BackgroundThe choline‐derived metabolite trimethylamine N‐oxide (TMAO) has been demonstrated to contribute to atherosclerosis and is associated with coronary artery disease risk.Methods and ResultsWe explored the impact of TMAO on endothelial and smooth muscle cell function in vivo, focusing on disease‐relevant outcomes for atherogenesis. Initially, we observed that aortas of LDLR −/− mice fed a choline diet showed elevated inflammatory gene expression compared with controls. Acute TMAO injection at physiological levels was sufficient to induce the same inflammatory markers and activate the well‐known mitogen‐activated protein kinase, extracellular signal–related kinase, and nuclear factor‐κB signaling cascade. These observations were recapitulated in primary human aortic endothelial cells and vascular smooth muscle cells. We also found that TMAO promotes recruitment of activated leukocytes to endothelial cells. Through pharmacological inhibition, we further showed that activation of nuclear factor‐κB signaling was necessary for TMAO to induce inflammatory gene expression in both of these relevant cell types as well as endothelial cell adhesion of leukocytes.ConclusionsOur results suggest a likely contributory mechanism for TMAO‐dependent enhancement in atherosclerosis and cardiovascular risks.

Background: ApoA1, a component of HDL, promotes anti-inflammatory, immunomodulatory, and cardioprotective functions. Results: ApoA1 suppresses tumor growth and metastasis, primarily via modulation of innate and adaptive immune responses. Conclusion: ApoA1 impacts tumor biology at multiple levels, which appear to be linked to immunomodulatory function. Significance: ApoA1 redirects elicited immune cells toward tumor suppression and rejection and may hold benefit as a cancer therapeutic.

SV, Dasarathy S. Hyperammonemia-mediated autophagy in skeletal muscle contributes to sarcopenia of cirrhosis. Am J Physiol Endocrinol Metab 303: E983-E993, 2012. First published August 14, 2012; doi:10.1152/ajpendo.00183.2012.-Hyperammonemia and sarcopenia (loss of skeletal muscle) are consistent abnormalities in cirrhosis and portosystemic shunting. We have shown that muscle ubiquitin-proteasome components are not increased with hyperammonemia despite sarcopenia. This suggests that an alternative mechanism of proteolysis contributes to sarcopenia in cirrhosis. We hypothesized that autophagy could be this alternative pathway since we observed increases in classic autophagy markers, increased LC3 lipidation, beclin-1 expression, and p62 degradation in immunoblots of skeletal muscle protein in cirrhotic patients. We observed similar changes in these autophagy markers in the portacaval anastamosis (PCA) rat model. To determine the mechanistic relationship between hyperammonemia and autophagy, we exposed murine C2C12 myotubes to ammonium acetate. Significant increases in LC3 lipidation, beclin-1 expression, and p62 degradation occurred by 1 h, whereas autophagy gene expression (LC3, Atg5, Atg7, beclin-1) increased at 24 h. C2C12 cells stably expressing GFP-LC3 or GFPmCherry-LC3 constructs showed increased formation of mature autophagosomes supported by electron microscopic studies. Hyperammonemia also increased autophagic flux in mice, as quantified by an in vivo autophagometer. Because hyperammonemia induces nitration of proteins in astrocytes, we quantified global muscle protein nitration in cirrhotic patients, in the PCA rat, and in C2C12 cells treated with ammonium acetate. Increased protein nitration was observed in all of these systems. Furthermore, colocalization of nitrated proteins with GFP-LC3-positive puncta in hyperammonemic C2C12 cells suggested that autophagy is involved in degradation of nitrated proteins. These observations show that increased skeletal muscle autophagy in cirrhosis is mediated by hyperammonemia and may contribute to sarcopenia of cirrhosis.

Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200 µM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 µM), mid (5 µM) and high (100 µM) levels of 98.2%, 97.3% and 101.6%, respectively. Additional assay performance metrics include intra-day and inter-day coefficients of variance of < 6.4% and < 9.9%, respectively, across the range of TMAO levels. Stability studies reveal TMAO in plasma is stable both during storage at −80 °C for 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n=349) shows a range of 0.73 – 126 µM, median (interquartile range) levels of 3.45 (2.25–5.79) µM, and increasing values with age. The LC/MS/MS based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic and environmental factors on TMAO levels.

Reactive aldehydes derived from reducing sugars and lipid peroxidation play a critical role in the formation of advanced glycation end (AGE) products and oxidative tissue damage. We have recently proposed another mechanism for aldehyde generation at sites of inflammation that involves myeloperoxidase, a heme enzyme secreted by activated phagocytes. We now demonstrate that human neutrophils employ the myeloperoxidase-H 2 O 2 -chloride system to produce ␣ -hydroxy and ␣ , ␤ -unsaturated aldehydes from hydroxy-amino acids in high yield. Identities of the aldehydes were established using mass spectrometry and high performance liquid chromatography. Activated neutrophils converted L -serine to glycolaldehyde, an ␣ -hydroxyaldehyde which mediates protein cross-linking and formation of N ⑀ -(carboxymethyl)lysine, an AGE product. L -Threonine was similarly oxidized to 2-hydroxypropanal and its dehydration product, acrolein, an extremely reactive ␣ , ␤ -unsaturated aldehyde which alkylates proteins and nucleic acids. Aldehyde generation required neutrophil activation and a free hydroxy-amino acid; it was inhibited by catalase and heme poisons, implicating H 2 O 2 and myeloperoxidase in the cellular reaction. Aldehyde production by purified myeloperoxidase required H 2 O 2 and chloride, and was mimicked by reagent hypochlorous acid (HOCl) in the absence of enzyme, suggesting that the reaction pathway involves a chlorinated intermediate. Collectively, these results indicate that the myeloperoxidase-H 2 O 2 -chloride system of phagocytes converts free hydroxy-amino acids into highly reactive ␣ -hydroxy and ␣ , ␤ -unsaturated aldehydes. The generation of glycolaldehyde, 2-hydroxypropanal, and acrolein by activated phagocytes may thus play a role in AGE product formation and tissue damage at sites of inflammation. ( J. Clin. Invest. 1997. 99:424-432.)