Background Mortality in sepsis is most often attributed to the development of multiple organ failure. In sepsis, inflammation-mediated endothelial activation, defined as a proinflammatory and procoagulant state of the endothelial cells, has been associated with severity of disease. Thus, the objective of this study was to test the hypothesis that AMPK activation limits inflammation and endothelium activation to protect against organ injury in sepsis. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is an AMP analogue, has been used to upregulate activity of AMPK. Compound C is a cell-permeable pyrrazolopyrimidine compound that inhibits AMPK activity. Methods Wild-type mice underwent CLP or Sham surgery. Mice were randomized to vehicle, AICAR, or Compound C. Mouse kidney endothelial cells were used for in vitro experiments. Renal and liver function, were determined by serum Cystatin C, BUN, creatinine, and ALT. Serum cytokines were measured by ELISA. Microvascular injury was determined using Evan’s blue dye and electron microscopy. Immunohistochemistry was used to measure protein levels of p-AMPK, LC3, and ICAM. LC3 levels were used as a measure of autophagosome formation. Results AICAR decreased liver, and kidney injury induced by CLP and minimized cytokine elevation, in vivo and in vitro. CLP increased renal and hepatic phosphorylation of AMPK and autophagic signaling as determined by LC3. Inhibition of AMPK with Compound C prevented CLP-induced autophagy and exacerbated tissue injury. Additionally, CLP led to endothelial injury as determined by electron microscopy and Evan’s blue dye extravasation, and AICAR limited this injury. Furthermore, AICAR limited CLP and LPS induced upregulation of ICAM in vivo and in vitro, and decreased LPS induced neutrophil adhesion in vitro. Conclusion In this model, activation of AMPK was protective and AICAR minimized organ injury by decreasing inflammatory cytokines and endothelial activation. These data suggest that AMPK signaling influences sepsis or LPS induced endothelial activation and organ injury.

Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in industrialized countries. Understanding the mechanisms of APAP‐induced liver injury as well as other forms of sterile liver injury is critical to improve the care of patients. Recent studies demonstrate that danger signaling and inflammasome activation play a role in APAP‐induced injury. The aim of these investigations was to test the hypothesis that benzyl alcohol (BA) is a therapeutic agent that protects against APAP‐induced liver injury by modulation of danger signaling. APAP‐induced liver injury was dependent, in part, on Toll‐like receptor (TLR)9 and receptor for advanced glycation endproducts (RAGE) signaling. BA limited liver injury over a dose range of 135‐540 μg/g body weight or when delivered as a pre‐, concurrent, or post‐APAP therapeutic. Furthermore, BA abrogated APAP‐induced cytokines and chemokines as well as high‐mobility group box 1 release. Moreover, BA prevented APAP‐induced inflammasome signaling as determined by interleukin (IL)‐1β, IL‐18, and caspase‐1 cleavage in liver tissues. Interestingly, the protective effects of BA on limiting liver injury and inflammasome activation were dependent on TLR4 signaling, but not TLR2 or CD14. Cell‐type–specific knockouts of TLR4 were utilized to further determine the protective mechanisms of BA. These studies found that TLR4 expression specifically in myeloid cells (LyzCre‐tlr4−/−) were necessary for the protective effects of BA. Conclusion: BA protects against APAP‐induced acute liver injury and reduced inflammasome activation in a TLR4‐dependent manner. BA may prove to be a useful adjunct in the treatment of APAP and other forms of sterile liver injury. (Hepatology 2014;60:990–1002)

Traumatic injury is a significant cause of morbidity and mortality worldwide. Microcirculatory activation and injury from hemorrhage contributes to organ injury. Many adaptive responses occur within the microcirculatory beds to limit injury including up regulation of heme oxygenase (HO) enzymes, the rate limiting enzymes in the breakdown of heme to carbon monoxide (CO), iron, and biliverdin. Here we tested the hypothesis that CO abrogates trauma induced injury and inflammation protecting the microcirculatory beds. Methods. C57Bl/6 mice underwent sham operation or hemorrhagic shock to a mean arterial pressure of 25mmHg for 120 minutes. Mice were resuscitated with Lactated Ringer’s at 2X the volume of maximal shed blood. Mice were randomized to receive CO-releasing molecule (CO-RM) or inactive CO-RM at resuscitation. A cohort of mice was pretreated with tin protoporphyrin-IX (SnPP) to inhibit endogenous CO generation by heme oxygenases (HO). Primary mouse liver sinusoidal endothelial cells were cultured for in vitro experiments. Results. CO-RM protected against hemorrhagic shock/resuscitation (HS/R) organ injury and systemic inflammation and reduced hepatic sinusoidal endothelial injury. Inhibition of HO activity with SnPP exacerbated liver hepatic sinusoidal injury. HS/R in vivo or cytokine stimulation in vitro resulted in increased endothelial expression of adhesion molecules that was associated with decreased leukocyte adhesion in vivo and in vitro. Conclusions. HS/R is associated with endothelial injury. HO enzymes and CO are involved in part in diminishing this injury and may prove useful as a therapeutic adjunct that can be harnessed to protect against endothelial activation and damage.

The objective of this study is to better define induction of the heat shock response by arsenite, and to evaluation if induction of heat shock proteins (HSPs) contributes to the carcinogenic activity of arsenite. We show here that arsenite is a ubiquitous inducer of the heat shock response in mammalian cells: that it activated heat shock transcription factor 1 (HSF1) DNA-binding activity, enhanced hsp 70 promoter, and induced hsp70mRNA and synthesis of HSP chaperones. Using a high throughput hsp70 promoter-luciferase reporter assay, we observed a hormetic dose response where low concentrations of arsenite stimulated and high concentrations inhibited. Further, the response was time-dependent such that with longer times of incubation, the dose response shifted to the left. The effect of arsenite in inducing the hsp 70-luciferase reporter absolutely required a functional HSF1 as it was not observed in HSF1 minus cells but re-instated by expression of HSF1. Consistent with the suggestion that arsenic targets vicinal cysteine-SH, we showed that dithiothreitol blocked the effect of arsenite. Assays of cell viability and caspase showed that arsenite caused a dose-dependent increase in cell death by activation of caspase 3/7 and pre-induction of HSPs blunted these effects. Using anchorage independent cell growth as a late stage tumor promotion assay, we showed that low concentrations of arsenite had a growth promoting effect, which was enhanced by moderate heat shock. Our study provides evidence that induction of the heat shock response is a sensitive biomarker of arsenic exposure and that induction of HSPs likely contributes to the tumor promotion effect of arsenic.

Ischemia-reperfusion reduces the negative functional effects of cyclic GMP in cardiac myocytes. In this study, we tested the hypothesis that upregulation of hypoxic inducible factor-1 (HIF-1) would improve the actions of cyclic GMP signaling following simulated ischemia-reperfusion. HIF-1α was increased with deferoxamine (150 mg/kg for 2 days). Rabbit cardiac myocytes were subjected to simulated ischemia [15 min 95% N₂-5% CO₂] and reperfusion [reoxygenation] to produce myocyte stunning. Cell function was measured utilizing a video-edge detector. Shortening was examined at baseline and after brain natriuretic peptide (BNP, 10^-8, 10^-7M) or S-nitroso-N-acetyl-penicillamine (SNAP, 10^-6, 10^-5M) followed by KT5823 (cyclic GMP protein kinase inhibitor, 10^-6M). Kinase activity was measured via a protein phosphorylation assay. Under control conditions, BNP (-30%) and SNAP (-41%) reduced percent shortening, while KT5823 partially restored function (+18%). Deferoxamine treated control myocytes responded similarly. In stunned myocytes, BNP (-21%) and SNAP (-25%) reduced shortening less and KT5823 did not increase function (+2%). Deferoxamine increased the effects of BNP (-38%) and SNAP (-41%) in stunning and restored the effects of KT5823 (+12%). The cyclic GMP protein kinase increased phosphorylation of several proteins in control HIF-1 +/- cells. Phosphorylation was reduced in stunned cells and was restored in deferoxamine treated stunned cells. This study demonstrated that simulated ischemia-reperfusion reduced the negative functional effects of increasing cyclic GMP and this was related to reduced effects of the cyclic GMP protein kinase. Increased HIF-1α protects the functional effects of cyclic GMP thorough maintenance of cyclic GMP protein kinase activity after ischemic-reperfusion.