophil infiltration nor histological signs of liver injury are altered by adipocyte STAT3 inhibition during sepsis, in male or female mice. Lastly, adipocyte STAT3 inhibition did not affect survival rate of male or female mice during sepsis. Conclusions: Our study demonstrates that sex influences white adipose tissue STAT3 activation and morphology during sepsis, which is not dependent on the presence of functional STAT3 in mature adipocytes. Furthermore, genetic inhibition of adipocyte STAT3 activation in male, but not female mice, results in reduced lung neutrophil infiltration and lung injury during sepsis. The results from our study demonstrate the importance of considering biological sex and the white adipose tissue as potential sources and targets of inflammation during sepsis.

Olfactomedin 4 as a novel loop of Henle-specific acute kidney injury biomarker.

Acute kidney injury (AKI) is associated with morbidity and mortality. Urinary biomarkers may disentangle its clinical heterogeneity. Olfactomedin 4 (OLFM4) is a secreted glycoprotein expressed in stressed neutrophils and epithelial cells. In septic mice, OLFM4 expression localized to the kidney's loop of Henle (LOH) and was detectable in the urine. We hypothesized that urine OLFM4 (uOLFM4) will be increased in patients with AKI and sepsis. Urine from critically ill pediatric patients was obtained from a prospective study based on AKI and sepsis status. uOLFM4 was quantified with a Luminex immunoassay. AKI was defined by KDIGO severe criteria. Sepsis status was extracted from the medical record based on admission diagnosis. Immunofluorescence on pediatric kidney biopsies was performed with NKCC2, uromodulin and OLFM4 specific antibodies. Eight patients had no sepsis, no AKI; 7 had no sepsis but did have AKI; 10 had sepsis, no AKI; 11 had sepsis and AKI. Patients with AKI had increased uOLFM4 compared to no/stage 1 AKI (p = 0.044). Those with sepsis had increased uOLFM4 compared to no sepsis (p = 0.026). uOLFM4 and NGAL were correlated (r2 0.59, 95% CI 0.304-0.773, p = 0.002), but some patients had high uOLFM4 and low NGAL, and vice versa. Immunofluorescence on kidney biopsies demonstrated OLFM4 colocalization with NKCC2 and uromodulin, suggesting expression in the thick ascending LOH (TALH). We conclude that AKI and sepsis are associated with increased uOLFM4. uOLFM4 and NGAL correlated in many patients, but was poor in others, suggesting these markers may differentiate AKI subgroups. Given OLFM4 colocalization to human TALH, we propose OLFM4 may be a LOH-specific AKI biomarker.

Colivelin, a synthetic derivative of humanin, ameliorates endothelial injury and glycocalyx shedding after sepsis in mice.

Endothelial dysfunction plays a central role in the pathogenesis of sepsis-mediated multiple organ failure. Several clinical and experimental studies have suggested that the glycocalyx is an early target of endothelial injury during an infection. Colivelin, a synthetic derivative of the mitochondrial peptide humanin, has displayed cytoprotective effects in oxidative conditions. In the current study, we aimed to determine the potential therapeutic effects of colivelin in endothelial dysfunction and outcomes of sepsis in vivo. Male C57BL/6 mice were subjected to a clinically relevant model of polymicrobial sepsis by cecal ligation and puncture (CLP) and were treated with vehicle or colivelin (100-200 µg/kg) intraperitoneally at 1 h after CLP. We observed that vehicle-treated mice had early elevation of plasma levels of the adhesion molecules ICAM-1 and P-selectin, the angiogenetic factor endoglin and the glycocalyx syndecan-1 at 6 h after CLP when compared to control mice, while levels of angiopoietin-2, a mediator of microvascular disintegration, and the proprotein convertase subtilisin/kexin type 9, an enzyme implicated in clearance of endotoxins, raised at 18 h after CLP. The early elevation of these endothelial and glycocalyx damage biomarkers coincided with lung histological injury and neutrophil inflammation in lung, liver, and kidneys. At transmission electron microscopy analysis, thoracic aortas of septic mice showed increased glycocalyx breakdown and shedding, and damaged mitochondria in endothelial and smooth muscle cells. Treatment with colivelin ameliorated lung architecture, reduced organ neutrophil infiltration, and attenuated plasma levels of syndecan-1, tumor necrosis factor-α, macrophage inflammatory protein-1α and interleukin-10. These therapeutic effects of colivelin were associated with amelioration of glycocalyx density and mitochondrial structure in the aorta. At molecular analysis, colivelin treatment was associated with inhibition of the signal transducer and activator of transcription 3 and activation of the AMP-activated protein kinase in the aorta and lung. In long-term outcomes studies up to 7 days, co-treatment of colivelin with antimicrobial agents significantly reduced the disease severity score when compared to treatment with antibiotics alone. In conclusion, our data support that damage of the glycocalyx is an early pathogenetic event during sepsis and that colivelin may have therapeutic potential for the treatment of sepsis-associated endothelial dysfunction.

Deficiency of AMPKα1 Exacerbates Intestinal Injury and Remote Acute Lung Injury in Mesenteric Ischemia and Reperfusion in Mice.

Mesenteric ischemia and reperfusion (I/R) injury can ensue from a variety of vascular diseases and represents a major cause of morbidity and mortality in intensive care units. It causes an inflammatory response associated with local gut dysfunction and remote organ injury. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of metabolic homeostasis. The catalytic α1 subunit is highly expressed in the intestine and vascular system. In loss-of-function studies, we investigated the biological role of AMPKα1 in affecting the gastrointestinal barrier function. Male knock-out (KO) mice with a systemic deficiency of AMPKα1 and wild-type (WT) mice were subjected to a 30 min occlusion of the superior mesenteric artery. Four hours after reperfusion, AMPKα1 KO mice exhibited exaggerated histological gut injury and impairment of intestinal permeability associated with marked tissue lipid peroxidation and a lower apical expression of the junction proteins occludin and E-cadherin when compared to WT mice. Lung injury with neutrophil sequestration was higher in AMPKα1 KO mice than WT mice and paralleled with higher plasma levels of syndecan-1, a biomarker of endothelial injury. Thus, the data demonstrate that AMPKα1 is an important requisite for epithelial and endothelial integrity and has a protective role in remote organ injury after acute ischemic events.

Hepatocyte-Specific Deletion of AMPKα1 Results in Worse Outcomes in Mice Subjected to Sepsis in a Sex-Specific Manner.

Alterations in the energy homeostasis contribute to sepsis-mediated multiple organ failure. The liver plays a central role in metabolism and participates to the innate immune and inflammatory responses of sepsis. Several clinical and experimental studies have suggested that females are less susceptible to the adverse outcome of sepsis. However, underlying mechanisms of organ damage in sepsis remain largely undefined. AMP-activated protein kinase (AMPK) is an important regulator of mitochondrial quality control. The AMPK catalytic α1 isoform is abundantly expressed in the liver. Here, we determined the role of hepatocyte AMPKα1 in sepsis by using hepatocyte-specific AMPKα1 knockout mice (H-AMPKα1 KO) generated with Cre-recombinase expression under the control of the albumin promoter. Using a clinically relevant model of polymicrobial sepsis by cecal ligation and puncture (CLP), we observed that male H-AMPKα1 KO mice had higher plasma levels of tumor necrosis factor-α and interleukin-6 and exhibited a more severe liver and lung injury than male H-AMPKα1 WT mice, as evaluated by histology and neutrophil infiltration at 18 h after CLP. Plasma levels of interleukin-10 and the keratinocyte-derived chemokine were similarly elevated in both KO and WT male mice. At transmission electron microscopy analysis, male H-AMPKα1 KO mice exhibited higher liver mitochondrial damage, which was associated with a significant decrease in liver ATP levels when compared to WT mice at 18 h after sepsis. Mortality rate was significantly higher in the male H-AMPKα1 KO group (91%) when compared to WT mice (60%) at 7 days after CLP. Female H-AMPKα1 WT mice exhibited a similar degree of histological liver and lung injury, but significantly milder liver mitochondrial damage and higher autophagy when compared to male WT mice after CLP. Interestingly, H-AMPKα1 KO female mice had lower organ neutrophil infiltration, lower liver mitochondrial damage and lower levels of cytokines than WT female mice. There was no significant difference in survival rate between WT and KO mice in the female group. In conclusion, our study demonstrates that AMPKα1 is a crucial hepatoprotective enzyme during sepsis. Furthermore, our results suggest that AMPK-dependent liver metabolic functions may influence the susceptibility to multiple organ injury in a sex-dependent manner.

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.

Matrix Metalloproteinase-8 Augments Bacterial Clearance in a Juvenile Sepsis Model.

Genetic ablation or pharmacologic inhibition of matrix metalloproteinase-8 (MMP8) improves survival in an adult murine sepsis model. Because developmental age influences the host inflammatory response, we hypothesized that developmental age influences the role of MMP8 in sepsis. First, we compared sepsis survival between wild type (WT, C57BL/6) and MMP8 null juvenile-aged mice (12-14 days) after intraperitoneal injection of a standardized cecal slurry. Second, peritoneal lavages collected at 6 and 18 hours after cecal slurry injection were analyzed for bacterial burden, leukocyte subsets, and inflammatory cytokines. Third, juvenile WT mice were pretreated with an MMP8 inhibitor prior to cecal slurry injection; analysis of their bacterial burden was compared to vehicle-injected animals. Fourth, the phagocytic capacity of WT and MMP8 null peritoneal macrophages was compared. Finally, peritoneal neutrophil extracellular traps (NETs) were compared using immunofluorescent imaging and quantitative image analysis. We found that juvenile MMP8 null mice had greater mortality and higher bacterial burden than WT mice. Leukocyte counts and cytokine concentrations in the peritoneal fluid were increased in the MMP8 null mice, relative to the wild type mice. Peritoneal macrophages from MMP8 null mice had reduced phagocytic capacity compared to WT macrophages. There was no quantitative difference in NET formation, but fewer bacteria were adherent to NETs from MMP8 null animals. In conclusion, in contrast to septic adult mice, genetic ablation of MMP8 increased mortality following bacterial peritonitis in juvenile mice. The increase in mortality in MMP8 null juvenile mice was associated with reduced bacterial clearance and reduced NET efficiency. We conclude that developmental age influences the role of MMP8 in sepsis.

Role of matrix metalloproteinase-8 as a mediator of injury in intestinal ischemia and reperfusion.

Acute mesenteric ischemia is associated with high morbidity and mortality. In recent studies, we found that the intestine is an important source of matrix metalloproteinase (MMP)8 during intestinal injury. We hypothesized that genetic ablation or pharmacological inhibition of MMP8 would reduce intestinal injury in mice subjected to intestinal ischemia-reperfusion (I/R) injury. Male mice aged 8-12 wk were subjected to intestinal I/R injury by transient occlusion of the superior mesenteric artery for 30 min. MMP8 was inhibited by genetic and pharmacological approaches. In vivo study endpoints included several functional, histological, and biochemical assays. Intestinal sections were assessed for barrier function and expression of tight junction proteins. I/R injury led to increased intestinal and systemic expression of MMP8. This increase was associated with increased intestinal neutrophil infiltration, epithelial injury, and permeability. I/R injury was associated with increased systemic inflammation and weight loss. These parameters were ameliorated by inhibiting MMP8. I/R injury caused a loss of the tight junction protein claudin-3, which was ameliorated by genetic ablation of MMP8. MMP8 plays an important role in intestinal I/R injury through mechanisms involving increased inflammation and loss of claudin-3. Inhibition of MMP8 is a potential therapeutic strategy in this setting.-Daly, M. C., Atkinson, S. J., Varisco, B. M., Klingbeil L., Hake, P., Lahni, P., Piraino, G., Wu, D., Hogan, S. P., Zingarelli, B., Wong, H. R. Role of matrix metalloproteinase-8 as a mediator of injury in intestinal ischemia and reperfusion.

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.