A Dietary Mixture Containing Fish Oil, Resveratrol, Lycopene, Catechins, and Vitamins E and C Reduces Atherosclerosis in Transgenic Mice123

Chronic inflammation and proatherogenic lipids are important risk factors of cardiovascular disease (CVD). Specific dietary constituents such as polyphenols and fish oils may improve cardiovascular risk factors and may have a beneficial effect on disease outcomes. We hypothesized that the intake of an antiinflammatory dietary mixture (AIDM) containing resveratrol, lycopene, catechin, vitamins E and C, and fish oil would reduce inflammatory risk factors, proatherogenic lipids, and endpoint atherosclerosis. AIDM was evaluated in an inflammation model, male human C-reactive protein (CRP) transgenic mice, and an atherosclerosis model, female ApoE*3Leiden transgenic mice. Two groups of male human-CRP transgenic mice were fed AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 6 wk. The effects of AIDM on basal and IL-1β–stimulated CRP expression were investigated. AIDM reduced cytokine-induced human CRP and fibrinogen expression in human-CRP transgenic mice. In the atherosclerosis study, 2 groups of female ApoE*3Leiden transgenic mice were fed an atherogenic diet supplemented with AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 16 wk. AIDM strongly reduced plasma cholesterol, TG, and serum amyloid A concentrations compared with placebo. Importantly, long-term treatment of ApoE*3Leiden mice with AIDM markedly reduced the development of atherosclerosis by 96% compared with placebo. The effect on atherosclerosis was paralleled by a reduced expression of the vascular inflammation markers and adhesion molecules inter-cellular adhesion molecule-1 and E-selectin. Dietary supplementation of AIDM improves lipid and inflammatory risk factors of CVD and strongly reduces atherosclerotic lesion development in female transgenic mice

Time-Resolved and Tissue-Specific Systems Analysis of the Pathogenesis of Insulin Resistance

BACKGROUND: The sequence of events leading to the development of insulin resistance (IR) as well as the underlying pathophysiological mechanisms are incompletely understood. As reductionist approaches have been largely unsuccessful in providing an understanding of the pathogenesis of IR, there is a need for an integrative, time-resolved approach to elucidate the development of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Male ApoE3Leiden transgenic mice exhibiting a humanized lipid metabolism were fed a high-fat diet (HFD) for 0, 1, 6, 9, or 12 weeks. Development of IR was monitored in individual mice over time by performing glucose tolerance tests and measuring specific biomarkers in plasma, and hyperinsulinemic-euglycemic clamp analysis to assess IR in a tissue-specific manner. To elucidate the dynamics and tissue-specificity of metabolic and inflammatory processes key to IR development, a time-resolved systems analysis of gene expression and metabolite levels in liver, white adipose tissue (WAT), and muscle was performed. During HFD feeding, the mice became increasingly obese and showed a gradual increase in glucose intolerance. IR became first manifest in liver (week 6) and then in WAT (week 12), while skeletal muscle remained insulin-sensitive. Microarray analysis showed rapid upregulation of carbohydrate (only liver) and lipid metabolism genes (liver, WAT). Metabolomics revealed significant changes in the ratio of saturated to polyunsaturated fatty acids (liver, WAT, plasma) and in the concentrations of glucose, gluconeogenesis and Krebs cycle metabolites, and branched amino acids (liver). HFD evoked an early hepatic inflammatory response which then gradually declined to near baseline. By contrast, inflammation in WAT increased over time, reaching highest values in week 12. In skeletal muscle, carbohydrate metabolism, lipid metabolism, and inflammation was gradually suppressed with HFD. CONCLUSIONS/SIGNIFICANCE: HFD-induced IR is a time- and tissue-dependent process that starts in liver and proceeds in WAT. IR development is paralleled by tissue-specific gene expression changes, metabolic adjustments, changes in lipid composition, and inflammatory responses in liver and WAT involving p65-NFkB and SOCS3. The alterations in skeletal muscle are largely opposite to those in liver and WAT

Macrovesicular steatosis is associated with development of lobular inflammation and fibrosis in diet-induced non-alcoholic steatohepatitis (NASH)

Non-alcoholic steatohepatitis (NASH) is characterized by liver steatosis and lobular inflammation. It is unclear how the development of liver steatosis and the formation of inflammatory cell aggregates are related to each other. The present study investigated the longitudinal development of two forms of steatosis, micro- and macrovesicular steatosis, as well as lobular inflammation. ApoE*3Leiden.CETP (E3L.CETP) transgenic mice were fed a high-fat diet containing 1% w/w cholesterol (HFC) for 12 weeks to induce NASH. Livers were harvested in intervals of 4 weeks and analyzed by histological and biochemical techniques, as well as transcriptome and subsequent pathway analysis. Major findings were validated in independent NASH studies using other rodent models, i.e. HFD-treated C57BL/6J and LDLr‑/-.Leiden mice. In E3L.CETP mice, microvesicular steatosis was rapidly induced and reached plateau levels after already 4 weeks of HFC treatment. By contrast, macrovesicular steatosis developed more gradually and progressed over time. Lobular inflammation increased after 4 weeks with a significant further progression towards the end of the study (12 weeks). Macrovesicular, but not microvesicular, steatosis was positively correlated with the number of inflammatory aggregates. This correlation was confirmed in a milder (C57BL/6J) and a more severe (LDLr‑/-.Leiden) NASH model. Furthermore, collagen staining showed onset of perihepatocellular fibrosis in E3L.CETP mice after 12 weeks of HFC treatment and transcriptome analysis substantiated the activation of pro-fibrotic pathways and genes. Macrovesicular steatosis correlated positively with liver fibrosis in LDLr-/-.Leiden mice with pronounced fibrosis. Collectively, this study shows that macrovesicular steatosis is associated with lobular inflammation and liver fibrosis in rodent models and highlights the importance of this form of steatosis in the pathogenesis of NASH

Characterization of Human Induced Pluripotent Stem Cell-Derived Hepatocytes with Mature Features and Potential for Modeling Metabolic Diseases

There is a strong anticipated future for human induced pluripotent stem cell-derived hepatocytes (hiPS-HEP), but so far, their use has been limited due to insufficient functionality. We investigated the potential of hiPS-HEP as an in vitro model for metabolic diseases by combining transcriptomics with multiple functional assays. The transcriptomics analysis revealed that 86% of the genes were expressed at similar levels in hiPS-HEP as in human primary hepatocytes (hphep). Adult characteristics of the hiPS-HEP were confirmed by the presence of important hepatocyte features, e.g., Albumin secretion and expression of major drug metabolizing genes. Normal energy metabolism is crucial for modeling metabolic diseases, and both transcriptomics data and functional assays showed that hiPS-HEP were similar to hphep regarding uptake of glucose, low-density lipoproteins (LDL), and fatty acids. Importantly, the inflammatory state of the hiPS-HEP was low under standard conditions, but in response to lipid accumulation and ER stress the inflammation marker tumor necrosis factor α (TNFα) was upregulated. Furthermore, hiPS-HEP could be co-cultured with primary hepatic stellate cells both in 2D and in 3D spheroids, paving the way for using these co-cultures for modeling non-alcoholic steatohepatitis (NASH). Taken together, hiPS-HEP have the potential to serve as an in vitro model for metabolic diseases. Furthermore, differently expressed genes identified in this study can serve as targets for future improvements of the hiPS-HEP.CC BY 4.0</p

Macrovesicular steatosis is associated with development of lobular inflammation and fibrosis in diet-induced non-alcoholic steatohepatitis (NASH): DOI: 10.14800/ics.804

Non-alcoholic steatohepatitis (NASH) is characterized by liver steatosis and lobular inflammation. It is unclear how the development of liver steatosis and the formation of inflammatory cell aggregates are related to each other. The present study investigated the longitudinal development of two forms of steatosis, micro- and macrovesicular steatosis, as well as lobular inflammation. ApoE*3Leiden.CETP (E3L.CETP) transgenic mice were fed a high-fat diet containing 1% w/w cholesterol (HFC) for 12 weeks to induce NASH. Livers were harvested in intervals of 4 weeks and analyzed by histological and biochemical techniques, as well as transcriptome and subsequent pathway analysis. Major findings were validated in independent NASH studies using other rodent models, i.e. HFD-treated C57BL/6J and LDLr?/-.Leiden mice. In E3L.CETP mice, microvesicular steatosis was rapidly induced and reached plateau levels after already 4 weeks of HFC treatment. By contrast, macrovesicular steatosis developed more gradually and progressed over time. Lobular inflammation increased after 4 weeks with a significant further progression towards the end of the study (12 weeks). Macrovesicular, but not microvesicular, steatosis was positively correlated with the number of inflammatory aggregates. This correlation was confirmed in a milder (C57BL/6J) and a more severe (LDLr?/-.Leiden) NASH model. Furthermore, collagen staining showed onset of perihepatocellular fibrosis in E3L.CETP mice after 12 weeks of HFC treatment and transcriptome analysis substantiated the activation of pro-fibrotic pathways and genes. Macrovesicular steatosis correlated positively with liver fibrosis in LDLr-/-.Leiden mice with pronounced fibrosis. Collectively, this study shows that macrovesicular steatosis is associated with lobular inflammation and liver fibrosis in rodent models and highlights the importance of this form of steatosis in the pathogenesis of NASH

Mirtoselect, an anthocyanin-rich bilberry extract, attenuates non-alcoholic steatohepatitis and associated fibrosis in ApoE*3Leiden mice

Background & AimsAnthocyanins may have beneficial effects on lipid metabolism and inflammation and are demonstrated to have hepatoprotective properties in models of restraint-stress- and chemically-induced liver damage. However, their potential to protect against non-alcoholic steatohepatitis (NASH) under conditions relevant for human pathogenesis remains unclear. Therefore, we studied the effects of the standardised anthocyanin-rich extract Mirtoselect on diet-induced NASH in a translational model of disease.MethodsApoE∗3Leiden mice were fed a Western-type cholesterol-containing diet without (HC) or with 0.1% (w/w) Mirtoselect (HCM) for 20weeks to study the effects on diet-induced NASH.ResultsMirtoselect attenuated HC-induced hepatic steatosis, as observed by decreased macro- and microvesicular hepatocellular lipid accumulation and reduced hepatic cholesteryl ester content. This anti-steatotic effect was accompanied by local anti-inflammatory effects in liver, as demonstrated by reduced inflammatory cell clusters and reduced neutrophil infiltration in HCM. On a molecular level, HC diet significantly induced hepatic expression of pro-inflammatory genes Tnf, Emr1, Ccl2, Mpo, Cxcl1, and Cxcl2 while this induction was less pronounced or significantly decreased in HCM. A similar quenching effect was observed for HC-induced pro-fibrotic genes, Acta2 and Col1a1 and this anti-fibrotic effect of Mirtoselect was confirmed histologically. Many of the pro-inflammatory and pro-fibrotic parameters positively correlated with intrahepatic free cholesterol levels. Mirtoselect significantly reduced accumulation and crystallisation of intrahepatic free cholesterol, providing a possible mechanism for the observed hepatoprotective effects.ConclusionsMirtoselect attenuates development of NASH, reducing hepatic lipid accumulation, inflammation and fibrosis, possibly mediated by local anti-inflammatory effects associated with reduced accumulation and crystallisation of intrahepatic free cholesterol

Anticonvulsant Effectiveness and Hemodynamic Safety of Midazolam in Full-Term Infants Treated with Hypothermia

Background: Midazolam is used as an anticonvulsant in neonatology, including newborns with perinatal asphyxia treated with hypothermia. Hypothermia may affect the safety and effectiveness of midazolam in these patients. Objectives: The objective was to evaluate the anticonvulsant effectiveness and hemodynamic safety of midazolam in hypothermic newborns and to provide dosing guidance. Methods: Hypothermic newborns with perinatal asphyxia and treated with midazolam were included. Effectiveness was studied using continuous amplitude-integrated electroencephalography. Hemodynamic safety was assessed using pharmacokinetic-pharmacodynamic modeling with plasma samples and blood pressure recordings (mean arterial blood pressure) under hypothermia. Results: No effect of therapeutic hypothermia on pharmacokinetics could be identified. Add-on seizure control with midazolam was limited (23% seizure control). An inverse relationship between the midazolam plasma concentration and mean arterial blood pressure could be identified. At least one hypotensive episode was experienced in 64%. The concomitant use of inotropes decreased midazolam clearance by 33%. Conclusions: Under therapeutic hypothermia, midazolam has limited add-on clinical anticonvulsant effectiveness after phenobarbital administration. Due to occurrence of hypotension requiring inotropic support, midazolam is less suitable as a second-line anticonvulsant drug under hypothermia. (C) 2015 S. Karger AG, Base