Global Profiling of Lysine Acetylation and Lactylation in Kupffer Cells.

Among the various cell types that constitute the liver, Kupffer cells (KCs) are responsible for the elimination of gut-derived foreign products. Protein lysine acetylation (Kac) and lactylation (Kla) are dynamic and reversible post-translational modifications, and various global acylome studies have been conducted for liver and liver-derived cells. However, no such studies have been conducted on KCs. In this study, we identified 2198 Kac sites in 925 acetylated proteins and 289 Kla sites in 181 lactylated proteins in immortalized mouse KCs using global acylome technology. The subcellular distributions of proteins with Kac and Kla site modifications differed. Similarly, the specific sequence motifs surrounding acetylated or lactylated lysine residues also showed differences. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to better understand the differentially expressed proteins in the studies by Kac and Kla. In the newly identified Kla, we found K82 lactylation in the high-mobility group box-1 protein in the neutrophil extracellular trap formation category using KEGG enrichment analyses. Here, we report the first proteomic survey of Kac and Kla in KCs.

Liver-derived extracellular vesicles: A cell by cell overview to isolation and characterization practices.

BACKGROUND: Extracellular vesicles (EVs) are a diverse group of membrane-bound nanovesicles potentially released by every cell. With the liver's unique ensemble of cells and its fundamental physiological tasks, elucidating the role of EV-mediated hepatic cellular crosstalk and their role in different pathologies has been gaining the attention of many scientists. SCOPE OF REVIEW: The present review shifts the perspective into practice: we aim to critically discuss the methods used to purify and to biochemically analyse EVs from specific liver resident cells, including hepatocytes, hepatic stellate cells, cholangiocytes, liver sinusoidal endothelial cells, Kupffer cells, liver stem cells. The review offers a reference guide to current approaches. MAJOR CONCLUSIONS: Strategies for EV isolation and characterization are as varied as the research groups performing them. We present main advantages and disadvantages for the methods, highlighting common causes for concern, such as FBS handling, reporting of cell viability, EV yield and storage, differences in differential centrifugations, suboptimal method descriptions, and method transferability. We both looked at how adaptable the research between human and rodent cells in vitro is, and also assessed how well either of them translates to ex vivo settings. GENERAL SIGNIFICANCE: We reviewed methodological practices for the isolation and analysis of liver-derived EVs, making a cell type specific user guide that shows where to start, what has worked so far and to what extent. We critically discussed room for improvement, placing a particular focus on working towards a potential standardization of methods.

Yes-Associated Protein in Kupffer Cells Enhances the Production of Proinflammatory Cytokines and Promotes the Development of Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Yes-associated protein (YAP) plays an important role in hepatocarcinogenesis, although the potential role of YAP in non-neoplastic liver diseases remains largely unknown. We report herein that YAP in Kupffer cells (KCs) enhances the production of proinflammatory cytokines and promotes the development of nonalcoholic steatohepatitis (NASH). Our data show that the expression of YAP is significantly increased in KCs of wild-type mice fed a high-fat diet (HFD). APPROACH AND RESULTS: We generated mice with macrophage/monocyte-specific deletion of YAP (YAPϕKO ) or Toll-like receptor 4 (TLR4; TLR4ϕKO ), and animals were fed an HFD or treated with lipopolysaccharide (LPS). Our data showed that YAPϕKO mice fed an HFD exhibited lower serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels and less hepatic inflammation when compared to their littermate controls. LPS treatment induced accumulation of YAP in KCs in vitro and in mice, which was prevented by macrophage/monocyte-specific deletion of TLR4 (TLR4ϕKO ). LPS transcriptionally activates YAP through activator protein 1 in macrophages/KCs. LPS-induced YAP further enhances expression of proinflammatory cytokines (including monocyte chemoattractant protein 1, tumor necrosis factor alpha, and interleukin 6) through YAP association with the TEA domain-binding motif in the promoter region of inflammatory cytokines. Forced overexpression of active YAP (YAP5SA) in KCs enhanced the production of proinflammatory cytokines. Treatment of HFD-fed mice with verteporfin inhibited KC activation, reduced liver inflammation, and decreased serum ALT/AST levels. Analyses of liver tissues from NASH patients reveal that YAP is increased in KCs and that level of YAP in human liver tissues is positively correlated with expression of proinflammatory cytokines. CONCLUSIONS: This study describes an important role of YAP in KCs for regulation of liver inflammation in NASH. Our findings suggest that inhibition of YAP may represent an effective therapeutic strategy for NASH treatment.

The effects of hepatic steatosis on thromboxane A2 induced portal hypertension / Efectos de la esteatosis hepática en la hipertensión portal inducida por tromboxano A2

Introduction and aim: Thromboxane (TX) A2 was identified as an important vasoconstrictor during Zymosan induced portal perfusion pressure (PP) increase. We aimed at investigating whether hepatic steatosis influences the extent of TXA2-induced portal hypertension. Materials and methods: Sprague-Dawley rats were randomly divided into control and steatosis (induced by the special diet) groups. PP and TXB2 (stable degradation product of TXA2) in the perfusate were measured after in situ liver perfusion with Zymosan (150μg/ml, 40-46min) or U46619 (TXA2 analog, 0.1μM/ml, 40-46min). The number of Kupffer cell (KC) was measured by immunohistochemistry with CD163. Results: Zymosan induced more TXB2 production and a higher PP increase in control group than in steatosis group despite more CD163 positive KCs in fatty livers. PP and TXB2 efflux revealed a strong correlation in control group and a moderate correlation in steatosis group. Contrary to the effect of Zymosan, U46619 induced a much higher PP increase in steatosis group than in control group. Conclusion: Severe steatosis increased number of KCs, however, PP increase and TXB2 efflux caused by Zymosan infusion in fatty livers were lower than those in healthy livers. In contrast, TXA2 analog caused higher PP increase in fatty livers. Targeting the more sensitive response to TXA2 in fatty livers might be a potential therapy of severe steatosis

Introducción y objetivos: Se ha identificado al tromboxano (TX) A2 como importante vasoconstrictor durante el aumento de la presión de perfusión portal (PP) inducida por zymosan. El objetivo ha sido analizar si la esteatosis hepática influye en el grado de hipertensión portal inducida por TXA2. Materiales y métodos: Las ratas Sprague-Dawley(R) se han dividido aleatoriamente en grupos de control y esteatosis (inducida por una dieta especial). Se midieron la PP y el TXB2 (producto de degradación estable de TXA2) en la perfusión después de la perfusión hepática in situ de zymosan (150μg/ml, minuto 40-46) o U46619 (análogo de TXA2, 0,1μM/ml, minuto 40-46). El número de células de Kupffer (CK) se midió mediante inmunohistoquímica con CD163. Resultados: Zymosan provocó más producción de TXB2 y mayor aumento de la PP en el grupo de control que en el grupo de esteatosis a pesar de hallar más CK positivas para CD163 en hígados grasos. El flujo de salida de la PP y el TXB2 reveló una fuerte correlación en el grupo de control y una correlación moderada en el grupo de esteatosis. De manera diferente al efecto de zymosan, U46619 indujo un aumento de la PP mucho mayor en el grupo de esteatosis que en el grupo de control. Conclusión: La esteatosis grave aumentó el número de CK; sin embargo, el aumento de la PP y el flujo de TXB2 provocado por la perfusión de zymosan en hígados grasos fueron menores que en los hígados sanos. En cambio, el análogo de TXA2 provocó un aumento de la PP en hígados grasos. Centrarse en la respuesta más sensible al TXA2 en hígados grasos podría convertirse en un tratamiento potencial de la esteatosis grave

The effects of hepatic steatosis on thromboxane A2 induced portal hypertension.

INTRODUCTION AND AIM: Thromboxane (TX) A2 was identified as an important vasoconstrictor during Zymosan induced portal perfusion pressure (PP) increase. We aimed at investigating whether hepatic steatosis influences the extent of TXA2-induced portal hypertension. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into control and steatosis (induced by the special diet) groups. PP and TXB2 (stable degradation product of TXA2) in the perfusate were measured after in situ liver perfusion with Zymosan (150µg/ml, 40-46min) or U46619 (TXA2 analog, 0.1µM/ml, 40-46min). The number of Kupffer cell (KC) was measured by immunohistochemistry with CD163. RESULTS: Zymosan induced more TXB2 production and a higher PP increase in control group than in steatosis group despite more CD163 positive KCs in fatty livers. PP and TXB2 efflux revealed a strong correlation in control group and a moderate correlation in steatosis group. Contrary to the effect of Zymosan, U46619 induced a much higher PP increase in steatosis group than in control group. CONCLUSION: Severe steatosis increased number of KCs, however, PP increase and TXB2 efflux caused by Zymosan infusion in fatty livers were lower than those in healthy livers. In contrast, TXA2 analog caused higher PP increase in fatty livers. Targeting the more sensitive response to TXA2 in fatty livers might be a potential therapy of severe steatosis.

Probing the unseen structure and function of liver cells through atomic force microscopy.

With the arrival of atomic force microscopy (AFM) about thirty years ago, this new imaging tool opened up a new area for the exploration of biological samples, ranging from the tissue and cellular level down to the supramolecular scale. Commercial instruments of this new imaging technique began to appear in the five years following its discovery in 1986 by Binnig, Quate & Gerber. From that point onwards the AFM has attracted many liver biologists, and the number of publications describing structure-function relationships on the diverse set of liver cells has grown steadily ever since. It is therefore timely to reflect on the achievements of AFM in disclosing the cellular architecture of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, stellate cells and liver-associated natural killer cells. In this thematic paper, we present new data and provide an in-depth overview of the current AFM literature on liver cell biology. We furthermore include a future outlook on how this scanning probe imaging tool and its latest developments can contribute to clarify various structural and functional aspects of cells in liver health and disease.

Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy.

It is central to the field of bacterial pathogenesis to define how bacteria are killed by phagocytic cells. During phagocytosis, the microbe is localized to the phagolysosome where crucial defense mechanisms such as acidification and production of reactive oxygen species (ROS) are initiated. This process has extensively been studied in vitro, however many resident tissue phagocytes will phenotypically change upon isolation from their natural environment. Therefore, interrogation of phagocytosis and phagosomal function of cells in the context of their natural tissue environment enhances our understanding of the biological process in vivo. This article outlines a real-time intravital microscopy protocol that utilizes fluorescent dyes to study the process of phagocytosis, which reveals acidification and oxidation of individual bacteria inside host cells of living animals. The novelty of this technique exists in use of bacteria that are covalently labelled with the fluorescent dyes Oxyburst and pHrodo, which respectively report on oxidation or acidification. Intravital microscopy is applied to visualize the uptake and subsequent oxidation or acidification of reporter bacteria in the organ of interest. Fluorescently labelled antibodies can be used to counter stain for host immune cells such as neutrophils and macrophages, along with reference stains to identify all bacteria. Although these assays were originally developed to assess the uptake and survival ofStaphylococcus aureusin liver resident macrophages (Kupffer cells), this protocol may be adapted to investigate any bacterium-host cell interaction.

Distribution of FcRn Across Species and Tissues.

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I type molecule that binds to, transports, and recycles immunoglobulin G (IgG) and albumin, thereby protecting them from lysosomal degradation. Therefore, besides the knowledge of FcRn affinity, FcRn protein expression is critical in understanding the pharmacokinetic behavior of Fc-containing biotherapeutics such as monoclonal antibodies. The goal of this investigation was to achieve for the first time a comparative assessment of FcRn distribution across a variety of tissues and species. FcRn was mapped in about 20 tissues including placenta from human and the most frequently used species in non-clinical safety testing of monoclonal antibodies (mouse, rat, cynomolgus monkey). In addition, the FcRn expression pattern was characterized in two humanized transgenic mouse lines (Tg32 and Tg276) expressing human FcRn under different promoters, and in the severe combined immunodeficient (SCID) mouse. Consecutive sections were stained with specific markers, namely, anti-CD68 for macrophages and anti-von Willebrand Factor for endothelial cells. Overall, the FcRn expression pattern was comparable across species and tissues with consistent expression of FcRn in endothelial cells and interstitial macrophages, Kupffer cells, alveolar macrophages, enterocytes, and choroid plexus epithelium. The human FcRn transgenic mouse Tg276 showed a different and much more widespread staining pattern of FcRn. In addition, immunodeficiency and lack of IgG in SCID mice had no negative effect on FcRn expression compared with wild-type mice.

[Nicotine alleviates the liver inflammation of non-alcoholic steatohepatitis induced by high-fat and high-fructose in mice].

OBJECTIVE: To investigate the anti-inflammation effects by activation of the cholinergic anti-inflammatory pathway and its mechanisms in non-alcoholic steatohepatitis (NASH) model mice. METHODS: 6-week-old male C57BL/6J (B6) mice were randomly divided into four groups: the first group was normal mice, injected with saline; the second group was normal mice, injected with nicotine; the third group was NASH model mice, injected with saline; the fourth group was NASH model mice, injected with nicotine. The experimental mice were fed with either standard chow (SC) or high-fat and high-fructose (HFHF) for 17 weeks to generate an NASH model mice. The mice received injection once daily for 3 weeks [nicotine dose, 400 µg/kg]. Then, their pathological characteristics and function of the liver were assessed. The expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum were analyzed by enzyme linked immunosorbent assay (ELISA). The expressions of alpha 7 nicotinic acetylcholine receptors (α7nAChR), Toll-like receptors-4 (TLR-4) and nuclear factor κB of phosphory-lation (p-NF-κB) in Kupffer cells were determined by Western blot and immunofluorescence assays. RESULTS: We successfully generated NASH model mice by imitating the high-fat and high-fructose dietary style of NASH patients. The results of our investigation demonstrated that nicotine could reduce significantly the levels of IL-6, and TNF-α in serum (P<0.05). The expression of p-NF-κB protein in the group which was NASH model mice injected with nicotine declined significantly as compared with the group which was NASH model mice injected with saline (P<0.05). And the expression of α7nAChR protein elevated significantly conversely (P<0.05). CONCLUSION: Activation of the cholinergic anti-inflammatory pathway could inhibit the release of inflammatory factors as TNF-α and IL-6 in NASH model mice, and the mechanism for the inhibition of inflammatory was mediated by NF-κB pathway.

[Cholesterol metabolism and non-alcoholic steatohepatitis].

The disease spectrum of non-alcoholic fatty liver disease (NAFLD) includes non-alcoholic simple fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), as well as liver cirrhosis and hepatocellular carcinoma, with the most serious type being NASH. The morbidity of NAFLD is seeing an increase year by year in the world, and it is a common cause of chronic hepatic disease and lacks effective treatment. The pathogenesis of NASH is still unknown, and the "two-hit" hypothesis was used to explain the mechanism of NASH. Recent research has found that cholesterol metabolism is closely related to the pathogenesis and severity of NASH. The validity of the "two-hit" hypothesis has been recently challenged, which gives rise to "multi-parallel hit" hypothesis. Cholesterol affects membrane fluidity and membrane protein function through genetic factors, and it can also induce unfolded protein response, and generate toxic oxysterol. Free cholesterol can activate hepatic Kupffer and stellate cells to produce inflammatory cytokines and collagen. The formation of cholesterol crystallization and crown-like structures can damage liver cells and activate Kupffer cells. The above processes can all aggravate liver damage, thus accelerating the development and making the clinical manifestations of NASH even worse. In the present review we summarize the association between cholesterol metabolism and pathogenesis of NASH.

Utilization of HPASubC for the Identification of Sinusoid-Specific Proteins in the Liver.

Mass spectrometry-based proteomes of human organs and tissues are powerful tools but fail to capture protein localization and expression at the cellular level. For example, the proteome signal in liver represents the combined protein expression across diverse cellular constituents that include hepatocytes, Kupffer cells, endothelial cells, and others. We utilized HPASubC and the Human Protein Atlas (HPA) to identify the sinusoidal component of protein liver expression to further subset and organize this homogeneous signal. We evaluated 51 109 liver images covering 13 197 proteins from the HPA and discovered 1054 proteins that were exclusive to sinusoidal cells. Sinusoidal staining patterns were identified in a Kupffer cell (n = 247), endothelial cell (n = 358), or lymphocyte (n = 86) specific pattern. Two-hundred and thirty-nine of these proteins were not present in the NextProt or Human Proteome Map liver data sets, potentially expanding our knowledge of the liver proteome. We additionally demonstrate unique endothelial cell expression patterns that distinguish between portal vein, hepatic artery, capillary sinusoids, and central vein regions. These findings significantly improve our understanding of the liver proteome with insight into the endothelial complexity across the hepatic vascular network.

ß-catenin signaling in murine liver zonation and regeneration: a Wnt-Wnt situation!

UNLABELLED: Liver-specific ß-catenin knockout (ß-Catenin-LKO) mice have revealed an essential role of ß-catenin in metabolic zonation where it regulates pericentral gene expression and in initiating liver regeneration (LR) after partial hepatectomy (PH), by regulating expression of Cyclin-D1. However, what regulates ß-catenin activity in these events remains an enigma. Here we investigate to what extent ß-catenin activation is Wnt-signaling-dependent and the potential cell source of Wnts. We studied liver-specific Lrp5/6 KO (Lrp-LKO) mice where Wnt-signaling was abolished in hepatocytes while the ß-catenin gene remained intact. Intriguingly, like ß-catenin-LKO mice, Lrp-LKO exhibited a defect in metabolic zonation observed as a lack of glutamine synthetase (GS), Cyp1a2, and Cyp2e1. Lrp-LKO also displayed a significant delay in initiation of LR due to the absence of ß-catenin-TCF4 association and lack of Cyclin-D1. To address the source of Wnt proteins in liver, we investigated conditional Wntless (Wls) KO mice, which lacked the ability to secrete Wnts from either liver epithelial cells (Wls-LKO), or macrophages including Kupffer cells (Wls-MKO), or endothelial cells (Wls-EKO). While Wls-EKO was embryonic lethal precluding further analysis in adult hepatic homeostasis and growth, Wls-LKO and Wls-MKO were viable but did not show any defect in hepatic zonation. Wls-LKO showed normal initiation of LR; however, Wls-MKO showed a significant but temporal deficit in LR that was associated with decreased ß-catenin-TCF4 association and diminished Cyclin-D1 expression. CONCLUSION: Wnt-signaling is the major upstream effector of ß-catenin activity in pericentral hepatocytes and during LR. Hepatocytes, cholangiocytes, or macrophages are not the source of Wnts in regulating hepatic zonation. However, Kupffer cells are a major contributing source of Wnt secretion necessary for ß-catenin activation during LR.

The relationship between hepatic resistin overexpression and inflammation in patients with nonalcoholic steatohepatitis.

BACKGROUND: The relationship between resistin and non-alcoholic steatohepatitis (NASH) is not clear, some studies claimed that serum resistin levels were associated with neither the presence of NASH nor its severity, others declared that serum resistin was related with inflammation and fibrosis in NASH. Our animal study verified that the distribution of resistin in the liver is correlated with inflammation in NASH. However, there is no pertinent study in humans. METHODS: Thirty patients with NASH, 28 simple steatosis, and 43 controls were recruited. Blood was collected for resistin, liver chemistries, fasting insulin and some metabolic parameters. Liver histology was scored according to NAFLD activity scoring system. Hepatic resistin expression was examined by real-time polymerase chain reaction, immunohistochemistry. Resistin protein expression was confirmed by western blotting in 13 patients with concomitant NAFLD and gallstone. RESULTS: Serum resistin was significantly elevated in both NASH and simple steatotic subjects compared with controls (all P < 0.05). Hepatic resistin was significantly increased in NASH patients in both mRNA and protein levels than those in simple steatosis and control subjects (all P < 0.05). Both serum and hepatic resistin had a correlation with obesity, but not with insulin resistance. The distribution of resistin positive cells was predominantly in perisinusoidal cells (such as Kupffer cells and hepatic stellate cells) in human NASH. Multivariate analysis revealed that waist-hip ratio, higher serum triglyceride, and hyperresistinemia were independent factors related to higher grade of steatosis; whereas hepatic resistin and serum cytokeratin predict NASH and severity of liver fibrosis. CONCLUSIONS: Hepatic resistin overexpression in NASH patients is associated with the severity of liver inflammation and fibrosis. Liver-derived resistin may be involved in the pathogenesis of human NASH.

Persistent hepatic structural alterations following nanoceria vascular infusion in the rat.

Understanding the long-term effects and possible toxicity of nanoceria, a widely utilized commercial metal oxide, is of particular importance as it is poised for development as a therapeutic agent based on its autocatalytic redox behavior. We show here evidence of acute and subacute adverse hepatic responses, after a single infusion of an aqueous dispersion of 85 mg/kg, 30 nm nanoceria into Sprague Dawley rats. Light and electron microscopic evidence of avid uptake of nanoceria by Kupffer cells was detected as early as 1 hr after infusion. Biopersistent nanoceria stimulated cluster of differentiation 3(+) lymphocyte proliferation that intermingled with nanoceria-containing Kupffer cells to form granulomata that were observed between days 30 and 90. Ultrastructural tracking of ceria nanoparticles revealed aggregated nanoceria in phagolysosomes. An increased formation of small nanoceria over time observed in the latter suggests possible dissolution and precipitation of nanoceria. However, the pathway for nanoceria metabolism/secretion remains unclear. Although frank hepatic necrosis was not observed, the retention of nanoceria increased hepatic apoptosis acutely, this persisted to day 90. These findings, together with our earlier reports of 5-nm ceria-induced liver toxicity, provide additional guidance for nanoceria development as a therapeutic agent and for its risk assessment.

Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.

We sought to determine whether hepatic cholesterol crystals are present in patients or mice with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NASH), and whether their presence or distribution correlates with the presence of NASH as compared with simple steatosis. We identified, by filipin staining, free cholesterol within hepatocyte lipid droplets in patients with NASH and in C57BL/6J mice that developed NASH following a high-fat high-cholesterol diet. Under polarized light these lipid droplets exhibited strong birefringence suggesting that some of the cholesterol was present in the form of crystals. Activated Kupffer cells aggregated around dead hepatocytes that included strongly birefringent cholesterol crystals, forming "crown-like structures" similar to those recently described in inflamed visceral adipose tissue. These Kupffer cells appeared to process the lipid of dead hepatocytes turning it into activated lipid-laden "foam cells" with numerous small cholesterol-containing droplets. In contrast, hepatocyte lipid droplets in patients and mice with simple steatosis did not exhibit cholesterol crystals and their Kupffer cells did not form crown-like structures or transform into foam cells. Our results suggest that cholesterol crystallization within hepatocyte lipid droplets and aggregation and activation of Kupffer cells in crown-like structures around such droplets represent an important, novel mechanism for progression of simple steatosis to NASH.

Roles of hepatic progenitor cells activation, ductular reaction proliferation and Notch signaling in morbid obesity.

BACKGROUND/AIMS: Hepatic progenitor cells (HPCs) activation, proliferative ductular reaction (DR), replicative arrest and Notch signaling have been demonstrated in a variety of human liver diseases. The relationships are poorly understood in morbid obesity. We investigated factors responsible for the HPCs/DR, replicative arrest and Notch signaling in non-NASH and NASH groups. METHODOLOGY: Cytokeratin 7 (and 19), p21, CD34, Ki67 and different Notch receptors and ligands immunohistochemical stained biopsies from morbid obese patients with non-NASH (n=10) and NASH (n=25) were studied. These results were correlated with clinicopathological variables. RESULTS: NASH patients presented with abnormal liver function tests and had higher HbA1c percentage. Strong association between HPCs and DR was seen (r=0.785, p<0.000). BMI, interface activity and replicative arrest were associated with HPCs expansion and DR in NASH patients. A strong association between CD34 with HPCs and DR was found in non-NASH patients. In NASH group, Notch 3 was important in bile ductular proliferation; whereas Notch 4 was associated with sinusoidal neovessels proliferation and Kupffer cell activation. CONCLUSIONS: HPCs and DR played an important role in hepatic regeneration in fatty liver disease of morbid obesity. An altered replication pathway in NASH promotes HPCs activation and DR. Notch-3 and Notch-4 were significantly different between non-NASH and NASH groups.

An experimental study on MR imaging of atherosclerotic plaque with SPIO marked endothelial cells in a rabbit model.

PURPOSE: To investigate how to label macrophages in atherosclerotic plaques with superparamagnetic iron oxide (SPIO) nanoparticles and trace SPIO with MR imaging. MATERIALS AND METHODS: Atherosclerotic lesions of a rabbit model were induced by a combination of high-fat and high-cholesterol diet and subsequent endothelial abrasion of the abdominal aorta. SPIO particles were pretreated with poly-L-lysine. SPIO nanoparticles and SPIO-labeled human endothelial cells (ECV-304) were IV injected into model animals, respectively. The MRI scans and histopathological examination were performed 12 h and 24 h after the injection. The imaging and histopathological data were analyzed. RESULTS: Prussian blue staining of the vessel specimens indicated that SPIO particles were not found in the atheroma but in the Kupffer's cells of the liver after SPIO injection. However, the accumulation of SPIO particles in the atheroma was confirmed in animals received SPIO-labeled endothelial cell transplantation. The best quality MR scan sequences of rabbit abdominal aorta were T(2) WI fat suppression, T(1) WI, and DIR series, on which of MR image had a higher quality. Signal loss of the original incrassate plaque in the vessel wall on T(2) WI was found in 6 of 10 animals received SPIO-labeled endothelial cell transplantation. CONCLUSION: SPIO-labeled endothelial cells were superior to SPIO for MR imaging of atherosclerotic plaques.

Iron distribution in the liver and duodenum during seasonal iron overload in Svalbard reindeer.

Seasonal iron overload in Svalbard reindeer was studied by light and electron microscopy and by X-ray microanalysis. The hepatic iron overload was of two types. The first type was characterized by massive siderosis of both parenchymal and non-parenchymal cells caused by a diet very rich in iron but low in energy and protein. Hepatocytes contained a moderate amount of free ferritin particles in the cytosol together with numerous siderosomes. This pattern is similar to that seen in primary haemochromatosis and thalassaemia. Kupffer cells contained large quantities of cytosolic ferritin, siderosomes and lysosomes with disintegrating red blood cells as seen in thalassaemia. The second type was characterized by massive non-parenchymal siderosis caused by an energy- and protein-poor diet with normal iron concentration. Hepatocytes contained little cytosolic ferritin and few siderosomes, but there were abundant electron-dense bodies without iron (i.e., autophagosomes). Kupffer cells were as described above. Ferritin was also present within the duodenal mucosa of these animals, located within enterocytes and lamina propria macrophages, as well as in the extracellular space and capillary and lacteal lumina. Ferritin was also present in the acinar cells of submucosal Brunner's glands. Changes consistent with exchange of ferritin particles between different cell types were observed. The role of ferritin as a possible iron transporter in this condition is discussed.

A comparison between whites and blacks with severe multi-organ iron overload identified in 16,152 autopsies.

BACKGROUND & AIMS: Little is known about differences in the prevalence of severe iron overload at death in whites and blacks. We evaluated data and samples from 16,152 autopsies (8484 whites, 7668 blacks) performed at a single university hospital. METHODS: Cases of severe multi-organ iron overload were identified by review of autopsy protocols and Perls-stained tissue specimens, analysis of hepatocyte and Kupffer cell iron levels, and measurement of liver tissue iron concentrations. RESULTS: We analyzed autopsy data from 10,345 adults (age > or =21 years), 1337 children (1-20 years), and 4470 infants (<1 year). Iron overload without reports of excessive exogenous iron was observed in 18 adults; the prevalence in whites and blacks was 0.0019 and 0.0015, respectively (P = .6494). Twenty-nine adults and 2 children had iron overload with reports of excessive exogenous iron. In adults, the prevalences of iron overload with reports of excessive exogenous iron in whites and blacks were 0.0040 and 0.0013, respectively (P = .0107). Among adults, the prevalence of cirrhosis was 6-fold greater in those with iron overload. In adults with severe iron overload, 67% without reports of excessive exogenous iron and 14% with reports of excessive exogenous iron died of hepatic failure or cardiomyopathy caused by siderosis. The overall prevalence of deaths caused by severe iron overload in whites and blacks was 0.0021 and 0.0009, respectively (P = .0842). CONCLUSIONS: The prevalence of severe iron overload without reports of excessive exogenous iron did not differ significantly between whites and blacks. The prevalence of iron overload with reports of excessive exogenous iron was greater in whites. Hepatic failure and cardiomyopathy were common causes of death in severe iron overload cases.