Ultrasonic Perfluorohexane-Loaded Monocyte Imaging: Toward a Minimally Invasive Technique for Selective Detection of Liver Inflammation in Fatty Liver Disease.

OBJECTIVES: To investigate the utility of ultrasonic (US) perfluorohexane (PFH)-loaded monocyte imaging for detection of liver inflammation in fatty liver disease. METHODS: C57Bl6 mice were injected intraperitoneally with tumor necrosis factor α and assessed by US PFH-loaded monocyte imaging 3 hours later. Echogenic monocytes were injected intravenously, leading to a transient increase in liver tissue intensity on a US perfusion scan. The contrast wash-out time constant was hypothesized to reflect the degree of inflammation. Next, we evaluated US PFH-loaded monocyte imaging in Ldlr-/- mice fed a 1-week high-fat/high-cholesterol diet as model for early developing nonalcoholic steatohepatitis. Adjunct analyses included tissue markers of liver inflammation. RESULTS: Tumor necrosis factor α-injected mice showed a reduced wash-out time constant (mean ± SEM, 0.013 ± 0.003; n = 8) compared to controls (0.054 ± 0.009; n = 7; P = .0006), indicative of increased inflammatory adhesion molecule expression on the endothelium. The Ldlr-/- mice fed the high-fat/high-cholesterol diet showed liver inflammation, as reflected by increased (3- to 4-fold) infiltration of inflammatory cells and increased (3- to 4-fold) gene expression of tumor necrosis factor α, integrin αM, intracellular adhesion molecule, and vascular cell adhesion molecule. However, in these mice, no difference was detected in the wash-out time constant as assessed by US PFH-loaded monocyte imaging (high-fat/high-cholesterol, 0.050 ± 0.017; n = 5; chow, 0.048 ± 0.006; n = 6; P = .91). CONCLUSIONS: Our results indicate that US PFH-loaded monocyte imaging is able to detect vascularly expressed inflammatory adhesion molecules in the mouse liver on direct endothelial stimulation. However, in our mouse model of early developing nonalcoholic steatohepatitis, we did not detect inflammation by this method, which may suggest that the time-dependent relationship between parenchymal and endothelial inflammation remains a fundamental issue to be addressed.

Hematopoietic overexpression of Cyp27a1 reduces hepatic inflammation independently of 27-hydroxycholesterol levels in Ldlr(-/-) mice.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and inflammation. Currently, the underlying mechanisms, leading to hepatic inflammation, are still unknown. The breakdown of free cholesterol inside Kupffer cells (KCs) by the mitochondrial enzyme CYP27A1 produces 27-hydroxycholesterol (27HC). We recently demonstrated that administration of 27HC to hyperlipidemic mice reduced hepatic inflammation. In line, hematopoietic deletion of Cyp27a1 resulted in increased hepatic inflammation. In the current manuscript, the effect of hematopoietic overexpression of Cyp27a1 on the development of NASH and cholesterol trafficking was investigated. We hypothesized that Cyp27a1 overexpression in KCs will lead to reduced hepatic inflammation. METHODS: Irradiated Ldlr(-/-) mice were transplanted (tp) with bone marrow from mice overexpressing Cyp27a1 (Cyp27a1(over)) and wild type (Wt) mice and fed either chow or a high-fat, high-cholesterol (HFC) diet for 3 months. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) from Cyp27a1(over) and Wt mice. RESULTS: In line with our hypothesis, hepatic inflammation in HFC-fed Cyp27a1(over)-tp mice was reduced and KCs were less foamy compared to Wt-tp mice. Remarkably, these changes occurred even though plasma and liver levels of 27HC did not differ between both groups. BMDM from Cyp27a1(over) mice revealed reduced inflammatory gene expression and increased expression of cholesterol transporters compared to Wt BMDM after lipopolysaccharide (LPS) stimulation. CONCLUSIONS: Our data suggest that overexpression of Cyp27a1 in KCs reduces hepatic inflammation independently of 27HC levels in plasma and liver, further pointing towards KCs as specific target for improving the therapy of NASH.

Plasma cathepsin D levels: a novel tool to predict pediatric hepatic inflammation.

OBJECTIVES: Nonalcoholic steatohepatitis (NASH) is the most severe form of a hepatic condition known as nonalcoholic fatty liver disease (NAFLD). NASH is histologically characterized by hepatic fat accumulation, inflammation, and ballooning, and eventually coupled with fibrosis that, in turn, may progress to end-stage liver disease even in young individuals. Hence, there is a critical need for specific noninvasive markers to predict hepatic inflammation at an early age. We investigated whether plasma levels of cathepsin D (CatD), a lysosomal protease, correlated with the severity of liver inflammation in pediatric NAFLD. METHODS: Liver biopsies from children (n=96) with NAFLD were histologically evaluated according to the criteria of Kleiner (NAFLD activity score) and the Brunt's criteria. At the time of liver biopsy, blood was taken and levels of CatD, alanine aminotransferase (ALT), and cytokeratin-18 (CK-18) were measured in plasma. RESULTS: Plasma CatD levels were significantly lower in subjects with liver inflammation compared with steatotic subjects. Furthermore, we found that CatD levels were gradually reduced and corresponded with increasing severity of liver inflammation, steatosis, hepatocellular ballooning, and NAFLD activity score. CatD levels correlated with pediatric NAFLD disease progression better than ALT and CK-18. In particular, CatD showed a high diagnostic accuracy (area under receiver operating characteristic curve (ROC-AUC): 0.94) for the differentiation between steatosis and hepatic inflammation, and reached almost the maximum accuracy (ROC-AUC: 0.998) upon the addition of CK-18. CONCLUSIONS: Plasma CatD holds a high diagnostic value to distinguish pediatric patients with hepatic inflammation from children with steatosis.

Reprogramming macrophages to an anti-inflammatory phenotype by helminth antigens reduces murine atherosclerosis.

Atherosclerosis is a lipid-driven inflammatory disease of the vessel wall, characterized by the chronic activation of macrophages. We investigated whether the helminth-derived antigens [soluble egg antigens (SEAs)] could modulate macrophage inflammatory responses and protect against atherosclerosis in mice. In bone marrow-derived macrophages, SEAs induce anti-inflammatory macrophages, typified by high levels of IL-10 and reduced secretion of proinflammatory mediators. In hyperlipidemic LDLR(-/-) mice, SEA treatment reduced plaque size by 44%, and plaques were less advanced compared with PBS-injected littermate controls. The atheroprotective effect of SEAs was found to be mainly independent of cholesterol lowering and T-lymphocyte responses but instead could be attributed to diminished myeloid cell activation. SEAs reduced circulating neutrophils and inflammatory Ly6C(high) monocytes, and macrophages showed high IL-10 production. In line with the observed systemic effects, atherosclerotic lesions of SEA-treated mice showed reduced intraplaque inflammation as inflammatory markers [TNF-α, monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD68], neutrophil content, and newly recruited macrophages were decreased. We show that SEA treatment protects against atherosclerosis development by dampening inflammatory responses. In the future, helminth-derived components may provide novel opportunities to treat chronic inflammatory diseases, as they diminish systemic inflammation and reduce the activation of immune cells.

Weekly Treatment of 2-Hydroxypropyl-ß-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice.

Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Two-hydroxypropyl-ß-cyclodextrin (HP-B-CD) is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs). Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr(-/-)) mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE) storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7α-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.

The cholesterol derivative 27-hydroxycholesterol reduces steatohepatitis in mice.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis is characterized by hepatic steatosis with inflammation. Although steatosis is benign and reversible, inflammation can increase liver damage. Hepatic inflammation has been associated with accumulation of cholesterol in lysosomes of Kupffer cells. 27-Hydroxycholesterol (27HC), a derivative of cholesterol formed by CYP27A1, can mobilize cholesterol from the lysosomes to the cytoplasm. We investigated whether 27HC can change the intracellular distribution cholesterol and reduce hepatic inflammation in mice. METHODS: We transplanted bone marrow from irradiated wild-type or Cyp27a1(-/-) mice to mice that do not express the low density lipoprotein receptor (Ldlr(-/-)), which are hyperlipidemic; 9 weeks later, mice were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 3 months. In a separate experiment, Ldlr(-/-) mice were given subcutaneous injections of 27HC and placed on regular chow or HFC diets for 3 weeks. Blood and liver tissues samples were collected and analyzed for intracellular cholesterol distribution and inflammation. RESULTS: In Ldlr(-/-) mice that received bone marrow transplants from Cyp27a1(-/-) mice, lysosomes of Kupfer cells had a greater accumulation of cholesterol than those of mice that received bone marrow from wild-type mice, after the HFC diet. Liver histology and gene expression analyses showed increased inflammation and liver damage in mice given bone marrow transplants from Cyp27a1(-/-) mice and placed on the HFC diet. Administration of 27HC to Ldlr(-/-) mice, following the HFC diet, reduced the accumulation of lysosomal cholesterol and hepatic inflammation, compared with mice that were not given 27HC. CONCLUSIONS: Accumulation of cholesterol in lysosomes of Kupfer cells promotes hepatic inflammation in mice. The cholesterol derivative 27HC reduces accumulation of cholesterol in lysosomes and might be used to treat non-alcoholic steatohepatitis.

Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis.

RATIONALE: Nuclear receptor Nur77, also known as NR4A1, TR3, or NGFI-B, is expressed in human atherosclerotic lesions in macrophages, endothelial cells, T cells and smooth muscle cells. Macrophages play a critical role in atherosclerosis and the function of Nur77 in lesion macrophages has not yet been investigated. OBJECTIVE: This study aims to delineate the function of Nur77 in macrophages and to assess the effect of bone marrow-specific deficiency of Nur77 on atherosclerosis. METHODS AND RESULTS: We investigated Nur77 in macrophage polarization using bone marrow-derived macrophages (BMM) from wild-type and Nur77-knockout (Nur77(-/-)) mice. Nur77(-/-) BMM exhibit changed expression of M2-specific markers and an inflammatory M1-phenotype with enhanced expression of interleukin-12, IFNγ, and SDF-1α and increased NO synthesis in (non)-stimulated Nur77(-/-) BMM cells. SDF-1α expression in nonstimulated Nur77(-/-) BMM is repressed by Nur77 and the chemoattractive activity of Nur77(-/-) BMM is abolished by SDF-1α inhibiting antibodies. Furthermore, Nur77(-/-) mice show enhanced thioglycollate-elicited migration of macrophages and B cells. The effect of bone marrow-specific deficiency of Nur77 on atherosclerosis was studied in low density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Ldlr(-/-) mice with a Nur77(-/-)-deficient bone marrow transplant developed 2.1-fold larger atherosclerotic lesions than wild-type bone marrow-transplanted mice. These lesions contain more macrophages, T cells, smooth muscle cells and larger necrotic cores. SDF-1α expression is higher in lesions of Nur77(-/-)-transplanted mice, which may explain the observed aggravation of lesion formation. CONCLUSIONS: In conclusion, in bone marrow-derived cells the nuclear receptor Nur77 has an anti-inflammatory function, represses SDF-1α expression and inhibits atherosclerosis.

Specific immunization strategies against oxidized low-density lipoprotein: a novel way to reduce nonalcoholic steatohepatitis in mice.

UNLABELLED: Nonalcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation combined with inflammation, which can ultimately progress into cirrhosis. Recently, we demonstrated that deletion of scavenger receptors (SRs) CD36 and SR-A in hematopoietic cells reduced hepatic inflammation. In addition to uptake of modified lipoproteins, CD36 and SR-A are also involved in other functions that can activate the inflammatory response. Therefore, the actual trigger for SR activation during NASH is unclear. Here, we hypothesized that hepatic inflammation is triggered by recognition of oxidized LDL (oxLDL) by Kupffer cells (KCs). To inhibit recognition of oxLDL by KCs, low-density lipoprotein receptor (Ldlr(-/-) ) mice were immunized with heat-inactivated pneumococci, which were shown to induce the production of anti-oxLDL immunoglobulin M (IgM) antibodies, due to molecular mimicry with oxLDL. The mice received a high-fat, high-cholesterol diet during the last 3 weeks to induce NASH. Immunization with pneumococci increased anti-oxLDL IgM levels and led to a reduction in hepatic inflammation, as shown by reduced macrophage, neutrophil, and T cell infiltration, and reduced gene expression of tumor necrosis factor (Tnf), interleukin-6 (Il-6), interleukin-1ß (Il-1b), monocyte chemoattractant protein 1 (Mcp1), and fibrosis-related genes. In immunized mice, KCs were smaller and showed fewer cholesterol crystals compared with nonimmunized mice. CONCLUSION: Antibodies to oxLDL play an important role in the pathogenesis of NASH. Therefore, the potential of phosphorylcholine-based vaccination strategies as a novel tool for the prevention and therapy of NASH should be tested in the future.

Trapping of oxidized LDL in lysosomes of Kupffer cells is a trigger for hepatic inflammation.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation. The transition from steatosis towards NASH represents a key step in pathogenesis, as it will set the stage for further severe liver damage. Under normal conditions, lipoproteins that are endocytosed by Kupffer cells (KCs) are easily transferred from the lysosomes into the cytoplasm. Oxidized LDL (oxLDL) that is taken up by the macrophages in vitro is trapped within the lysosomes, while acetylated LDL (acLDL) is leading to normal lysosomal hydrolysis, resulting in cytoplasmic storage. We have recently demonstrated that hepatic inflammation is correlated with lysosomal trapping of lipids. So far, a link between lysosomal trapping of oxLDL and inflammation was not established. We hypothesized that lysosomal trapping of oxLDL in KCs will lead to hepatic inflammation. METHODS: Ldlr(-/-) mice were injected with LDL, acLDL and oxLDL and sacrificed after 2, 6 and 24 h. RESULTS: Electron microscopy of KCs demonstrated that after oxLDL injection, small lipid inclusions were present inside the lysosomes after all time points and were mostly pronounced after 6 and 24 h. In contrast, no lipid inclusions were present inside KCs after LDL or acLDL injection. Hepatic expression of several inflammatory genes and scavenger receptors was higher after oxLDL injections compared with LDL or acLDL. CONCLUSIONS: These data suggest that trapping of oxLDL inside lysosomes of KCs in vivo is causally linked to increased hepatic inflammatory gene expression. Our novel observations provide new bases for prevention and treatment of NASH.

Role of scavenger receptor A and CD36 in diet-induced nonalcoholic steatohepatitis in hyperlipidemic mice.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a disorder that consists of steatosis and hepatic inflammation. It is not known why only some people with steatosis develop NASH. Recently, we identified dietary cholesterol as a factor that directly leads to hepatic inflammation and hepatic foam cell formation. We propose a mechanism by which Kupffer cells (KCs) take up modified cholesterol-rich lipoproteins via scavenger receptors (SRs). KCs thereby accumulate cholesterol, become activated, and may then trigger an inflammatory reaction. Scavenging of modified lipoproteins mainly depends on CD36 and macrophage scavenger receptor 1. METHODS: To evaluate the involvement of SR-mediated uptake of modified lipoproteins by KCs in the development of diet-induced NASH, female low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice were lethally irradiated and transplanted with bone marrow from Msr1(+/+)/Cd36(+/+)or Msr1(-/-)/Cd36(-/-) mice and fed a Western diet. RESULTS: Macrophage and neutrophil infiltration revealed that hepatic inflammation was substantially reduced by approximately 30% in Msr1(-/-)/Cd36(-/-)-transplanted mice compared with control mice. Consistent with this, the expression levels of well-known inflammatory mediators were reduced. Apoptotis and fibrosis were less pronounced in Msr1(-/-)/Cd36(-/-)-transplanted mice, in addition to the protective phenotype of natural antibodies against oxidized low-density lipoprotein in the plasma. Surprisingly, the effect on hepatic inflammation was independent of foam cell formation. CONCLUSIONS: Targeted inactivation of SR pathways reduces the hepatic inflammation and tissue destruction associated with NASH, independent of hepatic foam cell formation.

Chorioamnionitis induced hepatic inflammation and disturbed lipid metabolism in fetal sheep.

Chorioamnionitis frequently induces a fetal inflammatory response syndrome (FIRS), characterized by an elevation of proinflammatory mediators and systemic inflammation. Although there is increasing evidence that inflammation and lipid metabolism influence each other, the effects of chorioamnionitis-induced FIRS on fetal lipid homeostasis are currently not known. Accordingly, we hypothesize that chorioamnionitis induces an inflammatory response in the fetal liver, consequently leading to metabolic disturbances. Chorioamnionitis was induced by intra-amniotic injection of 10 mg endotoxin (control) for 2 d or 2 wk before delivery. Saline injections were given to controls. The effect of chorioamnionitis on hepatic inflammation and metabolic parameters was analyzed in ovine fetuses at the GA of 125 d (normal GA = 150 d). We found that 2 d after the endotoxin injections, inflammatory markers were significantly higher compared with controls. In addition, lipid and glucose metabolism were disturbed in response to endotoxin. Moreover, the antioxidant state capacity was reduced, and hepatic damage was apparent. Two weeks after the endotoxin injections, the fetal livers were still inflamed and had higher glucose concentrations in the blood. In addition, the levels of markers for hepatic damage (alanine aminotransferase and aspartate aminotransferase) were increased. In conclusion, chorioamnionitis induces liver inflammation leading to metabolic disturbances in the fetus.

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR-/-) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.

Dietary cholesterol, rather than liver steatosis, leads to hepatic inflammation in hyperlipidemic mouse models of nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic steatohepatitis (NASH) involves liver lipid accumulation (steatosis) combined with hepatic inflammation. The transition towards hepatic inflammation represents a key step in pathogenesis, because it will set the stage for further liver damage, culminating in hepatic fibrosis, cirrhosis, and liver cancer. The actual risk factors that drive hepatic inflammation during the progression to NASH remain largely unknown. The role of steatosis and dietary cholesterol in the etiology of diet-induced NASH was investigated using hyperlipidemic mouse models fed a Western diet. Livers of male and female hyperlipidemic (low-density lipoprotein receptor-deficient [ldlr(-/-)] and apolipoprotein E2 knock-in [APOE2ki]) mouse models were compared with livers of normolipidemic wild-type (WT) C57BL/6J mice after short-term feeding with a high-fat diet with cholesterol (HFC) and without cholesterol. Whereas WT mice displayed only steatosis after a short-term HFC diet, female ldlr(-/-) and APOE2ki mice showed steatosis with severe inflammation characterized by infiltration of macrophages and increased nuclear factor kappaB (NF-kappaB) signaling. Remarkably, male ldlr(-/-) and APOE2ki mice developed severe hepatic inflammation in the absence of steatosis after 7 days on an HFC diet compared with WT animals. An HFC diet induced bloated, "foamy" Kupffer cells in male and female ldlr(-/-) and APOE2ki mice. Hepatic inflammation was found to be linked to increased plasma very low-density lipoprotein (VLDL) cholesterol levels. Omitting cholesterol from the HFC diet lowered plasma VLDL cholesterol and prevented the development of inflammation and hepatic foam cells. CONCLUSION: These findings indicate that dietary cholesterol, possibly in the form of modified plasma lipoproteins, is an important risk factor for the progression to hepatic inflammation in diet-induced NASH.

Anticoagulant effect of dietary fish oil in hyperlipidemia: a study of hepatic gene expression in APOE2 knock-in mice.

OBJECTIVE: In hyperlipidemia, dietary fish oil containing n-3 polyunsaturated fatty acids (PUFA) provokes plasma triacylglycerol lowering and hypocoagulant activity. Using APOE2 knock-in mice, the relation of these fish-oil effects with altered gene expression was investigated. METHODS AND RESULTS: Male APOE2 knock-in mice, fed regular low-fat diet, had elevated plasma levels of triacylglycerol and coagulation factors. Plasma lipids and (anti)coagulant factors reduced on feeding the mice with fish oil (n-3 PUFA) or, to a lesser degree, with sunflower seed oil (n-6 PUFA). The fish-oil diet provoked a 40% reduction in thrombin generation. Microarray (Affymetrix) and single-gene expression analysis of mouse livers showed that fish oil induced: (1) upregulation of genes contributing to lipid degradation and oxidation; (2) downregulation of genes of gamma-glutamyl carboxylase and of transcription factors implicated in lipid synthesis; (3) unchanged expression of coagulation factor genes. After fish-oil diet, vitamin K-dependent coagulation factors accumulated in periportal areas of the liver; prothrombin was partly retained in uncarboxylated form. Only part of the changes in gene expression were different from the effects of sunflower seed oil diet. CONCLUSIONS: The hypocoagulant effect of n-3 PUFA is not caused by reduced hepatic synthesis of coagulation factors, but rather results from retention of uncarboxylated coagulation factors. In contrast, the lipid-lowering effect of n-3 PUFA links to altered expression of genes that regulate transcription and fatty acid metabolism.

Identification of TUB as a novel candidate gene influencing body weight in humans.

Previously, we identified a locus on 11p influencing obesity in families with type 2 diabetes. Based on mouse studies, we selected TUB as a functional candidate gene and performed association studies to determine whether this controls obesity. We analyzed the genotypes of 13 single nucleotide polymorphisms (SNPs) around TUB in 492 unrelated type 2 diabetic patients with known BMI values. One SNP (rs1528133) was found to have a significant effect on BMI (1.54 kg/m(2), P = 0.006). This association was confirmed in a population enriched for type 2 diabetes, using 750 individuals who were not selected for type 2 diabetes. Two SNPs in linkage disequilibrium with rs1528133 and mapping to the 3' end of TUB, rs2272382, and rs2272383 also affected BMI by 1.3 kg/m2 (P = 0.016 and P = 0.010, respectively). Combined analysis confirmed this association (P = 0.005 and P = 0.002, respectively). Moreover, comparing 349 obese subjects (BMI >30 kg/m(2)) from the combined cohort with 289 normal subjects (BMI <25 kg/m(2)) revealed that the protective alleles have a lower frequency in obese subjects (odds ratio 1.32 [95% CI 1.04-1.67], P = 0.022). Altogether, data from the tubby mouse as well as these data suggest that TUB could be an important factor in controlling the central regulation of body weight in humans.

Cathepsin D regulates lipid metabolism in murine steatohepatitis.

Due to the obesity epidemic, non-alcoholic steatohepatitis (NASH) is a prevalent liver disease, characterized by fat accumulation and inflammation of the liver. However, due to a lack of mechanistic insight, diagnostic and therapeutic options for NASH are poor. Recent evidence has indicated cathepsin D (CTSD), a lysosomal enzyme, as a marker for NASH. Here, we investigated the function of CTSD in NASH by using an in vivo and in vitro model. In addition to diminished hepatic inflammation, inhibition of CTSD activity dramatically improved lipid metabolism, as demonstrated by decreased plasma and liver levels of both cholesterol and triglycerides. Mechanistically, CTSD inhibition resulted in an increased conversion of cholesterol into bile acids and an elevated excretion of bile acids via the feces, indicating that CTSD influences lipid metabolism. Consistent with these findings, treating Wt BMDMs with PepA in vitro showed a similar decrease in inflammation and an analogous effect on cholesterol metabolism. CONCLUSION: CTSD is a key player in the development of hepatic inflammation and dyslipidemia. Therefore, aiming at the inhibition of the activity of CTSD may lead to novel treatments to combat NASH.

Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet.

Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis. ApoE(-/-) mice were fed a Western-type (WD) or control diet. In subgroups, tungsten (700 mg/L) was administered to inhibit XO. XO is a secreted enzyme which is formed in the liver as xanthine dehydrogenase (XDH) and binds to the vascular endothelium. High expression of XDH was found in the liver and WD increased liver XDH mRNA and XDH protein expression. WD induced the conversion of XDH to the radical-forming XO. Moreover, WD increased the hepatic expression of CD40, demonstrating activation of hepatic cells. Aortic tissue of ApoE(-/-) mice fed a WD for 6 months exhibited marked atherosclerosis, attenuated endothelium-dependent relaxation to acetylcholine, increased vascular oxidative stress, and mRNA expression of the chemokine KC. Tungsten treatment had no effect on plasma lipids but lowered the plasma XO activity. In animals fed a control diet, tungsten had no effect on radical formation, endothelial function, or atherosclerosis development. In mice fed a WD, however tungsten attenuated the vascular superoxide anion formation, prevented endothelial dysfunction, and attenuated KC mRNA expression. Most importantly, tungsten treatment largely prevented the development of atherosclerosis in the aorta of ApoE(-/-) mice on WD. Therefore, tungsten, potentially via the inhibition of XO, prevents the development of endothelial dysfunction and atherosclerosis in ApoE(-/-) mice on WD.

Macrophage Stimulating Protein Enhances Hepatic Inflammation in a NASH Model.

Non-alcoholic steatohepatitis (NASH) is a common liver disease characterized by hepatic lipid accumulation (steatosis) and inflammation. Currently, therapeutic options are poor and the long-term burden to society is constantly increasing. Previously, macrophage stimulating protein (MSP)-a serum protein mainly secreted by liver-was shown to inhibit oxidized low-density lipoprotein (OxLDL)/lipopolysaccharides (LPS)-induced inflammation in mouse macrophages. Additionally, MSP could reduce palmitic acid (PA)-induced lipid accumulation and lipogenesis in the HepG2 cell line. Altogether, these data suggest MSP as a suppressor for metabolic inflammation. However, so far the potential of MSP to be used as a treatment for NASH was not investigated. We hypothesized that MSP reduces lipid accumulation and hepatic inflammation. To investigate the effects of MSP in the early stage of NASH, low-density lipoprotein receptor (Ldlr-/-) mice were fed either a regular chow or a high fat, high cholesterol (HFC) diet for 7 days. Recombinant MSP or saline (control) was administrated to the mice by utilizing subcutaneously-implanted osmotic mini-pumps for the last 4 days. As expected, mice fed an HFC diet showed increased plasma and hepatic lipid accumulation, as well as enhanced hepatic inflammation, compared with chow-fed controls. Upon MSP administration, the rise in cholesterol and triglyceride levels after an HFC diet remained unaltered. Surprisingly, while hepatic macrophage and neutrophil infiltration was similar between the groups, MSP-treated mice showed increased gene expression of pro-inflammatory and pro-apoptotic mediators in the liver, compared with saline-treated controls. Contrary to our expectations, MSP did not ameliorate NASH. Observed changes in inflammatory gene expression suggest that further research is needed to clarify the long-term effects of MSP.

Prevention of oxLDL uptake leads to decreased atherosclerosis in hematopoietic NPC1-deficient Ldlr-/- mice.

BACKGROUND AND AIMS: Atherosclerosis is a chronic inflammatory disease of medium and large vessels and is typically characterized by the predominant accumulation of low-density lipoprotein (LDL)-cholesterol inside macrophages that reside in the vessel walls. Previous studies clearly demonstrated an association specifically between the oxidized type of LDL (oxLDL) and atherosclerotic lesion formation. Further observations revealed that these atherosclerotic lesions displayed enlarged, lipid-loaded lysosomes. By increasing natural antibodies against oxLDL, pneumococcal vaccination has been shown to reduce atherosclerosis in LDL receptor knockout (Ldlr-/-) mice. Relevantly, loss of the lysosomal membrane protein Niemann-Pick Type C1 (NPC1) led to lysosomal accumulation of various lipids and promoted atherosclerosis. Yet, the importance of lysosomal oxLDL accumulation inside macrophages, compared to non-modified LDL, in atherosclerosis has never been established. METHODS: By transplanting NPC1 bone marrow into lethally irradiated Ldlr-/- mice, a hematopoietic mouse model for lysosomal cholesterol accumulation was created. Through injections with heat-inactivated pneumococci, we aimed to demonstrate the specific contribution of lysosomal oxLDL accumulation inside macrophages in atherosclerosis development. RESULTS: While there were no differences in plaque morphology, a reduction in plaque size and plaque inflammation was found in immunized NPC1mut-transplanted mice, compared to non-immunized NPC1mut-transplanted mice. CONCLUSIONS: Lysosomal oxLDL accumulation within macrophages contributes to murine atherosclerosis. Future intervention strategies should focus specifically on preventing oxLDL, unlike non-modified LDL, from being internalized into lysosomes. Such an intervention can have an additive effect to current existing treatments against atherosclerosis.

Myeloid DLL4 Does Not Contribute to the Pathogenesis of Non-Alcoholic Steatohepatitis in Ldlr-/- Mice.

Non-alcoholic steatohepatitis (NASH) is characterized by liver steatosis and inflammation. Currently, the underlying mechanisms leading to hepatic inflammation are not fully understood and consequently, therapeutic options are poor. Non-alcoholic steatohepatitis (NASH) and atherosclerosis share the same etiology whereby macrophages play a key role in disease progression. Macrophage function can be modulated via activation of receptor-ligand binding of Notch signaling. Relevantly, global inhibition of Notch ligand Delta-Like Ligand-4 (DLL4) attenuates atherosclerosis by altering the macrophage-mediated inflammatory response. However, the specific contribution of macrophage DLL4 to hepatic inflammation is currently unknown. We hypothesized that myeloid DLL4 deficiency in low-density lipoprotein receptor knock-out (Ldlr-/-) mice reduces hepatic inflammation. Irradiated Ldlr-/- mice were transplanted (tp) with bone marrow from wild type (Wt) or DLL4f/fLysMCre+/0 (DLL4del) mice and fed either chow or high fat, high cholesterol (HFC) diet for 11 weeks. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) of DLL4f/fLysMCreWT and DLL4f/fLysMCre+/0 mice. In contrast to our hypothesis, inflammation was not decreased in HFC-fed DLL4del-transplanted mice. In line, in vitro, there was no difference in the expression of inflammatory genes between DLL4-deficient and wildtype bone marrow-derived macrophages. These results suggest that myeloid DLL4 deficiency does not contribute to hepatic inflammation in vivo. Since, macrophage-DLL4 expression in our model was not completely suppressed, it can't be totally excluded that complete DLL4 deletion in macrophages might lead to different results. Nevertheless, the contribution of non-myeloid Kupffer cells to notch signaling with regard to the pathogenesis of steatohepatitis is unknown and as such it is possible that, DLL4 on Kupffer cells promote the pathogenesis of steatohepatitis.