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.

Single and multidimensional measurements underestimate neuroblastoma response to therapy.

BACKGROUND: Changes in three-dimensional (3D) measurements of neuroblastoma are used to assess response. Linear measurements may not accurately characterize tumor size due to the infiltrative character of these tumors. The purpose of this study was to assess the accuracy of one-dimensional (1D), two-dimensional (2D), and 3D measurements in characterizing neuroblastoma response compared to a reference standard of tumor volume. PROCEDURE: We retrospectively reviewed imaging for 34 patients with stage 3 or 4 neuroblastoma. Blinded readers contoured or made linear measurements of tumors. Correlation coefficients were used to compare linear measurements to volumetric and 3D measurements. Bland-Altman analyses were used to assess bias between measurements. Sensitivity and specificity for patient events and survival were calculated for each measurement technique. RESULTS: Mean patient age was 2.9 ± 3.0 years (range 0-15 years). There was strong correlation between volumetric and 1D (r = 0.78, P < 0.0001), 2D (r = 0.86, P < 0.0001), and 3D (r = 0.88, P < 0.0001) measurements. Mean bias between volumetric measurements and 1D, 2D, and 3D measurements was 37.1% (95% limits: 6.2-67.9%), 16.1% (95% limits: -11.7-43.8%), and 7.7% (95% limits: -19.7-35.1%), respectively. 1D and 2D measurements undercategorized response versus volumetric change in 88.2% (30/34) and 29.4% (10/34) of cases. 3D measurements incorrectly characterized response in 16.7% (4/24) of cases versus volumetric change. 3D measurements were highly sensitive for patient events and survival, but all measurement techniques had poor specificity. CONCLUSIONS: 3D measurements most accurately quantify neuroblastoma size response versus volumetric change in patients with stage 3 and 4 neuroblastoma. 1D and 2D measurements underrepresent tumor response.

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.

Intestine-Derived Matrix Metalloproteinase-8 Is a Critical Mediator of Polymicrobial Peritonitis.

OBJECTIVE: Inhibition of matrix metalloproteinase-8 improves survival following cecal ligation and puncture in mice, making it a potential therapeutic target. In the current study, we expand our understanding of the role of matrix metalloproteinase-8 in sepsis by using an adoptive transfer approach and alternative sepsis models. DESIGN: We used three different sepsis models: cecal ligation and puncture, cecal slurry, and intestinal implantation. In our first model, adoptive transfer experiments were followed by cecal ligation and puncture to test the hypothesis that matrix metalloproteinase-8-containing myeloid cells are a critical factor in sepsis following cecal ligation and puncture. Our second model, cecal slurry, used intraperitoneal injections of cecal contents to induce polymicrobial peritonitis without tissue compromise in the recipient. Our third model, intestinal implantation, involved ligating and puncturing a cecum from a donor, and then removing the cecum and placing it into the recipient's peritoneal cavity. Clinically, blood samples were drawn from pediatric patients within 24 hours of meeting criteria for septic shock. SETTING: Basic science laboratory. SUBJECTS: Wild type and genetically modified mice. INTERVENTIONS: Experimental models of sepsis. MEASUREMENTS AND MAIN RESULTS: In our adoptive transfer experiments, matrix metalloproteinase-8 null mice receiving wild-type marrow had a survival advantage when compared with wild-type mice receiving matrix metalloproteinase-8 null marrow, suggesting that matrix metalloproteinase-8-containing myeloid cells are not a critical factor in sepsis following cecal ligation and puncture. In our cecal slurry model, no survival advantage was seen among matrix metalloproteinase-8 null mice. Our third model, intestinal implantation, found that mice receiving matrix metalloproteinase-8 null intestine had a survival advantage when compared with mice receiving wild-type intestine, regardless of recipient genotype. Clinically, median matrix metalloproteinase-8 serum concentrations were higher in patients with sepsis and primary intestinal pathology than in septic patients without primary intestinal pathology. CONCLUSIONS: Intestine-derived matrix metalloproteinase-8 is a critical component of septic peritonitis secondary to intestinal compromise.

Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli.

RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.