A biomimetic porous hydrogel of gelatin and glycosaminoglycans cross-linked with transglutaminase and its application in the culture of hepatocytes.

The development of blended gelatin and glycosaminoglycan (GAG) scaffolds can potentially be used in many soft tissue engineering applications since these scaffolds mimic the structure and biological function of native extracellular matrix (ECM). In this study, we were able to obtain a gelatin-GAG scaffold by using a concentrated emulsion templating technique known as high internal phase emulsion (HIPE), in which a prevailing in volume organic phase is dispersed in the form of discrete droplets inside an aqueous solution of three biopolymers represented by gelatin, hyaluronic acid (HA) and chondroitin sulfate (CS) in the presence of a suitable surfactant. In order to preserve the bioactive potential of the biopolymers employed, the cross-linking procedure involved the use of transglutaminase (MTGase) that catalyzes the formation of covalent N-ε-(γ-glutamyl) lysine amide bonds. Since neither HA nor CS possess the necessary primary amino groups toward which MTGase is active, they were functionalized with the dipeptide glycine-lysine (GK). In this way the introduction of foreign cross-linking bridging units with an unpredictable biocompatibility was avoided. These enzymatic cross-linked gelatin-GAG scaffolds were tested in the culture of primary rat and C3A hepatocytes. Results underlined the good performance of this novel support in maintaining and promoting hepatocyte functions in vitro.

The Cosmic Silence experiment: on the putative adaptive role of environmental ionizing radiation.

Previously we reported that yeast and Chinese hamster V79 cells cultured under reduced levels of background environmental ionizing radiation show enhanced susceptibility to damage caused by acute doses of genotoxic agents. Reduction of environmental radiation dose rate was achieved by setting up an underground laboratory at Laboratori Nazionali del Gran Sasso, central Italy. We now report on the extension of our studies to a human cell line. Human lymphoblastoid TK6 cells were maintained under identical in vitro culture conditions for six continuous months, at different environmental ionizing radiation levels. Compared to "reference" environmental radiation conditions, we found that cells cultured in the underground laboratories were more sensitive to acute exposures to radiation, as measured both at the level of DNA damage and oxidative metabolism. Our results are compatible with the hypothesis that ultra-low dose rate ionizing radiation, i.e. environmental radiation, may act as a conditioning agent in the radiation-induced adaptive response.

Retinoic acid modulates the cell-cycle in fetal rat hepatocytes and HepG2 cells by regulating cyclin-cdk activities.

Retinoic acid (RA), the most biologically active metabolite of vitamin A, is known to modulate cell proliferation, apoptosis and differentiation, with different effects depending on the cellular context. Retinoic acid can exert its effects by directly or indirectly influencing the expression of genes involved in the control of cell proliferation. In the present report we investigate the possible correlation between the antiproliferative, differentiative and apoptotic effects previously observed on rat hepatocytes and HepG2 cells, with a possible modulation of cell-cycle regulators. We demonstrate that RA induces growth arrest and differentiation in HepG2 cells by influencing the activities of cyclin-cdk complexes involved in the regulation of G1/S transition and S-phase progression, in particular by modifying the binding of these complexes to p21 and p27 inhibitors. In fetal cells, however, the induction of apoptosis and differentiation by RA was obtained via inhibition of cyclin D1-cdk4 activity, as result of an increased binding to the p16 inhibitor. Retinoic acid also modulates c-myc and Bcl-2 expression. In conclusion, our data suggest that RA could be useful to regulate the reversion of transformed phenotype and could also be utilized as a chemiopreventive agent in cells of hepatic origin.

Influence of a low background radiation environment on biochemical and biological responses in V79 cells.

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanità (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.

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.

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.

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 (31)P 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 treatment induces apoptosis or expression of a more differentiated phenotype on different fractions of cultured fetal rat hepatocytes.

The present study reports the effects of retinoic acid (RA) on cultured fetal rat hepatocytes. We show that RA treatment induces both differentiation and apoptosis. Hepatocytes cultured for 48 hours in the presence of 5 microl/L RA form junctional complexes in the areas of contact between neighboring cells and develop bile canaliculi, typical features of mature and well-differentiated cells. At the same time, about 20% of cells are induced to die by apoptosis, and the percentage of apoptotic cells increases according to the concentration of RA used and the duration of treatment. The induction of apoptosis, studied at the morphological and biochemical levels, revealed that, in our system, the classical compaction of chromatin occurs only during the final stages of the process; instead of the common marker of apoptosis, i.e., the "DNA ladder" pattern of fragmentation, megabase-sized fragments were found. These observations provide further evidence of the existence of fundamental differences in the mechanisms of apoptosis among cell types. To investigate the molecular mechanism of the effects of RA, we evaluated the expression of two proteins, c-myc and p53, which are known to be involved in both cell differentiation and apoptosis. The data obtained show that the amount of p53 remained unchanged after RA treatment. On the contrary, a dose-dependent reduction in c-myc levels was found, suggesting that RA action may be mediated by modulation of this oncogene. Our findings regarding the apoptosis-inducing effect of RA, which was not found in adult hepatocytes, suggest a possible relationship between this phenomenon and the proliferative capacity and/or differentiation state of hepatocytes.

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

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.

Modulation of proliferative activity and amino acid transport in chick embryo hepatocytes by EGF and retinoic acid.

In this work, the proliferative activity of chick embryo hepatocytes and its regulation by factors affecting cell growth, such as epidermal growth factor (EGF) and retinoic acid, were studied. The transport of nutritional molecules, like amino acids, was also investigated; in particular, the uptake mediated by the Na+ -dependent system A, known to be under hormonal and growth factor control. Moreover, some steps in the transduction pathway of mitogenic signal were analyzed, both for EGF and retinoic acid treatment. Results obtained suggest that chick embryo hepatocytes growth in an exclusive serum dependent way shows an early sensibility to EGF stimulation as regards to the proliferative activity and amino acid transport, responds to retinoic acid treatment and lack of contact inhibition. Moreover, as far as the signal transduction is concerned, at early stages, they do not seem to be able to utilize the same signal molecules as observed in adult life.

Expression of the asialoglycoprotein receptor in cultured rat hepatocytes is modulated by cell density.

The influence of cell density on expression of the asialoglycoprotein receptor system in primary cultures of rat hepatocytes was evaluated by measuring the level of the receptor specific mRNA. When the hepatocytes are cultured at high cellular density and are not in a proliferative condition, the transcript molecules of the receptor appear increased about 50% with respect to the low plating density, indicating a modulation of asialoglycoprotein receptor expression at transcriptional level. Such control may be dependent on surface molecules involved in cell specific reassociation, since it is well known that cell contacts play a significant regulatory role in differentiated cells.

Regulation of asialoglycoprotein receptor expression in rat hepatocytes cultured under proliferative conditions.

The expression of many glycoproteins on the surface of hepatocytes has been related to the state of differentiation of the liver. In particular, the asialoglycoprotein receptor (ASGP-R) is modulated in many physiological and pathological conditions in which hepatocyte proliferative activity is modified. We studied ASGP-R expression in primary monolayer cultures of rat hepatocytes stimulated to proliferate by the addition of epidermal growth factor and insulin. In proliferating hepatocytes the receptor concentration on the cell surface was lowered, with no modifications in its distribution; similarly, both the binding activity and the amount of specific transcripts were decreased. The decreases were related to the peak of cellular growth, as judged by [3H]thymidine incorporation into DNA. The results suggest the presence of a cell cycle-dependent ASGP-R expression also in in vitro systems; this control could be exerted by a decreased availability of receptor-specific mRNA molecules or on the stability of transcripts.

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes.

[1-14C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP3) and calcium, is concerned, substantial differences were found: EGF increased IP3 production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP3 and calcium transients, suggesting that this transduction pathway is not activated during fetal life.

Modulation of the expression of galactose-specific receptors on Kupffer cells after partial hepatectomy and zymosan stimulation.

Partial hepatectomy (PH) and a single intravenous injection of zymosan were used to provoke expansion of the Kupffer cell population in rat liver. The number of Kupffer cells per unit of area of liver increased after both stimulations. During these growth phases the number of binding sites for galactose-exposing ligands was studied on Kupffer cell plasma membranes. Both partial hepatectomy and zymosan stimulation affected the binding of lactosylated bovine serum albumin conjugated to gold particles (LacBSA-Au5). The reduction observed depended on the time after the PH or zymosan injection studied. Low levels of binding sites were found at 24 h from PH and 5 days after zymosan stimulation and represented only 1/4 or 1/10 of the binding sites expressed on the cell surface of Kupffer cells from normal adult rats. In addition, in both experimental situations we observed that the clustered distribution of gold particles typical of adult Kupffer cell was changed.

Amino acid uptake regulation by cell growth in cultured hepatocytes isolated from fetal and adult rats.

Amino acid uptake mediated by system A was studied in cultured fetal and adult hepatocytes, subjected to growth stimulation by EGF and insulin, or to growth inhibition by high cell density. The mitogenic stimulation induced a strong transport increase only in fetal cells, while the cell density-dependent growth inhibition, probably mediated by molecules present on adult hepatocyte membranes, provoked the decrease of amino acid uptake only in the adult cells. The results indicate that the different modulation of amino acid transport by cell growth is dependent on the age and the differentiation stage of hepatocytes.

Epinephrine regulation of amino acid transport in rat hepatocytes isolated during development.

The effect of epinephrine on the amino acid transport mediated by system A was investigated by determining the uptake of 2-amino [1-14C]isobutyric acid (AIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the hormone increased AIB uptake, enhancing the Vmax, while Km was unchanged. This effect was evident in cells from adult, 18- to 20-day-old fetus, and neonate rat. Actinomycin D or cycloheximide abolished the hormone dependent increase. Experiments carried out with alpha- and beta-antagonists showed that the effect of epinephrine was beta-mediated in fetal life and alpha-mediated in adult life. Membrane binding experiments showed a higher value for epinephrine and beta-agonist dihydroalprenolol in the fetus versus the adult. The calcium depletion obtained after cell incubation with EGTA or calcium ionophore A23187 reduced the hormonal stimulation in the adult, and was ineffective in the prenatal period. An involvement of cAMP was present in the epinephrine modulation of AIB transport, both in adult and in fetal life.

Modulation of asialoglycoprotein binding activity in livers of pregnant or estrogen-treated rats.

This report deals with the modulation of activity and expression of the hepatic asialoglycoprotein receptor, in pregnant or diethylstilbestrol-treated rats. The results show a two-fold increase in the total cell associated binding activity, both in pregnant and in estrogen-treated animals, with respect to normal values. On the contrary the surface expression was shown to be strongly enhanced only in the liver of pregnant rat. Therefore the modulation shown by this receptor system in pregnancy seems to be only partially estrogen-dependent.

Age-related changes in the galactose recognition system in rat liver cells.

In the present report, the galactose recognition system in 3- and 24-month-old rat livers has been studied with in vitro and with in situ binding experiments and in vivo ligand uptake. The galactose-specific receptors were visualized by using colloidal gold particles of different sizes (5 nm, 17 nm and 50 nm mean diameter), coated with lactosylated bovine serum albumin (LacBSA) as electron dense ligands. The data show that aging affects the expression of galactose-specific receptors and the rate of endocytosis. In the in vitro and in situ experiments hepatocytes and liver macrophages from old rats on the plasma membrane express a decreased number of binding sites with respect to those present on the adult rat liver cells. Approximately 80% of the total number of liver macrophages from aged rats show a binding distribution which is very different from the typical clustered receptor arrangement: the binding sites appear as single or small clusters of gold granules. As a direct consequence of the altered pattern of the receptor distribution, the capacity of liver macrophages from 24-month-old rats to internalize the larger ligands (17 nm and 50 nm) is decreased, as compared with adult rats. Aging, therefore, influences the galactose recognition system in two ways: (i) by decreasing the number of binding sites expressed on the liver cell surfaces; and (ii) by modifying the receptor distribution on liver macrophages and consequently affecting the internalization of galactose exposing particles.

Some aspects of glycosylation in rat liver during pregnancy.

The aim of the present report was to analyze the pattern of glycoprotein synthesis in rat liver on 19th and 22nd day of pregnancy by following the incorporation of 14C-glucosamine and 3H-galactose into isolated rat hepatocytes, the N-acetylglucosaminyl-1-P and galactosyl transferase activities, and the liver content of dolichol and dolichyl-phosphate. The data obtained show a decrease of precursor incorporation into glycoproteins during the last period of pregnancy; this decrease is independent of enzyme activities. The dolichol content increases and the dolichyl-phosphate content, usually considered as rate limiting for glycosylation, decreases. These results, present in other conditions of proliferation and differentiation of rat liver, could explain the differences in membrane organization, the increase of hepatic proteolysis and the alteration in secretory activity during the last phase of gestation.

Changes in lipoprotein binding and uptake by hepatocytes during rat liver regeneration.

The binding and uptake of cholesterol enriched lipoproteins by isolated hepatocytes was decreased at 16 hours after partial hepatectomy, with a tendency to return to control values as the regeneration proceeds. The number of lipoprotein binding sites of total cellular membranes remained similar to control at 16 and 24 hours. The plasma lipoprotein pattern, determined by electrophoretic analysis, showed a lower per cent of very low density lipoproteins (VLDL) and a higher per cent of low density lipoproteins (LDL) at 16 and 24 hours post-partial hepatectomy. At these times, plasma lecithin: cholesterol acyltransferase (LCAT) activity was decreased. It is intriguing to suggest that the regenerating liver could regulate the blood lipoprotein pattern and the uptake of lipoproteins by modulating the surface expression of the receptors.