3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro

Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation

Rapid and Efficient Clearance of Blood-borne Virus by Liver Sinusoidal Endothelium

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity

Self-assembled nanogel made of mannan : synthesis and characterization

Amphiphilic mannan (mannan-C16) was synthesized by the Michael addition of hydrophobic 1-hexadecanethiol (C16) to hydroxyethyl methacrylated mannan (mannan-HEMA). Mannan-C16 formed nanosized aggregates in water by selfassembly via the hydrophobic interaction among C16molecules as confirmed by hydrogen nuclearmagnetic resonance (1H NMR), fluorescence spectroscopy, cryo-field emission scanning electron microscopy (cryo-FESEM), and dynamic light scattering (DLS). The mannan-C16 critical aggregation concentration (cac), calculated by fluorescence spectroscopy with Nile red and pyrene, ranged between 0.04 and 0.02mg/mL depending on the polymer degree of substitution ofC16 relative to methacrylated groups. Cryo-FESEM micrographs revealed that mannan-C16 formed irregular spherical macromolecular micelles, in this work designated as nanogels, with diameters ranging between 100 and 500 nm. The influence of the polymer degree of substitution, DSHEMA andDSC16, on the nanogel size and zeta potential was studied byDLS at different pH values and ionic strength and as a function of mannan-C16 and urea concentrations. Under all tested conditions, the nanogel was negatively charged with a zeta potential close to zero. Mannan-C16 with higher DSHEMA and DSC16 values formed larger nanogels andwere also less stable over a 6month storage period and at concentrations close to the cac.When exposed to solutions of different pH and aggressive conditions of ionic strength and urea concentration, the size of mannan-C16 varied to some extent but was always in the nanoscale range.International Iberian Nanotechnology Laboratory (INL)Fundação para a Ciência e a Tecnologia (FCT

Activation of ERK and NF-κB during HARE-Mediated Heparin Uptake Require Only One of the Four Endocytic Motifs

We thank Bruce A. Baggenstoss and Jennifer L. Washburn for technical support in many experiments and Emma K. Blank, Andrew W. Egger, Brianna M. Kellar and Helen T. Russom for assistance with experiments supporting Fig 2.Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068–79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hep-mediated signaling. Deletion of M1, M2 or M4 singly had no effect on Hep-mediated ERK1/2 activation, whereas signaling (but not uptake) was eliminated in HARE(ΔM3) cells lacking NPLY2519. ERK1/2 signaling in cells expressing WT HARE(Y2519A) or HARE(Y2519A) lacking M1, M2 and M4 (containing M3-only) was decreased by 75% or eliminated, respectively. Deletion of M3 (but not M1, M2 or M4) also inhibited the formation of HARE•Hep•ERK1/2 complexes by 67%. NF-κB activation by HARE-mediated uptake of Hep, HA, dermatan sulfate or acetylated LDL was unaffected in single-motif deletion mutants lacking M1, M2 or M4. In contrast, cells expressing HARE(ΔM3) showed loss of HARE-mediated NF-κB activation during uptake of each of these four ligands. NF-κB activation by the four signaling ligands was also eliminated in HARE(Y2519A) or HARE(M3-only;Y2519A) cells. We conclude that the HARE NPLY2519 motif is necessary for both ERK1/2 and NF-κB signaling and that Tyr2519 is critical for these functions.Yeshttp://www.plosone.org/static/editorial#pee

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0+/-0.4x107 hepatocytes, 1.8+/-0.5x106 Kupffer cells, 4.3+/-1.9x105 liver sinusoidal endothelial cells, and 3.2+/-0.5x105 stellate cells. Hepatocytes were identified by albumin (95.5+/-1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5+/-1.2%) and exhibited phagocytic activity, as determined with 1mum latex beads. Endothelial cells were CD146+ (97.8+/-1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of alpha-smooth muscle actin (97.1+/-1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease

The Prometastatic Microenvironment of the Liver

The liver is a major metastasis-susceptible site and majority of patients with hepatic metastasis die from the disease in the absence of efficient treatments. The intrahepatic circulation and microvascular arrest of cancer cells trigger a local inflammatory reaction leading to cancer cell apoptosis and cytotoxicity via oxidative stress mediators (mainly nitric oxide and hydrogen peroxide) and hepatic natural killer cells. However, certain cancer cells that resist or even deactivate these anti-tumoral defense mechanisms still can adhere to endothelial cells of the hepatic microvasculature through proinflammatory cytokine-mediated mechanisms. During their temporary residence, some of these cancer cells ignore growth-inhibitory factors while respond to proliferation-stimulating factors released from tumor-activated hepatocytes and sinusoidal cells. This leads to avascular micrometastasis generation in periportal areas of hepatic lobules. Hepatocytes and myofibroblasts derived from portal tracts and activated hepatic stellate cells are next recruited into some of these avascular micrometastases. These create a private microenvironment that supports their development through the specific release of both proangiogenic factors and cancer cell invasion- and proliferation-stimulating factors. Moreover, both soluble factors from tumor-activated hepatocytes and myofibroblasts also contribute to the regulation of metastatic cancer cell genes. Therefore, the liver offers a prometastatic microenvironment to circulating cancer cells that supports metastasis development. The ability to resist anti-tumor hepatic defense and to take advantage of hepatic cell-derived factors are key phenotypic properties of liver-metastasizing cancer cells. Knowledge on hepatic metastasis regulation by microenvironment opens multiple opportunities for metastasis inhibition at both subclinical and advanced stages. In addition, together with metastasis-related gene profiles revealing the existence of liver metastasis potential in primary tumors, new biomarkers on the prometastatic microenvironment of the liver may be helpful for the individual assessment of hepatic metastasis risk in cancer patients