Stages of activation of hepatic stellate cells: effects of ellagic acid, an inhibiter of liver fibrosis, on their differentiation in culture

To further explore that hepatic stellate cell (HSC) activation results in physiological protection against environmental insult, the profile of differentiation of HSC has been examined upon treatment with ellagic acid (EA), a plant-derived antioxidant that shows multiple protective effects during liver disease. Sparse rat liver cell cultures were grown in media containing EA (3, 6, 30 and 100 microg/ml) and, as controls, without EA, and inspected until day 7 in culture. The cells were double-labelled with antibodies against glial fibrillary acidic protein (GFAP) and smooth muscle alpha-actin (SMAA), marker proteins of quiescent and activated HSC, respectively. In EA-free culture conditions, the quiescent (SMAA-/GFAP+) HSC transiently acquired a semi-activated (SMAA+/GFAP+), phenotype and were further transformed into activated (SMAA+/GFAP-), pleomorphic HSC. Up to a concentration of 30 microg/ml, EA induced an early synthesis of SMAA in all HSC and inhibited their morphologic differentiation and individual growth throughout the culture period. At a concentration of 6 microg/ml, EA supported the semi-activated (SMAA+/GFAP+) phenotype of HSC throughout the culture period, whereas treatment with high EA concentrations (30 microg/ml) resulted in an early loss of GFAP expression. In conclusion: (i) the uniform response of HSC to EA by mild activation adds functional significance to cellular features preceding the transformation of HSC to myofibroblasts; (ii) the high sensitivity of HSC to EA treatment suggests their involvement in any mechanisms of protection by this antioxidant; (iii) the maintenance of HSC morphology might be one of the factors playing a role in the prevention or slowing down of liver fibrosis; (iv) because the effects of EA are concentration- and time-dependent, an arbitrary usage of this antioxidant is a matter of potential concern; (v) the various patterns of HSC activation observed might correspond to distinct activities of these cells, which, in turn, might lead to different outcomes of liver fibrosis.     

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo.

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)-positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.     

Hemodynamic changes in immobilization stress

In chronic experiments on 75 Wistar, August and randombred rats hemodynamic changes were examined during 30-hour immobilization stress. The ECG was recorded and arterial blood pressure measured. The basic hemodynamic characteristics were determined with the help of the previously implanted ultrasonic blood flow probes. Analysis of hemodynamic changes in animals resistant, adapted and prone to stress demonstrated that changes in the total peripheral resistance play the leading role in the disturbance of the arterial blood pressure control. It was established that a progressive lowering of arterial blood pressure resulting from the abruptly reduced total peripheral resistance is the main and the most frequent cause of death of animals exposed to immobilization stress. At the same time the cardiac hemodynamic component may play an essential role in the mechanism of death. This component may include either progressive bradycardia or a combination of an ischemic myocardial damage and reduced total peripheral resistance.     

Acquisition of blood--tissue barrier--supporting features by hepatic stellate cells and astrocytes of myofibroblastic phenotype. Inverse dynamics of metallothionein and glial fibrillary acidic protein expression

A number of similarities between astrocytes and hepatic stellate cells (HSC) rose the question whether or not the protective barrier features of blood-tissue interface may be provided by HSC as well. To test this hypothesis, we investigated the presence of metallothionein (MT), a functional marker of blood--brain barrier, in HSC in situ and in cell culture and compared the results with those obtained with astrocytes. The dynamics of MT expression in cultured astrocytes and HSC was investigated by simultaneous labelling of the cells with a monoclonal antibody (MAb MT) against a lysine-containing epitope of the cadmium-induced monomer of MT-I from rat liver and antiserum against glial fibrillary acidic protein (GFAP). Cell activation was estimated by the presence of smooth muscle alpha-actin (SMAA). In immunoblotting, MAb MT recognized monomeric MT protein and proteins in the 30-kDa range; both bands were pronounced in brain and barely visible in liver homogenates. In situ, MAb MT reacted with very few perivascular cells situated in the parenchyma of the liver. Double immunolabelling of brain slices with MAb MT and antiserum against GFAP showed large areas of brain containing cells expressing both MT and GFAP. However, there were also regions in the brain where the cells produced solely GFAP or MT. In liver cell culture, MT was absent from HSC and hepatocytes in early periods of cultivation, during which the cells maintained their original features; however, MT was expressed strongly in HSC during their activation under prolonged culture conditions. Inversely, in astrocytes MT was expressed during early culturing and disappeared from the cells together with SMAA in late culture when GFAP was upregulated. These results suggest that the acquisition of myofibroblastic features by perivascular cells empowers them to establish a protective blood-tissue permeability barrier. In addition, this study shows that, at least in cell culture, an enrichment of perivascular cells in GFAP results in the disappearance of protective functions.     

An extremely acidic copper-containing protein from chromaffin granules

The contribution to magnetic shieldings of the anisotropy of the atomic susceptibility tensors is calculated and added to the ring current effect for nuclei located in a plane at z = 0.35 nm above the surface of the conjugated rings of the aromatic amino acids of proteins and of porphyrin. The variation of this contribution with the value of z is compared to the variation of the ring current effect.     

The immunoreactivity of glial fibrillary acidic protein in mesangial cells and podocytes of the glomeruli of rat kidney in vivo and in culture

In this study the presence of glial fibrillary acidic protein (GFAP) in kidney is for the first time demonstrated in cryostat sections and cultures of isolated glomerular explants derived from rat kidneys. In double immunolabelling analysis of adult rat kidney sections using antiserum against GFAP and monoclonal antibody (mAb) against vimentin or desmin, the presence of immunoreactivity for GFAP could be observed in the glomerulus of the kidney and vascular cells situated in the peritubular space which expressed vimentin and desmin. Labelling of the sections with absorbed antiserum against GFAP completely abolished the staining in all these cells. The mAb against GFAP, clone GF12.24 which is known to label GFAP both in neural and non-neural cells, recognised its antigen only in the cells located in glomeruli. The investigations performed on early 2- or 3-day-old cultures from glomerular explants revealed different patterns of staining for GFAP in mesangial cells and podocytes: weak filamentous in mesangial cells and a strong non-filamentous perinuclear pattern in podocytes. Due to prominent perinuclear expression in podocytes GFAP may be considered as a marker of these cells. A different pattern of distribution of immunoreactivity for GFAP in podocytes and mesangial cells might be due to function-related posttranslational modifications of GFAP resulting in assembly or disassembly of GFAP filaments. The different pattern of staining for GFAP in the podocytes and mesangial cells, cells which exert a different influence on the capillaries of the glomeruli, suggests a role for GFAP in regulation of the tension and permeability of vascular walls. Previous investigations and present studies hint at GFAP as being a general marker of perivascular cells.     

Two copper-containing proteins from white and gray matter of brain.

The procedure for the isolation of two water soluble copper-containing proteins from the white and gray matter of bovine brain is described. One of the proteins, cerebrocuprein I, is superoxide dismutase; and three molecular forms of this enzyme are to be found in brain. The other protein present in gray and white matter is devoid of superoxide dismutase and amine oxidase activities. The amino acid composition, molecular weight, isoelectric point and copper content of this protein were determined. The effect of some agents, pH and thermal treatment of the optical and EPR spectra of the protein were also studied. The copper of the protein may be removed and the holoprotein reconstituted again from apoprotein and copper. The results obtained led to the conclusion that in brain a new copper protein is discovered, which is named neurocuprein.