Laura Conti Devirgiliis

Hepatocytes Entrapped in Alginate Gel Beads and Cultured in Bioreactor: Rapid Repolarization and Reconstitution of Adhesion Areas

The maintenance of the differentiated hepatocyte phenotype and its specific physiological properties is known to depend on several factors, such as chemical signals, cell-cell and extracellular matrix molecular interactions, as well as the use of three-dimensional matrices. The entrapment of hepatocytes within Ca-alginate at high cell density and the culture under continuous flow favour the development of three-dimensional organization and promote expression of the differentiated hepatic phenotype. This system could represent an improvement in hepatocyte cultivation for basic studies of liver physiology and metabolism; it could also be applicable in toxicology, hepatocyte transplantation or development of bioartificial organs. This report describes the effect of alginate entrapment and culture in a bioreactor on hepatocyte aggregate formation, with particular attention to the re-establishment of cell polarity, cell junctions and three-dimensional re-organization of the cytoskeleton. Oxygen supply and cell oxygen consumption rate were monitored in order to evaluate possible changes in hepatocyte energy requirement. Our data show that after only 6 h of perfusion in the bioreactor, actin and cytokeratin localize along the adhesion areas of the plasma membrane, in which reconstituted bile canaliculi were also observed. Moreover, the presence of connexin at the level of joined membranes of neighbouring cells suggests the establishment of gap junctions between hepatocytes. After the first 30 min of perfusion the oxygen consumption rate remained constant throughout the experimental period.

The effect of retinoic acid on the re-establishment of differentiated hepatocyte phenotype in primary culture

Abstract The usefulness of cultured hepatocytes is limited by the gradual loss of their typical physiological functions that occurs in vitro, mainly due to the absence of microenviromental conditions found in vivo. In this study we describe the effect of retinoic acid on the re-establishment of morphological characteristics and on the reorganization of the cytoskeletal network in cultured rat hepatocytes. Results obtained demonstrate that retinoic acid can influence hepatocyte differentiation, as regards the recovery of cell polarity, polyhedric shape and reformation of bile canaliculi and junctional complexes. The main target of this action appears to be the cytoarchitecture of cytoskeletal components, particularly cytokeratin filaments, which regain the configuration present in intact liver. The reorganization of the intermediate filaments does not seem to be dependent on the induction of higher levels of cytokeratin proteins, but rather appears to be due to post-translational regulation. The effect of retinoic acid on the cytoskeletal organization could determine the stabilization of intercellular contacts by means of junctions, leading to the appearance of morpho-functional characteristics typical of well-differentiated hepatocytes.

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells

Aims: In this work, we aimed to investigate the possible modulation of cell–matrix interactions by retinoic acid (RA), in view of the well‐known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy.

Influence of Retinoic Acid on Adhesion Complexes in Human Hepatoma Cells: A Clue to Its Antiproliferative Effects

Retinoic acid exerts antiproliferative and differentiative effects in normal and transformed in vitro hepatocytes. In order to verify whether these effects are related to a modulation of adhesion molecules, we used Western blot analysis and immunofluorescence microscopy to investigate the E-cadherin/β-catenin complex, the main system of adherens junctions, and the occludin/ZO-1 complex present in the tight junctions in HepG2 cells cultured in the presence or absence of retinoic acid. Results showed that retinoic acid treatment increases the amount of β-catenin bound to E-cadherin by decreasing its tyrosine-phosphorylation level. Similar results were obtained with the tight junction system, in which the amount of occludin/ZO-1 complex is increased by a similar mechanism that reduced the level of ZO-1 phosphorylation on tyrosine. Immunofluorescence images also confirm these results, showing the localization on the cell surface of both adhesion complexes. Their insertion into the plasma membrane could be suggestive of an optimal reassembly and function of adherens and tight junctions in hepatoma cells, indicating that retinoic acid, besides inhibiting cell proliferation, improves cell-cell adhesion, sustaining or inducing the expression of a more differentiated phenotype.

Multiple parameters are involved in the effects of cadmium on prenatal hepatocytes

Cadmium, a toxic heavy metal, expresses its toxicity by affecting several cellular functions, such as enzyme activities, DNA repair systems, redox state of the cell and signal transduction. Although the liver is a known target organ, the mechanisms involved in cadmium toxicity are not yet clarified, especially during prenatal development. Here we consider the effects of cadmium on viability, proliferation, adhesion and defence mechanisms in primary adult and fetal rat hepatocytes. Fetal hepatocytes are less sensitive to cadmium toxicity, they appear to be unaffected or even stimulated by treatments that strongly inhibit DNA synthesis in adult cells. The behaviour of proteins involved in cell cycle regulation also differs from adult cells, according to the proliferative state. In addition, following Cd exposure, E-cadherin/beta-catenin complex disassembles in both cell types, with fetal cells being influenced at higher doses. The beta-catenin is not found in the nucleus, ruling out a direct role on DNA synthesis stimulation. Finally, metallothionein is more easily inducible in fetal hepatocytes, while Cd intracellular concentrations and HSP protein levels are not differentially affected. In conclusion, multiple cellular targets are affected by Cd in primary hepatocytes and the adverse effects of the metal are always better counteracted by fetal cells.

Energy metabolism and re-establishment of intercellularadhesion complexes of gel entrapped hepatocytes

We studied the effect of continuous medium flow on the viabilityand structural organization of hepatocytes high density entrapped inalginate gel beads in the first few hours after isolation.The metabolic energy status of the entrapped cells, monitored invivo by 31P NMR spectroscopy, was stable during theexperimental time and a physiological redox ratio was reachedafter the first three hours of culture. The morphologicalanalysis revealed that the entrapped hepatocytes placed in a fixed-bed bioreactor under continuous flow showed a polyhedricalshape with numerous microvilli on cell surface and reconstitutedtight junctions as well as bile canalicular structures, closelyresembling those present in the liver.These results suggest that continuous flow allows the culture ofhepatocytes at very high cell density within a matrix withoutloss of viability and accelerates cellular tissue reconstructionat very short times after isolation. This type of culture couldrepresent a very useful model for physiological andtoxicological studies as well as a promising approach toward thedevelopment of a bioartificial hybrid support device in acuteliver failure.

Retinoic acid modulates the asialoglycoprotein receptor expression in cultured fetal rat hepatocytes

The influence of retinoic acid on the expression of a typical marker of hepatocyte differentiation, i.e. the asialoglycoprotein receptor, has been studied. Cultured hepatocytes, isolated from adult rats, a model of quiescent, mature cells and from 20-day-old fetuses, a model of proliferating and less differentiated cells, were used. The asialoglycoprotein receptor expression appears to be affected by retinoic acid during prenatal life; both mRNA level and protein amount increased in fetal hepatocytes, but no modification has been found in adult cells, suggesting a regulative effect of retinoic acid during prenatal life, acting at transcriptional and/or translational level. Surprisingly, the receptor binding activity of adult hepatocytes is decreased after retinoic acid treatment, indicating a possible further modulation by this molecule on receptor activity at the post-translational level.

The lobular expression of the rat asialoglycoprotein receptor is regulated at the posttranscriptional level

Abstract: The purpose of this study was to define the distribution of the asialoglycoprotein receptor (ASGP‐R) main peptide, rat hepatic lectin (RHL)‐1, within the rat liver lobule and to investigate its possible modulation in physiological states characterised by marked changes of receptorial expression. In particular, we chose livers from rats partially hepatectomised or at the end of pregnancy, as models, respectively, of decreased or increased expression of the ASGP‐R, and used the in situ hybridisation and immunocytochemistry techniques to analyse in parallel the lobular distributions of RHL‐1 mRNA and protein. In normal rat liver, although the RHL‐1 mRNA was homogeneously distributed, the RHL‐1 peptide was predominantly localised on the surface of pericentral hepatocytes with a gradient of expression towards the periportal zone. This gradient of expression of RHL‐1 peptide was reduced in regenerating livers, in which the positive stain was restricted to a few layers of cells around the central vein. In contrast, livers at the end of pregnancy showed an overall increase of the peptide with a concomitant flattening of the gradient across the liver plate. In all the conditions, we never observed important changes in the pattern of expression of the specific mRNA. These findings indicate that the distribution of ASGP‐R is heterogeneous across the liver lobule, with a pattern of expression prevalently modulated at the posttranscriptional level.