Evie H. Carchman

Heme oxygenase‐1–mediated autophagy protects against hepatocyte cell death and hepatic injury from infection/sepsis in mice

Adaptive responses to sepsis are necessary to prevent organ failure and death. Cellular signaling responses that limit cell death and structural damage allow a cell to withstand insult from sepsis to prevent irreversible organ dysfunction. One such protective pathway to reduce hepatocellular injury is the up‐regulation of heme oxygenase‐1 (HO‐1) signaling. HO‐1 is up‐regulated in the liver in response to multiple stressors, including sepsis and lipopolysaccharide (LPS), and has been shown to limit cell death. Another recently recognized rudimentary cellular response to injury is autophagy. The aim of these investigations was to test the hypothesis that HO‐1 protects against hepatocyte cell death in experimental sepsis in vivo or LPS in vitro via induction of autophagy. These data demonstrate that both HO‐1 and autophagy are up‐regulated in the liver after cecal ligation and puncture (CLP) in C57BL/6 mice or in primary mouse hepatocytes after treatment with LPS (100 ng/mL). CLP or LPS results in minimal hepatocyte cell death. Pharmacological inhibition of HO‐1 activity using tin protoporphyrin or knockdown of HO‐1 prevents the induction of autophagic signaling in these models and results in increased hepatocellular injury, apoptosis, and death. Furthermore, inhibition of autophagy using 3‐methyladenine or small interfering RNA specific to VPS34, a class III phosphoinositide 3‐kinase that is an upstream regulator of autophagy, resulted in hepatocyte apoptosis in vivo or in vitro. LPS induced phosphorylation of p38 mitogen‐activated protein kinase (p38 MAPK), in part, via HO‐dependent signaling. Moreover, inhibition of p38 MAPK prevented CLP‐ or LPS‐induced autophagy. Conclusion: Sepsis or LPS‐induced autophagy protects against hepatocellular death, in part via an HO‐1 p38 MAPK‐dependent signaling. Further investigations are needed to elucidate how autophagic signaling prevents apoptosis and cell death. (HEPATOLOGY 2011;)

Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats

Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.

Experimental sepsis-induced mitochondrial biogenesis is dependent on autophagy, TLR4, and TLR9 signaling in liver

Organ injury in sepsis is initially characterized by dysfunction without cell death and structural damage, and thus with the ability to recover organ function. Adaptive metabolic responses to sepsis can prevent bioenergetic failure and death. These studies were aimed at investigating the influence of sepsis on mitochondrial homeostasis, focusing on removal of dysfunctional mitochondria and restitution of a healthy mitochondrial population. These data demonstrate decreased hepatic oxidative phosphorylation by 31 ± 11% following murine cecal ligation and puncture (CLP) at 8 h and 34 ± 9% following LPS treatment in vitro at 12 h (P<0.05). In addition, there was a loss of mitochondrial membrane potential. Mitochondrial density and number initially decreased (relative area per micrograph of 64±10% at baseline vs. 39±13% at 8 h following LPS; P<0.05) and was associated with an increase in autophagy and mitophagy. CLP‐induced markers of mitochondrial biogenesis and mitochondrial number and density recovered over time. Furthermore, these data suggest that mitochondrial biogenesis was dependent on an autophagy and mitochondrial DNA/Toll‐like receptor 9 (TLR9) signaling pathway. These results suggest that hepatocyte survival and maintenance of function in sepsis is dependent on a mitochondrial homeostasis pathway marked by mitophagy and biogenesis.—Carchman, E. H., Whelan, S., Loughran, P., Mollen, K., Stratamirovic, S., Shiva, S., Rosengart, M. R., Zuckerbraun, B. S., Experimental sepsis‐induced mitochondrial biogenesis is dependent on autophagy, TLR4, and TLR9 signaling in liver. FASEB J. 27, 4703–4711 (2013). www.fasebj.org

CaMKIα Regulates AMP Kinase–Dependent, TORC-1–Independent Autophagy during Lipopolysaccharide-Induced Acute Lung Neutrophilic Inflammation

Autophagy is an evolutionarily conserved cytoplasmic process regulated by the energy rheostats mammalian target of rapamycin and AMP kinase (AMPK) that recycles damaged or unused proteins and organelles. It has been described as an important effector arm of immune cells. We have shown that the cytoplasmically oriented calcium/calmodulin-dependent protein kinase (CaMK)Iα regulates the inflammatory phenotype of the macrophage (Mϕ). In this study, we hypothesize that CaMKIα mediates Mϕ autophagy. LPS induced autophagy in RAW 264.7 cells and murine peritoneal Mϕ that was attenuated with biochemical CaMK inhibition or CaMKIα small interfering RNA (siRNA). Inhibition of CaMKIα reduced LPS-induced p-Thr172AMPK and target of rapamycin complex-1 activity, and expression of a constitutively active CaMKIα but not a kinase-deficient mutant induced p-Thr172AMPK and autophagy that was attenuated by the AMPK inhibitor compound C. Coimmunoprecipitation and in vitro kinase assays demonstrated that CaMKIα activates AMPK, thereby inducing ATG7, which also localizes to this CaMKIα/AMPK complex. During LPS-induced lung inflammation, C57BL/6 mice receiving CaMKIαsiRNA displayed reduced lung and bronchoalveolar immune cell autophagy that correlated with reduced neutrophil recruitment, myeloperoxidase activity, and air space cytokine concentration. Independently inhibiting autophagy, using siRNA targeting the PI3K VPS34, yielded similar reductions in lung autophagy and neutrophil recruitment. Thus, a novel CaMKIα/AMPK pathway is rapidly activated in Mϕ exposed to LPS and regulates an early autophagic response, independent of target of rapamycin complex-1 inhibition. These mechanisms appear to be operant in vivo in orchestrating LPS-induced lung neutrophil recruitment and inflammation.

Polymicrobial sepsis is associated with decreased hepatic oxidative phosphorylation and an altered metabolic profile

BACKGROUNDnOrgan failure in sepsis accounts for significant mortality worldwide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus of the organ and organism. Adaptive responses in metabolism are critical to the recovery at the cellular level. The purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration and significant metabolic changes in the liver.nnnMETHODSnC57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or an operation without CLP. Mice were euthanized from 0-24 h after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity were measured. Global biochemical profiles of 311 metabolites were performed at the 8-h time point (n = 8/group) and analyzed by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry platforms by Metabolon (Durham, North Carolina). The influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated.nnnRESULTSnCLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measured at 12 h. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every tricarboxylic acid cycle intermediate and derivative, suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids.nnnCONCLUSIONSnSepsis results in a metabolic response and profile consistent with increased anaerobic respiration, which occurs prior to significant changes in hemodynamics. The metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.

Sepsis results in an altered renal metabolic and osmolyte profile

BACKGROUNDnSepsis remains a major health-care burden and source of morbidity and mortality. Acute kidney injury and failure frequently accompanies severe sepsis and contributes to this burden. Despite a great deal of research, the exact mechanisms underlying renal failure in sepsis are poorly understood. This study aims to further understand metabolic changes in renal tissue during sepsis.nnnMATERIALS AND METHODSnExperimental sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Serum and organs were harvested 8 h after CLP. Markers of renal function including serum creatinine, blood urea nitrogen, and cystatin C were measured. Whole kidneys were analyzed for a global biochemical profile via liquid chromatography/tandem mass spectrometry by Metabolon.nnnRESULTSnCLP induced renal injury as evidenced by elevated serum creatinine, blood urea nitrogen, and cystatin C. Global energetic profile in sepsis showed an increase in glycolytic intermediates with decreased flux through the tricarboxylic acid (TCA) cycle. Multiple inflammatory markers were elevated in response to CLP. Levels of osmotic regulators varied, with an overall increase in pinitol, urea, and taurine in response to CLP.nnnCONCLUSIONSnCLP resulted in dramatic changes in the renal macromolecular milieu. There appears to be an increased dependence on glycolysis and diminished flush through the TCA cycle. In addition, changes in renal osmolytes including pinitol, urea, and taurine were observed, perhaps uncovering an additional change with implications on renal function during sepsis.